Biopharma Expression Systems 080926

FIRST EDITION
Biopharmaceutical Expression Systems

and Genetic Engineering Technologies
Current and Future Manufacturing Platforms
by Ronald A. Rader
Title: Biopharmaceutical Expression Systems and Genetic Engineering
Technologies: Current and Future Manufacturing Platforms
Edition: 1st edition
ISBN: ISBN: 1-934106-14-3
Author: Ronald A. Rader (President, Biotechnology Information Institute;
Rockville, MD)
Publisher: Bioplan Associates, Inc.
2275 Research Blvd, Suite 500, Rockville, MD 20878
Web site: www.bioplanassociates.com
Managing Editor: Eric S. Langer
Layout and Cover Design: ES Illustration and Design, Inc., Arlington, VA
Date: November 2008
Copyright 2008 BioPlan Associates, Inc.
All rights reserved, including the right to reproduce in whole or in part in any form. No part of this publica-
tion may be reproduced, stored, in a retrieval system, or transmitted in any form or by any means, electronic,
mechanical, photocopying, recording or otherwise, without the written permission of the publisher.
This work and all of its contents are protected under U.S. and international copyright.
Contact the publisher to request permission to copy or extract any text, information or data.
The author and publisher cannot be held responsible for any consequences arising from any errors or omissions,
nor for any consequences arising from use of the information presented.
For information on special discounts or permissions contact:
BioPlan Associates, Inc. at 301-921-9074 or info@bioplanassociates.com
NOTE!
This reference is based on published and unpublished information. It is
recommended that readers confrm information, and obtain updates from
license holders.
Introduction and User Guide
T
his is the 1st edition of BiopharmaceuticalExpressionSystemsandGeneticEngineeringTechno-
logies:CurrentandFutureManufacturingPlatforms. Expression systems encompass the
technologies - biological materials and associated know-how - needed to genetically modify organisms
for the manufacture of recombinant proteins (including glycoproteins and antibodies). This book is
designed to be the single most informative source concerning commercial biopharmaceutical product
manufacturing-related expression systems and basic engineering technologies, with emphasis on those
currently used for biopharmaceutical manufacture and those available for commercial licensing for
this purpose; providing basic information for the knowledgeable user to determine relevance for their
applications; conduct further research and/or contact technology licensing sources.
The primary goal is to inform the user of the many technologies in commercial use and those claimed
to be useful for commercial-scale manufacture of biopharmaceutical products, rather than provide
detailed or comparative information about each. This directory is the result of multiple man-months
of cumulative effort in information acquisition, organization and analysis. As such, it is a high value-
added product that should save you considerable time and effort in finding technologies relevant to
your interests. It should reliably cover relevant technologies currently being used commercially, those
being actively offered for licensing, those discussed in industry news sources and review articles, and
those offered by leading genetic engineering and bioprocessing technology licensors. However, it does
not cover every relevant published or patented technology.
Coverage - Simply stated, coverage concentrates on host cells/organisms, basic genetic engineering
methods, recombinant constructs and the many technologies available to enable or improve expression
of desired proteins, including glycoproteins and antibodies. This directory concentrates on the
core genetic materials (e.g., host cell lines and organisms) and related methods and materials, e.g.,
vectors, promoters, selection and amplification methods, chaperones, etc., used or claimed useful for
commercial-scale manufacture of biopharmaceutical products, primarily recombinant proteins and
monoclonal antibodies. Thus, this directory concentrates only on what is used or needed for upstream
manufacture (and nothing else).
This directory includes broad platform technologies, generally defined by the living host cells/
organisms being used, which may be natural or genetically modified to begin with; and the basic
genetic engineering technologies needed to get the desired gene sequence(s) into these hosts and get
these genes efficiently expressed (transcribed and translated) for commercial-scale manufacture. Thus,
this directory includes a number of specific genetic engineering technologies, e.g., vectors, promoters,
chaperones, affinity fusion protein purification schemes, etc., useful with all, some or specific platform
technologies/host systems.
B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es
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Technologies involve or can be defined or viewed in many ways or on many different levels. For
example, one may broadly refer to yeast or baculovirus expression vector technologies, actually a
grouping or classification of multiple technologies. And very often, what is referred to as a specific
technology actually involves multiple components, each of which may be considered a technology, e.g,
be separately available for licensing. In most or nearly all cases, technologies have been described in or
exemplified by patents. Technologies involve know-how or enabling knowledge and related information.
With biopharmaceutical manufacturing and genetic engineering technologies, this invariably involves
information, e.g., methods and gene/protein sequences, often embodied in genetic constructs and culture
collection deposits. In the biopharmaceutical area, just about every technology of interest has been or is
in the process of being patented; and most technology acquisition or other technology transfer involves
patent licensing. In many cases, all one needs to effectively acquire rights and implement a desired
technology is to license related patents. In many other cases technology acquisition/licensing should
involve or requires initial or even continuing technical assistance from the inventors or the organization
handling licensing.
Coverage includes both technologies currently in predominant use for biopharmaceutical product
manufacture, with these primarily based on use of E.coli, Chinese hamster ovary (CHO) cells and yeasts,
primarily Saccharomycescerevisiae, and new and upcoming alternative platforms/hosts, most of which
have not yet been adopted/adapted for commercial-scale manufacture. Much of the older technologies,
particularly those in use since the 1980s (including most E.coli, CHO and yeast technologies), have
in recent years either lost or will soon lose patent protection. Many users of this directory will likely
be interested in these proven, regulatory agency-familiar, cheap (now or soon no licensing expenses
involved) but, in many respects, inefficient technologies. Most, if not most, directory users are presumed
to be interested in new alternatives and/or significantly improving current in-house platform technologies,
e.g., by adopting newer technologies offering higher yields.
What is not included - If a technology does not involve genetic materials and their manipulation,
generally host cells/organisms and genetic constructs or methods, it has not been included, no matter how
relevant to biopharmaceutical manufacture. Thus, this directory does not include;
a) technologies relevant to specific products, e.g., product-specific gene/protein sequences; only
technologies relevant to manufacture of all or broad classes of proteins, including glycoproteins and
antibodies.
b) protein engineering or other molecule design technologies, unless substantially involving commercial-
scale protein expression. Thus, nearly all methods for designing and predicting protein structures are not
included.
c) protein screening technologies, including selecting for desired active agent/product characteristics. An
incredible number and diversity of screening technologies are available, but are not included.
d) some rather generic genetic engineering, molecular biologic laboratory technologies, with these
sometimes discussed in brief generic entries. For example, there are hundreds of different chemical
and physical agents and related methods used for transfection or modifying cells so that vectors or other
genetic constructs get to and act upon genetic material within cells. Most of the related reagents and
materials are readily available from multiple commercial vendors/reagent sellers, and most of the methods
are readily available in standard references for molecular biology procedures.
Various basic, broad genetic engineering and molecular biology methods have been included where these
have been patented and/or require taking a license for commercial use. Many directory users will be
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unpleasantly surprised to learn that many of the most basic genetic engineering and molecular biologic
method and reagents are patented, and require taking royalty-bearing licenses for commercial use.
e) generic, non-genetic engineering-based methods for host cell/organism modification, e.g., non-
targeted or random mutation-based methods; e.g., exposure to mutagens and selection for desired
characteristics or adaptation to specific; e.g., protein-free, culture media by growth, repeated passages
and selection of adapting cells/organisms.
d) microorganism and cell culture, culture media, fermentation, and related bioreactor and fermenter
technologies. This directory does include various microbes, other organisms and plant, insect and
animal cell lines, many adapted to specific types of culture or bioreactors, e.g., adherent or suspension
culture, and/or adapted to specific types of culture media, e.g., serum- or protein-free media; but does
not include technologies related to bioreactors, fermentors, bioreactor/fermentor control, etc.
e) downstream technologies, including purification. This directory does not include separation,
purification, formulation, viral inactivation or other downstream technologies. This directory does
include genetic and protein expression-based methods for downstream processing, particularly
purification using fusion proteins for affinity-based purification, but does not include chromatography
and other technologies for protein purification.
In many cases, technologies, particularly those being offered for licensing, were described as such
by their owners/licensors; and the author generally followed how inventors/licensors described their
technologies. In many other cases in which a technology description is not clear, this author had to
identify and define technologies. Thus, the author often defined and developed his own technology
descriptions, presuming that essentially every/any technology, particularly those fully publicly disclosed
(e.g,. in patents), is available for licensing (for the right price).
Luckily, the biotechnology industry is a relatively open market for technology licensing, i.e., most every
non-product-specific technology is available for licensing, if one bothers to ask. However, there are
some exceptions in certain areas, notably with higher plants, e.g., field crops, and transgenic mammals,
where some companies simply hoard (do not license) their technologies or are very selective in their
licensing., e.g. not licensing them to companies that might be worthy competitors.
Monographs Content - Descriptive entries are provided for ~340 technologies. Data fields are:
1) Title - The various names of associated with technologies and major components are included,
optionally followed by a hyphen and the author's annotation of the host/organism system(s) used and/or
special capabilities of the technology (e.g., glycosylation; antibodies manufacture).
2) Organizations involved - The major organizations involved are listed along with characterization of
their role or involvement in the technology, e.g,. licensor, patent assignee, research, etc.
3) Description - A summary of available information about the technology concentrating on
functionality, improvements provided, etc.
4) Use with - Brief characterization of the main host cells/organisms used with the technology.
5) Use to make - A brief characterization of the types of products the technology is designed or claimed
to be useful for.
6) Background - An optional field presenting claimed benefits or desirable characteristics of the
technology.
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7) Patents - Information about relevant patents.
8) Licensing information - Information about licensing contact(s), optionally with information about
related commercial actitivies, e.g., know licensees.
9) Products made with this tech. - An optional field presenting information about biopharmaceutical
products made using/incorporating the technology.
10) Further info. - An optional field usually presenting citations to related publications.
Organization of Monographs - The monographs are divided into two main sections:
1) The first section presents broadly-enabling, platform-type technologies, particularly novel host cells
and organisms.
2) The second section presents more specific, supporting and component technologies. These may be
broadly generic, applying to diverse hosts/platforms, or applying to multiple or just one major host/
platform.
Note, these divisions represent purely subjective decisions on the part of the author! It is often very
difficult to determine the relevance and utility of these technologies. What may be presented as a more
specific, supporting or component technology, e.g., vectors or promoters useful with a specific organism
or class of organisms, may along with other technologies be the single critical components enabling or
defining a new manufacturing platform technology.
Within each of the two main technology sections, monographs are loosely classified or grouped by
broad platform technologies, generally host cell/organism classes, e.g., E. coli, yeasts, mammalian
cells, etc. However, keep in mind that most technologies are or can be presumed to either be relevant to
multiple broad platforms, as is often presented in monographs, or may actually be relevant to just one
specific platform, e.g., vectors claimed useful with yeasts may actually be only or primarily useful with
S.cerevisiae or another yeast (but available information does not make this clear). The author generally
followed the lead of available information from the licensor, including patents, in terms of describing the
utility of specific technologies.
Indexes - The following indexes are provided:
1) Company/Organization
2) Subject
3) Primary Host Systems
See the text at the beginning of each of these indexes for further information about their coverage,
conventions and limitations.
Information Sources Used - The primary source for the information in this directory was documents
collected by the author specifically for this purpose over an approximate 3-year period. The author of
this directory is also the author of BiopharmaceuticalProductsintheU.S.andEuropeanMarkets, the
only reference book/database concerning biopharmaceuticals (now in its 6th edition, 2 vol., 1602 pages.
Besides deriving information about biopharmaceutical products manufacture from this source, as part of
developing/maintaining this publication the author has long engaged in a continuous intensive competitive
intelligence gathering and analysis program (i.e., he intensively reviews the world's press releases,
industry newsletters, meeting abstracts and every other relevant publicly available information source).
Even before starting recent work on this directory, the author had over 2,500 documents collected for use
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in developing this directory. Thus, the author is confident that relevant technologies discussed in industry
publications and at industry-oriented conferences in recent years have been included.
The monographs were largely assembled by modifying and piecing together text retrieved from diverse
sources, mostly those available on the Internet (and within the bounds of fair use). Thus, those using this
directory and doing their own research will likely be able to recognize text adapted from or extracted from
Web sites, patents, articles, etc. However, in all cases, the author made sure to provide additional, value-
added information and analysis. This includes providing what should be useful contact information,
including use of the membership directory of the Licensing Executive Society (LES), the database
of registered patent attorneys/agents at the U.S. patent office Web site, and otherwise finding E-mail
addresses for relevant corporate contacts.
Other information sources and methods used in developing this directory include:
1) meeting announcements and abstacts - The world's major biotechnology-related conferences,
particularly those with a commercial orientation or involving relevant sessions, have been monitored for
several years.
2) literature searching - Some basic searching of the peer-reviewed biomedical literature, e.g.
PUBMED, was performed, including searching for overview and review-type articles concerning broad
platform technologies. In many cases, the biomedical literature was also searched concerning specific
technologies.
3) patent searching - Much searching of U.S. patents and applications (primarily using U.S. patent
office full-text databases) and international patents/ applications (primarily using EspaceNet) was
performed. Besides patents often being required to explain or obtain basic descriptive information
concerning technologies, patents very often provide analyses of related prior art (previous or competing
technologies).
4) Web sites - The Web sites of essentially every company/organization included in this reference, and
many others, were examined for technology-related information and to determine optimal contacts for
licensing-related inquiries. This included checking the online technologies available for licensing listings
of those organizations well known as sources of bioprocessing and genetic engineering technologies/
patents. For example, the University of California and NIH have consistently been among the leaders in
obtaining U.S. genetic engineering patents; RCT is the licensor for several basic platform technologies,
and the Boyce Thomson Institute and Texas A&M University are sources for various insect cell/
baculovirus expression technologies.
5) federal research funding and contracts - CRISP and other databases covering NIH and other federal
agency research funding and contracts were searched. Thus, the various expression systems being
developed largely with federal funding, mostly related to biodefense, are included, e.g., the DARPA,
DOD, and NIAID, NIH, grants and contracts seeking to develop systems for rapid manufacture of large
amounts of recombinant proteins, e.g., millions of doses of vaccines in just several months.
6) licensors/technology sources - The licensing contacts of hundreds of organizations, including the
majority of those mentioned, were contacted by the author by E-mail, requesting public/publishable
information about relevant technologies, particularly those available for licensing.
As further discussed, there are various reasons why many companies (vs. universities) are hesitant
to provide information for directories. Many are unprepared for anyone requesting nonproprietary
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information about their technologies available for licensing (making this directory all the more valuable).
And despite it being counter-productive, technology transfer/licensing professionals, and many scientists/
inventors involved in licensing and invention marketing simply prefer to avoid disseminating information
about licensing opportunities. Many licensing professionals feel that technology transfer/licensing is best
practiced, with public information dissemination viewed as a less sophisticated approach.
Information Sources Not Used; Limitations/Caveats- Bound by limitations of time and expenses, the
author did not use a number of relevant information resources and acquisition methods (that directory
users may want to follow-up with). For example, with over 300 technologies, if an information resource
or acquisition method was not free, i.e., involved spending money, it almost certainly was not used. Thus,
the author did not use high-end, fee-based online databases, e.g., DERWENT patent databases, online
versions of ChemicalAbstracts, etc. Some fee-based databases were searched using the online databases
at a local university library, along with document delivery services, but primarily to retrieve review
articles, not to retrieve information about specific technologies. Otherwise, the author concentrated
on finding and summarizing information to provide useful, but not all, information about technologies'
functions/characteristics, advantages and ownership. Thus, information retrieval was not exhaustive - the
author stopped looking when seemingly adequate descriptive and ownership information was retrieved.
Users should exercise caution in interpreting what technologies are actually relevant or useful for! The
author generally describes technologies much as described by their licensors and/or inventors. Many
times, licensors/inventors tend to restrict their claims about functionality and utility only to what they
have studied or documented, while other times they may be too expansive in their claims. The author's
descriptions reflect the content of inventions-available-for-licensing descriptions, patent descriptions and
claims, i.e., available information. For example, some descriptions (reflecting their source) are probably
too broad in their claims, e.g., may be primarily or actually relevant to one or a few members of a class
of organisms, while licensors/inventors claim utility for an entire class of organisms (e.g., an invention
actually relevant to only human cells may be claimed as relevant to all mammalian cells or eukaryotes).
Conversely, inventions may be described as relevant to xyz specific organisms or uses, but may actually
be relevant to many others (e.g., an invention claiming relevance to E. coli may actually be useful with
all bacteria or all organisms).
Also, be aware that many major sources of biopharmaceutical processing technology simply make it
hard for anyone to find and approach them or figure out what licensable technologies they have. And,
many technology sources are seemingly only interested in dealing with major players. For example,
essentially all of the long-surviving biotechnology/biopharmaceutical companies, i.e., those around
for several decades, have amassed considerable portfolios of patented and also unpatented proprietary
manufacturing-related technologies. However, few technologies from these major companies, e.g.,
Genentech, Amgen, Biogen Idec, Wyeth/Genetics Inst., J&J/Centocor, etc., are included in this directory.
Adequate information is simply unavailable, with essentially none of these companies publicly disclosing
their manufacturing- or basic genetic engineering-related technologies available for licensing, their
licenses granted or responding to the author's inquiries. Similarly, most every contract manufacturing
organization (CMO) has likely developed in-house proprietary technology and/or licensed-in and is
able to offer sublicenses or access to technologies from others. These technologies have been included
where information was available, but following the general pattern, seemingly few CMOs bother to
disclose their proprietary technologies in their public information or only do it in vague generalities [yet
another paradox in the marketing (or lack of it) of biopharmaceutical manufacturing and related genetic
engineering technologies].
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For many users, examining all entries will be the most effective way to use this directory, in addition
to using its organization into topical sections and its indexing. Knowledgeable persons will likely be
able to see and make their own connections and conclusions about the relevance of technologies. Many
technologies that may seem irrelevant, e.g, from their titles, placement and/or indexing, may actually
provide new ways of approaching problems or provide improvements that you had not been looking for.
Finding Further Information - So, with this directory designed to get you started, what can or should
you do after finding technologies of interest. Obviously, much depends on your particular interests. Your
options include:
a) Search the world's publications, Web sites, patents, etc. Use Google and one or more complimentary
Web search engines. Search the biological and chemical literature, e.g., PUBMED, use the online
versions of ChemicalAbstracts, BiologicalAbstracts, and do not ignore the chemical engineering
literature, which may also have relevant information. Whether from going through their Web site and/or
searching the Web, read up about the licensor organization and any related licensing track record. Use
patent databases, including better fee-based ones, to retrieve further information about patents, e.g.,
what is their status, which countries are patents being sought in, etc. For old(er) technologies, e.g., those
invented in the 1980s or with patents granted about 17 or more years ago, check to whether patents
have expired in countries of interest. If so, you may not need to take a formal license. If a technology
involves a biological material, e.g., cell line, and even if it is in the public domain, it cannot hurt to license
this from the original source, vs. getting a derivative from a culture collection or commercial vendor.
This may save considerable testing and avoid documentation problems with FDA and other regulatory
agencies.
b) Network with and/or delegate or pass-upwards your inquiries to others in your organization,
particularly your own technology transfer office or professionals. Licensor contacts, particularly licensing
professionals, are more likely to respond to inquiries from other technology transfer professionals, patent
attorneys, corporate executives, etc., vs. inquiries from scientists or mid-level managers.
c) Make contact with the licensor - The Company/Organization Index includes a contact point to initiate
licensing-related inquiries. The response you get from these or other contacts may depend on your
organizational affiliation, e.g., whether you are perceived as a potential client or competitor, and whether
you are perceived as having licensing negotiation authority. Of course, it is always best to be prepared
and as knowledgeable as one can be when interacting with licensor contacts, many of whom are already
overworked with dealing with obtaining patents on inventions, negotiating licenses. It is probably best
to volunteer up-front to sign non-disclosure agreements, even if you are only seeking public information,
with this showing that yours is more a genuine licensing vs. simply an information or competitive
intelligence gathering request. If you don't get a prompt response, make personal contact because many
technology transfer professionals prefer personalized information dissemination and they prefer personal
contacts before they respond.
Most every licensor will or should be able to offer serious inquirers various options, ranging from sending
out information (which may require a non-disclosure agreement), material transfer agreements (MTAs) or
other standard agreements allowing access/release of materials, e.g, cell lines or vectors, for further study,
usually with many explicit limitations on use; licenses allowing limited in-house technology evaluation;
and other options short of a full licensing with big up-front licensing payments and royalties on sales.
d) Contact the inventor(s). Besides being the most knowledgeable, they are more likely to be scientists,
and will likely be more responsive at least in terms of providing you with public/published information
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and even discussing your potential interest/application. And, many inventors make themselves available
for you to hire as consultants or contractors.
e) If you want an outside expert(s)/consultant(s) to do further research and make initial contact, which
could include not disclosing your identity contact the publisher, BioPlan Associates, Inc.;
info@bioplanassociates.com; 301-921-9074.
Monographs Table of Contents
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Broad/Platform Technologies
Entry Number Monograph Titles
Cell-free systems
100 Cell-free expression, ATP regeneration system ...........................................................................19
101 Cell-Free Protein Synthesis (Cell-Free) ........................................................................................19
102 Large ribosomal subunit proteins, E. coli - cell-free systems ......................................................22
Broad, cross- or multi-platform technologies
103 In vivo linearization; InVoLin; Meganuclease Recombination System (MRS) - improved
transformation ..............................................................................................................................22
104 Vectors, site-specifc recombinase assembly - eukaryotes .........................................................23
105 Glycosylation, mammalian [Generic entry] ...................................................................................23
106 Aminoglycoside phosphotransferase marker; Neomycin phosphotransferase I (nptI) marker;
Geneticin (G-418) selection - universal ........................................................................................24
107 Cellulose binding domain (CBD) fusion protein affnity tags; pET-CBD - universal ....................25
108 Controllable Self-Cleaving Intein Derivative; IMPACT-CN; pH-sensitive self-cleaving fusion
protein affnity purifcation tag - universal ....................................................................................25
109 Cotransformation, Columbia University - eukaryotes, selection ..................................................27
110 Cre-lox mediated in vitro recombination; Cyclization Recombination/locus of X-over P1;
site-specifc recombination (SSR) - universal; genetic recombination .........................................29
111 deltaPhase; Elastin-like polypeptide (ELP) fusion protein tags - chromatography-free
purifcation; universal ...................................................................................................................30
112 Directed Nuclease Editor (DNE); Meganuclease Design - universal ............................................31
113 Dual expression vectors; Dual Affnity ReTargeting (DART) - antibodies; bacteria and
mammalian cells ...........................................................................................................................32
114 Expression enhancers, oligonucleotides - universal ....................................................................33
115 Expression factory; baculoworkstation - automated parallel expression ....................................33
116 Fusion protein expression systems; Affnity purifcation tags and chaperones - universal .........34
117 Fusion proteins, self-cleaving, pH-sensitive - universal ...............................................................34
118 Glutathione-S-transferase (GST) fusion protein affnity tags; pGEX vectors ...............................35
119 GUS reporter system (beta-glucuronidase); GUS control vector - prokaryotes; plant cells;
mammalian cells, non-vertebrate .................................................................................................36
120 Heat shock Promoters (HSPs); HSP chaperones ........................................................................37
121 His tags; Poly-Histidine fusion affnity tag technology; 6xHistidine-tag - univeral;
purifcation tags ............................................................................................................................37
122 His-tags; Poly-Histidine fusion affnity tag technology - univeral; purifcation tags .....................38
123 Hybridomas (Khler and Milstein) - monoclonal antibodies .........................................................38
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124 Lambda recombination protein; Homologous recombination......................................................39
125 Meganuclease Recombination System (MRS)/I-SceI; homologous recombination -
universal genetic recombination ..................................................................................................39
126 Mucin promoters; IIM14 and IIM22 - vector enhancers ...............................................................41
127 New Cabilly; Cabilly-Boss; Monoclonal antibody 2-chains expression - universal .....................41
128 phCMV vectors; GenePORTER - universal ..................................................................................46
129 Poly(A) polymerase .......................................................................................................................46
130 Promoters .....................................................................................................................................46
131 Recombinant DNA; Protein Expression; Cohen-Boyer ................................................................47
132 Reconstituting chemically orthogonal directed engineering (ReCODE) -
Unnatural amino acids; UAAs; E. coli; yeasts; mammalian cells .................................................49
133 Selectable markers; Selection of transformed cells; Reporter genes ..........................................50
134 Small Ubiquitin-like Modifer (SUMO) fusion protein tags; Split SUMOpro System;
Ubl-specifc protease - universal .................................................................................................51
135 Strep-tag affnity fusion protein tag; Strep-Tactin purifcation - universal ...................................53
136 Super core promoters; SCP1; Core promoters - the strongest [promoters] ever made ...........54
137 TAGZyme; dipeptidyl aminopeptidase I (DPPI; DAPase Enzyme; cathepsin C) -
cleavage of His tags; universal .....................................................................................................54
138 TEV Protease; Tobacco Etch Virus (TEV) NIa protease - cleavage of fusion
protein tags; universal ..................................................................................................................55
139 Transfection - universal ................................................................................................................56
140 Translation Engineering expression; CODA; Computationally Optimized DNA
Assembly; Hot Rod Genes; Controlled Ribosomal Pausing - universal ......................................57
141 White collar complex (WCC) promoter; UV light induction - universal ........................................59
142 Minos transposon cell transformation - insect larvae; also eukaryotes .......................................60
143 Coconut Express cell-free translation - plants .............................................................................60
Prokayotes
144 Bacterial cell expression technology (BCE); araB promoter - antibody fragments;
E. coli; prokaryotes .......................................................................................................................61
145 Subtilisin (psub) fusion protein tag expression; Profnity eXact expression - E. coli;
bacteria; yeasts; CHO cells ..........................................................................................................62
146 Tetracycline-induced expression repression - prokaryotes .........................................................63
Bacteria
147 Bacillus megaterium expression - E. coli alternative ...................................................................63
148 Bacillus subtilis, super-oxidizing strains; Thiol-disulfde oxidoreductases (TDORs);
Thioredoxin A (TrxA) depletion - disulfde bridge optimization.....................................................64
149 Bacterial artifcial chromosome (BAC) expression; pBAC vectors - bacteria ..............................65
150 Caulobacter crescentus expression; PurePro Caulobacter Expression System -
Caulobacter bacteria hosts ..........................................................................................................66
151 Clostridium Expression System; NTNH promoter from Clostridium botulinum ...........................67
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152 Flavobacterium heparinum expression - glycoproteins ...............................................................68
153 Lactococcus lactis htrA- expression - protease depletion ..........................................................68
154 Lactose (lac) promoters; Isopropyl-beta-galactosidase (IPTG) induction - bacteria ...................70
155 NIsin-Controlled gene Expression (NICE); Lactococcus lactis expression;
nisA promoter - antibiotic selection .............................................................................................70
156 P170 Expression System;Lactococcus lactis expression ............................................................71
157 Pfenex Expression; Pseudomonas fuorescens biovar I (MB101) - E. coli alternative .................72
158 Plasmids stabilization ...................................................................................................................74
159 Pseudoalteromonas haloplanktis TAC125 (PhTAC125) - cold expression ...................................74
160 Quasi-synthetic vectors; Synthetic gene sequences in vectors - bacteria ..................................75
161 Ralstonia eutropha expression; Alcaligenes eutrophus expression .............................................76
162 Rhodospirillum rubrum (bacterial) expression - membrane proteins ...........................................77
163 Staphylococcus carnosus expresion ...........................................................................................78
164 Subtilin Regulated Gene Expression; SURE competency - B. subtilis ........................................79
165 E. coli expression/vectors ............................................................................................................80
166 CASCADE expression; pALEX1 plasmids - E. coli.......................................................................81
167 Choline-binding fusion affnity tags - bacteria .............................................................................82
168 Clean Genome E. coli; Stripped-down E. coli .............................................................................82
169 GroEL,GroES chaperones; Chaperonins - proper folding; universal; E. coli ................................83
170 Methylobacterium extorquens (bacterium) expression ................................................................84
171 CANGENUS; Streptomyces (lividans and griseus) expression ....................................................85
172 Saccharomyces cerevisiae expression ........................................................................................86
173 Streptomyces stationary phase expression; SPE system; Streptomyces vectors;
Secreted Protease Production (SPP) System ..............................................................................86
174 Streptomycetes hyper-inducible expression; PnitA-NitR system; Caprolactam
induction - Streptomycetes .........................................................................................................87
Yeasts
175 ApoLife Yeast Expression; S. cerevisiae Twin Cassette Plasmids - antibodies in yeast ..............88
176 Arxula adeninivorans expression - alternative yeast ....................................................................89
177 Chrysosporium lucknowense expression; C1 Express Hyperproducing Protein
Expression System - fungi ...........................................................................................................90
178 CoMed system; Universal Yeast vectors; pCoMed vectors; Arxula
adeninivorans-derived TEF1 promoter .........................................................................................91
179 Fungal expression systems ..........................................................................................................92
180 GlycoFi technology; Next Generation Biotherapeutics - Pichia pastoris; yeasts;
glycosylation; antibodies ..............................................................................................................92
181 Hansenula expression (yeast) .......................................................................................................93
182 Hansenula polymorpha (yeast) expression - E. coli alternative ...................................................94
183 Kluyveromyces lactis expression; pKLAC1; Acetamidase/Acetamide selection;
K. lactis GG799 - yeast ................................................................................................................95
x|v
184 NeuBIOS expression; Neurospora crassa expression; NeuKARYON - flamentous fungi;
glycosylation; Cabilly-Boss workaround ......................................................................................96
185 Neurospora expression; cotA promoter - fungi ............................................................................98
186 Ophiostoma expression - ascomycetes fungi ..............................................................................98
187 Yeast expression systems ............................................................................................................99
188 Zygosaccharomyces bailii expression; Zbleu2 strain - yeast ...................................................100
189 EASYEAST; Saccharomyces cerevisiae strains - easy protein release ......................................101
190 Yeast cell lines and vectors; Saccharomyces cerevisiae cell lines - proper folding and
glycosylation ...............................................................................................................................101
191 Pichia pastoris expression .........................................................................................................102
192 VelociMab; EESYR expression system; FASTR cell lines - CHO expression optimization;
antibodies ...................................................................................................................................104
Plants
193 Plastid Transformation; Translation-based vectors (TBV) - plants .............................................105
194 Chlamydomonas reinhardtii chloroplast expression; promoter - algae, single-cell ...................106
195 Chlamydomonas reinhardtii expression - algae, single-cell .......................................................106
196 Drosophila Expression System (DES) - insect cell culture .........................................................108
197 Streptomyces lividans ................................................................................................................109
Eukaryotes
198 Antibiotic inducible promoters - eukaryotes ..............................................................................109
199 AttSite recombinases - precise gene insertion; eukaryotes .......................................................110
200 Bovine growth hormone (bGH) polyadenylation sequence - eukaryotes; expression
enhancement ..............................................................................................................................110
201 Expression enhancers; Copy number increase - eukaryotic cells .............................................111
202 Homologous Recombination; Knock-out/knock-in animals and cells .......................................112
203 Internal Ribosome Entry Sequences (IRES) RNA translation enhancers; pCITE vectors;
Cardiovirus 2A IRES; pIRES - eukaryotes ..................................................................................112
204 Light-switchable promoters .......................................................................................................114
205 Tetracycline (Tc; Tet) Expression Systems - eukaryotes.............................................................114
206 Zinc fnger DNA-binding proteins (ZFPs); ZFP Transcription Factors - gene modifcation;
mammalian cells; plant cells ......................................................................................................115
Protozoa
207 Ciliate Performance Expression System; CIPEX system;
Tetrahymena (protozoa) expression ...........................................................................................117
208 LEXSY; Leishmania tarentolae expression system - protozoa ...................................................118
209 Perkinsus marinus expression - protozoa express large proteins .............................................119
210 TetraExpress; Tetrahymena (protozoan) expression ...................................................................119
211 Tetrahymena thermophila (protozoan) expression .....................................................................120
xv
Animals, misc.
212 Milk protein promoter - proteins in milk of transgenic animals ..................................................121
213 Shrimp expression; Penaeus stylirostris expression; Taura Syndrome Virus (TSV)
IRES vectors - glycoproteins; antibodies ...................................................................................121
214 Transgenic rabbits - humanized polyclonal antibodies ..............................................................122
Mammalian
215 AmProtein vectors - stongest mammalian vector set; universal .............................................123
216 Anti-apoptosis expression system; BCL-xL or BCL-2 expressing cell lines -
CHO, NS0, BHK, SP2/0-Ag14 ..................................................................................................123
217 Autocatalytic cleavage sites; Mengo virus vectors; Scission cassettes - mammalian cells ......124
218 Cell adhesion optimization; cdkl3, siat7e, and lama4 genes - antibody-expressing cells.........124
219 CMV (human) promoter; Cytomegalovirus promoter; Complete Control Inducible
Mammalian Expression System - mammalian cells ...................................................................125
220 Cumate gene-switch; Q-mate Inducible Expression - mammalian cells ...................................125
221 Dihydrofolate reductase (DHFR) System - selectable marker/amplifcation;
CHO and NS0 cells ....................................................................................................................127
222 Flp-In expression system; FLP-Mediated Gene Modifcation in Mammalian Cells; FLP
recombinase - mammalian cells.................................................................................................128
223 Gene-Activated (GA) expression, in vivo - mammalian cells .....................................................129
224 Glutamine synthetase (GS) System - NS0, CHO, mammalian cells ..........................................130
225 GPEx Gene Product Expression Technology - mammalian; CHO .............................................131
226 MARtech; Matrix Attachment Regions; Scaffold Attachment Regions;
SARs - mammalian cells ............................................................................................................133
227 RheoSwitch Mammalian Inducible Expression System; RheoSwitch Ligand RSL1
promoter; Ecdysone receptor induction - mammalian cells; adjustable expression .................134
228 Selexis Genetic Elements (SGEs) - mammalian cell lines ..........................................................135
229 STabilizing Anti-Repression; STAR elements - expression enhancement; mamalian cells ........136
230 Ubiquitous chromatin opening element (UCOE) expression - mammalian cells .......................136
231 Whey acidic protein (WAP) milk promoters - mammals .............................................................138
Chinese hamster ovary (CHO) cells
232 ACE Expression System - MAb-expressing CHO cells lines .....................................................139
233 Boehringer Ingelheim High Expression System (BI HEX); CHO-DG44 ......................................139
234 CHO cell line (Puck); CHO-K1 cell line .......................................................................................140
235 CHO DG44 cells, DHFR-; CHO-DG44; DUK-XB11; CHO K1 DUX B11 (DHFR-) cells;
Dihydrofolate reductase selection/amplifcation, CHO cells ......................................................141
236 CHO SSF cell lines - adherent CHO cells; protein-free media ...................................................142
237 CHO Supercell; Targeted transfection; CHO DG44 cell line ......................................................143
238 CHOZn CHO DG44 cell lines - antibodies .................................................................................143
239 GS-CHO Protein Free System; CHOK1SV cell lines - protein-free media .................................143
xv|
240 Sympress expression; Human polyclonal antibodies (rpAB) - CHO cells ..................................144
241 Baby hamster kidney (BHK) 21 cells; ATCC CCL 10 .................................................................145
Hybridomas
242 Human MORPHODOMA; Morphogenics; Hypermutation; PMS2 gene screening,
modifcation - human monoclonal antibodies from hybridomas................................................145
243 Cell-free system - glycoproteins; hybridoma .............................................................................146
244 Ex-Cell EBx expression; EBx cells; Chicken embryonic stem cells;
Chicken EBx cells; Duck EBx cells ............................................................................................147
Chickens/Poultry
245 Chicken egg expression; Transgenic animals ............................................................................148
246 Chicken rimordial germ cells (PGCs) - Human proteins/monoclonal antibodies in chicken eggs ...
149
247 OVA System; Avian Transgenic Biomanufacturing - chicken eggs expresssion ........................150
248 Transgenic poultry; avian transgenesis and nuclear transfer - proteins in chicken eggs ..........152
249 Windowing Technology - transgenics avians/chickens..............................................................152
Human
250 CEVEC Amniocyte Production (CAP) expression; Human amniocytes, immortalized ...............153
251 bcl-2 (p21) overexpressing NS0 cell lines - NS06A1(100)3 cell line - antagonize
apoptosis; Mabs .........................................................................................................................153
252 Cell fusion, NS0 cells - antibodies .............................................................................................154
253 Cholesterol/3-ketosteroid reductase (p3-KSR) expression - cholesterol
selection/induction of NS0 cells .................................................................................................155
254 GlycoExpress human cell lines; NM-F9 cell line - glycolysis .....................................................155
255 Human primary preB lymphocytes; HupreB cells - human monoclonal antibodies ..................156
256 NS0 murine myeloma cell line ....................................................................................................157
257 NS0-PFCF cells; NS0 cells, protein- and cholesterol-free - antibodies .....................................158
258 PER.C6 expression; Extreme density (XD) Technology - glycoproteins; antibodies ..................159
259 Retrotransposon vectors - transgenic avian/chicken cell culture ..............................................161
260 Sp2/0-Ag14 cells - protein-free media; antibodies ....................................................................161
261 HEK 293 cell line, protein- and peptide-free (Hektor G) media; human
embryonic kidney (HEK 293) cell line - .....................................................................................162
262 HEK 293 expression ...................................................................................................................162
263 HEK 293 expression ...................................................................................................................163
264 HEK293 cell lines ........................................................................................................................164
265 HEK293SFE cell line ...................................................................................................................165
266 HKB-11 (HKB11) expression; Hybrid of kidney and B cells - HEK-293 alternative ...................165
Plants
267 Agrobacterium tumefaciens; Ti plasmids - transgenic plants ....................................................166
268 Antibiotic resistance markers - plants ........................................................................................167
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269 Chloroplast expression - plants .................................................................................................168
270 Chloroplast Transformation Technology (CTT) - plant cells .......................................................168
271 Coupled regeneration/ transformation, plants ...........................................................................169
272 GENEWARE expression; Tobacco mosaic virus (TMV) vectors - tobacco; plants ....................170
273 Glyco-Engineered Moss; Physcomitrella patens expression; moss expression
promoting regions (MEPRs) - glycosylation; antibodies ............................................................171
274 iBioLaunch expression; Launch vectors - proteins and Mabs in plants ....................................173
275 LEX System; Lemna (duckweed) expression - algae, whole plants ...........................................174
276 Nuclear transfer Cultured inner cell mass cells (CICM) - transgenic animals;
cloning from somatic cells..........................................................................................................176
277 Nuclear Transformation Suite, plants .........................................................................................177
278 Plant expression - glycosylation ................................................................................................177
279 pPIPRA vectors - plants; public domain ....................................................................................178
280 ProCellEx Plant Expression - plant cells; glycosylation .............................................................178
281 Profcia expression - transient expression, plants .....................................................................179
282 RNA-dependent RNA polymerase (RdRP) - universal ...............................................................179
283 Stratosome Biologics System; Oilbody expression; Saffower plant seed expression ..............180
284 Super-mas Plant Gene Promoter; Gelvin promoter; (Aocs)3AmasPmas - plants ......................181
285 TransBacter Gene Transfer System -plants; royalty-free ...........................................................182
286 Ubiquitin linkage domain - multiple proteins in transgenic plants .............................................183
287 Ubiquitin plant promoters ...........................................................................................................184
288 Zara technology; Protein body-inducing sequence (PBIS) fusion proteins;
Recombinant protein body-like assembly (RPBLA); StorPro organelles
(protein encapsulation); Prolamin fusion proteins - eukaryotes .................................................184
289 CleanGene plant transformation ................................................................................................185
290 Plastids (chloroplasts) expression - plant cell culture ................................................................186
Insects
291 High Five cell line (BTI-TN-5B1-4, ATCC CRL 10859); Trichopulsia ni cell line -
baculovirus host cells; insect cell culture ...................................................................................187
292 Insect cells glycosylation ............................................................................................................187
293 Insect cells glycosylation ............................................................................................................188
294 Insect cells/Baculovirus expression systems; Baculovirus expression
vector systems (BEVS) ...............................................................................................................189
295 PERLXpress; TRANSPILLAR larvae; Trichoplusia ni larvae expression -
transformed caterpillars .............................................................................................................190
296 Trichoplusia ni (cabbage looper) cell lines; BTI-TN-MG1; ATCC CRL 10860;
BTI-TN-5B1-4; ATCC CRL 10859 ..............................................................................................192
297 Trichoplusia ni (cabbage looper) cell lines; H5CL-B and H5CL-F;
BTI-TN-5B1-4-derived insect cell lines ......................................................................................193
298 Baculovirus expression vector systems (BEVS) .........................................................................193
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299 InsectSelect Protein Expression System - insect cells; avoid baculoviruses ............................194
300 Mimic Sf9 Insect Cells - mammalian-like glycosylation .............................................................195
301 Polydnavirus vectors - insect cells; baculovirus altnerative .......................................................195
302 Pre-occluded Virus (POV) baculovirus vectors; Insect cells, per os (oral)
baculovirus infection ..................................................................................................................196
303 Spodoptera frugiperda Sf-21 cell line - baculovirus host insect cells .......................................196
304 Spodoptera frugiperda Sf-9 cell line - baculovirus host insect cells .........................................197
305 NusA E. coli fusion proteins - eukaryotes; protein solubilization ...............................................197
Other broad/universal and older technologies
306 Enterokinase - fusion protein cleavage ......................................................................................198
307 Phage lambda promoters; PL promoter; PR promoter - E. coli .................................................198
308 Phage T5 promoter ...................................................................................................................199
309 WI-38 cell line, Normal human fetal lung fbroblasts ..................................................................199
310 Alkaline Phosphatase; Calf Intestinal Alkaline Phosphatase (CIAP) - prevent vector
recircularization ..........................................................................................................................200
311 Benzonase; Serratia marcescens endonuclease - polynucleotides breakdown ........................200
312 DNA Ligase (E. coli) ....................................................................................................................200
313 Site-directed mutagenesis .........................................................................................................201
314 T4 DNA Ligase ...........................................................................................................................202
315 T4 RNA Ligase ............................................................................................................................202
More Specifc and Component Technologies
316 Altogen transfection; RNAi gene silencing .................................................................................203
Bacteria/Prokaryotes
317 Profuse vectors; Cisperone chaperone fusion tags - E. coli; Saccharomyces cerevisiae .........203
318 Bacterial transcription promoters ...............................................................................................204
319 BresaGen fusion protein expression - E. coli. ............................................................................205
320 Cold-Induced expression - Bacillus subtilis ...............................................................................205
321 desA promoter, iron-regulated; DmdR repressors - Actinomycetes ..........................................205
322 E. coli. vectors; Mnt-Arc promoters; T1 and T2 rrnB ribosomal terminators - bacteria ............206
323 pAVEway expression - E. coli and Pseudomonas .....................................................................206
324 pTAT-HA plasmids - E. coli; bacteria ..........................................................................................207
325 Twin-arginine translocation (Tat) system; Tat nanomachine - protein folding, then secretion;
bacteria .......................................................................................................................................207
326 VegI promoters - Bacillus subtilis and E. coli .............................................................................208
327 Xer-cise gene excision; Xer recombinases - Bacillus subtilis ....................................................209
328 Agrobacterium tumefaciens RpoA co-expression transcriptional activator - E. coli .................210
x|x
329 Avidin affnity fusion protein affnity tags; Biotin purifcation; PinPoint Xa
Protein Purifcation System - E. coli ...........................................................................................211
330 Biogenerics manufacturing technology packages - E. coli .......................................................212
331 BL21(DE3) competent E. coli cells .............................................................................................212
332 C-LYTAG affnity fusion protein tag; Streptococcus pneumoniae
N-acetylmuramoyl-L-alanine amidase LytA - E. coli; purifcation ..............................................213
333 Campylobacter jejuni glycosylation genes; OTase of C. jejuni - glycosylation; E. coli ..............214
334 Chaperone expression plasmids; DnaK, DnaJ and GrpE chaperones - E. coli .........................215
335 cis-Acting Peptide chaperones - E. coli .....................................................................................216
336 Codon-Optimized, Expression-Ready E. coli Clones ................................................................216
337 Continuous culture - bacteria; E. coli .........................................................................................216
338 Disulfde isomerase coexpression; DsbC and DsbG - E. coli; disulfde bonds and folding ......217
339 Elastin-like polypeptide (ELP) self-cleaving fusion protein tags - chromatography-free
purifcation; E. coli ......................................................................................................................218
340 ExpressProtect; p26, SicA, and alpha-crystallin-type fusion protein tags - E. coli ...................219
341 High copy number plasmids; pBGP120 - E. coli .......................................................................219
342 High transformation effciency (Hte) competency - E. coli cells ................................................220
343 His-Patch ThioFusion System; pThioHis vector - E. coli; fusion proteins .................................220
344 N(pro) fusion protein tag, self-cleaving; Swine fever virus N(pro) autoproteolysis;
EDDIE - E. coli ............................................................................................................................221
345 OverExpress C41(DE3) and C43(DE3) - E. coli strains ...............................................................222
346 OxlT plasmids; Oxalate/Formate Exchange Protein - E. coli .....................................................222
347 pBR322 - E. coli plasmid; antibiotic selection ...........................................................................223
348 pCold vectors; Cold Shock Protein A (cspA) promoters - E. coli, cold induction ......................224
349 pET Expression System; T7 promoter; pET Directional TOPO Cloning; Champion pET
Expression Vectors; T7 RNA polymerase (T7 RNAP) - E. coli ....................................................224
350 pMAL Protein Fusion and Purifcation System; Maltose binding (MBP) fusion
affnity tags; pMAL plasmids - antibody fragments; E. coli .......................................................227
351 Polyhydroxybutyrate (PHB) fusion tags, self-cleaving coexpressed with
affnity medium - E. coli; universal .............................................................................................228
352 Red/ET recombination; ET cloning/ET recombination; GET recombination;
Recombineering; f Red-mediated recombination; lambda-mediated
recombination - E. coli ...............................................................................................................229
353 Skp and DsbC chaperone fusions - E. coli; secretion control ...................................................230
354 Tac promoters - E. coli ...............................................................................................................231
355 Tryptophan (trp) promoters - E. coli ...........................................................................................231
356 WACKER Secretion System; E. coli K12-based secretion system - antibody fragments .........232
357 Flavivirus vectors; Kunjin replicon vectors - prokaryotes; Streptomyces; prokaryotes .............232
358 Streptomyces inducers ..............................................................................................................233
359 Streptomyces lividans strains; xysA promoters .........................................................................233
xx
Yeasts
360 AlcoFree Yeasts; Saccharomyces cerevisiae KOY.TM6* strains ................................................234
361 ALEU2 marker; AHSB4 promoter; Arxula adeninivorans expression .........................................235
362 Aspergillus niger expression; A. niger A4 promoters - humanized antibodies ..........................235
363 Aureobasidin A vectors (pAUR) - selectable marker in yeasts ...................................................236
364 Calnexin chaperone - Hansenula polymorpha; yeasts ..............................................................236
365 Chitin synthase (CHS1), Yeast growth factor, chitin synthase (CHS1) -
yeast promoter discovery ...........................................................................................................237
366 Estradiol-dependent enhancer; Gal4-ER-VP16 - yeasts............................................................237
367 Formaldehyde dehydrogenase (FLD1) promoter; Formaldehyde selection -
Pichia pastoris; yeasts ...............................................................................................................238
368 GAPFL promoter; Glyceraldehyde-3-phosphate dehydrogenase-derived
promoter - yeasts .......................................................................................................................239
369 Gene Design, algorithmic - yeasts .............................................................................................239
370 Hypermutable yeast ..................................................................................................................240
371 PH05 promoter; Phosphate induction -yeasts ...........................................................................240
372 Vesicular fusion factor 2 protein (Vff2p) enhancer - yeast; bacteria; CHO cells ........................241
373 Vesicular fusion factor 2 protein (Vff2p) enhancer - yeasts ........................................................241
374 Xplor Vector System, yeast optimization....................................................................................241
375 Antibody fragment expression - Saccharomyces cerevisiae .....................................................242
376 Saccharomyces cerevisiae, cold induction ................................................................................243
377 Secretion Enhancer Vector System (SEVS); SecHancer vector - Saccharomyces cerevisiae ...243
378 Mab Xpress Antibody Production System; Pichia pastoris - glycosylated antibodies .............244
379 Pichia expression, rhamnose induction .....................................................................................245
380 Pichia GlycoSwitch System; Glycoswitch plamids - yeasts; glycosylation ...............................245
381 Pichia pastoris antibody expression ..........................................................................................245
382 Pichia pastoris AOX1 promoters ................................................................................................246
383 Pichia pastoris super yeast; Pichia pastoris AOX1 promoters ................................................247
384 Aminoglycoside adenylyltransferase (aadA1) promoter - bacteria; eukaryotes .........................247
385 ColE1 plasmids, E. coli - prolonged viability ..............................................................................248
Mammals
386 CMV/R Promoter - eukaryotes ...................................................................................................248
387 piggyBac transposon - eukaryotes; insect cells ........................................................................249
388 Tax-inducible expression; Bovine leukemia virus (BLV) promoter - mammalian cells ...............250
389 BacMam; pBacMam vectors - mammalian vectors, baculovirus-based ...................................250
390 Calnexin, calreticulin, Erp57, Hsp40, and Hsp70 chaperones - mammalian cells ....................251
391 CCT promoters - mammalian cells ............................................................................................251
392 ClonePixFL Selection - antibodies, mammalian cells ................................................................252
393 Hsp60, Hsp70, Hsp90, Hsp100 chaperones - mammalian cells ...............................................252
xx|
394 IRF-1 estrogen receptor promoter; Estradiol induction - mammalian cells ...............................253
395 Osteoclast-associated receptor (OSCAR) promoter - mammalian; CHO cells ..........................253
396 pAccAB vectors - antibodies; mammalian cells ........................................................................253
398 RP Shift; Senescence induction; PACE Expression Vector - mammalian
cells; expression enhancement; antibodies ...............................................................................254
399 Semliki forest virus (SFV) vectors - mammalian and insect cells ...............................................255
Chinese Hamster Ovary (CHO) cells
400 CHEFl expression; CHO elongation factor-la (EF-Ialpha) promoter - CHO cells .......................256
401 CHO cells - antibodies; serum-free media .................................................................................256
402 CHO cells dhfr RNA interference; RNA silencing vectors - CHO cells ......................................257
403 CSL4S-342 CHO cells (CHO-K1 CSL4S-342) ...........................................................................258
404 Osteoclast-associated receptor (OSCAR) promoter - mammalian; CHO cells ..........................258
405 Pangen CHO expression ............................................................................................................258
406 StableFast Biomanufacturing System; pBFdfhr.2 Expression Vector -
CHO cells; antibodies .................................................................................................................259
407 Tandem Chimeric Antibody Expression (TCAE) vectors; ANEX vectors;
CHO cell line TCAE 8 (ATCC 9119); Kozak sequences, impaired - antibodies..........................260
408 UTR Clone Generation; UTRtech; Cell Factories; Gaussia luciferase signal
peptides - Mabs supersecretion; CHO cells ..............................................................................261
409 DNA microinjection - transgenic animals, chickens ...................................................................261
HEK-293 cells
410 293ST-3F6 cell line; HEK-293 adapted to SFM .........................................................................262
411 CRE-inducible expression; cyclic AMP response elements (CREs) - HEK-293 cells ................262
412 HEK-293 expression...................................................................................................................263
413 HEK-293 expression...................................................................................................................263
414 pTT vectors for HEK-293E cells .................................................................................................263
Plants
415 Magnifection; magnICON; Transgene Operating System (TOS) - antibodies;
plants and plant cells .................................................................................................................264
416 MARs PLUS; Matrix attachment regions PLUS - plants ............................................................266
417 Concert Plant-Cell-Produced system - tobacco plant cell culture ............................................267
418 Phyton plant cell fermentation ...................................................................................................268
419 ExpressTec expression; ExpressPro; ExpressMab - rice and barley; antibodies ......................268
Insects
420 AcMNPV p35 apoptosis inhibition; Sf9P35AcV5-1 and Sf9P35AcV5-3 -
insect cells, apoptosis resistance ..............................................................................................269
421 AF 99 insect cell line ..................................................................................................................270
422 BL-Sf-21AE-Cl 3 cell line - Insect cell lines, baculovirus hosts .................................................270
xx||
423 Cre/loxP Recombination-Mediated Cassette Exchange (Cre/loxP RMCE) - Drosophila
(mosquitos) .................................................................................................................................270
424 Drosophila expression ................................................................................................................271
425 Drosophila melanogaster S2 cells; Drosophila-SFM.D.Mel-2 Cells; Schneider S2
Drosophila cells; S2 cells, SFM - insect cell culture ..................................................................271
426 IE-1 (BmNPV 1.2 kb fragment) promoters; Bombyx mori actin promoters - insect cells ..........271
427 Insect cell lines - baculovirus hosts ...........................................................................................272
428 Insect cells, per os (oral) baculovirus infection ..........................................................................273
429 Lymantria diapar nucleopolyhedrovirus and L. dispar 652Y (Ld652Y) cell lines -
baculovirus host cells .................................................................................................................274
430 pIEx baculovirus vectors; hr5 enhancer; ie1 immediate early promoter - insect cells;
avoid baculovirus pathogenicity .................................................................................................274
431 Rhopalosiphum padi virus Internal Ribosome Entry Sequence (IRES);
Picorna-like virus IRES; Drosophila IRES - insect cells; plant cells ...........................................275
432 Tni PRO; Trichoplusia ni cell line ................................................................................................276
433 BAC-TO-BAC Baculovirus Expression System; baculovirus shuttle vectors;
bacmids - insect vectors produced in E. coli .............................................................................277
434 BacTen System; p10 promoter vectors - insect cells ................................................................277
435 Baculovirus vectors and promoters - glycosylation ...................................................................278
436 BestBac vectors; Autographa californica nuclear polyhedrosis virus (AcNPV) vectors -
insect cells ..................................................................................................................................278
437 Drosophila sialyltransferases - insect cells; glycosylation .........................................................279
438 Sapphire baculovirus expression - disulfde bond formation .....................................................279
439 Vankyrin enhanced baculovirus expression vector system (VE-BEVS);
Vankyrin-enhanced cell lines (VE-CL) .........................................................................................280

Indexes
Company/Organization Index .........................................................281
Subject Index ....................................................................................305
Primary Host/Organism Index .........................................................313
1
Introduction:
N
ew expression systems and recent improvements available for current systems have the potential
to revolutionize the biopharmaceutical industry! As reflected by currently marketed products,
since the advent of genetic engineering in the 1970s, there has been little basic change in the
technologies used for commercial-scale manufacture of biopharmaceutical products. Nearly all current
products are manufactured using much the same old, familiar technologies primarily using Esherichia
coli(E.coli bacterium), Chinese hamster ovary (CHO) cells and the yeast Saccharomycescerevisiae(S.
cerevisiae) as hosts technologies invented in the 1970s and commercialized in the 1980s.
Today, a number of factors are rapidly changing the biopharmaceutical manufacturing environment.
Scientific and technological advances, including both totally new platforms (new host cells/organisms;
some you never even imagined using for biopharmaceutical manufacture) and genetic engineering
advances applied to traditional platforms, offer significant advances and advantages. This includes
not just multiples or even orders of magnitude improvements in product yield, itself a major or
revolutionary advance, but also improved product quality, lower operating and infrastructure costs, and
provide opportunities for innovators to make their products unique (difficult or impossible to copy) and
allow biosimilar/follow-on biologics manufacturers to compete (make their products at low cost).
Combined with the trend towards use of disposable equipment and advances in purification and other
downstream technology, it is obvious that biopharmaceutical manufacturing will be undergoing a major
paradigm shift in coming years. Recombinant protein manufacture that typically has involved one
or more multi-1000 liter bioreactors along with a dedicated facility/infrastructure can now or soon be
accomplished using bioreactors an order of magnitude or smaller, e.g., a 100 liter or somewhat larger
bioreactor, which may be modular, portable or even disposable. Rather than a dedicated building
or wing, commercial-scale manufacture can be done in a smaller room or suite. Also, many newer
technologies offer speed, including much shorter times required to go from gene to transformed
host cell line/organism to commercial-scale manufacture; and many can produce the same or higher
amounts of products in much shorter periods.
Besides offering advantages and savings for product manufacture, new biopharmaceutical
manufacturing systems/platforms and genetic engineering technologies offer new opportunities for
further corporate development, licensing, investment and profit. Whether you are a large or small
Expression Systems and Genetic
Engineering Technologies:
Opportunities for Innovators,
CMOs and Product Developers
by Ronald A. Rader
2
innovator, CMO or biogenerics company and seek to develop new licensing/income streams,
dream of starting a new company or simply are looking for a good investment, there are ample
opportunities to get involved now, particularly before the advantages of getting involved in earlier
stages of technology development and licensing are lost. Currently, there are just a few major
players promoting new technologies, and as shown in this directory, there are an incredible number
of technological advances ready for adoption, adaptation and further development, providing ample
opportunities for process improvements and profit.
Already, most major large (bio)pharmaceutical companies have made some moves in this area,
with some paying millions dollars to acquire companies whose only or primary assets are new
manufacturing platforms, while others are quietly but actively investigating, optioning, licensing-in
and implementing new technologies. For example, for about $400 million Merck & Co. acquired
GlyoFi, a developer of methods for manufacture of antibodies with human-like and modifiable
glycosylation using the yeast Pichiapastoris; and for $56.5 million, Hoffmann-La Roche acquired
Therapeutic Human Polyclonals Inc., a developer of methods for manufacture of polyclonal
humanized antibodies in transgenic rabbits. The opportunities for profit are also illustrated by past
technology licensing, e.g., Columbia University took in nearly $370 million over 17 years (average
over $20 million/year) from licensing of co-transformation technology (see related entry) at less than
a 1% royalty; Stanford University and the University of California each took in over $200 million
from licensing of the Cohen-Boyer patents covering basic aspects of recombinant protein manufacture
(see related entry); and the City of Hope National Medical Center is still arguing in court with
Genentech about an additional ~$300 million in royalties due for now off-patent E.coli vectors from
a licensing agreement originally initiated in 1976. Unlike these early recombinant technologies which
largely originated from public sector/university research, as can be seen from examining entries in this
directory, the private sector now largely dominates expression systems and related genetic engineering
technology invention and development (with this often involving university spin-off-type companies).
Why a Directory?
This directory was designed to provide you with access to the most important new technologies
relevant to the commercial-scale manufacture of biopharmaceutical products, particularly
recombinant proteins, including glycoproteins and antibodies. If you have not recently fully assessed
your commitment to your current systems (quite possibly the same basic technologies you and others
are familiar with, e.g., used in graduate school or since entering the industry; the same ones in use for
decades), you probably should. If you are not considering implementing totally systems/platforms or
at least improving the one(s) you currently use, you are missing significant opportunities. If nothing
else, new systems and improvements to traditional ones offer compelling increases in product yields
and associated reductions in manufacturing costs.
Soon enough, most every biopharmaceutical developer (or the more successful ones) will likely
be using 2-3 or more different expression systems/platforms in-house, including using multiple
diverse systems at early stages of R&D to determine advantages each offers in terms of product
characteristics (improved efficacy and safety) and switching between different systems/platforms
product manufacture for preclinical studies, early and mid-stage trials and, perhaps, another for
commercial manufacture.
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What are Expression Systems and How are They Acquired?
Expression systems encompass the technologies biological materials and associated know-how
needed to genetically modify organisms for the manufacture of recombinant proteins and other
products. This includes vectors used to transfer genetic material for expression of desired proteins into
host cells/organisms as well as the source and transformed cells themselves, along with various other
required and expression-enhancing genetic elements incorporated into vectors and host cells/organisms.
In some cases, vectors may be commercial products on their own, such as those used for gene therapy
or live viral vaccines, but these are outside of the scope of this directory. Although usually involving
culture of microorganisms or cells, expression systems can also involve macroscopic and higher
organisms, such as transgenic animals and plants. As core technologies for genetic modification, many
expression systems are often ubiquitous research and drug discovery tools as well, e.g., use of E.coli
for recombinant protein production is routine throughout modern life sciences research. The component
technologies for protein expression can include core and backbone vectors for gene delivery; activation
and promoter sequences to drive transcription of inserted genes; selection and amplification methods
and reagents to get just the optimal clone producing the right product; chaperone proteins to assure
proper protein folding and conformation; expression of desired proteins as fusion proteins to facilitate
purification; and methods for adding and controlling glycosylation in organisms inherently deficient in
these abilities. Of course the host cell lines are the starting point and main constituents, and themselves
may include genetic modifications to improve product yield, stability and alter environmental
sensitivities, nutritional profile or other characteristics.
Expression systems, like most other commercial manufacturing and biotechnologies, are generally
patented and often available for licensing from their owners (patent assignees) and/or from companies
having licensed the patents for further (sub)licensing, including CMOs that offer related manufacturing
services. Thus, in many respects they are defined and exemplified by related patents which define their
ownership (or rather, a time-limited right to prevent others from using infringing technologies). As can
be seen from examining the entries in this directory, just about everything related to genetic engineering
and recombinant protein manufacture has been patented. That is simply the nature of high technology.
Many researchers and executives will be distressed to learn that some of the technologies they use in
product manufacture have been patented and should be licensed. This is especially true for many basic
molecular biology methods and reagents that researchers are used to purchasing from catalogs and using
routinely, but that require taking a patent license for commercial use.
Patents on early technologies, those developed in the 1980s or earlier, have already or will soon expire.
Already, one can manufacture recombinant proteins at large-scale without having to license technologies
(pay royalties generally based on product sales), e.g., using E.coli technology from the 1970s/1980s.
However, by current standards, this will likely be inefficient and costly to implement, e.g., requiring
large steel fermentors/bioreactors, associated dedicated facilities and expenses, e.g., consuming mass
quantities of culture media, and the handling of large volumes of dilute product requiring multiple
purification steps. Although adopting such classic technologies for commercial biopharmaceutical
manufacture, using what is familiar to FDA and every other regulatory agency worldwide, may be
the safety route from a regulatory perspective, there are many trade-offs involved in doing this. The
efficiencies and advantages offered by newer technologies and or simply incorporating improvements
into (upgrading) classic technologies have or are becoming too compelling to ignore.
One consequence of ubiquitous patenting can be the need to use (and license) a number of component
technologies, known as patent stacking, to manufacture just about any biopharmaceutical. For example,
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licensing fees and royalties can easily start to become a significant portion of product sales as one
licenses in a basic platform (host cells/organisms) along with use of a vector, promoter, amplification
and selection method(s), chaperone, etc. Generally, downstream process technologies, e.g.,
chromatography, tend more to be in the public domain (not patented), with the reagents used, e.g.,
chromatography media, more likely to be covered by patents. However, in these cases, licensing is
often transparent or not an issue, with the licensing or right to use these products for commercial use
generally included with the purchase of these materials. But, this is not the case with most molecular
biology reagents and, certainly, not with expression systems or other upstream manufacturing-
enabling technologies.
This frequent need to license multiple technologies to make a product is a reality but is rarely
discussed openly in the biotech industry (suggesting avoidance of the issue of patent stacking and/or
that patent infringement is common). The alternative to not taking a license to a patented technology
owned by (assigned to) someone else is patent infringement using patented technologies without
permission (not taking a license and not paying related royalties). This is illegal and never a prudent
course for any company. If caught after launching a product, the offending firm could be liable for
compensatory and punitive damage awards; the patent assignee could refuse to grant a license, forcing
the company to remove your product from the market; the licensor could demand outlandish licensing
fees and royalties; and/or whoever is responsible could even go to jail (although this is highly
unlikely). Despite these downsides, it seems that infringement of biopharmaceutical manufacturing
technologies may be common in industry not the basic platforms/host systems (the cells/organisms
used), which are impossible to hide with a marketed product, but the various genetic engineering
methods and improvements likely never publicly disclosed. For example, various vectors, promoters,
selection and amplification methods, chaperones, etc., commonly available from reagent/catalog
companies could easily be used for commercial product manufacture, infringing related patents.
Various factors contribute to an environment and attitude in many companies that may well be
contributing to widespread patent infringement with process-related technologies. The status of
patents and what they actually cover can be difficult to determine. Few organizations with expression
systems or genetic engineering technologies available for licensing have effective information
dissemination or technology transfer marketing programs (as discussed below). Few manufacturers
ever conduct rigorous due diligence to determine the need to license all of the technologies they use
in biopharmaceutical manufacture. In fact, dont ask, dont tell or plausible deniability (ability
to plead ignorance vs. premeditated infringement), combined with strict non-disclosure agreements,
appears to be a common approach, particularly in larger companies. The author has spoken to patent
attorneys within multiple major innovator biopharmaceutical companies who proudly state that their
scientists and managers know enough not even to bring up the issue of the patent/licensing status of
manufacturing-related technologies (much as they likely also avoid asking about the patent/licensing
of many therapeutics discovery, screening and design technologies used in-house). Also, the actual
processes used for manufacturing marketed biopharmaceuticals are almost never fully disclosed, so
it is often difficult for licensors to enforce their patents. For licensors, one way around this problem
is to rely on trade secrets in addition to patents, e.g., keep one or a few key aspects (not needing
to be included in relevant patents) totally secret among select employees, i.e., never published or
patented; and, ideally, make this clear in any technology-related publications or communications with
prospective licensees.
5
Old Technologies Still Dominate the Industry, But This Will Change
Looking at the expression systems used to manufacture current biopharmaceuticals (and those in
development), most are still being manufactured using old technologies developed in the 1970s and
adapted for biopharmaceutical manufacture in the 1980s, even recently-approved products and also
those in late-stage development. Table 1 classifies expression systems used in manufacturing the
~130 recombinant protein products in the United States and European markets (1). Over half (55%)
of these products are expressed using microbes, either bacteria (40% of the total/all products) or
yeasts (15%), with nearly all those bacteria involving Escherichiacoli (E.coli, 39% of the total).
Another 45% of products are expressed in mammalian cells, primarily Chinese hamster ovary (CHO)
cells (35% of the total). E.coli, yeasts, and CHO cells have the longest history of use, beginning with
commercialization of the first recombinant proteins in the 1980s.
Together, E.coli, yeasts, and CHO cell platforms account for 89% of the expression systems used in
manufacturing currently marketed recombinant therapeutics (in major markets, i.e., U.S. and Europe).
The concentration on just a few systems is even higher if you also include the 11 products (all
monoclonal antibodies) made using murine myeloma cell lines. Similarly, among the 24 recombinant
proteins with blockbuster revenue ($1 billion in total revenue/year), 89% use E.coli, yeast, or CHO
cells. Examination of products currently in later-stage development shows much the same pattern.
These classic expression systems have been substantially improved over the years e.g., newer
host cell and genetic engineering methods have significantly increased yield and stability, as can be
easily seen from the many related entries in this directory. However, the failure to adopt new(er)
platform technologies often offering considerable advantages is indicative of problems that have
plagued the industry. Despite their availability, truly novel platform technologies (new to human
biopharmaceutical manufacture) such as fungi (non-yeast), insect cells, plants and transgenic animals,
have yet to make a significant impact on marketed products or those in later-stage development.
Novel expression systems (in terms of human biopharmaceuticals) include those based on bacteria
other than E.coli (e.g., Pseudomonasfluorescens,Staphylococcuscarnosus,Bacillussubtilis and
Caulobactercrescentus); fungi and yeasts (e.g., Chrysosporiumlucknowenseand Arxula); improved
human cell lines (e.g., PER.C6 or use of amniocytes/stem cells); microorganisms culturable in simple
media, e.g., glucose or ethanol, and/or that provide human-like glycosylation; bacteria that express
high levels of protein at low, near freezing temperature; algae ranging from single cells through
whole plants (e.g., Lemna); various insect cells and insect larvae; a wide variety of terrestrial plants
(e.g., safflower and tobacco); and various transgenic animals. Completely cell-free systems are
also becoming available. Some novel systems (for biopharmaceuticals) are now commonly used
for nonpharmaceutical recombinant protein manufacture (e.g., most industrial enzymes), and some
are used for food, nutritional supplement and large-scale pharmaceutical manufacture, but only in
the diagnostic and veterinary areas. Some of the most promising platform systems for recombinant
protein manufacture are those already used industrially at very large scale, e.g., for food and
nutritional products, just not for biopharmaceutical manufacture.
Many novel expression systems offer significant improvements in yields or other performance
factors; increased simplicity; and lower costs for manufacture, equipment, facilities, and
infrastructure, including less dependency on large, fixed bioreactors. Many offer potential product
improvements, e.g., more human-like or better control of protein glycosylation, protein folding, and
higher purity. Many are considered novel solely because they remain largely unused for producing
human biopharmaceuticals, although the technologies themselves may be as old as the current
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dominant expressions systems. For example, Pichia yeast, baculovirus vectors and insect cells, and
non-E.coli bacteria (e.g., Bacillussubtilis) were initially developed decades ago and are widely used,
but rarely or simply not yet used for manufacture of marketed human biopharmaceuticals. When
newer technologies are considered in a more global context, including looking at improvements in
culture media, bioreactors/fermentors, including disposable and even pipe-free systems, and advances
in chromatography and fluid handling, the case for integrated adoption of new(er) technologies
becomes compelling. But, all of this technology still comes back to or is dependent on the choice of
manufacturing platform the host cells or organisms being used. Thus, expression systems are the
primary controlling technological factor in recombinant protein manufacturing systems.
In many cases, older expression systems have been incrementally modified over the years, often
leading to significant improvements, as can be readily seen in this directory. For example, some
yeast, insect-based and other non-mammalian systems now offer post-translational modifications
(e.g., glycosylation and folding) that closely mimic those of mammalian and human cells. Host cell
line and genetic engineering, combined with culture media and other incremental improvements offer
increased yields with many older systems by an order of magnitude or more, e.g., from a fraction
of a gram to multiple grams per liter. But many newer systems offer even further advantages.
Even so, the basic aspects and components of the expression systems used in current large-
scale biopharmaceutical manufacture have changed little over recent decades. This lack of active
industry involvement and investment in newer technologies often carries over to other aspects of
bioprocessing, as well, e.g., downstream processing. This attitude is changing, and those who do not
bother to investigate, if not constantly work, to improve product manufacture risk being left behind,
missing out on related efficiencies and the flexibility available when a company has a wider choice
and expertise in expression systems and related genetic engineering technologies. (Table 1)
Why the Failure to Adopt New(er) Technologies?
The failure of biopharmaceutical companies to adopt more modern expression systems is not new
(2). Simply stated, human biopharmaceutical developers do not want to be the ones to pioneer
a new manufacturing technology, and face a Catch-22 situation if they do. Even if a novel
manufacturing technology provides significant competitive advantages and product improvements,
biopharmaceuticals manufactured using novel technology can be expected to come under increased
scrutiny and potentially encounter delays in regulatory approval. To date, the innovator-dominated
industry has obviously decided that such potential delays and problems are not worth the cost savings,
improvements and potential protection from biogenerics offered by newer technologies. Many,
if not all, manufacturers currently using classic platform technologies have likely upgraded their
original processes by incorporating new(er) technologies, e.g., to increase yields. As can be seen in
this directory, there are many options available for those seeking this route. However, most of the
industry, particularly the major players with the most capacity, remain committed to older platform
technologies for product manufacture, with many having considerable investments in large-scale
bioreactors and facilities. New emergent innovator and also follow-on protein/biosimilar companies
and contract manufacturers of all sizes have been and will continue to be among the first adopters of
a broader range of expression systems and other bioprocessing technologies for commercial-scale
product manufacture.
Beyond regulatory uncertainty and inertia, a problem holding back adoption of new, better
technologies is that their widespread commercial visibility and adoption first requires scale-up
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and demonstration at suffciently large scale. In order to prove commercial viability of a new
manufacturing platform, something has to actually be manufactured. The only organizations for
whom such projects are economically justifable are biopharmaceutical companies. Thus, those
developing new manufacturing technologies must either partner with one or more manufacturers
for technology development; new technology developers must license their unproven technologies
at low rates to attract licensees; or as can be seen in this directory, it is common for manufacturing
technology developers to also be developing one or more early-stage therapeutics manufactured
using their technology; or the contrary, small companies developing early-stage therapeutics adopt or
themselves are also developing new manufacturing technologies and using these with their products
in development. Similarly, various companies seeking primarily to develop new manufacturing
Table 1: Expression systems/transformed hosts for recombinant products in the
U.S. and European markets
Microbial 79
Bacteria 58
Escherichia coli (E. coli) 56
Streptococci 2
Yeasts 21
Saccharomyces cerevisiae 19
Pichia pastoris 2
Insect cells/baculovirus vectors 3
Plants 0
Mammalian 77
Mammalian cells, non-primate 65
chicken embyros (whole eggs) 2
chicken embryo cell culture 1
hamster, Chinese ovary (CHO) 50
murine myeloma cells 11
murine cells other 1
Mammalian cells, primate 9
monkey cells, diploid, kidney or fetal lung 4
Human cells 5
human cells, Epstein-Barr virus transformed 1
human cells, Gene Activation (TKT) 2
human kidney cells, embryonic (HEK) 1
human cells, unspecifed 1
Mammals, transgenic 3
goats, transgentic 1
rabbits, transgenic 1
mice, transgenic XenoMouse (used in dev. only) 1
Viruses, recombinant, as products (live vaccines) 6
Yellow fever virus vector 1
natural recombinant viruses 5
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technologies have or are adopting a contract manufacturing organization (CMOs) business model,
with contract manufacture supporting technology and infrastructure development and proving
commercial feasibility. Many smaller companies and academic researchers offering or developing
novel manufacturing-related technologies lack the funding, facilities, and other resources needed for
scaling up and have no viable products to manufacture, if only for use in clinical trials; and many
of the technologies originating from academia/public sector and small companies have yet to find a
champion (commercial user/developer). This results in slow development of these technologies, but
provides opportunities for those with the interest and resources to get involved.
Established companies, notably those few profitable large biopharmaceutical companies and
large international pharmaceutical companies with multiple marketed recombinant products, have
historically shown a clear preference to continue using familiar manufacturing technologies. This is
exemplified by the handful of companies that currently control the great majority of the worlds large-
scale biopharmaceutical manufacturing capacity. Essentially all of that capacity involves established
systems: mostly E.coli, yeast, and CHO expression systems using fixed, large-scale bioreactors often
10,000 L or larger. This is particularly true for manufacture of recombinant monoclonal antibodies,
which require large amounts of protein, because they are administered in repeated high dosages. The
classic technologies have worked well for the industry, existing facilities work well enough and need
to be put to use, and many managers throughout the industry see no need to think critically about
their expression systems or the other biomanufacturing technologies their companies use.
In this industry it is unreasonable to blame company executives for using what has worked in
the past, and continues to be used. The biopharmaceutical industrys reluctance to adopt modern
expression systems is not due to lack of availability or access, mostly inertia and timidity, although
those offering novel technologies do a poor job of marketing, as discussed below, and there are few
resources available to assist those with an interest in finding alternatives or updating their processes
(with this being the first directory of any type in this area). Most novel technologies are available
for licensing, and licensing fees for older and newer technologies are not significantly different, so
licensing fees should not play much of a role in selecting older vs. newer technologies. Many newer
platform and associated genetic engineering technologies are more cost-effective and require less up-
front investment than traditional systems, and offer flexibility and other compelling reasons for their
adoption.
Newer Technologies Use in Early Product Development
Expression of the same protein (or glycoprotein or antibody), i.e., the same primary structure/
sequence, using the same gene sequence and even the exact same vector constructs in different host
cells or organisms invariably results in different active agents/products, although differences may be
subtle and unquantifiable using current analytical technology. This is embodied in the process =
product paradigm originally promulgated by innovators as a defense against biogenerics/biosimilars,
particularly when arguing that products from different manufacturers have inherently different
manufacturing processes, making the products inherently different, and that these different products
should not be considered interchangeable/substitutable. The unique structural properties conferred
by different expression systems, even if not detectable using conventional technology, can open
opportunities. For example, a candidate product could be manufactured in multiple very different
expression systems for preclinical studies, with the one with the most optimal properties selected for
further development. This could involve optimized biological activity, reduced immunogenicity (or
9
increasing it for vaccines), increased circulating half-life, and other improvements. Some expression
systems in development promise the ability to make recombinant proteins so cheaply that even
inefficient oral delivery may be viable. Many of those developing field crop-based transgenic plant
platforms are working towards this.
It is becoming practical and prudent to use (or have the option to use) multiple manufacturing
platforms for manufacture of candidate products as they progress through different stages of
development. For example, one might do preclinical studies with a quick and easy E.coli-expressed
protein, move to insect cells/BEVS for early clinical studies and do commercial manufacture using
yet another platform. For many, minimizing time in development is the primary or among their top
priorities. Many of the technologies in this directory claim to be significantly faster in going from a
designed or identified gene/protein sequence to expressing milligrams or grams of protein for early
studies and later commercial scale-up, making it much more feasible to use multiple systems to make
candidate products. Also contributing to reducing time in development, various platforms claim to be
fully scaleable, such as from standard 96-well plates to tons of protein/year. This can greatly reduce
development and scale-up time and expenses. For example, with insect larvae, all one has to do is
cultivate more larvae which can be simply stored for downstream processing whenever convenient.
Biogeneric/Biosimilars Make Expression Systems More Essential
Newer and improved expression systems are becoming more important in the context of generic
biopharmaceuticals (biosimilars, follow-on biologics, biogenerics, etc.) (3). Innovators, having
spent untold $10s or $100s of millions in product development and having invested years in building
up markets and brand recognition do not want competition from copies or similar versions of their
products as their product-specific patents expire (despite innovators almost always having a 2nd or
even 3rd generation replacement product on the market by the time their original product goes off-
patent). Biogeneric/biosimilar/follow-on biologics developers will need to manufacture their product
efficiently and cheaply, with their profit depending on this, and probably also often in higher quality
than reference products likely developed two decades previous (i.e., they will be expected to meet
current products standards, not those decades old).
How can new(er) technologies help innovators? For innovators developing novel biopharmaceuticals,
use of unique expression systems and genetic engineering technologies can be a potent defense
against later development of follow-on/biosimilar products, particularly if combined with exclusive
licensing (or outright ownership/full control of technology) and/or trade secrets (still the best way to
maintain secrecy about industrial technologies, with patents requiring trading-off full disclosure in
return for limited-time monopoly for commercial use). This is particularly true for those technologies
capable of conferring unique molecular properties that would hard or impossible to copy using
most any different technology. Expression systems and related genetic engineering technologies
can add value in the form of insurance against copying by providing unique molecules that could
only be duplicated using the same or similar technologies in a similar manufacturing process. For
example, this directory includes a number unique cell lines, organisms, glycosylation add-ons and
modifications, chaperons, fusion protein systems, etc. that would each confer unique properties
to any expressed proteins, with many or most of these useful with multiple host systems. Even if
related technology being used must or will be disclosed, e.g., in regulatory review documents, the
range of technologies available and their complexity, including the many ways in which they can be
implemented, could well make it very difficult, if not impossible, for even knowledgeable competitors
10
to duplicate or make a biopharmaceutical product sufficiently similar to another product such that
regulators can approve it on the basis of similarity.
Innovators seeking to protect established products approaching loss of patent protection from
biosimilar/follow-on competition can also adopt new(er) technologies as a defensive measure. With
no regulatory agencies worldwide publicly disclosing substantive information about supplemental
approvals, e.g., BLA supplements with FDA, unless it involves safety or product use, e.g.,
indications, manufacturers are free to upgrade their product manufacturing processes with assurances
of secrecy. This is illustrated by frequent claims by biopharmaceutical industry leaders asserting that
manufacturing processes for biopharmaceuticals undergo continual improvements, with the industry
adopting appropriate technologies as they become available or practical. However, despite this
likely being generally true, other than such generalized statements, there is simply no or negligible
evidence in the public domain to support this. This author, also the author of the reference book/
database, Biopharmaceutical Products in the US and European Markets, can attest that process-related
improvements and changes concerning marketed biopharmaceuticals are rarely ever disclosed.
For example, most of the major biopharmaceutical companies with the most manufacturing capacity
have reported that their processes have been significantly upgraded over the years, e.g., with
yields now in the low grams/L range, but it remains entirely unknown which of the many available
technologies they have adopted either in terms of specific facilities or manufacturing processes
and certainly not in the context of specific products. While this practice of neither companies nor
regulators ever disclosing product manufacture process changes is not good in the long term for
maintaining public trust in the industry, this is unlikely to change significantly and regulators can
continue to be relied upon to not disclose product manufacturing upgrades. This all presumes
that process alterations can support supplemental approvals, i.e., the resulting product from an
changed manufacturing process has to be shown sufficiently identical or comparable to the prior
product to receive supplemental approval. In most cases, a relatively small bioequivalency-type
trial, combined with laboratory and other preclinical comparative testing will be enough to gain
supplemental approval. For many, the costs of adopting/adapting a new manufacturing or related
genetic engineering technology and the need for clinical trials will be well worth the difficulties this
will pose for biosimilar/follow-on developers. For example, if timed right, biosimilar/follow-on
developers will either end up trying to emulate and conducting comparative testing, including clinical
trials, with innovator products no longer manufactured/marketed when their product finally comes to
market (confounding their product launches and marketing; with innovators being able to point out
improvements in their product and/or be able to claim that, despite approval, the biosimilar has never
been tested in comparative studies against the current product). The biosimilar/follow-on developers
may have to scramble to figure out and then try to emulate whatever process-imparting product
characteristics may have been altered, which will often be difficult, if not impossible.
The value in controlling access to unique manufacturing platform technologies, particularly
those supporting unique properties in final products, is being recognized by the mainstream
(bio)pharmaceutical industry. This appears to be a significant factor in the recent acquisitions
and exclusive licensing of novel expression systems technologies by many larger companies. For
example, as mentioned above, Merck acquired GlycoFi, which has developed yeast expression
systems for controlled human-like protein glycosylation (see related entry), for about $400 million;
and for $56.5 million, Hoffmann-La Roche acquired Therapeutic Human Polyclonals Inc. with its
unique manufacture of polyclonal humanized antibodies in transgenic rabbits (see related entry). It
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seems reasonable to expect that the business/industry of biopharmaceutical process developers follow
that of biopharmaceutical product developers, with big companies acquiring small innovators, and
company success often considered to be acquisition, rather than growth into an independent company.
For the largest companies, such as those with blockbuster ($1 billion/year sales) recombinant
humanized-type antibody products, generally requiring mass quantities of product because of repeated
high doses, it may make more sense to simply acquire a company whose technology is wanted/
needed, vs. having to pay a royalty likely for a decade or much more. For example, just a 2% royalty
on $2 billion/year is $40 million/year. In this context, it is easier to understand why Merck would
payout $400 million for GlycoFi, with this likely being less than what the company would pay out in
licensing fees; and now Merck owns a company that will likely be collecting comparable royalties
from other companies (besides Merck being able to shut out direct competing products from using this
technology).
How can new(er) technologies help biosimilar/follow-on developers? New and improved expression
systems are being adopted by many follow-on/biosimilar developers. For those in major markets and
developed countries, this is an imperative, despite different expression systems and clearly different
manufacturing processes potentially confounding regulators who may perceive insufficient similarity
between their product and the innovator/reference product, which by definition is an older product
usually manufactured using a platform (host cells/organisms) technology that is several decades old.
Many, if not most, biosimilar/follow-on developers are adopting the most cost-effective manufacturing
technologies useful for their products. In most cases, this is a necessity. These companies will have to
compete against multiple others of their kind as well as against innovators, who as discussed below,
may well decide to do whatever it takes, i.e., reduce product prices, to maintain market share (and
out of basic principle and ingrained habits, aggressively defend their established markets). Many
established innovator companies already have 20 years of experience and world-scale manufacturing
facilities making the reference product, and have already been providing the entire worlds supply of
their products, with any related financing long ago paid off in full. Biogeneric companies will have to
compete on the cost of manufacture, with innovators often having lower costs of manufacture, market
dominance, and ability to simply relabel and market (bio)generic versions of their own products or
have proxies do this. Innovators will be more able and may be predisposed to undercut the price of
follow-on proteins/biosimilars, if only to maintain their market share.
Many, if not most, biosimilar/follow-on proteins are likely to be manufactured using novel expression
systems, or use classic systems incorporating multiple yield-improving technologies. This is the
dominant presumption, and many developers of such products have already adopted newer high-
tech systems. However, it remains to be seen how many of their products will actually be approved
under follow-on protein/biosimilar regulatory mechanisms. The manufacturing process still largely
controls and defines biotech products (the process = product paradigm). So those using a significantly
different manufacturing process risk making a product that will be considered by regulators to be
inherently dissimilar to the innovator product such that comparative and abbreviated testing,
applications and approvals may not be allowed. This does not currently appear to be a likely scenario
in the U.S. or European Union. However, there are indications that some countries may follow a
rather narrow view of what constitutes sufficient similarity to support abbreviated approvals; and U.S.
and EU regulators could always buckle under pressure from innovators, politicians or public opinion
and decide to narrowly view manufacturing processes and radically favor safety (i.e., require full or
near-full clinical and other testing for biosimilar/follow-on products).
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In some countries, biosimilar approvals may even be restricted to only reverse-engineered copies
using the same old manufacturing technologies as their (likely ~20-year-old) reference products. For
example, recently issued draft Canadian guidelines for subsequent entry biologics (SEBs) require
the use of analogous manufacturing technologies, seemingly with particular emphasis on use of
very similar or the same expression systems and manufacturing processes (4). It would appear that a
Canadian SEB version of recombinant EPO, i.e., products comparable to Epoetin and Procrit, both
containing erythropoietin expressed by CHO cells cultured in 1,000s of roller bottles, would have to
emulate this now relatively-antiquated but effective manufacturing process. Presumably, this proposed
policy will not be implemented, i.e., biosimilar/follow-on regulation should be based on considering
the products similarity, not comparisons of manufacturing processes. Despite this restrictive
Canadian proposal, many companies worldwide are developing biosimilar/follow-on versions of
EPO, with all of them reported or fully expected to use modern bioreactors, not roller bottles, and
many adopting alternative expression systems. The European Union, which has implemented
biosimilar regulations, does not explicitly require process-based similarity, just product similarity.
In the U.S., the FDA has yet to issue guidelines that were due years ago for the simpler follow-on
proteins it regulates as drugs, and Congress has yet to enact a law enabling abbreviated approvals of
biologics, with full FDA implementation likely requiring years after that happens. In the meantime
in the U.S. and elsewhere where biosimilar/follow-on regulations have yet to be implemented, once
product-covering patents expire, which in many cases will include companies claiming coverage by
process patents, there is nothing to stop any company from bringing out a similar or even identical
(interchangeable/substitutable) biopharmaceutical product, provided that they receive traditional full
approval. In fact, with many, if not most, biopharmaceuticals worldwide being biogeneric copies in
many respects, some of the first products being developed as biosimilars/follow-ons may well receive
full rather than abbreviated approvals, with absolutely no need for comparisons or analogies with any
prior product.
Biosimilar/follow-on biologics developers generally cannot simply copy decades old manufacturing
schemes, even if these no longer involve licensing fees. These companies generally cannot afford or
justify the large up-front expenses associated with older, less efficient manufacturing platforms; and
they have every reason or simply must use optimally efficient manufacturing technologies, particularly
those with lower up-front investment/infrastructure costs, e.g., not requiring large dedicated
equipment, facilities, land, those conducive to use maximal us of disposables vs. fixed equipment,
etc., with old technologies generally providing lower yields or more dilute products requiring more
resource- and labor-intensive purification steps.
This author does not agree with the predominant belief that biosimilar/follow-on biologics companies
will gain large market share by beating the prices charged by innovators for their original products.
The success of biosimilar/follow-on products and companies may well come down to price
competition, with biosimilar/follow-on manufacturers needing to produce their products at as low
cost as possible (the same situation with generic drugs). Something is wrong, if innovator companies,
after manufacturing and marketing their product for a decade or often much longer, and having
supplied the worlds markets for their product for this entire period, cannot meet or even beat the
prices to be charged by biosimilar/follow-on biologics manufacturers (and still make a respectable
profit). Even with inefficient, old processes and equipment, innovators making older products should
have economies-of-scale that cannot be beat by any upstart competitor. This ability and likelihood
for innovators to compete on price is particularly true when one considers that innovators will almost
13
always already have a 2nd or later generation replacement on the market, and they have a strong
motivation, if not imperative (often demanded by stock holders) to above all maintain market share
for their broad product portfolios, usually including multiple products marketed for the same/similar
indications. Thus, any company planning to market a biosimilar version of erythropoietin (EPO) in
the U.S. could face serious or devastating price competition from Amgen and Centocor/Ortho/J&J, the
original innovators and market leaders in the U.S. From prior actions, it also seems likely that many
innovator (bio)pharmaceutical companies will do whatever it takes to maintain market dominance
simply as a matter of basic principles, and if that means discounting old products, not much is lost.
What Is the Market for Expression Systems and Related Technology?
How Will It Evolve?
The industry and markets for novel and improved expression systems and related biopharmaceutical
manufacturing technologies are still in their infancy, but one can start to see some patterns, trends
and likely winners. The industry/market will likely take off, e.g., with licensing and acquisitions of
many of the technologies and companies in this directory, in coming years, coincidentally along with
the expected implementation of biosimilars/follow-on biologics regulations in the U.S. Much like
other early and evolving research-based high tech industries, as largely exemplifed by the broader
biopharmaceutical and biotechnology industries, this industry/market is characterized by diversity
and follows many of the same patterns familiar in the biotech/biopharma industries. Thus, most
research and innovations tend to either come from academic researchers; small companies, including
those solely dedicated to commercializing one or a few technologies/products; and large companies
that can afford to do things right, i.e., invest the needed resources to either license in or develop new
technologies themselves, rapidly scale these up and prove commercial viability and market these
effectively.
The market for licensing of expression systems and related genetic engineering technologies largely
follows that for biotechnologies and product acquisition and licensing, in that sense that there is a
relatively open market with most every technology available for license, particularly nonexclusively,
if one asks and at the right price. Thus, essential every technology in this directory is available for
licensing. Major exceptions and problems persist primarily in the areas of higher plants, particularly
those involving major agricultural crops, and transgenic animals; with patent disputes common, with
many still to be resolved. Many of the patents in these areas are broad and/or vague in terms of what
they do and do not cover, and many key, if not requisite, technologies are controlled by one or just
a few companies, many of which simply refuse or severely limit their licensing (they dont want to
share). Many of the large international companies with dominant patents have exclusively licensed
or cross-licensed these among themselves. So, those interested in using or licensing a higher plant or
animal platform technology would likely be best served by licensing this from one of the companies
that has positioned itself well in terms of licensing or acquiring a full portfolio of needed technologies
and/or contract for development and manufacturing services. In contrast, most microbial and lower
organism platforms and basic genetic engineering patents are more straightforward, related patents
easier (relatively) to decipher, and if not, most have had their patent ownership and coverage resolved
over the years, often as the result of patent disputes.
What is the current market for expression systems and related genetic engineering technologies? This
is very diffcult to answer. Unlike with marketed biopharmaceutical products, where total end-user
sales are hard to conceal and where most companies publicly report annual or even quarterly sales, most
14
everything related to new technologies, particularly concerning product manufacture, and licensing
is kept proprietary by companies, including technology inventors and developers and licensees, with
license agreements likely including non-disclosure requirements. Of course, some information is
disclosed about what has been licensed. For example, one could search for SEC documents and/or
foreign equivalents filed by public companies that may even include copies of licensing contracts
explicitly delineating fees and royalty payments or otherwise survey or search for this information, but
this was beyond the scope of this directory project. But few companies, as reflected in this directory,
actually publicly disclose their technology licensing royalty rate structures.
The market for licensing of technologies appears much like that of licensing candidate products.
Presuming one is dealing with a broad platform technology, e.g., novel host cells or organisms in
early development, i.e., largely unproven at commercial scale, platform expression systems, like
products in early/preclinical development, may be available for licensing with royalties on total
revenues of about one or a few percent. More mature systems or those offering proven compelling
advantages may charge more. For example, licensing of the Pichiapastoris expression system (see
related entry) from RCT involves royalties of about 3-5%. However, in some cases, particularly with
platform technologies that provide a more complete package and/or unique advantages, licenses may
involve about 10% royalties. At this level, one should expect to receive not just host organisms/cells,
but also supporting vectors, promoters, maybe a selection/-related documentation, e.g., copies of
studies, or access to a Biologics Master File. As mentioned above, patent stacking, i.e., licensing of
various pieces of complementary technologies from different sources, can rapidly result in rising total
royalties.
So, how big is the market? Current annual worldwide recombinant biopharmaceutical product sales are
on the order of $70 billion, with most of this, say $50 billion, in major markets and other industrialized/
developed countries (where patents are most likely to be filed and enforceable). Presuming that on
aggregate just 4% of $50 billion/year total revenue is spent on expression systems and related genetic
engineering technology acquisition and, particularly, licensing fees, the current market or industry
is on the order of $2 billion, with this scattered among a large number of companies and public
sector research organizations. For comparison, the industry of therapeutics discovery, screening and
optimization, nearly all involving contracting/outsourcing and licensing by established, large, drug and
biopharmaceutical companies is estimated to be about $7 billion/year (i.e., the pharmaceutical industry
outsources about $7 billion in product discovery and related early R&D). Several $billion/year seems
reasonable, when one considers that it is essentially impossible not to have to licensing one, often more,
technologies to manufacture a recombinant protein, e.g., host cells/organisms, vectors, promoters,
selection/amplification methods, chaperones, etc. But, in some or many cases (no one, either licensees
or licensors, discuss their royalty agreements publicly), total expression systems/genetic engineering
licensing royalties closer to 10% may be more realistic, and in this case, the current industry/market size
could well be about $3-$4 billion.
What is a technology worth? That is a very difficult question. For example, presuming other factors
are equal or comparable, would it be worthwhile to improve product yield by 200-300% but have
to pay an additional 1% royalty on sales, or would this level of royalties require more like a 10-fold
increase? How much is it worth to license a technology that will make it very difficult or impossible
for biogeneric companies to copy your product, both immediately in many lesser-developed countries
(where relevant patents have not been filed/granted and/or where pharmaceutical process-related
patents are simply not enforced), and later, probably well over a decade in the future, when product-
15
protecting patents expire in major markets/developed countries? How much it is worth to save months
in getting from identification of a candidate product to manufacturing enough of it to get it into
preclinical and clinical trials? Many of the more prominent and popular new platform technologies,
including those actually marketed, those publicly licensed to multiple companies and those affiliated
with large companies, seemingly charge higher fees/royalties. But how much additional is it worth
to have the assurance that other companies are using and will likely pioneer regulatory approvals
with a new technology, that meaningful user support is routinely available and to have the security in
knowing that others have evaluated a technology and similarly taken a license? Is it worth it (in your
situation) to license a more fully-developed technology, e.g., proven at commercial-scale manufacture,
vs. probably paying less and licensing one where you will be investing more time and effort in final
development and scale-up?
The current industry (if one can call it that; it is still rather early, with as discussed above, just a
few technologies still dominating current product manufacture) for expression systems and related
genetic engineering technologies is highly diverse and parallels the broader biopharma/biotech
industries. Thus, small players very much dominate numerically, while a relatively few successful/
profitable companies dominate economically. Many inventions and technologies come from lone
researchers, with many of these associated with companies that may be largely virtual (i.e., lack their
own facilities, infrastructure, employees other than founders, etc.). There are also small, relatively
adequately funded companies developing and licensing these technologies, often developing one or
more therapeutics in parallel, either as their main goal or using these to demonstrate the viability of
their manufacturing-related technologies. And, as with the biopharmaceutical, biotechnology and
pharmaceutical industries, you always have a few relatively dominant major players. For example,
Dow Chemical is the commercial developer and licensor of Pfenex (Pseudomonas) technology;
Invitrogen and RCT each handle commercial licensing for multiple technologies; Crucell, a small
biotech-type company, has joined with DSM, a large international chemical/drug company, for
development and licensing of PER.C6 technology; and Merck owns GlycoFi with its technology for
glycosylation in yeasts. It is easy to see that companies such as these with credible technological
advances, financial and other technological support from large parent companies, and with aggressive
marketing programs will be among the winners/survivors. But, a large number of small companies
have viable technologies, and many of these will sooner or later attract licensees and become
successful, whether success involves becoming profitable, even public independent companies or
being acquired.
Is this a growth industry? Definitely. The industrys growth will closely track that of the
biopharmaceutical industry. A growing number and percentage of pharmaceuticals in development
and on the market are biopharmaceuticals, and this trend will continue. As discussed above, although
one can now manufacture recombinant proteins (but generally not yet glycoproteins or antibodies)
without having to license related manufacturing technologies, economics, regulatory and other aspects
do not support this in the long term. Newer technologies simply are usually worth their extra cost
and bother. Many sources cite total recombinant protein, broader biopharmaceutical sales and the
number of biopharmaceutical (vs. drug) products growing on the order of 15% of more annually. The
associated market for acquisition and licensing of expression systems and related genetic engineering
technologies can be expected to closely track this. While there may be a short-term drop in total
licensing/royalty outlays now or in the near future as many patents covering older technologies
(covering much or most marketed products) expire, new products essentially all using newer, licensed
16
technologies will eventually be coming online and many, if not all, of the manufacturers of mature
products have likely upgraded their manufacture in recent years or will do so, so they have been or
will soon be paying royalties on many newer technologies.
Access, Including Marketing and Information Dissemination, Needs to
Greatly Improve
One problem that is evident with this industry is marketing, or rather the lack of it. One would
presume that companies and even universities seeking to out-license these technologies would at least
take the minimal steps, e.g., put some minimal or useful information up at their Web site, present
and/or exhibit at relevant conferences, publish in industry periodicals, etc. to let themselves be found
by potential clients, but overall that is not the case with this industry. Other than a few organizations
that obviously do some marketing, even advertising, with these usually part of or affiliated with
large international companies (e.g., see some mentioned above), most of the companies with relevant
technologies available for license maintain low profiles, i.e., they and their technologies require
considerable effort to find. Many of the technologies in this directory might not have been found,
had the author not monitored the worlds commercial biotech/biopharma literature over the period of
many years, combined with recent searching for relevant technologies/companies. Many companies
simply have not made effective efforts at promoting technologies, and fewer are proactive, e.g.,
engage in effective marketing, advertising or exhibiting.
Hundreds of organizations, mostly companies, including those with business models based on out-
licensing as their primary income source, were contacted in connection with this project. In addition,
the information provided at licensor organizations Web sites, where this is available, is included. As
discussed in the Users Guide, these factors affect the content of the technology descriptions in this
directory.
One factor contributing to this lack of marketing is the conservative approach to licensing,
particularly predominant in larger companies. Here, most information not related to corporate
or products medical/use aspects is considered inherently proprietary/undiscloseable, and if it
involves technology and, particularly, product manufacture, companies avoid the issue. Even
companies whose income is dependent on out-licensing, which requires some effective marketing and
communications, do not do it.
Another factor likely restricting ready access is the perception that inclusion of their technologies
along with others in directories could reduce the perception of exclusivity. As such, technology
transfer/licensing is only being done on a face-to-face basis. Thus, in many cases, prospective
licensees need to be persistent, should volunteer to sign non-disclosure agreements (which at
least initiates interaction) and should expect to have to deal with legal and financial issues. Those
organizations that want to be successful at out-licensing their technologies might consider assigning
marketing, outreach and information dissemination responsibilities to professionals dedicated to
locating and interacting with serious, qualified, screened prospects who are passed on for license
negotiation.
The lack of marketing and information dissemination by many companies can be used to advantage
by prospective licensees. With fewer prospective licensees and with many technologies and
companies having low or negligible profiles (hard to find, even when looking), those with serious
licensing intentions may be in a better bargaining position. Also, many companies and technologies
17
may be ripe for outright acquisition by those with the resources, particularly by biopharmaceutical
companies in a position to apply and rapidly develop technologies and support their effective
marketing.
Currently, some of the most effective marketing and information dissemination is done by a
relatively few major players, primarily large international companies, that have gotten involved
in expression systems and related genetic engineering technologies, e.g., DOW, GE, RCT, DSM
(with Crucell), Invitrogen, etc. These companies have marketing staffs and budgets, put effort into
publishing their technologies, and present and exhibit at industry-oriented conferences. In contrast,
some organizations, offering relevant technologies that have limited marketing effort, may have
published in peer-reviewed scientific vs. more commercially and industry-oriented publications,
similarly may have presented at scientific vs. more industry-oriented conferences, have limited
promotional literature available, have limited Web presence, and are ill-prepared to disclose
nonproprietary information, even for appropriate venues.
The Future is Bright
So, what is going to happen? Will the hegemony of the same, old expression systems continue?
Probably not for long, but it may take a while. Although economics and often improved product
quality may favor newer technologies, the established innovator biopharmaceutical industry,
actually a relatively small number of companies, which currently predominantly controls large-scale
manufacturing capacity, appears content with the established technologies that have served it so
well, particularly since they have likely all been upgraded with new technology, e.g., more potent
promoters, in recent years. Many companies will likely continue to reject new technologies to avoid
associated regulatory delays and standing out from the pack, and continue to maximize their return
on the large-scale facilities they have constructed by keeping them busy manufacturing products.
However, many of these large (bio)pharmaceutical companies have been and will increasingly
be testing and optioning new technologies, often starting with using these to make products for
preclinical and early studies. Much, if not most, of the early adoption of new technology will likely
be at the lower end, i.e., involving small companies and CMOs.
Biosimilar/follow-on products will be a major factor driving the industrys adoption of new
expression systems by all types of companies (especially biogenerics makers) for their cost
savings and by innovators as a defense against future biosimilar competition. No matter how
regulatory agencies resolve (bio)similarity issues, the use of novel expression systems will increase.
If products are required to be similar in their manufacture, that will further induce innovators to
adopt (license) new technologies to confer unique properties to their products, thus gaining inherent
defenses against copying or close similarity. If, as this author expects, regulatory agencies ultimately
will be more concerned with comparing products rather than methods of manufacture, then follow-
on/biosimilar companies will be likely be the main pioneers and first to adopt many of the new,
improved, and as yet underused (at large scale) expression systems and related genetic engineering
technologies.
18
REFERENCES
1. Rader RA. Biopharmaceutical Products in the US and European Markets, volumes 1 and 2. BioPlan Associates,
Inc.: Rockville, MD, September 2007; www.biopharma.com.
2. Langer E. Manufacturers Must Cooperate to Compete: The Biomanufacturing Industry Faces a Catch-22.
Contract Pharma October 2002: 6670.
3. Rader R. What Is a Generic Biopharmaceutical? Biogeneric? Follow-On Protein? Parts 1 and 2. BioProcess Int.
5(3,5) 2007: 2838, 2038.
4. Draft Guidance for Sponsors: Information and Submission Requirements for Subsequent Entry Biologics (SEBs).
Health Canada: 14 March 2008; www.hc-sc.gc.ca/dhp-mps/brgtherap/activit/consulation/seb-pbu/2008-tc-tim_
e.html. c
Ronald A. Rader is president of the Biotechnology Information Institute, 1700 Rockville Pike, Suite 400,
Rockville, MD 20852, 1-301-424-0255, ron@biopharma.com; www.bioinfo.com.
19
Cell-free systems
100
Cell-free expression, ATP
regeneration system
Organizations involved:
National Institutes of Health (NIH) - Licensor,
primary
Description: An adenosine triphosphate (ATP)
regeneration system has been developed for
cell-free protein expression using one of the early
intermediates of the glycolytic pathway as the
secondary energy source. The system improves
efficiency of protein synthesis by several fold by
providing an improved energy regeneration sys-
tem and protein-folding machinery.
Use with: cell-free systems
Use to make: proteins; glycoproteins
Background: Cell-free protein expression is
becoming a valuable tool for rapid and economi-
cal production of recombinant proteins. In con-
ventional cell-free protein synthesis systems, the
ATP (high energy) supply is accomplished by
secondary energy regenerating sources contain-
ing high-energy phosphate bonds. The sources
include glucose (G), glucose-6-phosphate (G-
6P), phosphoenolpyruvate (PEP), acetyl phos-
phate (AP), creatine phosphate (CP) or pyruvate.
However, some of these systems (G, G-6P and
pyruvate) require the addition of exogenous
enzymatic cofactors such as NAD/NADH, adding
considerable expense to the system. In addition,
the conventional systems (PEP, AP or CP) are also
mired by unproductive enzymatic degradation of
energy sources and unproductive consumption of
ATP resulting in lower yields of protein.
Benefits: The ATP energy source costs only a
fraction of the conventional substrates, provides
chemical energy for protein synthesis without
the addition of an exogenous enzymatic cofactor,
thereby reducing the costs of the system.
Patents: U.S. application. 60/454,013, Chatterjee,
et al., was filed in March 2003 (DHHS Reference
No. E-328-2002/0-US-01); PCT/US04/07449 was
filed in March 2004.
Licensing information: Licensees are sought.
Contact, Fatima Sayyid, Licensing Specialist,
NIH.
101
Cell-Free Protein Synthesis
(Cell-Free)
Fundamental Applied Biology, Inc. (FAB)
- Licensor, primary
Stanford University - Assignee, patent
Description: Cell-Free Protein Synthesis (Cell-
Free) activates single protein expression from
combined transcription and translation reactions
without the need for living cells. This avoids
many significant limitations associated with in
vivo platforms.
Cell-Free is accomplished by extracting the
necessary catalytic machinery, including enzymes,
required for metabolism, transcription, translation,
and proper folding from living cells. The lack of a
cell wall or membrane barrier, and lack of need to
satisfy biochemical needs associated with growth
and maintenance of a living cell, provides critical
advantages over conventional recombinant DNA
protein expression technology. Cell-Free technol-
ogy uses highly efficient oxidative phosphoryla-
tion to produce a stable, plentiful, and lasting
energy source to drive protein synthesis. Energy
and other resources can be more focused and
efficiently utilized for processes directed toward
synthesis and folding of the target protein.
Cell-Free is engineered for disulfide bond for-
mation through sulfhydryl redox potential stabili-
zation, adjustment, and catalysis. This flexibility
allows Cell-Free to synthesize and properly fold
many targets that are difficult to produce using
Broad/Platform
Technologies
20
conventional recombinant protein expression plat-
forms. These attributes may result in improved
therapeutic efficacy, as well as reduced allergic re-
actions, immunogenicity, and viral contamination.
FABs Cell-Free technology is fully scalable
from lab bench to production scale volume in
batch mode. From preliminary product synthesis
in the lab, to development of an early-stage pro-
duction process, and making preclinical material,
Cell-Free technology conserves performance at
any scale. This allows a smoother transition from
process development to R&D production. At
larger scales, Cell-Free is designed to work with
conventional bacterial cell culture reactors mak-
ing the facility construction easy and development
of new reactor technology unnecessary.
Fundamental Applied Biology claims, The
unprecedented speed and systemic control pro-
vided by Cell-Free promises to change the entire
paradigm for protein production, from discovery
and development through final product manufac-
turing. This is particularly true for mammalian
protein pharmaceuticals with complex fold-
ing pathways. The cell-free platform slows the
translational rate of ribosomes to provide more
time for protein folding and reduces the overall
macromolecule concentration to further discour-
age aggregation and facilitate folding. The disul-
fide/sulfhydral redox potential is controlled and
disulfide isomerases added to enhance the proper
formation of disulfide bonds. Since only a single
protein is translated by the system, interference
from other nascent proteins is eliminated. All of
these factors are combined within a natural chemi-
cal environment to produce a system with unprec-
edented folding efficiency. However, efficient
folding alone is not enough. For cost reduction,
central metabolism is activated so that inexpen-
sive substrates provide an abundant energy supply
and also avoid the need for expensive nucleotide
triphosphates (NTPs). For scale-up, simple and
inexpensive technology allows Cell-Free to be
conducted in standard fermentors.
Use with: cell-free systems
Use to make: proteins; glycoproteins; antibody
fragments
Background: Cell-free protein synthesis methods
have been around for over 40 years. However,
recent advances made at Stanford University and
then FAB Inc. have transformed it from a bench-
top novelty into a viable manufacturing platform.
These advances include:
Creating an environment that more closely
mimics the natural intracellular environment,
dramatically lowering costs and increasing yields
from Cell-Free reactions;
Activating folding to enable Cell-Free to di-
rectly produce properly folded proteins;
Scaling-up in an off-the shelf stirred tank reac-
tor;
Successfully demonstrating of the ability to
produce difficult to produce proteins of pharma-
ceutical interest for industry collaborators.
Benefits: Cell-Free technology has advantages
that differentiate it from current cell-based sys-
tems, and may improve efficacy, safety, and speed
of production due to more natural protein folding
with better biologic activity, higher purity, lower
degradation, and higher yields.
Claimed Unique Cell-Free Advantages
include:
Degradation
Shorter reactions - avg. 4 hrs
Dilute environment - 20x lower protein con-
centration than intracellular environment.
Solubility
Slower translational rate of ribosomes - 2AA/s
Lower macromolecule concentration
Folding
Ability to control sulfhydral redox potential
Ability to add disulfide isomerases
Ability to add folding chaperone proteins
No Toxicity
Protein is produced in-vitro
Low Expression
No cell walls - direct control of environment
through additions of substrates/catalysts.
Extract can be modified through extract host
strain or upstream pre-treatment.
Purification
Fewer variants
Cleaner starting material
21
Process Scale-up
Homeostatic system
Scalable from 15?l to standard bioreactors.
Rapid process development
In contrast, cell-based technologies involve;
proteins that are often toxic to cell-based systems;
improper protein folding; require specialized post-
translational modification; are often limited by
recombinant DNA safety (RAC) guidelinesl have
potential for viral and prion contaminants; may
contain other antigenic contaminants; and often
provide low yield.
Patents: FABs proprietary cell free protein syn-
thesis technology was primarily developed by Dr.
James R. Swartz, School of Engineering, Stanford
University, and exclusively licensed to FAB.
Exemplary patents include U.S. 7,312,049,
Total amino acid stabilization during cell-free
protein synthesis, Dec. 25, 2007, assigned to
Stanford University, concering S30 cell-free ex-
tracts of a E. coli bacteria containing inactivated
genes for tryptophanase, arginine decarboxylase,
L-serine deaminase
and gamma-glutamylcysteine synthase;
7,041,479, Enhanced synthesis of active proteins
containing disulfide bonds, concerning enhanced
in vitro synthesis of polypeptides containing
disulfide bond and; 6,994,986, In vitro synthesis
of polypeptides by optimizing amino acid metabo-
lism.
Licensing information: Contact Fundamental
Applied Biology, Inc. (FAB).
Products made using this tech.: Cell-Free has
been successfully used by FAB and its partners
for the production of:
Novel vaccine constructs
Peptides prone to proteolysis
Therapeutic proteins that are difficult to ex-
press in soluble form
Therapeutic proteins that are prone to variants
and difficult to purify.
Further info.:
1. Kanter G, Yang J, Voloshin A, Levy S, Swartz
JR, Levy R. Cell-free production of scFv fusion
proteins: an efficient approach for personalized
lymphoma vaccines. Blood 2007, 109, 3393-

3399.
2. Yang J, Kanter G, Voloshin A, Michel-Reydel-
let N, Velkeen H, Levy R, Swartz JR. Rapid ex-
pression of vaccine proteins for B-cell lymphoma
in a cell-free system. Biotechnol. Bioeng. 2005,
89, 503-511.
3. Calhoun KA, Swartz JR. Energizing cell-free
protein synthesis with glucose metabolism. Bio-
technol. Bioeng. 2005, 90, 606-613
4. Michel-Reydellet N, Woodrow K, Swartz JR.
Increasing PCR fragments stability and protein
yields in a cell-free system with genetically modi-
fied Escherichia coli extracts. J. Mol. Microbiol.
Biotechnol. 2005, 9, 26-34.
5. Voloshin A, Swartz JR. Efficient and scalable
method for scaling up cell-free protein synthesis
in batch mode. Biotechnol. Bioeng. 2005, 91,
516-21.
6. Liu DV, Zawada JF, Swartz JR. Streamlining
Escherichia coli S30 extract preparation for eco-
nomical cell-free protein synthesis. Biotechnol.
Prog. 2005, 21, 460-465.
7. Jewett MC, Swartz JR. Mimicking the Esch-
erichia coli cytoplasmic environment activates
long lived and efficient cell-free protein synthesis.
Biotechnol. Bioeng. 2004, 86, 19-26.
8. Jewett MC, Swartz JR. Rapid expression and
purification of 100 nmol quantities of active pro-
tein using cell-free protein synthesis. Biotechnol.
Prog. 2004, 20, 102-109.
9. Zawada JF, Swartz JR. Maintaining rapid
growth in high-density Escherichia coli fermenta-
tions. Biotechnol. Bioeng. 2004, 89, 407-415.
10. Kim DM, Swartz JR. Regeneration of ATP
from glycolytic intermediates for cell-free protein
synthesis. Biotechnol. Bioeng. 2001, 74, 309-316.
11. Kim DM, Swartz JR. Oxalate improves
protein synthesis by enhancing ATP supply in
cell-free system derived from Escherichia coli.
Biotechnol. Lett. 2000, 22, 1537-1542.
12. Kim DM, Swartz JR. Prolonging cell-free
protein synthesis with a novel ATP regeneration
system. Biotechnol. Bioeng. 1999, 66, 180-188.
22
102
Large ribosomal subunit
proteins, E. coli - cell-free
systems
Johns Hopkins University (JHU) - Licensor,
primary
Description: Large ribosomal subunit proteins
from E. coli are useful in cell-free expression sys-
tems for in vitro reconstitution of functional 50S
subunits. Each of 34 individual large ribosomal
subunit proteins for E. coli for use in vitro ribo-
some reconstitution reactions have been cloned,
expressed and purified. The ready manipulation of
ribosomes assembled from these and other com-
ponent substituents permits completely synthetic
large ribosomal subunits and fine-tuning of the
activity of the ribosome population to enhance
particular desirable features such as speed, accu-
racy or unusual substrate permissivity.
These subunits could be used for large-scale
protein production in cell-free systems readily
subjected to specific environmental control.
Use with: E. coli cell-free systems
Use to make: proteins
Patents: Related applications include
WO/2006/085954) IN VITRO RECONSTITU-
TION OF RIBONUCLEOPROTEIN COM-
PLEXES, AND METHODS OF USE THERE-
FOR, Rachel Green and Katharina Semrad, Aug.
17, 2006, assigned to Johns Hopkins University
(JHU). The abstract states: In general, the inven-
tion provides methods and compositions for the in
vitro production of ribonucleoprotein complexes
and related multimolecular complexes useful for
cell-free methods of protein production. Claim
1 states, An isolated ribonucleoprotein complex
comprising a recombinant prokaryotic ribosomal
polynucleotide and a recombinant prokaryotic
ribosomal protein, or fragments thereof, wherein
the ribonucleoprotein complex has a biological
activity of a reference prokaryotic 50S ribosomal
subunit.
Licensing information: Contact Johns Hopkins
University (JHU Ref. 4466).
Further info.:
Dohme, F., Nierhaus, KH (1976) Total recon-
stitution and assembly of 50S subunits from
Escherichia coli ribosomes in vitro. JMB 107 (4),
585-99.
Culver, GM, Noller, HF (1999) Efficient reconsti-
tution of functional Escherichia coli 30S ribo-
somal subunits from a complete set of recombi-
nant small subunit ribosomal proteins. RNA 5(6),
832-43.
Semrad, K, Green, R (2002) Osmolytes stimulate
the reconstitution of functional 50S ribosomes
from in vitro transcripts of Escherichia coli 23S
rRNA. RNA 8(4), 401-11.
Green, R., Noller, HF (1999) Reconstitution of
functional 50S ribosomes from in vitro transcripts
of Bacillus stearothermophilus 23S rRNA. Bio-
chem. 38(6), 1772-9.
Green, R., Noller, HF (1996) In vitro complemen-
tation analysis localizes 23S rRNA posttranscrip-
tional modifications that are required for Esch-
erichia coli 50S ribosomal subunit assembly and
function. RNA 2(10), 1011-21.
Broad, cross- or multi-platform
technologies
103
In vivo linearization; InVoLin;
Meganuclease Recombination
System (MRS) - improved
transformation
Cellectis SA - Licensor, primary
Description: InVoLin, standing for in vivo lin-
earization is a specific Meganuclease Recombina-
tion System (MRS; see related entry) technology
where the transgene is excised from a circular
plasmid within the cell. This MRS does not rely
on homologous recombination and improves the
efficiency of classical transgenesis.
23
Classical transgenesis relies on the transfec-
tion of foreign sequences into the nucleus. The
best efficiencies are usually obtained after in vitro
linearization of the transgene. It is excised from
its plasmid vector, and the purified DNA frag-
ment is introduced into the cell. However, even in
these conditions, many problems are encountered,
including: efficiency, which can be very low,
depending on the organism; number of copies,
which can be very high, often resulting in silenc-
ing; and integrity of the copies. InVoLin improves
these three parameters, making transgenesis ac-
cessible to a number of resistant cells and species.
Impressive results have been reported in fish.
Use with: generic (universal)
Licensing information: Contact Cellectis.
104
Vectors, site-specifc
recombinase assembly -
eukaryotes
Protemation, Inc. - Licensor, primary
Description: Expression vectors constructed us-
ing site-specific recombinases are used in vitro or
in vivo with eukaryotic cells to directly fuse a lin-
ear segment of donor DNA (e.g., gene of interest)
with another linear segment of DNA comprising
various functional elements such as promoters, se-
lectable markers and a recombination site, result-
ing in a single circular donor DNA. This circular
donor DNA is then recombined into a circular ac-
ceptor vector which also contains a recombination
site through site-specific recombination catalyzed
by a recombinase. The recombination product can
be used to transform, transfect or transduce vari-
ous types of host cells, depending on the specific
type of acceptor vector used. See also the AttSite
recombinases entry.
Use with: eukaryotic cells
Patents: Exemplary patents include U.S.
6,953,689, Cloning system for construction of
recombinant expression vectors, Clark, assigned
to Protemation, Inc.; and 6,551,828, Compositions
and methods for generating expression vectors
through site-specific recombination, Clark, as-
signed to Protemation, Inc.
Licensing information: Contact Protemation,
Inc.
105
Glycosylation, mammalian
[Generic entry]
Description: Various technologies provide
mammalian or human-like glycosylation in
host organisms otherwise lacking requisite
enzymes or improve glycosylation, i.e., provide
more mammalian or human-like glycosylation,
in organisms, such as yeasts and insect cells,
possessing some inherent glycosylation activity.
Prokaryotes may also be engineered for
glycosylation. The function of many mammalian
proteins is depedent on their glycosylation.
Antigenicity, particularly the lack of it, is often
dependent on glycosylaton patterns.
Glycosylation of proteins normally takes place
post-translation by enzymes, glycotransferases
and glycosidases, in the ER and Golgi complex.
Thus, unlike protein production, glycosylation is
not guided by DNA, RNA or any other control-
ling mechanism. This results in a wide range of
glycoproteins forming from attachment of various
glycoforms to a protein, leading to considerable
(micro)heterogenicity of glycoproteins.
Oligosaccharides are covalently attached at
either a nitrogen (N-glycosylation) or oxygen
(O-glycosylation). N-linked glycan groups have
a core pentasaccharide of three mannose and
N-acetylglucosamine (Man3GlcNac2) to which
different sugars may be added and branch to form
varying side chains. O-linkage involves attach-
ment to thronyl or seryl residues.
Cell lines vary and each offers unique glyco-
sylation. For example, CHO cells do not normally
produce bisecting N-acetylglucosamine structures,
24
and murine cell lines often form terminal galac-
tose (Galalpha1-->3Gal) structures immunogenic
in humans.
Use to make: glycoproteins.
106
Aminoglycoside
phosphotransferase marker;
Neomycin phosphotransferase
I (nptI) marker; Geneticin
(G-418) selection - universal
Novartis AG - Licensor, primary
Chiron Corp., Novartis AG - Assignee, patent
Description: A truncated bacterial gene (aph-
I), e.g., neomycin phosphotransferase I (nptI),
coding for an aminoglycoside (e.g., neomycin)
phosphotransferase incorporated into vectors is
useful for selection of stable transformants surviv-
ing presence of the antibiotic geneticin G-418.
Expression of neomycin or other aminoglycoside
phosphotransferase confers resistance to the toxic
effects of geneticin.
A truncated coding sequence for APH-I (Kan
gene) contains convenient restriction sites imme-
diately upstream of the start codon. The coding
sequences of this marker can be used as a cassette
and integrated into plasmids containing control
sequences such as promoters and transcription
termination sequences which are effective in any
desired host organism. Thus, the coding sequence
can be shuttled from a carrier vector into a vector
adapted to replication and expression in yeast or
one which is adapted to expression and replication
in a number of other types of organisms such as
procaryotes, plants, and mammalian cells. There
are several aminoglycoside phosphotransferases
conferring resistance to aminoglycoside antibiot-
ics. The aminoglycoside phosphotransferase I
(aph-I) enzyme and the aminoglycoside (or neo-
mycin) phosphotransferase II (APH-II or NPTII)
are unrelated except for their ability to inactivate
the antibiotic G-418. The aph-I gene was origi-
nally found on transposon Tn601, also known as
Tn903. It has been reported that aph-I is approxi-
mately four times more effective than aph-II in
inactivating G418.
Two neomycin phosphotransferase genes are
used in selection of transformed organisms: the
neomycin phosphotransferase I (nptI) gene and the
neomycin phosphotransferase II (nptII) gene. The
second one is the more widely used. It was initial-
ly isolated from the transposon Tn5 present in the
bacterium strain Escherichia coli K12. The gene
codes for the aminoglycoside 3-phosphotrans-
ferase (denoted aph(3)-II or NPTII) enzyme,
which inactivates by phosphorylation a range of
aminoglycoside antibiotics including kanamycin,
neomycin, geneticin (G418), and paromomycin.
In animal cells, G418 and neomycin are used
as selectable agents.
NPTII is probably the most widely used select-
able marker for plant transformation. It is also
used in gene expression and regulation studies in
different organisms in part because N-terminal
fusions can be constructed that retain enzymatic
activity. Plants such as maize, cotton, tobacco,
Arabidopsis, flax, soybean and many others have
been successfully transformed with the nptII gene.
In plants, kanamycin is the most commonly used
selective agent, normally in concentrations ranging
from 50 to 500 mg/l. It is very effective in inhibit-
ing the growth of untransformed cells. The choice
of the selective agent is important and based on
the plant species to be transformed.
Use with: plants; mammalian cells; universal
(generic)
Patents: U.S. 4,784,949, Universal dominant
selectable marker cassette, Gelfand, et al., Nov.
15, 1988, assigned to Cetus Corp. (later became
Chiron Corp., acquired by Novartis), includes
claims for truncated coding sequence for APH-I.
This original and probably related patents have
likely expired.
A related patent 5,116,750, a division of
4,784,949, directed to a fusion protein containing
aph-I enzyme expired due to lack of payment of
fees on September 26, 2000.
25
Availability: Marketed for non-commercial uses
by various vendors with no footnotes about need
to license for commercial uses.
Licensing information: Almost certainly in the
public domain. But contact Chiron/Novartis, if
unsure about the patent status and the need to
license.
Further information: FEMS Microbiology Let-
ters, An inducible expression system of histi-
dine-tagged proteins in Streptomyces lividans for
one-step purification by Ni2+ affinity chroma-
tography, Volume 137 Issue 2-3 Page 135 - April
1996 [Besides use of S. lividans (see related
entry), aminoglycoside phosphotransferase gene
(aph) is used as a selective marker].
107
Cellulose binding domain (CBD)
fusion protein affnity tags;
pET-CBD - universal
CBD Technologies, Inc. - Licensor, primary
University of California - Assignee, patent
Yissum Research Development Co. - Assignee,
patent
Novagen - Retail seller
Description: Cellulose binding domain (CBD)
fusions with proteins facilitate downstream puri-
fication by CBD affinity chromatography. CBD
provides an expression system for the produc-
tion of a fusion protein comprising the CBD and
a second protein of interest. CBD has also been
incorporated into the pET-CBD vector (see pET
entry).
CBDs have high affinity for crystalline cel-
lulose and chitin, including when these are bound
to or used as chromatography media. CBD has a
high affinity for crystalline cellulose and chitin,
having a Kd ranging from about 1.4 to about 0.8.
In particular, with various samples of crystalline
cellulose, the CBD exhibits a Kd of about 1.2 or
less. CBD-fusion products comprising CBD and a
second protein retain the avid binding capacity of
the CBD to cellulose.
Use with: universal (generic)
Patents: Exemplary U.S. patents include
5,496,934; 5,202,247; 5,340,247; and 5,137,819,
coassigned to Yissum Research Development Co.
(Hebrew University of Jerusalem) and the Univer-
sity of California.
Availability: Marketed by Novagen for non-com-
mercial uses.
Licensing information: Contact CBD Technolo-
gies, Inc. regarding commercial use and licensing.
108
Controllable Self-Cleaving
Intein Derivative; IMPACT-
CN; pH-sensitive self-
cleaving fusion protein affnity
purifcation tag - universal
Health Research, Inc. - Licensor, primary
Rensselaer Polytechnic Institute - Assignee,
patent
Description: Autocatalytic cleaving elements
incorporated in vectors along with a gene for a
desired protein are useful for efficient, large-scale,
self-cleaving affinity fusion protein mediated
purification. Inclusion of autocatalytic cleaving
domains (inteins) between the binding domain
and product protein renders the binding domain
self-cleaving in response to an added nucleophile
during the purification procedure. Following
translation of the host protein-intein precursor
sequence, the intein excises itself and ligates the
flanking host protein segments (exteins) to form
the native host protein and released intein. Cleav-
age is induced purely through a mild pH shift in
the running buffer. The result is on-column cleav-
age, which eliminates the need for subsequent
downstream processing as well as addition of an
exogenous cleavage agent.
A major drawback for fusion protein-based
purification has been the time required for com-
pletion of the cleavage reaction. In the case of
C-terminal cleavage, where the product protein is
26
fused downstream from the binding domain, the
reaction time for many currently available sys-
tems is in excess of 3 days at 4C. This invention
provides a pH-sensitive mutant cleaving element
with greatly accelerated cleavage, decreasing the
time for C-terminal cleavage from days to several
hours at 4C, or to minutes at higher temperatures.
Furthermore, this intein mutant does not require
addition of any exogenous factors to the product
stream; the cleavage reaction is induced purely
through a mild pH shift in the running buffer.
The invention also describes the genetic selection
used in the isolation of these mutants, which can
be used to generate other mutants that are further
optimized for size and cleavage rate.
Benefits: Main advantages include:
1. The invention describes a genetic selection sys-
tem wherein activity of a modified intein results
in a selectable phenotype, allowing rapid genera-
tion of useful intein mutants through a combina-
tion of rational and random mutagenesis.
2. Affinity fusion-based protein purification
-- Self-cleavage, rather than splicing of the intein
releases the desired protein, thereby eliminating
the need for protease addition.
3. On-column cleavage, which eliminates the need
for subsequent downstream processing as well as
addition of an exogenous cleavage agent.
4. Acceleration of intein cleavage reactions --
Method can decrease C-terminal cleavage-based
affinity separation times and make this technique
more attractive for scaleup of intein-based protein
purifications.
5. Mutant mini-inteins isolated using this genetic
screen have elevated activities in vivo and in vi-
tro, and form the basis of a pH- and temperature-
dependent protein purification system.
6. Technology provides new insights into the
structural and functional roles of some conserved
residues in protein splicing.
Patents: Exemplary patents include 6,933,362,
GENETIC SYSTEM AND SELF-CLEAVING
INTEINS DERIVED THEREFROM, BIOSEPA-
RATIONS AND PROTEIN PURIFICATION EM-
PLOYING SAME, AND METHODS FOR DE-
TERMINING CRITICAL, GENERALIZABLE
AMINO ACID RESIDUES FOR VARYING
INTEIN ACTIVITY, Belfort, et al., August 23,
2005, assigned to Rensselaer Polytechnic Insti-
tute. The abstract states, A self-cleaving element
for use in bioseparations has been derived from
a naturally occurring, 43 kDa protein splicing
element (intein) through a combination of protein
engineering and random mutagenesis. A mini-in-
tein (18 kDa) previously engineered for reduced
size had compromised activity and was therefore
subjected to random mutagenesis and genetic se-
lection. In one selection a mini-intein was isolated
with restored splicing activity, while in another,
a mutant was isolated with enhanced, pH-sensi-
tive C-terminal cleavage activity. The enhanced
cleavage mutant has utility in affinity fusion-
based protein purification. The enhanced splicing
mutant has utility in purification of proteins such
as toxic proteins, for example, by inactivation
with the intein in a specific region and control-
lable splicing. These mutants also provide new
insights into the structural and functional roles of
some conserved residues in protein splicing. Thus,
disclosed and claimed are: a genetic system and
self-cleaving inteins therefrom; bioseparations
employing same; protein purification by inactiva-
tion with inteins in specific regions and control-
lable intein splicing; methods for determining
critical, generalizable residues for varying intein
activity; and products.
Availability: This forms the foundation of the
IMPACT-CN system marketed by New England
Biolabs for non-commercial uses.
Licensing information: Contact Health Re-
search, Inc. A Prototype of this technology is
available. A range of techniques is currently being
studied to provide a reliable, rapid, economical
laboratory method.
27
109
Cotransformation, Columbia
University - eukaryotes,
selection
Columbia University - Licensor, primary
Description: The broad genetic engineering
technology, cotransformation, involves inserting
multiple gene sequences - a sequence for expres-
sion of the desired protein along with an unlinked
DNA sequence coding for a selectable phenotype
not expressed by the host cell permitting survival
or identification of the transformed eukaryotic
cells.
Use with: universal, most any gene manipu-
lable organism
Benefits: Use of a gene for a selectible phenotype
(trait; characteristic), usually related to nutritional
requirements or resistance to toxicity, along with
the desired protein gene allows selection of suc-
cessfully transformed cells.
Patents: Simply stated, U.S. cotransformation
patents related to essentially all cell types except
CHO cells are now expired, in the public domain,
while the status of a more recent patent covering
CHO cells remains legally unresolved, but with
the university apparently requesting relatively
low, presumably <1%, royalties.
A broad recombinant DNA patentcovering co-
transformation processing assigned to Columbia
University was granted in 1983. U.S. 4,399,216,
Axel, et al., August 16, 1983, Processes for in-
serting DNA into eucaryotic cells and for produc-
ing proteinaceous materials, assigned to Colum-
bia Univ., filed on Feb. 25, 1980, expired on Aug.
16, 2000. This patent covered cotransforming
eukaryotic cells, involving inserting multiple
DNA sequences--a sequence for expression of
the desired protein along with an unlinked DNA
sequence coding for a selectable phenotype not
expressed by the host cell and permitting survival
or identification of transformed eukaryotic cells.
This included cotransformation with thymidine
kinase (rendering cells resistant to acyclovir
exposure), dihydrofolate reductase (DHFR;
rendering cells resistant to methotrexate), and
other selectable phenotypic markers allowing the
ready identification and isolation of transformed
cells. Two other patents, 4,634,665 and 5,179,017,
both originating from 4,399,216 and expiring
on the same day, include additional/extended
claims, e.g., adding Chinese hamster ovary (CHO)
expression. U.S. 6,455,275, issued in 2002, is
further discussed below.
Columbia Univ. collected nearly $370 mil-
lion in royalties on its original cotransformation
patents between 1983 and 2000. Through the 17
year life of the patent, the university collected 1%
royalty from 30 manufacturers, including royal-
ties related to 12-14 (undisclosed) recombinant
proteins. Licensees and the royalties cumulatively
paid to Columbia (from available recent court fil-
ings; see below) include:
Genentech estimating it paid Columbia over $70
million in royalties;
Biogen Idec, $35 million;
Genzyme, almost $25 million;
Wyeth (originally Genetics Inst.), over $70 mil-
lion;
Baxter, over $5 million (for Factor VIII supplied
to Wyeth); and
Ares/-Serono, over $6 million.
Genentech took a license on Oct. 12, 1987;
Amgen, now including Immunex, on Oct. 1,
1991; BASF, now Abbott Bioresearch, on June 1,
1995; Biogen (now Biogen Idec), in 1993; Wyeth
(Genetics Institute), in 1990; Ares/Serono S.A., on
May 1, 1992; Johnson & Johnson (Ortho), N.A;
and Baxter (for its own Factor VIII), in 1997.
The university initiated public policy and po-
litical controversies in early 2000 when it began
lobbying Congress to grant it a special 15-month
patent extension for 4,399,216, which would have
brought the university an extra $70-100 million
in royalty income. The university argued that it
was unfair that the Hatch-Waxman Act allows
extension of U.S. pharmaceutical composition-of-
matter patents for the amount of time the product
was under review by FDA (e.g., from BLA or
28
NDA filing until approval), but does not allow for
comparable extension of process patents.
On Sept. 24, 2002, Columbia Univ. received
its fourth, patent, 6,455,275, a divisional of the
original 1980 cotransformation patent, with a
17 year life (expiry in 2019). This patent, DNA
construct for producing proteinaceous materials
in eucaryotic cells, by Axel, et al., has claims
explicitly covering co-transformation of Chinese
hamster ovary (CHO) cell lines (not all eukaryotic
cells, as claimed by the 4,399,216). Simply stated,
the original patent primarily claimed the process
of inserting foreign genes into another cell, while
this new patent claims rights to transformed CHO
cells. This patent was quickly challenged in court
after Columbia started requesting new royalty
payments from relevant recombinant protein
manufacturers. For example, Biogen (now Biogen
Idec), Genzyme and Abbott jointly filed a suit
in federal court in Boston, and Genentech and
Amgen jointly filed a federal suit in San Francisco
to have the patent declared invalid (as an illegal
extension of the original patent). Other companies
also filed suits. The companies alleged that Co-
lumbia used bait and switch and other question-
able legal tactics to obtain the new patent that is
substantially the same as the original. The suit
filed in Boston cites 6,455,275 as a submarine
patent, because its application lay dormant and
hidden for so long. In response, Columbia noted
that the patent office properly reviewed this patent
application after its filing in 1995 and concluded
that it was a new invention. With the new and
original patents being very similar in content,
much legal wrangling involved interpreting the
semantics of claims and supporting documenta-
tion. In May 2004, the U.S. patent office granted
the Public Patent Foundation (PUBPAT; New
York, NY), acting on behalf of companies hav-
ing filed suits, a Request for Reexamination of
6,455,275, citing a substantial new question of
patentability regarding every claim of the patent.
On Nov. 5, 2004, a federal district judge in
Boston dismissed most of the companies claims
against Columbia University, with both sides
claiming victory. During hearings, Columbia
Univ. stated it would not assert claims or seek
royalties from alleged patent infringers, and the
case was dismissed. The companies portrayed this
as the university abandoning its patents. However,
a patent office reexamination requested by the
university was then in progress, and the ruling did
not exclude the possibility of the university reas-
serting infringement at a later date.
In Aug. 2005, Genzyme Corp. and Biogen Idec
Inc. settled their dispute with Columbia Univ. and
withdrew from their lawsuit regarding the univer-
sity allegedly illegally extending its original pat-
ent. The companies reported that their settlement
with the university allows them to continue sell-
ing affected products. Terms were not disclosed,
but Biogen Idec reported it was very pleased
by the settlement. At the time, Genentech and
Amgen were reported to be making progress in
settling their lawsuit against the university. Other
companies likely have similarly settled with the
university.
The U.S. Patent and Trademark Office (PTO)
has yet to rule regarding the universitys more
recent patent; and Genentech, Amgen and other
companies are still in dispute with the university.
Licensing information: Now in the public do-
main, except seemingly for CHO cells. Contact
Columbia University with any questions.
Products made using this tech.: The great ma-
jority of marketed recombinant protein products
expressed in eukaryotes are probably manufac-
tured using cells selected using this technology.
29
110
Cre-lox mediated in vitro
recombination; Cyclization
Recombination/locus of X-over
P1; site-specifc recombination
(SSR) - universal; genetic
recombination
Bristol-Myers Squibb Co. (BMS) - Licensor,
primary
DuPont Corp. - Assignee, patent
Description: Cre-Lox recombination is a special
type of site-specific recombination (SSR) using
a two-vector system that utilizes the Cre-lox site-
specific recombination system of bacteriophage
P1. Cre-LoxP recombination leads to directional
transfer of a DNA fragment from a single donor
vector into any of the variety of acceptor vectors,
bypassing traditional sub-cloning steps. Once a
gene is directionally cloned into a donor vector,
it may be easily shuttled to protein expression ac-
ceptor vectors. This technology allows transfer of
individual library inserts directly into expression
vectors with no subcloning.
SSR is an important process in viruses, such
as bacteriophages. Site-Specific Recombination
(SSR) involves specific sites for the catalyzing
action of recombinase enzymes, allowing them
to integrate their genetic material into that of the
infected host. Cre-Lox recombination involves
the targeting of a specific sequence of DNA and
splicing it with the help of an enzyme called Cre
recombinase. Recombination takes place at the
specific loci called loxP sites mediated by Cre
recombinase. Using this technology, specific tis-
sue types or cells of organisms can be genetically
modified, whilst other cells and tissues remain
unchanged. Cre-lox allows a variety of genetically
modified organisms to control gene expression,
delete undesired DNA sequences and modify
chromosomes. A Cre transgene under control of
an inducible promoter can be used to delete the
target DNA inside selected cells of a transgenic
organism.
Cre site-specific DNA recombinase can ca-
talyse the recombination of DNA between spe-
cific sites in a DNA molecule. These sites, loxP
sequences, contain specific binding sites for Cre
that surround a directional core sequence where
recombination can occur. When cells that have
loxP sites in their genome express Cre, a recipro-
cal recombination event will occur between the
loxP sites. The double stranded DNA is cut at
both loxP sites by the Cre protein and then ligated
back together in a quick and efficient process. The
efficiency of recombination depends on the ori-
entation of the loxP sites. For two lox sites on the
same chromosome arm, inverted loxP sites will
cause an inversion, while a direct repeat of loxP
sites will cause a deletion event. If loxP sites are
on different chromosomes it is possible for trans-
location events to be catalysed by Cre induced
recombination.
A number of conservative site-specific recom-
bination systems have been developed for use in
both prokaryotic and eukaryotic organisms.
Use with: prokaryotic and eukaryotic cells,
including yeasts. Commonly used to make knock-
out transgenic animals
Background: Cre/Lox is based on basic research
performed on the bacteriophage called P1. Cre
(Cyclization Recombination) protein consists of
4 subunits and two domains a larger carboxyl
(C-terminal) domain, and a smaller amino (N-ter-
minal) domain. The total protein has 343 amino
acids. Lox P (locus of X-over P1) is a site on the
Bacteriophage P1 consisting of 34 bp.
Sauer, B.L., Site-specific recombination of
DNA in eukaryotic cells, September 25, 1990,
assigned to DuPont, subsequently acquired by
Bristol-Myers Squibb. The exemplary claim (no.
1) states: A method for producing site-specific
recombination of DNA in eukaryotic cells, com-
prising, (a) introducing into the cells a first DNA
sequence comprising a first lox site and a second
DNA sequence comprising a second lox site, and
(b) contacting the lox sites with Cre, thereby pro-
30
ducing the site specific recombination.
The patent also states, In a preferred embodi-
ment, a third DNA sequence comprising a cre
gene is also introduced into the cells. Most prefer-
ably, the third DNA sequence further comprises a
regulatory nucleotide sequence and expression of
the cre gene is produced by activating the regula-
tory nucleotide sequence. In another preferred
embodiment, the first and second DNA sequences
are introduced into the DNA connected by a pre-
selected DNA segment. In one embodiment, the
eukaryotic cell is yeast.
Availability: Invitrogen is among many retail
suppliers of related reagents.
Licensing information: For commercial use
licensing, contact Bristol-Myers Squibb.
111
deltaPhase; Elastin-like
polypeptide (ELP) fusion
protein tags - chromatography-
free purifcation; universal
Duke University - R&D
PhaseBio - Licensor, primary
Description: deltaPhase technology is used with
vectors containing elastin-like polypeptide (ELP)
fusion tags along with a desired protein to provide
controllable self-cleaving fusion protein affinity
tags useful for purification of proteins expressed
in diverse hosts without use of chromatography.
See also the other ELP self-cleaving system. This
technology platform can be used to improve pro-
tein expression and rapid purification in a single
step.
ELP, a polypeptide composed of naturally-
occurring amino acids, transitions from highly
soluble to insoluble when exposed to modest
increases in solution temperature or increases
in ionic strength. A recombinant protein can be
expressed as a fusion of the ELP and the protein
of interest. After expression, the crude cell extract
is heated slightly or salt is added to increase the
ionic strength, causing the fusion protein to ag-
gregate and precipitate from the solution. Simple
centrifugation or filtration separates the purified
protein from the cellular impurities. The protein
can be further isolated by cleaving the ELP tag
and removing it using another round of thermal
cycling. The deltaPhase technology has been
extended to enzyme biocatalysis, using the ELP
as a soluble carrier for enzymes where the phase
transition is used to recover the enzyme from
solution.
The deltaPhase recombinant expression/puri-
fication system is based on the transition proper-
ties of ELPs and their ability to retain this inverse
temperature phase transition when conjugated
to other molecules. The phase transition offers
a new method of purification for therapeutic
proteins and peptides. The process consists of
fusing an ELP sequence to the N or C terminus of
the polypeptide of interest by recombinant DNA
techniques. The DNA coding for the polypeptide
or protein tagged with an ELP is introduced into
an expression system (e.g. E. coli or mammalian
cells) for production.
These elastin-like polypeptides (ELPs) are
oligomeric repeats of the pentapeptide Val-Pro-
Gly-Xaa-Gly, where Xaa is variable amino acid
which affects the transition temperature of the
ELP. ELPs have been designed to undergo an
inverse phase transition in solution in response
to a change in solution conditions. For example,
ELPs are highly soluble in water below the tran-
sition temperature (Tt) but, when heated, undergo
a rapid phase transition over a narrow temperature
range (2-3 C). The transition results from desol-
vation of the ELP and leads to an aggregation of
the ELP. This process is completely reversible so
that cooling of the solution results in resolubili-
zation of the aggregate as shown schematically
at right. This transition can also be triggered by
increasing the salt concentration of the solution.
The deltaPhase technology has already been
shown useful across a broad range of applications
in the drug discovery and life science markets.
These applications leverage the unique phase tran-
sition properties of ELPs.
31
Use with: E. coli; mammalian cells; plants;
universal (generic);
Use to make: proteins; glycoproteins; monoclo-
nal antibodies
Benefits: PhaseBio states, We believe that
the deltaPhase technology offers an innovative
method for protein/polypeptide purification and
avoids costly chromatography. PhaseBios delta-
Phase technology represents the first new method
for production and purification of biologics in
20 years and has led to a number of important
research collaborations with biotechnology and
pharmaceutical partners. Benefits include:
Simple, one-step purification
Purity equal to or better than His-IMAC chro-
matography
Scalable from bench-top to reactor with little
modification
Aids in protein solubility during expression
No immobilization chemistry required
No losses due to immobilization
Patents: Note, this technology is very similar to
the other ELP fusion tag technology (from Princ-
eton Univ.).
Fusion peptides isolatable by phase transition,
Chilkoti, Feb. 8, 2005, assigned to Duke Univ.
The abstract states Genetically-encodable,
environmentally-responsive fusion proteins
comprising ELP peptides. Such fusion proteins
exhibit unique physico-chemical and functional
properties that can be modulated as a function of
solution environment. The invention also provides
methods for purifying the FPs, which take advan-
tage of these unique properties, including high-
throughput purification methods that produce high
yields (e.g., milligram levels) of purified proteins,
thereby yielding sufficient purified product for
multiple assays and analyses. The high throughput
purification technique is simpler and less expen-
sive than current commercial high throughput
purification methods, since it requires only one
transfer of purification intermediates to a new
multiwell plate.
Availability: deltaPhase products incorporating
this technology are available from PhaseBio for

non-commercial uses.
Licensing information: Contact PhaseBio (and/
or Duke Univ.) regarding commercial use and
licensing.
Further info.: Self-Cleavable Stimulus Respon-
sive Tags for Protein Purification without Chro-
matography, Xin Ge, Daniel S. C. Yang, Kim-
berly Trabbic-Carlson, Bumjoon Kim, Ashutosh
Chilkoti, and Carlos D. M. Filipe, J. Am. Chem.
Soc., 127 (32), 11228 -11229, 2005.
Expression and purification of recombinant
proteins from Escherichia coli: Comparison of an
elastin-like polypeptide fusion with an oligohisti-
dine fusion, Protein Sci. 2004 Dec;13(12):3274-
84.
Purification of recombinant proteins by fusion
with thermally-responsive polypeptides, Meyer
DE, Trabbic-Carlson K, Chilkoti A., Biotechnol
Prog. 2001 Jul-Aug;17(4):720-8.
112
Directed Nuclease Editor
(DNE); Meganuclease Design -
universal
Duke University - R&D, of tech.
Precision BioSciences, Inc. - Licensor, primary
Description: Directed Nuclease Editor (DNE) en-
ables the rapid production of redesigned homing
endonucleases (also known as meganucleases),
capable of producing double-strand breaks at pre-
determined DNA sites, including in viruses (vec-
tors), bacteria and other microorganisms, animals
and in plants. This can be used to promote site-
specific gene insertion, knock-out, or sequence
modification. Directed Nuclease Editor (DNE)
technology may well allow for the identification,
insertion, removal, or alteration of virtually any
gene for therapeutic, agricultural, and diagnostic
applications.
DNE is based on the LAGLIDADG family
of meganucleases that make contacts with DNA
bases and the DNA backbone when the mega-
32
nucleases associate with a double-stranded DNA
recognition sequence. The sequence specificity
of the designed enzymes provided is sufficient
to define a single cleavage site within a genetic
background as large as the human genome. This
break in the DNA effectively marks that spot for
user-specified gene modification using the cells
natural DNA-repair machinery.
Patents: Exemplary applications include
20070117128, Rationally-designed meganucle-
ases with altered sequence specificity and DNA-
binding affinity, Smith, J.J., May 24, 2007. Its
abstract states, Rationally-designed LAGL-
IDADG (SEQ ID NO: 37) meganucleases and
methods of making such meganucleases are pro-
vided. In addition, methods are provided for using
the meganucleases to generate recombinant cells
and organisms having a desired DNA sequence
inserted into a limited number of loci within the
genome, as well as methods of gene therapy, for
treatment of pathogenic infections, and for in vitro
applications in diagnostics and research.
Licensing information: Contact Precision Bio-
sciences. The company has exclusively licensed
this technology from Duke University. The com-
pany claims, Our DNE technology is the new
gold standard in genome engineering and does
not make use of the methods claimed by Cellectis
[see related meganucleses entries]. However, in
March 2008, Cellectis filed an infringement suit
against Precision BioSciences.
113
Dual expression vectors; Dual
Affnity ReTargeting (DART)
- antibodies; bacteria and
mammalian cells
MacroGenics, Inc - Licensor, primary
Description: Dual Affinity ReTargeting (DART),
using dual expression vectors, is useful for
antibody expression in bacterial and mammalian
cells. DART uses dual specificity antibody-like
scaffold proteins capable of targeting multiple
different receptors with a single molecule. DART
affords a number of advantages over conventional
recombinant monoclonal antibody manufacture.
The dual expression vector system is capable
of expressing and secreting into the periplasmic
space antibody fragments in bacteria and express-
ing and secreting full-length IgG in mammalian
cells. In this novel vector, regulatory elements
required for expression and secretion of Fab
fragments in bacteria overlap sequence elements
required for both proper processing of IgG heavy
and light chain RNA transcripts and secretion of
IgG heavy and light chain polypeptides in mam-
malian cells. In particular, a bacterial promoter
and signal sequence are included in an intron
located within the sequence coding for the signal
sequence of a mammalian IgG heavy or light
chain gene, and a bacterial stop codon is included
in another intron between the CH1 and the hinge
region of the heavy chain gene (or, in an alterna-
tive embodiment, in an intron located between
the hinge region and the CH2 domain). Thus,
when expressed in bacteria, transcription from
the bacterial promoter and termination at the stop
codon in the second intron results in Fab fragment
expression in bacteria periplasmic space, while in
mammalian cells, splicing removes the bacterial
promoter and signal sequence and regenerates the
mammalian signal sequence, allowing expression
of the full-length IgG heavy or light chain poly-
peptide.
An important feature of the vector is the
structural and functional overlap of a bacterial
and mammalian regulatory sequence elements,
i.e., the mammalian signal sequence and splice ac-
ceptor site, and the bacterial promoter and signal
sequences, so that the functionality of all four of
these sequence elements is maintained. It is there-
fore critical in construction of the vector that any
changes made within this overlap region maintain
the functionality of these four sequence elements.
Use with: bacteria; E. coli; mammalian cells
Use to make: antibodies
33
7,112,439, Dual expression vector system for
antibody expression in bacterial and mammalian
cells, Johnson, et al., Sept. 26, 2006
Licensing information: Contact MacroGenics,
Inc., which is apparently using this technology
for manufacture of its own antibody products in
development.
114
Expression enhancers,
oligonucleotides - universal
University of Texas Southwestern Medical
Center - Licensor, primary
Description: RNA-based expression enhanc-
ers that bind to a targeted promoter sequence
are claimed to provide the most effective and
consistent method developed so far for prompt-
ing genes to make the proteins necessary for gene
expression. A polynucleotide oligomer of 12-28
bases complementary to a region within a target
promoter is used to selectively increases synthesis
of the target transcript. Genes in cultured cells
are activated using strands of RNA to stir up the
mixture of proteins that surrounds chromosomal
DNA, proteins that control whether genes are
switched on or off.
Patents: U.S. application 20070111963, Modu-
lation of gene expression by oligomers targeted to
chromosomal DNA, Corey, D.R., et al., May 17,
2007, has claim 1 stating, A method of selective-
ly increasing synthesis of a target transcript of a
gene in a mammalian cell, wherein the target tran-
script is predetermined to be in need of increased
synthesis, the method comprising the steps of:
contacting the cell with a polynucleotide oligo-
mer of 12-28 bases complementary to a region
within a target promoter of the gene under condi-
tions whereby the oligomer selectively increases
synthesis of the target transcript; and detecting
resultant selective increased synthesis of the target
gene; wherein the oligomer is double-stranded
RNA, and wherein the region is located between
nucleotides -100 to +25 relative to a transcription
start site of the gene.
Licensing information: Contact the University of
Texas Southwestern Medical Center.
115
Expression factory;
baculoworkstation - automated
parallel expression
NextGen Sciences Inc. - Licensor, primary
Description: expressionfactory is the first com-
mercially available technology to fully automate
protein expression, from gene to protein. Fol-
lowing the activation of a gene, gene fragment or
domain (using Gateway technology; see related
entry), the system expresses each gene in parallel,
in multiple vectors. Uniquely, the expressionfac-
tory also grows each of the newly created cell
lines in parallel, before purifying all of the dif-
ferent versions of each protein. This is achieved
without user supervision or intervention. The ex-
pressionfactory allows a matrix approach for the
cloning and expression of any gene, increasing the
chances of successfully isolating useful protein.
Associated with this NextGen has also de-
veloped the baculoworkstation which increases
throughput of recombinant baculovirus production
by automating key methods (insect cell seed-
ing, transfections, viral dilutions, and small-scale
protein production). The baculoworkstation is
compatible with a variety of commercially avail-
able baculovirus technologies.
Use with: universal (generic); insect cells
Licensing information: Contact NextGen Sci-
ences Inc. regarding CRO services, purchase and
licensing.
34
116
Fusion protein expression
systems; Affnity purifcation
tags and chaperones -
universal
Description: Fusing of genes, e.g., a sequence
coding for a peptide or protein with high affinity
for a specific chromatography medium or a mark-
er gene that is fused to the gene for the desired
protein, in vectors and subsequent expression of
fusion proteins is a common artifice to facilitate
expression and/or separation based on character-
istics of the appended protein. Rather pure protein
can be obtain using just one purification step, ,
e.g., by affinity, ion-exchange, hydrophobic cova-
lent or metal-chelate chromatography, depending
on the tag. Expression as fusion proteins can also
offer other advantages, e.g., increasing expression
levels, correct folding, etc. Fusion proteins may
by chimeric or hybrid and generally also incor-
porate a convenient site for enzymatic or chemi-
cal cleavage to remove the tag, e.g., cleavage by
endopeptidases, enterokinase or thrombin. Some
fusion proteins are self-cleaving.
Tags include glutathione-S-transferase, beta-
galactosidase, chloramphenicolacetyltransferase
or other whole enzymea; or shorter peptides, such
as poly-histidine sequences which have a high af-
finity for Ni-NTA chromatography resins.
These systems generally enhance expres-
sion, but are often innately hampered by two
fundamental flaws, 1) The proteases for removal
of tags (thrombin, enterokinase, TEV protease,
etc.) require linkers between the fusion tag and
the protein of interest, leading to protein with an
artificial N-terminus after cleavage; and 2) Within
the linker, the recognition sequences for the pro-
teases are often small and degenerate and hence,
identical amino acid sequences within the protein
of interest are also cleaved. Many newer systems,
e.g., SUMO, have been designed with these prob-
lems in mind.
Use with: universal
Use to make: proteins; glycoproteins.
117
Fusion proteins, self-cleaving,
pH-sensitive - universal
Health Research, Inc. - Licensor, primary
Description: An accelerated pH-sensitive auto-
catalytic cleaving element renders ligand binding
domains (e.g., affinity peptide/protein portions of
fusion proteins) self-cleaving with greatly accel-
erated cleavage (e.g., in affinity fusion-mediated
protein purification. The inclusion of autocatalytic
cleaving domains (inteins) between the binding
domain and product protein renders the binding
domain self-cleaving in response to an added
nucleophile during the purification procedure,
with the cleavage reaction induced purely through
a mild pH shift in the running buffer. Self-cleav-
age, rather than splicing of the intein releases the
desired protein, thereby eliminating the need for
protease addition.
This technology decreases the time for C-ter-
minal cleavage from several days to several hours
at 4C, or to minutes at higher temperatures. This
intein mutant does not require addition of any
exogenous factors to the product stream. Meth-
ods are provided for genetic selection used in the
isolation of these mutants, which can be used to
generate other mutants that are further optimized
for size and cleavage rate.
Patents: Related patents include U.S. 6,933,362,
GENETIC SYSTEM AND SELF-CLEAVING
INTEINS DERIVED THEREFROM, BIOSEPA-
RATIONS AND PROTEIN PURIFICATION EM-
PLOYING SAME, AND METHODS FOR DE-
TERMINING CRITICAL, GENERALIZABLE
AMINO ACID RESIDUES FOR VARYING
INTEIN ACTIVITY, Belfort, M., August 23,
2005. Claim 1 states, A non-naturally occurring
intein or cleavage or cleavage and splicing moiety
having splicing activity and/or controllable cleav-
age activity, wherein the intein is a truncated Mtu
recA intein with the endonuclease domain deleted,
35
and V67L and/or D422G mutation(s), wherein the
truncated Mtu recA intein is delta.I-CM or delta.
I-SM, or any full-length Mtu recA intein having a
V67L and/or a D422G mutation(s).
Licensing information: HRI seeks commercial
partners to develop the technology, and it is avail-
able for licensing. A Prototype of this technology
is available.
118
Glutathione-S-transferase
(GST) fusion protein affnity
tags; pGEX vectors
AMRAD Corporation Ltd. - R&D, of tech.
Chemicon International, Inc., Millipore Corp.
- Licensor, primary
Millipore Corp. - Parent co.
Description: The Glutathione S-transferase
(GST) Gene Fusion System is a system for the
expression, purification and detection of fusion
proteins produced in E. coli and various other
systems. Fusion proteins of GST with a desired
protein can be readily expressed in diverse expres-
sion systems. With GST having high affinity for
glutathiones, GST fusion proteins are easily puri-
fied from bacterial lysates by affinity chromatog-
raphy using glutathione-bound chromatography
media, e.g., Glutathione-Sepharose. Cleavage of
the desired protein from media-bound GST fusion
proteins is readily achieved using a site-specific
protease, e.g., thrombin. A variety of site-specific
proteases are available. The pGEX plasmids are
designed for inducible, high-level intracellular
expression of genes or gene fragments as fusions
with Schistosoma japonicum GST.
In the GST system, the sequence encoding the
GST protein is incorporated into an expression
vector, generally upstream of the multi-cloning
site. The sequence encoding the protein of inter-
est is then cloned into the vector. Induction of the
vector results in expression of a fusion protein
(the protein of interest fused to the GST protein).
The fusion protein can then be harvested and puri-
fied using glutathione-exposing chromatography
media. This strategy results in quick and efficient
production of highly purified proteins.
Use with: E. coli; bacteria; yeasts; insect cells;
mammalian cells
Background: GST occurs naturally as an 26 kDa
protein which can be expressed in E. coli with full
enzymatic activity. Fusion proteins which pos-
sess the complete amino acid sequence of GST
also demonstrate GST enzymatic activity and can
undergo dimerization similar to that observed in
nature.
Patents: CHEMICON International Inc., now
part of Millipore, has exclusively licensed Glu-
tathione S-transferase (GST) gene fusion system
worldwide patents from AMRAD. The technology
is patent protected in 21 countries. U.S. patent
coverage is claimed to expire in 2014.
Exemplary patents include 5,654,176, Fusion
proteins containing glutathione-s-transferase,
Smith, August 5, 1997, assigned to AMRAD. The
abstract states, A recombinant DNA molecule
comprising a nucleotide sequence which codes for
expression of a fusion protein in which a for-
eign protein or peptide is fused with the enzyme
glutathione-S-transferase, is disclosed, as well as
expression vectors or host cells containing such
a molecule. Optionally, the foreign protein or
peptide is fused to the enzyme through a cleavable
link. Also disclosed is an expression vector hav-
ing inserted therein a nucleotide sequence capable
of being expressed as the enzyme glutathione-
S-transferase followed by at least one restriction
endonuclease recognition site for insertion of a
nucleotide sequence capable of being expressed as
a foreign protein or peptide fused to the glutathi-
one-S-transferase.
Availability: Exclusively marketed by GE
Healthcare (formerly Amersham plc) for non-
commercial uses.
Licensing information: Contact CHEMICON/
Millipore regarding commercial use and licensing.
Further information:
Smith, D. B. and Johnson, K. S. Single-step puri-
36
fication of polypeptides expressed n Escherichia
colias fusions with glutathione S-transferase.
Gene 67, 31-40 (1988).
119
GUS reporter system (beta-
glucuronidase); GUS control
vector - prokaryotes; plant
cells; mammalian cells, non-
vertebrate
Cambia Biosystems, L.L.C. (CBL) - Licensor,
primary
Description: The GUS reporter system involves
expression of gene fusions of a desired gene and
a beta-glucuronidase gene. Beta-glucuronidase
(GUS) from E. coli naturally catalyzes the hydro-
lysis of a very wide variety of beta-glucuronides.
GUS gene fusions may be used to introduce GUS
activity into a wide variety of bacterial, microbial,
plant, and animal host organisms. With a variety
of useful color-generating substrates for beta-
glucuronidase enzyme, GUS gene fusions serve
as a superior reporter gene system as well as an
effective means of altering cellular phenotype,
with only successfully transformed cells (express-
ing fusion proteins from the linked sequence for
the desired gene and beta-glucuronidase gene)
having beta-glucuronidase activity on many color-
generating substrates. GUS activity is largely if
not completely absent from higher plants, mosses,
algae and ferns, making this system ideally usable
in these organisms. Similarly, GUS is not present
in most bacteria.
In addition to beta-glucuronidase enzyme, the
GUS operon of E. coli also encodes glucuronide
permease, providing a mechanism for transport
of beta-glucuronidase through the cell membrane,
and permiting the entrance of a surprisingly wide
variety of substrates, ranging from simple ali-
phatic compounds to large, complex heterocyclic
conjugates into the cytoplasm.
The GUS reporter system is very widely used,
and is probably the most widely used reporter
system for research, particularly for non-mam-
malian-based systems. An organism is suitable for
a GUS assay if it has no beta-glucuronidase or its
own activity is very low. For this reason the assay
is not useful in almost all vertebrates and many
molluscs. As in higher plants, mosses, algae,
ferns, fungi and most bacteria there is no detect-
able GUS activity, these organisms are perfectly
suitable for the assay and this is the reason why
the assay is widespread in plant, and also fungal
and bacterial-based research methods. A further
advantage of the GUS gene fusion system is the
wide variety of available substrates, the ease and
economy of GUS assay systems, and the fact that
GUS activity may be detected and accurately
measured in single cells.
Use with: prokaryotes; plant cells; mammalian
cells, non-vertebrate
Background: The system was originally devel-
oped by Richard Anthony Jefferson during his
Ph.D. research at the University of Colorado,
Boulder. He adapted the technique for the use
with plants as he worked in the Plant Breeding
Institute of Cambridge, between 1985 and 1987.
Since then thousands of labs have made use of the
system, making it probably the most widely used
tool in plant molecular biology, as indicated by
over 6000 citations in scientific literature.
Patents: When the University of Colorado had
declined to patent GUS, Dr. R.A. Jefferson did
this himself. Within a short space of time GUS
became the most widely used reporter gene in the
field, largely because it was freelly available (to
researchers).
Use of the uidA gene for GUS is covered by
U.S. 5,268,463; 5,432,081 and 5,599,670. U.S.
5,268,463, Plant promoter .alpha.-glucuronidase
gene construct, Jefferson, Dec. 7, 1993, with its
abstract stating, The present invention relates to
the _-glucuronidase (GUS) gene fusion system,
and to the cloning and characterization of the
_-glucuronidase and glucuronide permease genes
of Escherichia coli. It is based on the surprising
discovery that gene fusions comprising the _-
37
glucuronidase gene may be effectively expressed
in a wide variety of organisms to produce active
_-glucuronidase enzyme. Because of the abun-
dance and availability of useful substrates for
_-glucuronidase enzyme, GUS gene fusions may
serve as a superior reporter gene system as well as
an effective means of altering cellular phenotype.
In conjunction with recombinant glucuronide
permease, which may be used to render host cells
permeable to _-glucuronidase substrates, the GUS
gene fusion system offers almost unlimited ap-
plications in the fields of plant and animal genetic
engineering
Availability: Reagents available for non-commer-
cial use from Invitrogen and other vendors.
Licensing information: Contact Cambia regard-
ing commercial use and licensing.
120
Heat shock Promoters (HSPs);
HSP chaperones
Description: Heat shock proteins (HSPs) incor-
porated into vectors are useful in expressed fusion
proteins as chaperones to assure proper folding
and protein conformation (shape) and prevent
unwanted protein aggregation.of expressed pro-
teins. See related entries. HSPs also called stress
proteins, are a group of proteins that are present in
all cells in all species. HSP expression is upregu-
lated under conditions of cell stress. This increase
in expression is transcriptionally regulated.
HSPs are induced when a cell undergoes vari-
ous types of environmental stresses like heat,
cold and oxygen deprivation. Heat shock proteins
are also present in cells under normal conditions.
The function of heat-shock proteins is similar in
virtually all living organisms. HSPs act as chap-
erones, making sure that the cells proteins are in
the right shape and in the right place at the right
time. For example, HSPs help new or distorted
proteins fold into the structures essential for their
function. They also shuttle proteins from one
compartment to another inside the cell, and trans-
port old proteins to garbage disposals inside the
cell. Heat shock proteins are also believed to play
a role in the presentation of pieces of proteins (or
peptides) on the cell surface to help the immune
system recognize diseased cells.
HSPs are named according to their molecular
weights. The small 8 kilodalton protein, ubiquitin,
which marks proteins for degradation, also has
features of a heat shock protein.
121
His tags; Poly-Histidine
fusion affnity tag technology;
6xHistidine-tag - univeral;
purifcation tags
Roche Protein Expression Group (RPEG) - Li-
censor, primary
Hoffmann-La Roche Inc. - Parent co.
Description: Multiple histidine (His) residues,
usually 6 histidines, appended to the N-terminal
end of desired proteins are useful affinity purifica-
tion tags. Purification is based on the remarkable
selectivity of metal ion-based affinity chroma-
tography media for proteins with an affinity tag
of 6 histidine residues. This technology allows
one-step purification of almost any His-tagged
protein from most any expression system. The
gene sequence for multiple histidines (e.g., 6) is
placed next to the desired gene, and the desired
protein and with terminal histidines are expressed
as a fusion protein. Affinity peptides having at
multiple neighbouring histidine residues are espe-
cially suitable for the purification of recombinant
proteins by means of metal chelate affinity chro-
matography on nitrilotriacetic acid (NTA) resins,
which have high affinity for poly-His chains.
Once bound to the chromatography medium, an
enzyme specific for the link of the affinity tag
sequence to the N-terminal of the desired protein
is added, causing release of the desired protein in
a rather pure state. In some or many cases, this
one-step purification may be all that is required.
38
nal antibodies
Benefits:
Affinity peptides, Dobeli, et al., May 10, 1994,
assigned to Hoffman-La Roche Inc. Claim 1
states, A DNA sequence coding for a fusion pro-
tein comprising a biologically active polypeptide
or protein linked by its amino- or carboxy-termi-
nus to one or two affinity peptides, which peptides
have from 2 through 6 adjacent histidine residues
Roche also holds key patents on the use of Ni-
NTA chromatography resins, including 4,877,830,
and 5,047,513. Ni-NTA resin is exclusively manu-
factured by QIAGEN.
Availability: Qiagen markets His tag reagents for
non-commercial use.
Licensing information: Contact the Roche Pro-
tein Expression Group (RPEG) regarding com-
mercial use and licensing.
122
His-tags; Poly-Histidine fusion
affnity tag technology -
univeral; purifcation tags
Inproteo, Inc. - Licensor, primary
Lilly, Eli & Co. - Assignee, patent
Description: Multiple histidine (His) residues ap-
pended to the N-terminal end of desired proteins
are useful affinity purification tags. Purification is
based on the remarkable selectivity of metal ion-
based affinity chromatography media for proteins
with an affinity tag of 2-5 histidine residues.
See the His tags (licensed by Hoffmann-La
Roche) entry for a broader description of His-tag
fusion proteins and their use. A major difference
between the two technologies appears to be that
this method uses a tag of 2-5 amino acids (his-
tidine) while the newer technology, now widely
used, uses 6 histidines.
Patents: U.S. 4,569,794, Process for purifying
proteins and compounds useful in such process,
Smith, M.C., Feb. 11, 1986, assigned to Eli
Lilly & Co.. The abstract states, This invention
describes a process for separating a biologically
active polypeptide or protein in the form of its
precursor from a mixture containing said precur-
sor and impurities, which comprises contacting
said precursor with a resin containing immobi-
lized metal ions, said precursor comprising the
biologically active polypeptide or protein cova-
lently linked directly or indirectly to an immo-
bilized metal ion chelating peptide, binding said
precursor to said resin, and selectively eluting said
precursor from said resin. Such precursor com-
pounds are also described. This patent has most
likely expired.
Availability: GE Healthcare markets His-tag
products covered (or formerly covered) by the
Smith patent.
Licensing information: Inproteo, formerly
known as the Indiana Proteomics Consortium,
has exclusively licensed the Smith patent from
Eli Lilly. Although patent coverage likely has
expired, it would be prudent to contact Improteo
before using this method commercially.
123
Hybridomas (Khler and
Milstein) - monoclonal
antibodies
Medical Research Council, U.K. - R&D
Description: The original methods for hybridoma
generation, first reported in 1975 [G. Khler and
C. Milstein, Continuous cultures of fused cells
secreting antibody of predefined specificity, Na-
ture, 256:495, 1975] were never patented by the
U.K. Medical Research Council, and have always
been in the public domain.
Note, see the enties concerning novel applica-
tion of hydridomas, e.g., for recombinant protein
expression.
Use with: hybridomas; fused myelomas and
B-cells
39
Use to make: monoclonal antibodies
Benefits: The Pros
- Straightforward methodology (infect mouse or
other animal with antigen, extract B-cells, and
clone)
- High yield
- Highly specific antibodies
The Cons
- Long generation time (5-15 months)
- Not all epitopes are identified
- Not all antigens are solved
- No antibodies for nonimmunogenic or highly
toxic antigens
- Functional screens only after clone selection and
culture
- Therapeutic recombinant antibodies likely need
to be humanized
Licensing information: No license needed now
or in the past.
Products made using this tech.: There are very
few non-recombinant hydridoma-expressed
monoclonal antibodies current on the market in
the U.S. and other major markets. Those that
remain are nearly all conjugated to radioisotopes
and used for immunoradiodiagnostic imaging, and
have low sales.
124
Lambda recombination protein;
Homologous recombination
primary
Description: Methods using recombinases, lamb-
da recombination protein and similar proteins are
useful for generating recombinant DNA molecules
in cells using homologous recombination. These
methods offers superior recombination efficiency,
and require only one step for operations vs. two
steps with Red/ET (see related entry).
The methods promote high efficiency ho-
mologous recombination in bacterial cells, and
in eukaryotic cells such as mammalian cells. The
methods are useful for cloning, the generation
of transgenic and knockout animals, and gene
replacement. The methods are also useful for
subcloning large DNA fragments without the need
for restriction enzymes. The methods are further
useful for repairing single or multiple base muta-
tions to wild type or creating specific mutations
in the genome. Also disclosed are bacterial strains
and vectors which are useful for high-efficiency
homologous recombination.
Use with: bacteria; mammalian cells
7,144,734, Enhanced homologous recombina-
tion mediated by lambda recombination proteins,
Court , et al., Dec. 5, 2006, assigned to NIH
Licensing information: Contact NIH.
125
Meganuclease Recombination
System (MRS)/I-SceI;
homologous recombination
- universal genetic
recombination
Description: Meganuclease Recombination
System use the natural DNA maintenance mecha-
nisms present in all living cells to make homolo-
gous recombination efficient in some difficult spe-
cies such as plants or fish, and rendering it very
efficient in a number of species such as mammals.
This technology is based on meganucleases,
enzymes, e.g., I-Scel, which recognize and cut
specifically a unique DNA target site within a liv-
ing cell. Homologous recombination is a natural
DNA sequence exchange mechanism, which can
take place in any living cell. The mechanism is
based on recognition and exchange of sequences
between two similar nucleotide sequences. Once
the homologous sequences are paired, a strand
exchange takes place between the two molecules -
this event is homologous recombination. This has
been demonstrated and reproduced extensively in
40
a wide variety of organisms (yet its success varies
with the species to which it is applied).
Cellectis claims it is the first company to ap-
ply the Meganuclease Recombination System ap-
proach to in vivo genome engineering. The com-
pany is developing Meganucleases that can target
a unique DNA break in vivo, as a fundamentally
enabling technology for commercial applications
in human therapeutics, pharmaceutical discovery,
agriculture and industrial biotechnology.
A meganuclease is an endonuclease, a pro-
tein that binds and cuts DNA at specific target
sites. Meganucleases function similarly to re-
striction endonucleases with the exception that
their recognition sites are much larger (18 bp).
This means that meganucleases are very rare
and precise DNA cutters. For an 18 bp recogni-
tion site, the frequency is one out of 34 billion
base pairs. Meganuclease induced double-strand
breaks have been shown to enhance recombina-
tion 1,000 - 10,000 fold compared to spontaneous
homologous recombination. Cellectis currently
holds a portfolio/library of >18 meganucleases
with modified specificity, deriving from the I-CreI
meganuclease.
I-SceI was one of the first identified homing
endonucleases, and is today the most widely used
for research and genome engineering. I-SceI is a
homing endonuclease of the LAGLIDADG fam-
ily. I-Scel is encoded by the w intron of Saccha-
romyces cerevisiae (bakers yeast) mitochondria.
This intron was found in the large rRNA gene in a
number of strains, but not in others. When intron
minus strains are crossed with intron-plus strains,
the intron spreads into the intronless copies of
the gene by a gene conversion process, with a
coconversion of the flanking exons extending
over a few hundred base pairs. This conversion is
initiated by the endonuclease activity of I-SceI. I-
SceI delivers a DSB in the intronless copies of the
genes, which is repaired by homologous recom-
bination with the copy containing the intron. The
I-SceI DNA target site is an 18 bp sequence. On
a random basis, such sequences are found once
in 70 billion base pairs. This specificity makes
I-SceI an ideal tool for genome engineering in any
organism. I-SceI has become a paradigm for me-
ganuclease induced-recombination, and has been
used to induce recombination in bacteria, yeast,
mammalian cells, fish, worm, flies and plants.
Cellectis claims to be the first company to ap-
ply the Meganuclease Recombination System ap-
proach to in vivo genome engineering. The com-
pany is developing Meganucleases that can target
a unique DNA break in vivo, as a fundamentally
enabling technology for commercial applications
in human therapeutics, pharmaceutical discovery,
agriculture and industrial biotechnology.
Patents: Exemplary/relevant U.S. patents include
6,528,313; 6,528,314; 6,638,768; 5,474,896;
5,792,632; 5,866,361; 5,948,678; 5,962,327;
6,395,959; 6,238,924; 6,610,545; 6,822,137; and
6,833,252. U.S. 6,528,313 and 6,528,314 cover
the procedure for specific replacement of a copy
of a gene present in the recipient genome by the
integration of a gene different from that where the
integration is made.
Availability: I-SceI Meganuclease can be bought
at Fermentas for non-commercial use.
Licensing information: Contact Cellectis regard-
ing commercial uses and licensing.
Cellectis has reported forming a number of
nonexclusive licenses for MRS, including with
AstraZeneca (for in vitro and in vivo modification
of rodents), BASF Plant Science GmbH (plants),
Regeneron (for use with its Velocigene technol-
ogy/mice); GlaxoSmithKline; Genentech; Delta-
gen (rodents), Fermentas (manufacture of I-Scel);
Diversa (microbes), Limagrain Group (seeds),
Nucleis (mice), DuPont-Pioneer HiBred, Ozgene
Pty Ltd. (eukaryotes), and Xenogen Biosciences
(eukaryotes). Cellectis has also reported research
collaborations with companies including Biogen
Idec (mammalian cells).
In 2005, Cellectis formed a collaboration with
Celliance, now Millipore, to evaluate Meganucle-
ase Recombination System (MRS) technology for
the development of a new generation of I-SceI
41
meganuclease tagged host cell lines for biophar-
maceutical production.
126
Mucin promoters; IIM14 and
IIM22 - vector enhancers
Boyce Thompson Institute for Plant Research
- Licensor, primary
Description: Insect (Trichpulsia ni) intestinal mu-
cin promoters with two nearly identical isoforms,
IIM14 and IIM22, enhance the effectiveness of di-
verse viral expression vectors. Both IIM isoforms
have an approximate molecular mass of 400 kDa.
These sequences are useful for the production of
recombinant vectors for microorganisms, plant, or
animals.
Invertebrate intestinal mucin cDNA and related
products and methods, assigned to the Boyce
Thompson Institute for Plant Research, Inc.
Licensing information: Contact the Boyce
Thompson Institute for Plant Research, Inc. (tech.
no BTI-39).
127
New Cabilly; Cabilly-Boss;
Monoclonal antibody 2-chains
expression - universal
Celltech Biologics plc - Assignee, patent
Genentech, Inc. - Licensor, primary
Description: Genentech currently holds a patent,
New Cabilly or Cabilly-Boss (6,331,415; 415
patent), broadly covering methods widely used
for recombinant monoclonal antibody expres-
sion, secretion and subsequent reconstitution
of two-chain antibodies. The method is widely
used for manufacture of chimeric and humanized
monoclonal antibodies. As cited below, the patent
includes broad claims for separately co-express-
ing both antibody light and heavy chains in the
same E. coli or yeast cells and use of the pBR322
plasmid.
Use with: universal; particularly E. coli; Sac-
charomyce cerevisiae; CHO cells; NS0 cells
Background: The current patent in many re-
spects follows-on Genentechs Boss patent, U.S.
4,816,397 (later revoked); and Adair, et al., e.g.,
U.S. 5,859,205 and WO 91/09967, assigned to
Celltech Group [Note, Celltechs technology
is now essentially assigned to Genentech (see
below); Celltech was acquired by UCB Bioprod-
ucts in May 2004]. Cabilly, et. al., U.S. 6,113,415
(Cabilly-Boss; New Cabilly patent) is a continua-
tion of 4,816,567 assigned to Genentech, Inc.
Originally, patent disputes between Celltech
Group (now UCB Bioproducts) and Genentech
over recombinant monoclonal antibody design/
construction technologies began in 1983. Celltech
Group filed its original U.K. application for
methods of designing/constructing recombinant
antibodies and antibody fragments together with
vectors and host cells useful in these processes
on March 25, 1983. On April 8, 1983, about two
weeks after Celltechs filing of its priority U.K.
application, Genentech filed its Cabilly appli-
cation for similar technology in the U.S. Shortly
thereafter, Celltech filed its Boss application in
the U.S.
Celltechs filings, eventually granted as U.S.
4,816,397 (Boss patent), Multichain polypep-
tides or proteins and processes for their produc-
tion, Boss, M.A., et al., and European Patent
Application 120694, describe expression in E.
coli of chimeric antibodies (but with few specific
details). The patents describe recombinant expres-
sion of monoclonal antibodies by cells trans-
formed with nucleic acid sequences encoding the
two desired component immunoglobulin chains.
Granted Boss patents included: U.S. 4,816,397,
expiry 2006 (later revoked); European patent: 0
120 694, expiry 2004; Japan: 2,594,900, expiry
2004; and UK: 2 137 631, expiry 2004.
42
The exemplary claim of 4,816,397 (Boss)
states, A process for producing an Ig molecule
or an immunologically functional Ig fragment
comprising at least the variable domains of the
Ig heavy and light chains, in a single host cell,
comprising the steps of: (i) transforming said
single host cell with a first DNA sequence encod-
ing at least the variable domain of the Ig heavy
chain and a second DNA sequence encoding at
least the variable domain of the Ig light chain,
and (ii) independently expressing said first DNA
sequence and said second DNA sequence so that
said Ig heavy and light chains are produced as
separate molecules in said transformed single host
cell. The patents coverage included, according
to Celltech, Any antibody, or antibody fragment,
produced from cells into which the separate se-
quences for the antibodys heavy and light chains
have been introduced by transformation. The an-
tibody producing cell may have been transformed
with either a single or separate vectors. These
cells may be: eukaryotic hosts or cell lines, e.g.,
NS0 cells, CHO cells, transgenic animals, yeast
cells, or bacterial cells. The antibodies produced
may be: whole antibodies, or Fab fragments, or
di-Fab (F(ab)2) fragments, or any antibody frag-
ment incorporating both a heavy chain variable
domain and a light chain variable region, or chi-
meric antibodies in which the constant domains
of either or both heavy and light chains have been
derived from a different source to the variable
domains, or humanized anti-bodies...[truncated]
Celltech also received the Adair patents
covering aspects of the design and engineering
of recombinant humanized antibodies. Celltechs
Adair patents include U.S. 5,859,205, expiry date
2016, and European 0 460 167, expiry date 2010.
The Adair patents claim a series of structural
features necessary in order for most humanized
antibodies to obtain full pharmacological activity.
Genentechs original Cabilly patent applica-
tion, granted as U.S. patent 4,816,567, Recom-
binant immunoglobulin preparations, Cabilly,
et al., was issued on March 28, 1989, the same
day as Celltechs Boss patent. Claim no. 1 of this
patent is, A method comprising (a) preparing
a DNA sequence encoding a chimeric immuno-
globulin heavy or light chain having specificity
for a particular known antigen wherein a constant
region is homologous to the corresponding con-
stant region of an antibody of a first mammalian
species and a variable region thereof is homolo-
gous to the variable region of an antibody derived
from a second, different mammalian species; (b)
inserting the sequence into a replicable expres-
sion vector operably linked to a suitable promoter
compatible with a host cell; (c) transforming the
host cell with the vector of (b); (d) culturing the
host cell; and (e) recovering the chimeric heavy or
light chain from the host cell culture. Note, with
both of Celltechs Boss and Genentechs Cabilly
patents issued on the same day, both were origi-
nally set to expire on March 28, 2006.
Genentech subsequently exactly copied word-
for-word all of Celltechs Boss patent claims
into a previously-filed continuation filing of its
Cabilly patent, with this eventually granted and
termed the New Cabilly Patent. This resulted
in the U.S. patent office declaring an interference
between the Celltech Boss patent and Genentechs
continuation filing (with the first-to-invent the
technology slated to receive patent protection).
In Feb. 1991, Genentech initiated an interference
with the patent office to determine which party
was the first to invent. In Aug. 1998, a panel of
patent office judges ruled in favor of Celltech,
finding that Genentech had deficient laboratory
notebooks and corroborating evidence, i.e., that
Celltechs Boss patent was valid and that Genen-
techs newer Cabilly application (incorporating all
of the Boss patent claims) was invalid.
Genentech filed a suit in U.S. District Court
appealing the granting of Celltechs Boss patent.
In March 2001, the court ruled that Genentech,
not Celltech, had priority (was the first to invent);
vacated the 1998 patent office decision in favor
of Celltech/Boss; withdrew Celltechs Boss U.S.
patent; and cleared the path for Genentech to
eventually receive its continuation (later granted
as 6,331,415, the New Cabilly patent) of its
43
original Cabilly patent.
On Dec. 18, 2001, as a result of earlier inter-
ference proceeding, the patent office revoked
Celltechs Boss patent, and granted Genentech
(and coassigned to the City of Hope National
Medical Center) its New Cabilly Patent, (Ca-
billy II), 6,331,415, entitled Methods of produc-
ing immunoglobulins, vectors and transformed
host cells for use therein, with an expiration date
of Dec. 18, 2018. This essentially combined both
Genentechs Cabilly and Celltechs Boss original
patents claims regarding methods for design/con-
struction of recombinant humanized antibodies.
This new patent also extended the expiration date
for essentially the same technology by over a
decade (to 2018).
Also on Dec. 18, 2001, as the result of media-
tion ordered by the U.S. District Court, Genentech
and Celltech Group settled their disputes concern-
ing recombinant humanized antibodies, including
the New Cabilly Patent. Both companies cross-li-
censed their technologies and Genentech took the
lead in licensing the combined technologies. Ge-
nentech would also compensate Celltech for any
lost income it would have received from licensing
of its Boss patent or its own sales of products
until its patents expiration in 2006. For Celltechs
prior licensees, e.g., MedImmune, this effectively
meant over a decade extension of their need to
pay royalties for the same technology (repackaged
into U.S. 6,331,415, the New Cabilly patent), into
2018! Thus, Genentech now holds patents broadly
covering methods for preparing recombinant
chimeric antibodies in diverse expression systems,
including co-expression of immunoglobulin heavy
and light chains and their refolding into immu-
nologically active forms. Many companies have
been developing recombinant monoclonal anti-
bodies, and many of these companies had been
presuming that U.S. patent protection Genentech
and Celltech technologies would expire in 2006
(now extended to 2018).
MedImmune, Inc. responded to the Genen-
tech-Celltech cross-licensing agreement by filing
suits in federal court alleging antitrust violations
between Genentech and Celltech, and challenging
6,331,415. MedImmune alleged that Genentech
and Celltech conspired to gain a monopoly, with
Celltech agreeing to abandon defense of its 2006-
expiring Boss patent in exchange for continuing
(through 2018) licensing revenue from Genen-
techs licensing of its New Cabilly patent and ac-
cess (through cross-licensing) to the New Cabilly
patent. MedImmune asserted that the Genentech-
Celltech agreement resulted in 29 years of patent
protection for the same technology.
In May 2004, the U.S. District Court dismissed
MedImmunes suit, with the Genentech/Celltech
patents and agreements remaining intact. The
judge found in favor of Genentech, because a U.S.
District Court judge had previously approved the
Genentech-Celltech agreement.
On Sept. 13, 2005, in a reexamination case
brought by MedImmune Inc., the U.S. patent
office rejected an extension of the New Cabilly
patent (6,331,415) on the grounds that it covered
overlapping material to the first Cabilly patent.
The patent office rejected the patent under the
doctrine of obviousness-type double patenting
and said New Cabilly is unpatentable over claims
of U.S. 4,816,567, the first Cabilly patent issued
in 1989. MedImmunes counsel had argued that
claims 1-36 of the Cabilly II patent were obvious
variants of claims 1-7 of the Cabilly I patent and,
thus invalid for obviousness-type double patent-
ing. In the meantime and currently, despite this
ruling, the patent and Genentechs licenses remain
fully enforceable. Genentech described this rul-
ing as a routine and expected next step in the
reexamination procedure and filed an appeal. By
rejecting all of the existing New Cabilly claims,
the patent office would force Genentech to make
its case for each one explicitly and in a manner
that addresses the arguments that the patent office
has laid out in its judgment.
On Oct. 18, 2005, the U.S. Court of Appeals
for the Federal Circuit (CAFC) in Washington,
DC, ruled against MedImmune in its appeal to
have Genentechs Cabilly II patent ruled invalid
along with alleged violations of antitrust, pat-
44
ent and unfair competition laws. MedImmune, a
licensee in good standing, having kept up with its
royalties paid to Genentech from sales of Synagis,
had filed a declaratory judgment action challeng-
ing the patents validity and enforceability. The
court ruled that MedImmune lacked standing and
could not bring suit against Genentech, since there
was no legal dispute, since MedImmune had not
violated its license agreement (contract), e.g., by
refusing to pay royalties due. This and the lower
court ruling this affirmed based their decisions
on the Federal Circuit decision in Gen-Probe
Inc. v. Vysis. Inc., holding that a patent licensee
in good standing cannot establish the requisite
case or controversy under Article III to invoke
the Declaratory Judgment Act because the license
agreement removes any reasonable apprehension
of a suit for infringement. MedImmune had not
stopped paying royalties, because this would have
resulted in immediate revocation of its license,
threatening marketing of Synagis, its main prod-
uct, and exposed the company to severe finanacial
penalties. The court also upheld the dismissal of
MedImmunes antitrust claims.
On Jan. 9, 2007, the U.S. Supreme Court (in
MedImmune, Inc., v. Genentech, Inc., et al.; Case
No. 05-608) reversed the U.S. Court of Appeals
for the Federal Circuit finding that MedImmune
had no standing to contest its Cabilly license with
Genentech and to challenge the validity of the
New Cabilly patent. The Supreme Court ruled
that MedImmunes Declaratory Judgement Act
complaint pled a contract dispute, that it had no
obligation to pay royalties on an invalid patent
and that was sufficient to confer subject-matter
jurisdiction under the Act. The Supreme Court ar-
ticulated the specifics of the case as follows: the
question in each case is whether the facts alleged,
under all the circumstances, show that there is a
substantial controversy, between parties having
adverse legal interests, of sufficient immediacy
and reality to warrant the issuance of a declaratory
judgment. The issue was whether MedImmune,
having made royalty payments under protest and
with reservation of all of [its] rights, elilmianted
the existence a legal case or controversy. Justice
Scalia summarized the decision as holding that a
licensees failure to stop its royalty payments did
not render a dispute over the validity of the patent
non-justiceable, i.e., just because a patent licens-
ee is in good standing regarding royalty payments
and other license/contractual requirements, it can
still challenge the validity of the patents involved.
Note, the Supreme Court made no rulings regard-
ing the New Cabilly patent or its licensing, just
that MedImmune (and other licensees) could chal-
lenge the patent. Those having supported MedIm-
mune in its case by filing amicue briefs included
the U.S. Solicitor General; the General Counsel of
the U.S. Patent & Trademark Office; the Generic
Pharmaceuticals Association; the Natural Re-
sources Defense Counsel; and Medtronic, Inc
On Feb. 21, 2007, the U.S. patent office is-
sued a final Office action in its reexamination of
Cabilly, et al., U.S. 6,331,415 (New Cabilly) and
rejected the patentability of all of the claims of
the patent, effectively revoking/invalidating the
patent. Genentech is appealing the decision. In the
meantime, the patent remains valid and enforce-
able through the appeals process. Genentech has
estimated that the entire appeals process may take
one to two years, or longer.
Genentech is estimated to have received about
$220 million in Cabilly licensing royalties in 2005
(according to UBC analysts), with MedImmune
paying about $30 million in royalties related to
sales of Synagis (respiratory syncytial virus Mab).
Some have estimated Genentechs royalties to be
as high as $300 million/year. Genentech refuses to
disclose how many companies pay royalties for li-
censing of New Cabilly, but in Feb. 2007 reported
that Abbott Labs., Centocor/J&J Inc. and Imclone
Systems Inc. paid Genentech a combined $105
million in royalties related to the patent in 2006.
In Jan. 2007, Genentech reported that revenue
from the Cabilly patent license with MedImmune
totaled 6 cents a share in 2006 and would increase
to 7 cents in 2007. With the New Cabilly patent
potentially around into 2018 (until Genentech
gives up its appeals, which is highly unlikely, or
45
the patent validity is eventually ruled on by the
Supreme Court), Genentech stands to make bil-
lions from related licensing in coming years.
This dispute will likely take years to fully
resolve, and many expect the Supreme Court to
end up deciding the validity of New Cabilly. Most
analysts expect that Genentech has a good chance
of successfully appealing and retaining New
Cabilly. Some earlier challenges to the original
Cabilly patent were unsuccessful, and the Genen-
tech has a history at aggressively defending itself
in such cases.
Note, Genentechs intellectual property portfo-
lio concerning humanized and other recombinant
monoclonal antibodies is broader than just the
New Cabilly patent. Besides its own relevant
patents, Genentech has used its strong/dominant
position to obtain rights to other patents. For ex-
ample, in Jan. 2006, Genentech and Amgen cross-
licensed to each other multiple patents related to
recombinant antibodies and their manufacture,
including Amgen receiving rights to New Cabilly.
Amgen refused to provide this author any infor-
mation regarding what it licensed to Genentech
in return, and neither party would disclose what
other Genentech patents, besides New Cabilly,
were licensed to Amgen, or whether Genentech
and/or Amgen have rights to further license their
cross-licensed patents to other companies.
The MedImmune-led challenge to the valid-
ity of Genentechs New Cabilly patent continues
in federal courts, and Genentech is appealing the
U.S. patent office revocation of the patent. Final
resolution of the validity of New Cabilly and the
need for its licensing will likely take years. In
the meantime, companies developing humanized
monoclonal antibodies will either need to take a
license from Genentech or face dire consequenc-
es, if the patent is eventually reinstated and held
valid.
Patents: The first 7 of 36 claims of 6,331,415
(New Cabilly) state:
1. A process for producing an immunoglobulin
molecule or an immunologically functional im-
munoglobulin fragment comprising at least the
variable domains of the immunoglobulin heavy
and light chains, in a single host cell, comprising
the steps of:
(i) transforming said single host cell with a first
DNA sequence encoding at least the variable
domain of the immunoglobulin heavy chain and a
second DNA sequence encoding at least the vari-
able domain of the immunoglobulin light chain,
and
(ii) independently expressing said first DNA
sequence and said second DNA sequence so that
said immunoglobulin heavy and light chains are
produced as separate molecules in said trans-
formed single host cell.
2. The process according to claim 1 wherein said
first and second DNA sequences are present in
different vectors.
3. The process according to claim 1 wherein said
first and second DNA sequences are present in a
single vector.
4. A process according to claim 3 wherein the vec-
tor is a plasmid.
5. The process according to claim 4 wherein the
plasmid is pBR322.
host cell is a bacterium or yeast.
host cell is E. coli or S. cerevisiae.
Licensing information: Contact Genentech re-
garding commercial uses and licensing.
Products made using this tech.: Most likely
every manufacturer of marketed recombinant
monoclonal antibodies has licensed this patent.
With Genentech renown for aggressive patent
enforcement, any infringer would likely already
be subject to infringement suits in federal court.
The most significant Cabilly-related royalties
on U.S. sales are paid to Genentech by MedIm-
mune for Synagis; Abbott Labs. for Humira;
Centocor/Johnson & Johnson for Remicade; and
ImClone Inc. for Erbitux. However, the largest
portion, probably the majority, of New Cabilly
licensing fees received by Genentech come from
Hoffmann-La Roche (the majority owner of Ge-
nentech; now seeking to fully acquire Genentech)
46
for its ex-U.S. sales of Herceptin, Rituxan, and
Avastin.
128
phCMV vectors; GenePORTER
- universal
Genlantis - Licensor, primary
Gene Therapy Systems, Inc. (GTS) - Parent co.
Description: phCMV vectors are engineered to
produce significantly higher expression levels
than traditional human cytomegalovirus (CMV)
promoter based constitutive expression vectors.
The phCMV vectors contain a specially modified
promoter/enhancer sequence that has been sys-
tematically engineered for high-level expression.
In these vectors, the CMV promoter/enhancer
sequences that are redundant and deleterious for
high levels of expression have been removed,
while sequences that confer high transcriptional
potency have been retained. As a result, phCMV
high-expression vectors can provide the full po-
tential of the CMV promoter and yield the high-
est possible levels of expression. In addition, the
plasmid backbones of the phCMV vectors have
also been optimized to allow higher plasmid yield
and smaller vector size.
Benefits: Advantages/characteristics include:
High-level constitutive transgene expression
Suitable for both stable (G418) and transient
expression
Expression of native and HA-tagged proteins
Small size for increased transfection efficiency
Convenient multiple cloning site containing the
most commonly-used restriction sites
Patents: No published patents or applications
retrieved from simple searches.
Availability: Genlantis markets these vectors as
GenePORTER vectors for non-commercial uses.
Licensing information: Contact Genlantis re-
129
Poly(A) polymerase
Description: - Poly(A) polymerase is useful
to place adenine residues, e.g., poly-A signals,
into the 3-termini of RNA. Poly(A) Polymerase
catalyzes the incorporation of adenine residues
into the 3-termini of RNA, using single-stranded
RNA as a primer.
Use with: plasmids; vectors
Use to make: plasmids; vectors
Availability: Marketed by Takara and other ven-
dors.
Licensing information: These enzymes should
now eaily be in the public domain, although
it would be prudent to check with your source
regarding and patents/licensing they claim are
involved in commercial use.
Krug, M.S. and Berger, S.L. (1987) Methods
Enzymol. 152:316-25.
Winter, G. and Brownlee, G.G. (1978) Nucleic
Acids Res. 5:3129-39.
130
Promoters
Description: Simply stated, promoters are poly-
nucleotide sequences that direct the cell which
genes to transcribe, i.e., switch gene transcription
on/off. Promoters are either constitutive (switched
on permanently) or inducible, containing se-
quences allowing transcription to be switched on
or off in a regulated fashion. Promoter strength
is a primary determinant of the level of expres-
sion of a cloned gene. Promoters generally affect
frequency of gene sequence transcription initia-
tion, not the rate of transcription. Transcription is
facilitated by cloning a target gene downstream
of a promoter sequence and associated transloca-
tion signal sequences. Strong promoters generally
have high affinity fo RNA polymerase binding.
Use of strong promoters can result in metabolic
stress of host cells, so transcription activity may
need to be tightly controlled, e.g, using a tran-
47
scription termination element (TTE).
The strongest promoters have generally been
derived from viruses, and generally contain a
CAAT box and TATA box upstream of an mRNA
transcription initiation site. Examples include
SV40 and CMV often used in CHO cells; and
CMV and RSV used in myeloma/hybridoma cells.
Enhancer element are often used in conjunction
with promoters. Promoter sequences often in-
clude tsp and binding sites for regulatory proteins.
TATA boxes or TA-rich regions are involved in
determining the tsp and maintaining a base level
of transcription. But not all strong promoters
require a TATA box.
Promoters range from those tightly regulated to
those constitutively expressed. It is important to
select an appropriate promoter for high-level pro-
tein expression. Generally, an inducible promoter
is more preferable than a constitutive promoter.
A strong promoter can result in expression of
recombinant protein at levels about 50% of total
cell protein.
Some constitutively expressed promoters have
been designed to be induced by changes, e.g.,
increases, in temperature; others are sensitive
(induced at) to low temperature, low oxygen ten-
sion or by certain sugars. Sequences controlling
expression of inducible promoter may be within
several genes encoding proteins with unlinked
functions, all controlled by a single regulatory
protein. Such networks of co-regulated genes may
respond to physiological factors, e.g., pH, carbon
sources, and alter metabolism. Genes coregulated
need not be located adjacent, near or even on
same chromosome.
Many prokaryotic expression systems have
been developed for expression of recombinant
proteins. Most gene expression systems in gram-
negative bacteria such as Escherichia coli have
relied exclusively on a limited set of bacterial
promoters, most now old enough to be in the
public domain (any patents expired). The most
widely used bacterial promoters have included
the lactose (lac) (Yanisch-Perron et al. Gene 33:
103-109 {1985}), tryptophan (trp) (Tacon et al.
Mol. Gen. Genet. 177:427-38 {1980}), and hybrid
derivatives such as the tac (deBoer et al. Proc.
Natl. Acad. Sci. U.S.A. 80:21-25 {1983}) and trc
(Brosius. Gene 27: 161-172 {1984}; Amanna and
Brosius. Gene 40: 183-190 {1985}) promoters.
Easily regulated E. coli promoters include lac, tac
and trc. Other expression systems include use of
the phage lambda promoters (PL and PR) (Ber-
nard et al. Gene 5:59-76 {1979}; Elvin et al. Gene
37: 123-126 {1990}), the phage T7 promoter
(Studier et al. J. Mol. Biol. 189:113-130 {1986};
see related entry), and phage T5 promoter (Bujard
et al. Methods Enzymol. 155:416-433 {1987}).
While these systems are commonly used and
contain many desirable features, these expression
systems, in comparison with many newer ones,
are generally subject to leaky expression from the
promoters, which can prohibit cloning of extreme-
ly toxic proteins, RNA, or enzymes producing
toxic metabolites.
Licensing information: Essentially all of the
classic promoters, e.g., developed/used in the
1970s and 1980s, are decades old and now in the
public domain (off-patent). The patent status of
specific promoters may often be determined by
examining the full documentation provided by
reagent suppliers offering the product. Otherwise,
if information is not readily available, e.g., the
promoter of interest is not included in this refer-
ence, it would be prudent to search the patent
literature.
131
Recombinant DNA; Protein
Expression; Cohen-Boyer
Stanford University - Licensor, primary
Description: Recombinant DNA techniques
developed by Drs. Cohen and Boyer, with pat-
ents assigned to the University of California
and Stanford University, can be used to express
proteins from exogenous genes in various biologi-
cal systems.
48
Cohen-Boyer patents covered fundamental
aspects of modern recombinant DNA techniques,
e.g., use of restriction enzymes for cutting and
splicing nucleic acid sequences, and construction
of basic phage-type vectors. These technolo-
gies were originally developed in the mid-1970s
by Drs. Cohen and Boyer. The two universities
collected over $200 million from Cohen-Boyer
patent licensing through 1996 when patents
expired. This involved a large number of rela-
tively low-priced nonexclusive licensing fees plus
royalties on sales. Stanford Univ. has reported a
total of 369 companies took licenses, encompass-
ing just about all companies active in commercial
recombinant protein R&D and manufacturing in
the 1980s through the mid-1990s. Loss of Cohen-
Boyer revenue has resulted in a drop of about $12
million in revenue annually to each school.
Stanford Univ. took the lead in licensing the
Cohen-Boyer patents, and classed licenses (and
royalty base) as involving Basic genetic product,
Process improvement product, Bulk product, and
End product. Royalty rates ranged from 10% for
the basic genetic technology, e.g., use of genetic
constructs, to .5% for use with major end prod-
ucts. For example, use of a transformed organism
for manufacture of insulin is a basic genetic prod-
uct with a royalty of 10%. Bulk products were
products processed further by a manufacturer and
not used or consumed by the end user, with roy-
alty ranging from 3% for annual sales volume un-
der $5 million; 2% for sales of $5 to $10 million;
and 1% for annual sales over $10 million. End
product was a product for use by the ultimate
consumer, such as an insulin injection, with roy-
alties ranging from 1% for annual sales volume
under $5 million; .75% for sales of $5 to $10 mil-
lion; and .5% for annual sales over $10 million.
Process improvement product was a material
developed for or by a manufacturer to improve an
existing process, e.g., a recombinant enzyme to
catalyze a reaction, with royalties of 10% of the
costs savings or other economic benefit. Since the
end of Sept. 1996 (through the life of the relevant
patents), the new license end-product royalty rate
was a flat 1% based on sales volume.
Patents: Related patents (now expired) were
co-assigned to the University of California and
Stanford University. U.S. 4,237,224 issued on
Dec. 2, 1980, includes claims for the basic use
of plasmid or viral vectors to transform or splice
exogenous (foreign) DNA into unicellular organ-
isms for recombinant protein expression. Until
their expiration, essentially all manufacturers of
commercial recombinant proteins were required
to obtain a nonexclusive license to these patents.
The first patent application was filed by Stan-
ford University in November 1974 in the midst
of much soul-searching on the part of the scien-
tific community. Drs. Stanley Cohen and Herbert
Boyer, who developed the technique together at
Stanford and the University of California, San
Francisco (UCSF), respectively, were initially
hesitant to file a patent. Several years of discus-
sion involving the National Institutes of Health
(NIH) and Congress followed. By 1978, NIH
decided to support the patenting of recombinant
DNA inventions by universities. In December
1980, the process patent for making molecular
chimeras was issued. The product patent for
prokaryotic DNA was issued in 1984. The patents
were jointly awarded to Stanford and UCSF and
shared with Herbert Boyer and Stanley Cohen.
The first licensee signed agreements with Stan-
ford on Dec. 15, 1981.
The Cohen-Boyer patent is considered by
many to be the classic model of technology trans-
fer envisaged by supporters of the Bayh-Dole Act,
which was intended to stimulate transfer of uni-
versity-developed technology into the commercial
sector. Is is considered the most-successful patent
in the entire history of university licensing. All
licenses were nonexclusive.
Availability: Related reagents, e.g., restriction
enzymes, are generally available from many ven-
dors, with no license required for commercial use.
Licensing information: The Cohen-Boyer
patents are not expired, with no need to take a
49
license.
Products made using this tech.: Stanford Univ.
reports having received significant Cohen-Boyer
licensing royalty income from sales of the fol-
lowing recombinant human biopharmaceuticals,
presumably all classed as basic genetic product
licenses (~10% royalties), with company date
of taking a license: a) Epogen (erythropoietin),
Amgen (12/2/80); b) Procrit, Amgen partnered
with Ortho/Johnson & Johnson (12/2/80); c) Ne-
upogen (granulocyte-colony stimulating factor),
Amgen (12/2/80); d) Activase (tissue plasmino-
gen activator), Genentech (12/2/80); e) Nutropin
(human growth hormone), Genentech (12/2/80);
f) Pulmozyme, Genentech (12/2/80); g) Humu-
lin (insulin), Genentech partnered with Eli Lilly
& Co. (12/2/80); h) Protropin (human growth
hormone), Genentech (12/2/80); i) Nutropin AQ,
Genentech (12/2/80); j) Roferon A, Hoffmann-
La Roche Inc. (12/2/80); and Leukine (granu-
locyte-macrophage colony stimulating factor),
Immunex Corp. (9/1/86). Posilac (recombinant
bovine growth hormone) used in cattle, licensed
by Monsanto (12/2/80), also provided significant
licensing income. Presumably, other recombinant
products marketed in the U.S., particularly those
manufactured in the U.S., during the life of the
patents also involved licensing of the Cohen-
Boyer patents.
132
Reconstituting chemically
orthogonal directed
engineering (ReCODE) -
Unnatural amino acids; UAAs;
E. coli; yeasts; mammalian
cells
Ambrx, Inc. - Licensor, primary
Scripps Research Institute, The - Assignee,
patent
Description: ReCODE methods, vectors and
modified host cells have been developed for ex-
pression of proteins containing unnatural amino
acids (UAAs), i.e., amimo acids other than the
basic 20+ normally used in humans and most
organisms, in E. coli, yeast and other host cells. A
variety of methods are provided for making and
using translation systems that can incorporate un-
natural amino acids into proteins. Both known and
new unnatural amino acids can be incorporated
into proteins using the translation system.
ReCODE provides unprecedented control
over the site-directed placement of UAAs, and is
designed and demonstrated to be non-disruptive to
protein function. Methods for design, evolution,
and selection of amino acid and amino-acyl tRNA
synthetase pairs have enabled incorporation of
over 30 different chemically-specified amino ac-
ids into biosynthetic proteins in E. coli, yeast and
mammalian cell systems. Through directed evolu-
tion and selection, specialized orthogonal tRNA
synthetases (RSo) are evolved to selectively and
specifically amino-acylate a similarly orthogonal
amber codon-suppressing tRNA (tRNAo) with
unique, chemically-specified amino acids. The
cells translational apparatus then incorporates
the Ambrx amino acid into the elongating peptide
sequence at positions designated by the amber
codon. In the absence of the Ambrx aa-amino-acyl
tRNAo, protein elongation is terminated at amber.
Modified yeast host cells and methods have
been developed for expression of proteins con-
taining unnatural amino acids (UAAs). Parts
of the simple but highly efficient protein-mak-
ing machinery of E. coli bacteria were inserted
into the advanced but inefficient protein-making
machinery of yeast cells. The result was a geneti-
cally-engineered yeast cell that produces complex
proteins containing UAAs at levels 300 times
higher than normal yeast cells. A yeast strain
deficient in nonsense-mediated mRNA decay was
generated to prevent rapid degradation of target
mRNA containing premature stop codons, which
are the most frequently used to encode unnatural
amino acids. The methods enable efficient expres-
sion of orthogonal bacterial tRNA in yeast using
50
polymerase III promoters that are cleaved from
primary transcripts. These new strategies provide
a significant increase in yield of unnatural amino
acid containing proteins from tens of micrograms
to tens of milligrams per liter in yeast.
Use with: E. coli; yeasts
Patents: Exemplary patents include,
7,045,337, In vivo incorporation of unnatural
amino acids, 05/16/2006, Schultz, P., et al., coas-
signed to Scripps and the Univ. of California. The
patent broadly claims the in vivo incorporation of
unnatural amino acids into proteins, not limited by
the type of cell, tRNA synthetase, tRNA, unnatu-
ral amino acid, selector codon or resultant protein.
Claim 1 states, A cell comprising: (a.) an orthog-
onal tRNA synthetase that aminoacylates endog-
enous tRNAs of the cell with a reduced efficiency
as compared to aminoacylation of the endogenous
tRNAs by endogenous tRNA synthetases of the
cell, and (b.) an orthogonal tRNA that is aminoac-
ylated by the endogenous tRNA synthetases with
reduced efficiency as compared to aminoacyla-
tion of the endogenous tRNAs by the endogenous
tRNA synthetases, wherein said orthogonal tRNA
synthetase preferentially aminoacylates said
orthogonal tRNA, as compared to the endogenous
tRNAs, with an unnatural amino acid in said cell,
and wherein the orthogonal tRNA synthetase
preferentially aminoacylates aid orthogonal tRNA
with an unnatural amino acid, as compared to a
natural amino acid, the orthogonal tRNA synthe-
tase having a lower Km for the unnatural amino
acid than for the natural amino acid, and wheein
said orthogonal tRNA recognizes a selector codon
of an mRNA in said cell, with further claims
concerning specific UAAs and use in E. coli.
A related application is U.S. 20080166766,
Genetic incorporation of unnatural amino acids
into proteins in mammalian cells, Liu, W., et al.,
concerning mammalian host cell lines for expres-
sion of UAA-containing proteins.
Yeast UUA technology was first reported in
April 2008. No related published applications
were retrieved.
Licensing information: Ambrx has exclusively
licensed UAA technology from Scripps. Contact
Ambrx regarding commercial uses and licensing.
The company claims, Ambrxs initial focus has
been on commercialization of its E. coli expres-
sion system. Ambrx has evolved the technology
to the point of commercial practicality. Impor-
tantly, Ambrx has demonstrated that its ReCODE
technology is efficient, robust and scalable to
manufacturing.
Products made using this tech.: Ambrx is de-
veloping ARX-201, a long-acting human growth
hormone, containing UAAs.
Further info.:
New Methods Enabling Efficient Incorporation
of Unnatural Amino Acids in Yeast. Journal of
the American Chemical Society. J. Am. Chem.
Soc., 130 (19), 60666067, May 14, 2008.
(http://dx.doi.org/10.1021/ja800894n).
133
Selectable markers; Selection
of transformed cells; Reporter
genes
Description: Selectable marker genes are used
in conjunction with gene(s) for desired protein(s)
to allow for rapid and cost-effective selection or
isolation of cells with properly transformed genes.
As can be seen from the related entries, a number
of promoters are available for prokaryotic and
eukaryotic expression systems. Generally, the ex-
pression of the the marker protein allow overcom-
ing of cell growth inhibiton by a specific inhibitor
of an essential cellular enzyme or other essential
cellular metabolic function. One can use correc-
tion of a defect (deletion) in the host organism,
e.g., restoration of ability to metabolize a particu-
lar substrate or grow in absense of an otherwise
essential nutrient. Presuming the marker gene is
physically adjacent or near the gene for a desired
protein(s), expression of the marker is associated
with proper insertion of the desired vector se-
quence into target cells. Selectible markers, as the
names imply, generally provide a simple method
51
for selecting successfully transformed cells, e.g.,
with addition or not of drugs, toxic chemicals,
environmental conditions, etc., killing off cells
not expressing the marker gene (and apparently
not successfully transformed.
Perhaps, the simplest method is positive selec-
tion using an antibiotic-resistance marker gene
incorporated into a vector, so only transformed
bacteria can grow in presence of antibiotic. One
can also identify clones containing a specific gene
sequence by PCR, restriction endonuclease map-
ping or hybridization
Chromogenic, i.e., color-generating, fluores-
cent and luminescent reporters, e.g., E. coli lacZ,
encoding beta-galactosidase, may also be used,
but these arre used more for research rather than
commercial protein manufacture. Antibiotic re-
sistance genes, e.g, cat encoding chloramphenicol
acetyl transferase, confer resistance to chloram-
phenical or other specific antibiotics.
Chomogenic substrate cleavage enzyme sys-
tems that have been commonly used in bacteria
include: 1) beta-galactosidase (lacZ) gene of E.
coli, with chromogens used including 5-bromo-
4-chloro-3-indolyl-beta-D-galactopyranoside
(X-Gal; turns blue) and other beta-D-galactopy-
ranosides, e.g. ONPG, FDG, Cl-AMPGD and
MUG; 2) celB gene encoding thermostable beta-
glucosidase, which also has beta-galactosidase
activity; and 3) phoA (alkaline phosphatase) using
5-bromo-4-chloro-3-indoyl phosphate.
Selectable markers used with mammalian cells
include dominant markers, e.g., drug resistance,
which can be used with most cell lines. Recessive
markers used in combination with a specific host
cell line include enzymes of purine and pyrimi-
dine metabolism, drug resistance and amino acid
metabolism. The two most common systems are
glutamine synthetase (GS) and dihydrofolate
reductase (DHFR) systems. The earliest markers
in use were based on complementation using hy-
poxanthine phosphoribosyl transferase (HGPRT)
and herpes simplex virus (HSV) thymidine kinase
(TK) genes, with expression of TK in transformed
cells resulting in metabolisms of thymidine kinase
inhibitors to active forms, resulting in toxicity and
death of untransformed cells when acyclovir or
another nucleoside analog drug with thymidine
kinase activity is added. With HGPRT, cells are
cultured in 8-azaadenine, 6-thioguanine or other
nucleoside, and toxic metabolites kill untrans-
formed cells.
Some marker systems, e.g., GS and DHFR (see
related entries), are amenable to amplification.
Cells cultured in increasing levels of inhibitors
activated by the marker genes overcome toxic ef-
fects by selecting for cells expressing high levels
of DHFR or GS, with ability to amplify (increase
copy count) of large regions of DNA (100s or
1000s of kb), potentially containing 100s of cop-
ies of target gene.
Selectable markers are generaly not needed
with most transcient expression systems and
high-efficiency gene transfer systems, e.g., viral
vectors or microinjection. However, most com-
mercial production cell lines are now derived by
direct DNA transfection followed by selection and
amplification.
Patents: The oldest systems, including comonly
commercially-used DHFR, are now in the public
domain (off-patent).
Licensing information: A large number of select-
able marker and related reagents are available
from a large number of vendors. If in doubt as
to the patent status (need to license) a selectable
marker, check the full documentation available
from vendors regarding their product and/or ask
them about the need for licensing for commercial
use.
134
Small Ubiquitin-like Modifer
(SUMO) fusion protein tags;
Split SUMOpro System; Ubl-
specifc protease - universal
Cornell University - Licensor, secondary
LifeSensors Inc. - Assignee, patent
52
Description: The SUMO system, particularly
Split SUMO, provides fusion proteins from
expression of a sequence coding for a SUMO
protein linked to a gene for a desired protein, with
the resulting SUMO fusion readily purified using
an included terminal poly-His tag (using affnity
chromatography; see related entry) and the fusion
protein cleaved using SUMO protease. This versa-
tile system enhances protein expression, facilitates
affinity purification, and allows generation of
correct N-termini. SUMO Protease 1 cleaves puri-
fied recombinant SUMOfusion proteins with any
amino acid except proline at the +1 position of
the cleavage site. The system enables expression
of high quantities of soluble protein with desired
amino-termini.
Newer Split SUMO technology offers the flex-
ibility that a single fusion system can be used in
any organism, such as E.coli, yeast, insect, and
mammalian cells, with the expression cassette
fully interchangeable between different organ-
isms. In Split SUMO, yeast SUMO, a 103 AA
residue protein, is bifurcated to generate two
halves of SUMO. The inner core of the protein is
highly hydrophobic, with this partly responsible
for efficient translation and folding. The outer sur-
face of the protein is rather hydrophilic, with this
partly responsible for the solublization properties
of SUMO when fused to an insoluble protein.
Affinity purification using the poly-histidine
tags (see related entry) included in the fusion
protein can be achieved by two different methods,
Ni-NTA chromatography or CTHSNTHS hydro-
phobic interaction chromatography.
The SUMO system enables construction of
E. coli, yeast, and baculovirus vectors that allow
simple unidirectional cloning of PCR-amplified
genes.
SUMO Protease, a highly active cysteinyl pro-
tease also known as Ulp, is a recombinant frag-
ment of Ulp1 (Ubl-specific protease 1) from Sac-
charomyces cerevisiae. SUMO Protease cleaves
in a highly specific manner, recognizing the ter-
tiary structure of the ubiquitin-like (UBL) protein,
SUMO, rather than an amino acid sequence. The
protease cleaves SUMO from recombinant fusion
proteins. Following digestion, SUMO protease is
easily removed from the cleavage reaction by af-
finity chromatography using the polyhistidine tag
at the N-terminus of the protein.
Use with: prokaryotes; E. coli; yeasts; insect
cells; eukaryotes
Background: There is a drawback to the original
SUMO gene fusion system for eukaryotic sys-
tems. In eukaryotes, the SUMO fusion is cleaved
in vivo, and the facility to purify SUMO fusions
is lost. To circumvent this problem, the SUMO
tag was bifurcated (cut) into two halves (Split
SUMOpro System). The C-terminal half SUMO
(CTHS) is fused with a protein of interest to
enhance the level of protein expression while de-
tracting endogenous SUMO proteases in eukary-
otic cells from cleaving the fusion. Once purified
from the cell, the N-terminal half SUMO (NTHS)
is reconstituted with CTHS-fusion protein to form
a hydrophobic core complex. An added bonus
of the strong hydrophobic interaction between
two halves is the restoration of the full SUMO
structure. Addition of SUMO protease releases
the native protein by rapid cleavage of the fusion
protein.
Benefits: SUMO can provide at least a 10x in-
crease in expression.
Patents: LifeSensors holds worldwide patents
and pending applications for the use of SUMO-
fusion and applications of proteases to generate
novel N-termini.
Applications filed include: Butt, T. R., Mal-
akhova O.A and Malakhov M. P (2003) Meth-
ods and composition for protein expression and
purification. US Patent Application, 60/482817;
Butt, T.R., Weeks, S.D., Tran, H.T., Malakhova,
O.A., and Malakhov, M.P. (2002) Methods and
compositions for protein expression and purifica-
tion. International Patent Application No. PCT/
US03/00436; Tran, H.T., Askari, H., Schwartz,
M. and Butt, T.R. (2001) Methods and composi-
tion for identifying ligands for nuclear recep-
tors. International Patent Application No. PCT/
53
US01/05429 and; Butt, T.R. and Tran, H.T. (2001)
Biosensors for identifying gender at embryonic
stage. International Patent Application No. PCT/
US02/12590.
Exemplary patents include U.S. 7,220,576
(also 7,060,461), Methods and compositions for
protein expression and purification, Butt, et al.,
May 22, 2007, assigned to Lifesensors, Inc.
by Invitrogen Corp., Active Motif and LifeSen-
sors Inc. Invitrogen markets Champion pET
SUMO vector, incorporating SUMO into the pET
vector for E. coli.
Licensing information: Cornell University re-
garding licensing for commercial uses
Further info.: Enhanced protein expression in
mammalian cells using engineered SUMO fu-
sions: Secreted Phospholipase A2, Peroutka RJ,
Elshourbagy N, Piech TL, Butt TR., Protein Sci.
2008 Jun 26.
135
Strep-tag affnity fusion protein
tag; Strep-Tactin purifcation -
universal
IBA Gene Technologies - Licensor, primary
Institut Fur Bioanalytik Gemeinnutzige Gesell-
schaft MBH - Assignee, patent
Description: Fusion proteins from the joined
expression of a Strep-tag (either Strep-tag I or II)
sequence, i.e., mutant forms of streptavidin with
improved binding affinity, with that for a desired
protein are useful for purification by Strep-Tactin
(streptavidin variant) affinity chromatography,
with subsequent cleaving of the Strep-tag. Af-
ter expression of the Strep-tag II fusion protein,
Strep-tag II enables affinity chromatography on
immobilized Strep-Tactin yielding over 99% pu-
rity in a single step.
The Strep-tag II is an 8-amino acid peptide that
was originally developed for affinity purification
of corresponding fusion proteins on streptavidin
columns, but now may be used with improved
Strep-Tactin columns. The peptide recognizes
the same pocket of streptavidin where the natu-
ral ligand is normally bound so that biotin or its
chemical derivatives can be used for competitive
elution. The binding affinity of Strep-tag II to
Strep-Tactin is nearly 100 times higher than to
streptavidin. The tagged protein binds to immobi-
lized Strep-Tactin during affinity purification, an
elution of purified recombinant protein is per-
formed by addition of desthiobiotin. Desthiobiotin
is an inexpensive, reversibly binding and stable
analog of biotin - the natural ligand of streptavi-
din. This competitive elution is the second step
conferring specificity, enabling unparalleled puri-
fication factors. Purification steps are performed
under mild conditions.
Benefits: Simplified, cost-effective purification.
Patents: Strep-tag technology for protein puri-
fication and detection is covered by US patent
5,506,121, Fusion peptides with binding activity
for streptavidin, UK patent 2272698 and French
patent; assigned to Institut Fur Bioanalytik Ge-
meinnutzige GmbH, and exclusively licensed to
IBA Gene Technologies.
Strep-Tactin is covered by US patent
6,103,493, Streptavidin muteins, also assigned
to Institut Fur Bioanalytik Gemeinnutzige GmbH,
and exclusively licensed to IBA Gene Technolo-
gies.
Availability: EMD Biosciences markets these
products for non-commercial use.
Licensing information: Contact IBA Gene Tech-
nologies regarding commercial use and licensing.
Further info.:
Improved affinity of engineered streptavidin for
the Strep-tag II peptide is due to a fixed open con-
formation of the lid-like loop at the binding site,
Protein Science (2002), 11:883-893.
54
136
Super core promoters;
SCP1; Core promoters - the
strongest [promoters] ever
made
University of California - Licensor, primary
Description: Super core promoters, including
SCP1, with multiple core promoter motifs are the
strongest [promoters] ever made, five to 10 times
more potent than the adenovirus major late-core
or cytomegalovirus immediate early-core pro-
moters in cells. SCP1 could prove useful in any
application where increased protein expression is
needed.
Super core promoter 1 (SCP1) directs high
amounts of transcription by RNA polymerase II
in metazoans. SCP1 contains four core promoter
motifs--the TATA box, initiator (Inr), motif ten el-
ement (MTE) and downstream promoter element
(DPE)-in a single promoter. SCP1 is distinctly
stronger than the cytomegalovirus (CMV) IE1
and adenovirus major late (AdML) core promot-
ers both in vitro and in vivo. Each of the four core
promoter motifs is needed for full SCP1 activity.
SCP1 is bound efficiently by TFIID and exhibits
a high propensity to form productive transcription
complexes. Super core promoters include the 80
or so nucleotides encompassing the RNA start site
that direct the initiation of transcription by Pol II.
Use to make: proteins; glyocproteins
were retrieved from simple searches. The primary
inventor is James Kadonaga, chair of molecular
biology, University of California, San Diego.
California.
Further info.:
Rational design of a super core promoter that
enhances gene expression. T Juven-Gershon,
S Cheng, JT Kadonaga. Nature methods. 2006
Nov;3(11):917-22.
137
TAGZyme; dipeptidyl
aminopeptidase I (DPPI;
DAPase Enzyme; cathepsin C) -
cleavage of His tags; universal
QIAGEN GmbH - Licensor, primary
Novo Nordisk A/S - Licensor, secondary
UNIZYME Laboratories - Assignee, patent
Description: TAGZyme or dipeptidyl aminopep-
tidase I (DPPI; DAPase Enzyme; DAP I; cathep-
sin C) is useful for cleavage of poly-histidine fu-
sion protein tags (see relate entries). This enables
purification of all His-Tagged proteins, no matter
what their sequence, and is applicable to most
expression systems.
TAGZyme is a DPPI-based enzymatic system
for the efficient production and affinity purifica-
tion of therapeutic proteins using His-tags and
tag removal. Using TAGZyme, precise removal
of N-terminal His-tags is obtained without the
risk of degradation of the target protein. Using
TAGZyme, it is possible to go from identifica-
tion of target directly into production, reducing
development time and costs. This enzyme results
in complete removal of N-terminal poly-histidine
tags from recombinant proteins. It may be part
of a combined IMAC/TAGZyme strategy for the
efficient production of highly purified homoge-
neous protein. DAPase may be used in combina-
tion with glutamine cyclotransferase (Qcyclase
Enzyme) and pyroglutamyl aminopeptidase
(pGAPase Enzyme).
Benefits: The system enables standardization of
downstream processing, reducing the number of
unit operations. It offers the possibility of going
directly from identification of target into produc-
tion, thus it reduces development time. Other
advantages are:
a) Complete removal of N-terminal poly-histidine
purification tags without the risk of degradation of
the target protein encountered when using endo-
55
proteases.
b) Possibility of developing a standard method for
purification of pharmaceutical proteins which is
scalable from R&D to production.
c) Very simple method, competitively priced, with
suitable patent protection for commercial use.
Process for preparing a desired protein, Nov.
25, 1997; 5,783,413, Enzymatic process for
producing a desired protein from an amino ter-
minal extended protein, 1998; and E.U. patent
00759931B1, each assigned to Unizyme.
TAGZyme and DAPase are trademarks of
Unizyme.
Assignee: Novo Nordisk A/S (DK)
Availability: Marketed by GE Healthcare (for-
merly Amersham plc) and Qiagen for non-com-
mercial uses. TAGZyme is marketed worldwide
in both the R&D and large-scale markets by
QIAGEN, which also sells IMAC column matri-
ces and other reagents used in connection with
polyhistidine tags.
UNIZYME Laboratories offers contract down-
stream process development applying the TAG-
Zyme technology and also manufactures proteins
on a contract basis.
Licensing information: Contact Unizyme regard-
Products made using this tech.: TAGZyme has
been used and tested by more than 20 biophar-
maceutical firms.
138
TEV Protease; Tobacco Etch
Virus (TEV) NIa protease -
cleavage of fusion protein tags;
universal
University of Oregon - Licensor, primary
University of Texas - Licensor, secondary
Description: TEV Protease, recombinant (rTEV),
a plant-derived enzyme, is a site-specific protease
useful for the removal of glutathione-S-transfer-
ase (GST), poly-histidine and maltose binding
protein (MBP) fusion protein affinity tags (see
related entries) from fusion proteins. The seven-
amino-acid recognition site for rTEV is Glu-Asn-
Leu-Tyr-Phe-Gln-Gly (1-4) with cleavage occur-
ring between Gln and Gly. The enzyme can be
used at room or low, temperatures, e.g., 4C. Fol-
lowing digestion, TEV Protease can be removed
by affinity chromatography using its own attached
poly-histidine tag sequence.
TEV Protease is an improved 50kDa version of
the Nla protease from Tobacco Etch Virus (TEV),
which has been engineered to be more stable than
native TEV protease for prolonged enzymatic
stability.
5,532,142, Method of isolation and purification
of fusion polypeptides,: Johnston, et al., July 2,
1996, co-assigned to the University of Texas and
University of Oregon.
Availability: Marketed by Invitrogen, Promega
(as ProTEV Protease) and perhaps other vendors
for non-commercial use.
Licensing information: Contact the University
of Texas and/or University of Oregon regarding
commercial use and licensing..
Further info.:
Dougherty, W.G. et al. (1989) Characterization of
the catalytic residues of the tobacco etch virus 49-
kDa proteinase. Virology 172, 302-10.
Dougherty, W.G. and Parks, T.D. (1991) Post-
translational processing of the tobacco etch virus
49-kDa small nuclear inclusion polyprotein: iden-
tification of an internal cleavage site and delimi-
tation of VPg and proteinase domains. Virology
183, 449-56.
Carrington, J.C. and Dougherty, W.G. (1988)
A viral cleavage site cassette: identification of
amino acid sequences required for tobacco etch
virus polyprotein processing. Proc. Natl. Acad.
Sci. USA 85, 3391-5.
56
139
Transfection - universal
Description: Note, this directory does not include
full coverage of the many, seemingly hundreds, of
transfection methods available.
A large number of methods/technologies with
many or most in the public domain, are available
for transfection - the transient or stable introduc-
tion of exogenous molecules and genetic material,
DNA or RNA, into cultured cells. This commonly
involves physical disruption of teh cell mem-
brane. Current methods of fransfection include
use of electroporation, virus vectors, lipofection,
various transfection reagents (e.g., calcium phos-
phate), microinjection, gene guns and microinjec-
tion. Each has a tradeoff between the flexibility
of agents to be transfected and the risk of cellular
damage upon transfection. For example, although
electroporation allows great flexibility in the types
of agents and cells that can be used and can be
used on large numbers of cells simultaneously,
this technique engenders a high risk of cellular
damage, which can potentially alter cell pheno-
type. Virus vectors, while causing limited cell
damage can be a source of difficulty in construct-
ing customized delivery vectors. In addition, and
there are concerns about phenotype alteration
following virus transfection. Lipofection (using
liposome-like lipid bilayer membranes) suffers
from some of the customization challenges that
impact viral vector transfection and is also some-
what more limited in the variety of agents which
can be delivered. Gene guns offer low to moder-
ate throughput and are customized for nucleic
acid delivery. Manual microinjection remains
the stalwart of cellular transfection, functioning
mostly as a fallback technique that is used when
other techniques are inappropriate or ineffective.
However, manual microinjection is relatively low
throughput and requires a high level of operator
skill.
A large number of methods for transfection
of introducing exogenous DNA into mammalian
cells are available. These include: plasmids and
viral vectors; electroporation; fusion with bacte-
rial protoplast; lipofection (using liposomes);
carriers, e.g., calcium phosphate particles; mi-
croinjection into cells; and protoplast fusion.
Many, probably most, of these methods have been
known for decades and are in the public domain
(off-patent). Various cationic liposomal formula-
tions were developed in the early nineties.
In general, it is easier to introduce exogenous
genes into mammalian cells, but difficult to delete
specific genes. Heterologous recombination limits
ease of insertions and deletions (unlike yeasts
and E. coli). Adherent cells, e.g., CHO and BHK,
are often transfected by calcium phosphate and
cationic lipids; myelomas are often transfected by
electroporation or protoplast fusion. Transfected
DNA expression is often lost in days or weeks,
unless integrated into chromosomes (a good rea-
son to use selection/selective markers).
Benefits: Some pros and cons with various ap-
proaches include:
ELECTPORATION - Can use a variety of trans-
fection agents.
Can transfect large numbers of cells.
Cons
Risk of low efficiency from leaky or destroyed
cells.
Requires moderate operator skill.
VIRUS VECTORS - Low risk of cell damage.
Can transfect large numbers of cells.
Cons
Risk of low efficiency from virally infected cells.
Limited flexibility of transfection agents. Re-
quires
increased operator skill. May require special
biosafety processes. Creating virus vectors is
time-intensive.
LIPOFECTION - Low risk of cell damage. Can
transfect large numbers of cells. Requires moder-
ate operator skill.
Cons
Risk of low efficiency from cytotoxicity. Limited
flexibility of transfection agents. Consumables
57
can be expensive.
GENE GUN - Can transfect large numbers of
cells.
Requires moderate operator skill.
Con
Risk of low efficiency from destroyed cells and
cytotoxicity. Limited flexibility of transfection
agents.
DIRECT INJECTION - Unlimited flexibility of
transfection agents.
Can transfect adherent cells.
Cons
Risk of low efficiency from leaky or destroyed
cells.
Requires high level of operator skill.
Low throughput.
140
Translation Engineering
expression; CODA;
Computationally Optimized
DNA Assembly; Hot Rod Genes;
Controlled Ribosomal Pausing
- universal
CODA Genomics, Inc. - Licensor, primary
University of California - R&D
Description: Translation Engineering allows
optimization of expression vectors for use in any
host using (Computationally Optimized DNA As-
sembly; CODA), a computational process which
creates user defined, self-assembling genes from
commercial oligonucleotides. Translation Engi-
neering exclusively provides the ability to manip-
ulate pause signals to control the speed of protein
translation, a key factor in determining protein
yield and functionality. CODA allows synthetic
gene manufacture to be scaled so robustly that
the imprecise process of pooled oligo assembly,
through extensive computer optimization, yields
only one correct product that will reliably express
the desired protein....CODA is commercializing
its Translation Engineering technology to generate
improved heterologous expression in any well-
characterized host...CODAs translation engineer-
ing enables moving an open reading frame into a
heterologous host, opening up a whole range of
new possibilities...CODA proprietary algorithms
go far beyond mere melting temperature optimi-
zation and specify the oligo sets and laboratory
steps to achieve high thermodynamic probability
of perfectly-correct hybridization.
Translation Engineering is based on pairs of
codons, di-codons, within open reading frames
which result in translation pause signals at the
ribosomal level. CODA has the exclusive ability
to manipulate di-codons, a factor in determining
protein yield and functionality. The following
technologies are included: Control of translational
pauses, encoded as codon pairs; Codon usage
optimization; Control of secondary structure in
mRNA; Management of DNA sites (restriction
sites, internal Shine-Delgarno sequences, user
defined sequences etc.) and; Massive Computa-
tion, Global Optimization and Scaleable Synthetic
DNA Assembly.
CODA has developed design tools that place
codon pair encoded pause sites at selected targets
within the open reading frame. These pauses can
be placed with the purpose of enhancing domain
folding, secretion and other protein processing
- events that occur in real time with respect to
protein translation. This allows new approaches to
solving long standing problems in protein folding
and other aspects of maintaining function in the
production of biologically active products.
Correct assembly is achieved by making
silent codon substitutions into the DNA code to
strengthen correct hybridizations and to disrupt
erroneous ones, thus achieving a large DNA
melting temperature gap between the one cor-
rect versus all possible incorrect hybridizations.
While computationally demanding, this process
ensures a temperature range within which, with
virtual certain thermodynamic probability, correct
hybridizations form and incorrect ones melt.
Genes are fuse with the integrase (IN) gene
of the Saccharomyces cerevisiae transposable
58
element, Ty3. Perfect match hybridizations are
stabilized and moved to higher temperatures, far
above the melting temperatures of all mismatched
assemblies. Further point mutation/deletion error
reduction at an early stage yields a high prob-
ability of error-free fragments. Sub-assembly
preparation ensures, with high probability, that
perfect short sequences yield perfect larger ones
up to the limits of polymerase extension. Transla-
tional kinetics, codon usage preferences, and other
desirable expression traits are added to the general
optimization procedure using remaining degrees
of freedom in the sequence design. The result
is elimination of both macro and micro errors
because the component oligomers self-assemble
cleanly and correctly into the desired order. Using
CODA-optimized kits and protocols, users can
easily, economically, and reliably assemble their
own synthetic DNA constructs.
Hot Rod genes useful to express high levels
of desired protein can be engineered by remov-
ing the major pauses in the ORF at chi-squared
statistics higher than an organism-specific maxi-
mum. CODA optimization includes creating large
amounts of expression by the elimination of pause
sites.
Use to make: proteins; glycoproteins; antibod-
ies
Background: Regulating the speed of the step-
wise process of protein elongation during trans-
lation has a critical role in determining the final
amount and the functionality of the encoded
protein. Living organisms from bacteria, to yeasts,
to humans all exhibit extremes in their distribu-
tipn of codon pairs (codon context). The presence
of these biased signals is universal. These signals
have been shown to relate to the speed of protein
translation in E. coli and expression levels in
humans. Moving protein production into a new
organism often scrambles these signals, and may
cause unpredictable levels of gene expression and
impaired protein function, such as lack of solubil-
ity or other problems. Codon usage optimization
alone is simply not enough to reliably engineer
expression. Translation Engineering takes the
gene design process beyond codon usage to con-
trol of translation kinetics.
Benefits: This is claimed to be the first method
for gene synthesis eliminating the costly and
time-intensive intermediate stages of selection
and correction via biological methods, i.e., bio-
logical tesing of gene constructs is eliminated or
minimized. Benefits/characteristics include:
Improved protein expression in E. Coli, CHO,
yeast, human, insect, plant, rodent, and other cell
lines,
Improved secretion and function of a target
protein.
Flexible in a variety of cell lines,
Scaleable for large studies
Guaranteed to express proteins of interest
Patents: CODAs Translation Engineering
technology has been developed under exclusive li-
cense on issued and pending patents from the Uni-
versity of California, Irvine. Additional patents
around the technology are pending.
Exemplary U.S patents include 5,082,767,
Codon pair utilization, Hatfield, et al., Jan. 21,
1992. Applications include 20050106590 and
20040235035, both titled Method for producing
a synthetic gene or other DNA sequence.
Licensing information: CODA offers its im-
proved gene designs as synthetic gene kits or as
assembled genes (CRO services).
In Jan. 2007, CODA and Integrated Genomics
formed a collaboration to enhance protein pro-
duction in Pichia expression systems (see related
entry). In Dec. 2006, CODA Genomics expanded
its collaboration with Viventia Biotech to various
antibody projects within Viventia.
Further info.:
Translation Engineering and Synthetic Biology
David A. Roth, Liza S. Z. Larsen, and G. Wesley
Hatfield, In Cell-Free Expression. (Edited by W.
Antoni Kudlicki). Landes Bioscience, George-
town, Texas, 2007.
Gutman G. and Hatfield G.W. Nonrandom Uti-
lization of Codon Pairs in Escherichia coli. The
Proceedings of the National Academy of Sciences
59
USA, 86:3699-3703. (1989).
Hatfield G. W. and Gutman G. A. Codon Pair
Utilization Bias in Bacteria, Yeast and Mammals.
In Transfer RNA in Protein Synthesis. (Edited by
D. L. Hatfield, B. J. Lee, and R. M. Pirtle). Pub-
lished by CRC Press, Boca Raton, FL, 1993. A
comprehensive review of codon pair bias and its
relationship to codon context effects on transla-
tion.
Kittle, J.D. Radical Changes in the Engineering
of Synthetic Genes for Protein Expression. Bio-
Pharm International. 2006:12-18 (2006).
141
White collar complex (WCC)
promoter; UV light induction -
universal
Dartmouth College - Licensor, primary
Description: Vectors including the white collar
complex (WCC) promoter from the filamentous
fungus Neurospora can activate gene expression
in vivo using only light as an inducer. This pro-
vides a means to induce gene expression using a
reliable, readily available, inexpensive, non-toxic,
and non-pleiotropic inducer - ultraviolet light.
The WCC is comprised of two proteins, WC-1
and WC-2 that readily heterodimerize and strong-
ly bind the cofactor FAD. The WCC is able to ab-
sorb light and subsequently activate transcription
of a gene operatively-linked to a light-responsive
regulatory sequence which is bound by the WCC.
In a host cell, transcription of a gene, operatively-
linked to one or more light-responsive regulatory
sequences, is stimulated by WC-1/WC-2 by alter-
ing the fluence or wavelength of light exposed to
the host cell.
Use with: universal (generic); E. coli; plants;
mammalian cells; insect cells
Background: Current methods of controlling
gene expression such as inducible promoters, heat
shock, hormones, or drugs/chemicals such as tet-
racycline or IPTG, have generally suffered from
exogenous inducer molecules evoking pleiotropic
effects which complicate analyses. Dartmouth
researchers have identified ths two-protein com-
plex that reliably activates transcription of genes
linked to one or more short DNA light-responsive
regulatory sequences.
Patents: Exemplary U.S. patents include U.S.
6,733,996, Methods for regulating gene expres-
sion using light, Froehlich, et al., May 11, 2004,
assigned to Dartmouth College. The full claims
are:
1. A method of regulating expression of a gene in
a host cell comprising: a) contacting a host cell
containing FAD and a gene operatively-linked
to a light-responsive regulatory sequence with a
recombinant WC-1/WC-2 transactivator which
binds FAD and the light-responsive regulatory
sequence, and b) exposing the host cell to light to
stimulate activity of the recombinant WC-1/WC-2
transactivator so that the gene operatively-linked
to the light-responsive regulatory sequence is
expressed.
2. The method of claim 1 wherein the light-re-
sponsive regulatory sequence comprises SEQ ID
NO:7.
3. An isolated light-responsive regulatory se-
quence consisting of SEQ ID NO:2, SEQ ID
NO:3, or SEQ ID NO:4.
4. A kit comprising a first container means which
contains a recombinant WC-1/WC-2 transactiva-
tor and a second container means which contains
an isolated light-responsive regulatory sequence.
Licensing information: Dartmouth is seeking an
industrial partner to further refine and market this
technology.
Further info.: Froehlich, et al. ((2002) Science
297(5582):815-819) and He, et al. ((2002) Sci-
ence 297(5582):840-843).
60
142
Minos transposon cell
transformation - insect larvae;
also eukaryotes
Minos Biosystems - Licensor, primary
Institute for Molecular Biology and Biotech-
nology/FORTH - R&D
Description: An isolated DNA sequence which
encodes a transposase protein (or a portion of a
transposase protein) is useful for a transposon-
based method for eukaryotic cell transformation,
including for producing transgenic animals and
for stably transfecting cells in vitro. Applications
include the large scale, low cost production of
high value proteins in mass reared insect larvae.
Isolated transposable elements or the isolated
DNA sequences hybridize to the DNA sequence
of Minos 1 under stringent hybridization condi-
tions.
Transposable elements or transposons are
natural genetic elements capable of jumping or
transposing from one position to another within
the genome of all species. Some transposons will
only function within a limited number of host
organisms while others are more promiscuous
and will function in different organisms including
insects, worms, fish and mammals.
The Minos-1 transposable element is a 1775
bp dispersed repetitive sequence inserted within
the transcribed spacer in one of the repeats of
Drosophila hydei. The element is characterized by
255-bp long perfect inverted repeats and the pres-
ence of two long, non-overlapping open reading
frames (ORFs) on the same strand. The longest
of the ORFs shows approximately 30% sequence
identity with TcA, but does not begin with an
ATG codon. The cloned element represents a de-
fective member of the Minos family, as is the case
with all previously sequenced Tc1-like elements,
with the possible exceptions of Tc1 and TCb1.
Use with: insect larvae; dipteran insects includ-
ing Ceratitis capitata, Anastrepha, Dacus oleae,
Cochliomyla hominivorax, Lucila cuprina, Si-
mulium, Anopheles, Aedes, and Musca domes-
tica; eukaryotes
Benefits: Minos BioSystems uses proven trans-
poson technology to create recombinant insect,
e.g., Medfly, larvae producing functional human
protein therapeutic. Production of proteins in
insect larvae, has many advantages over compa-
rable plant, animal or cell culture based systems.
The process is contained, rapidly scalable, has
low capital and product cost, high volume output
and an excellent biosafety profile. Annual yields
are potentially over 10 tons of product for each
manufacturing facility capable of producing 1 bil-
lion flies per week.
Patents: Exemplary patents include Eukaryotic
transposable element, 5,348,874 and 6,225,121,
Savakis, et al., Sept. 20, 1994, assigned to Insti-
tute for Molecular Biology and Biotechnology/
FORTH and exclusively licensed to Minos.
Licensing information: Contact Minos Biosys-
tems.
Further information: Stable Expression of Hu-
man Growth Hormone Over 50 Generations in
Transgenic Insect Larvae, Transgenic Research,
16:99-107, 2007.
143
Coconut Express cell-free
translation - plants
Plant BioScience Ltd - Licensor, primary
Description: Coconut Express is a method for
low cost, cell-free, high level, protein expres-
sion using the liquid syncytium from coconut
endosperm (a.k.a., coconut water). This method
removes the need for time-consuming cell trans-
formation steps, and has the further advantage of
performing the glycosylation modifications appro-
priate for eukaryotic proteins.
Use to make: Protein, glycoproteins, likely includ-
ing Mabs
Patents: Reportedly filed, but no relevant U.S.
or international patents or published application
61
assigned to the company retrieved.
Licensing information: Contact Plant BioSci-
ence Ltd .
Prokayotes
144
Bacterial cell expression
technology (BCE); araB
promoter - antibody fragments;
E. coli; prokaryotes
Xoma Corp. - Licensor, primary
Description: The Bacterial cell expression tech-
nology (BCE), including the araB promoter, en-
ables bacterial, particularly E. coli, expression and
secretion of antibody domains, including antibody
fragments and single-chain antibodies, directly
from bacterial cells as fully functional, properly
folded molecules.
Use with: E. coli; prokaryotes
Background: XOMA scientists were the first to
demonstrate the secretion of antibody domains
directly from bacterial cells as fully functional,
properly folded molecules.
Besides large-scale protein expression, Bac-
terial cell expression technology (BCE) is an
enabling technology used to discover and screen
recombinant proteins and antibodies for commer-
cial purposes. BCE is also an essential technol-
ogy used in multiple systems for high-throughput
screening of antibody domains. Expression of
antibodies by phage display technology, for
example, depends on the expression and secretion
of antibody domains from bacteria as properly
folded, functional proteins.
Patents: XOMA has reported that it has received
ten U.S. patents to date relating to aspects of its
BCE system, including six patents that broadly
cover the secretion of immunoglobulins from
bacteria, including antibody fragments such as
Fab and single-chain antibodies. Corresponding
foreign patents have also been granted. XOMAs
patent estate is applicable to the practice of anti-
body phage display and other antibody screening
applications.
Exemplary patents include 7,094,579, Eu-
karyotic signal sequences for prokaryotic expres-
sion, Gray, et al., Aug. 22, 2006; and 7,396,661,
Eukaryotic signal sequences for polypeptide
expression and polypeptide display libraries,
Gray, et al., July 8, 2008. These claim methods
and compositions for expressing proteins or
polypeptides in prokaryotic hosts using eukaryotic
signal sequences.
Availability: BCE reagents are available form
Takara Mirus Bio (and perhaps others) for non-
commercial use.
Licensing information: Contact Xoma regarding
commercial use and licensing.
More than 45 companies have licensed BCE.
This seemingly includes most major biopharma-
ceutical developers, and particularly most every
company involved with developing recombinant
antibody fragments, single-chain antibodies, etc.
In some or many cases, BCE may be used for an-
tibody fragment discovery and early development,
switching to eukaryotic system for commercial
manufacture.
XOMA has also provided its BCE technology
in cross-license arrangements that have allowed
XOMA to gain access to other technologies criti-
cal to its antibody discovery and development
programs. XOMA generally does not disclose roy-
alty percentages, up-front or milestone payments,
or other financial terms of specific licenses.
Products made using this tech.: Marketed
biopharmaceuticals manufactured using BCE
include:
a) Cimzia [Certolizumab pegol - CDP 870; tumor
necrosis factor monoclonal antibody Fab, re-
combinant-polyethylene glycol (PEG) polymer
conjugate] marketed for rheumatoid arthritis and
Crohns disease by the UCB Group S.A. with
contract manufacture by Lonza Biologics plc and
BioReliance Corp. Xoma reportedly receives a
royalty of <1% (with Lucentis sales approaching
$1 billon/year).
62
b) Lucentis [ranibizumab - vascular endothelial
growth factor (VEGF) monoclonal antibody frag-
ment, recombinant] manufactured and marketed
(in the U.S.) for wet age-related macular degen-
eration (AMD), with international marketing by
Novartis.
145
Subtilisin (psub) fusion protein
tag expression; Profnity eXact
expression - E. coli; bacteria;
yeasts; CHO cells
Potomac Affinity Proteins - Assignee, patent
Description: Subtilisin prodomain (psub) fu-
sion protein tags are useful for improved expres-
sion and one-step purification using S189, an
unusually stable form ot the protease enzyme
subtilisin from Bacillus species bacteria. The 75
aa psub is fused to the N-terminal of the desired
protein. The subtilisin prodomain (psub) portion
of the fusion proteins binds to chromatography
media-immobilized subtilisin with high affinity,
and once an activating agent, fluoride, is added,
the subtilisin is activated and cleaves the fusion
protein, releasing the desired protein into solution.
The ligand, S189 subtilisin, both specifically rec-
ognizes and cleaves protein from the bound tag.
Cleavage is claimed to be much more complete
and reliable than other systems.
The systems involve two basic components:
1) a target protein fused to the C-terminus of an
engineered prodomain (protagged protein); 2) a
subtilisin mutant (psub) which is virtually inactive
in the absence of fluoride as a triggering agent.
The ability to isolate the binding and process-
ing steps with a triggering mechanism creates a
processing system with a virtual on-off switch and
allows psub to be used as both the affinity ligand
and processing enzyme for affinity purification
and processing of proteins fused to the tag.
The method consists of passing a crude cell
lysate containing the prodomain-target fusion
protein onto a column of immobilized subtilisin
resulting in strong binding of the target to the
resin. Once contaminating proteins are washed
away, the subtilisin can cleave the target protein
from the bound prodomain by the addition of a
fluoride that converts the enzyme from an inactive
to active form, generating the native protein in a
pure form.
The company has engineered forms of sub-
tilisin and prodomain regions of the enzyme
that possess strong and specific affinity for one
another, and have constructed plasmid vectors
that enable the high-level expression of virtually
any target protein as a fusion with the subtilisin
prodomain.
Background: This method uses a highly modified
form of subtilisin, S189, designed to selectively
break one specific bond in a strategic location---
-and only when it is triggered to do so. Modified
forms of subtilisin, a protease enzyme from Bacil-
lus subtilis, are widely used in laundry detergents.
A number of N-terminal extensions (prodo-
mains) are large enough to fold independently and
have been shown to bind tightly to the active site
of the mature subtilisin protease. Subtilisin BPN
is an extracellular serine proteinase from Bacillus
amyloliquefaciens having a primary translation
product which is a pre-pro-protein. A 30 amino
acid pre-sequence serves as a signal peptide for
protein secretion across the membrane and is hy-
drolyzed by a signal peptidase. The extracellular
part of the maturation process involves folding of
prosubtilisin, self-processing of a 77 amino acid
sequence to produce a processed complex and
finally degradation of the prodomain to create the
275 amino acid mature subtilisin sequence. The
77 amino acid prodomain (in this expression sys-
tem, fused to the protein of interest) is removed
autocatalytically.
Use with: E. coli and other bacteria; also yeasts
and CHO cells
Benefits: In addition to substantial cost savings,
the method makes it possible to rapidly purify a
variety of different protein targets with a single
type of affinity tag.
63
Patents: Exemplary U.S. applications include
20060134740, Engineered proteases for affinity
purification and processing of fusion proteins,
Bryan, Philip N., June 22, 2006
Availability: An E. coli-based expression system
is marketed as Profinity eXact by Bio-Rad for
Licensing information: Contact Potomac Affin-
ity Proteins regarding commercial use and licens-
ing.
146
Tetracycline-induced
expression repression -
prokaryotes
IBA Gene Technologies - Licensor, primary
Max-Planck-Gesellschaft Zur Forderung Der
Wissenschaften E.V. - Assignee, patent
Description: Much like the Tetracycline (Tc; Tet)
Expression Systems widely used in eukaryotes
(see related entry), this system enables tetracy-
cline-induced repression of prokaryotic expres-
sion.
Use with: prokayotes
Patents: This tetracycline promoter based pro-
karyotic expression system is covered by U.S.
5,849,576, Tetracycline promoter for the strin-
gently regulated production of recombinant
proteins in prokaryotic cells, assigneed to Max-
Planck-Gesellschaft Zur Forderung Der Wissen-
schaften E.V.; and EP759997 (BE, DE, FR, UK).
These and other Max-Planck patents have been
exclusively licensed by IBA.
The exemplary claim (1) of 5,849,576 states,
A prokaryotic vector comprising (a) a regulatable
expression control sequence which can be re-
pressed by a tetracycline repressor protein and (b)
a DNA encoding a tetracycline repressor protein
in operative linkage with an expression control
sequence which cannot be repressed by a tetracy-
cline repressor protein.
Availability: Reagents are available for non-com-
mercial use from IBA.
Licensing information: Contact IBA regarding
Bacteria
147
Bacillus megaterium
expression - E. coli alternative
Technical University Carolo-Wilhelmina at
Brunswick - R&D
Description: Bacillus megaterium expression
has been developed, and is an alternative to E.
coli. This includes a polylinker downstream of
the promoter that facilitates versatile cloning in
pHIS1522/pHIS1525 using the BsrGI site directly
before the ATG start codon, allowing cloning
without changing the N-terminus of the protein of
interest. pHIS1525 contains a sequence encoding
the signal peptide of the B. subtilis extracellular
esterase (LipA) enabling efficient secretion of
the target protein into the culture medium, and
target genes can be inserted directly after the
signal peptidase restriction site. Due to use of
the tightly regulated xylose operon, genes can be
induced 130- to 350-fold without proteolysis. A
parallel cloning strategy of the gene of interest in
pHIS1522 and pHIS1525 enables intracellular and
extracellular protein production and a 6xHis tag
(see related entry) upstream the polylinker allows
convenient purification and detection of target
proteins (see related entry). Vectors without tag
sequences (pMM1522 & pMM1525) and vectors
providing a Step-tag (see related entry) are also
available.
Use with: Bacillus megaterium
Benefits: Among bacilli strains (e.g., E. coli), B.
megaterium has the advantage that no alkaline
proteases are present, resulting in expression of
foreign proteins without degradation. In addi-
64
tion, there are no endotoxins found in the cell
wall. Protein yields are exceptionally good, even
if inexpensive substrates are used. All B. subtilis
vectors are compatible with B. megaterium as
well. Advantages include:
stable protein expression with high yield
suited for industrial large-scale protein produc-
tion
xylose operon: tightly regulated and efficiently
inducible by xylose (up to 350-fold)
extended polylinker downstream of promoter
allows versatile cloning - additional BsrGI site en-
ables cloning without modifying the N-terminus
no indication of proteolytic instability even up
to 5 hours after induction, since alkaline proteases
(such as e.g., in B. subtilis) are not produced
endotoxins are not found in the cell wall
pHIS1525, pMM1525, pSTREP1525 &
pSTREPHIS1525 vectors provide a signal se-
quence for efficient protein secretion
easy purification and detection using 6xHis-
and/or Streptagged target proteins
were retrieved from simple searching.
Availability: Reagents and kits are available from
MoBiTec for non-commercial uses.
Licensing information: Contact the Technical
University Carolo-Wilhelmina at Brunswick.
Products made using this tech.: HIV gp120 (or
160) for diagnostics is commercially produced by
B. megaterium.
Further information: Hollmann, R., Malten, M.,
Biedendieck, R., Yang, Y., Wang, W., Jahn, D.,
Deckwer, W.-D., (2006). Bacillus megaterium as
a production system for recombinant proteins,
Chemie-Ingenieur-Technik 78, 289-294.
Production and secretion of recombinant pro-
teins using Bacillus megaterium, Ph.D. thesis,
Dr. Yang Yang, Universitt Carolo-Wilhelmina
zu Braunschweig (online at http://bib1lp1.rz.tu-
bs.de/docportal/servlets/MCRFileNodeServlet/
DocPortal_derivate_00004176/Yang_Dissertation.
pdf;jsessionid=0000prPHOl4DoqSiCQeksai5Fso?
hosts=local).
Yang, Y., Biedendieck, R., Wang, W., Gamer, M.,
Jahn, D., Malten, M., Deckwer, W.-D. (2006).

High yield recombinant penicillin G amidase pro-
duction and export into the growth medium using
Bacillus megaterium. Microb Cell Fact.5:36.
Biedendieck, R., Yang, Y., Deckwer, W.-D., Jahn,
D., Malten, M. (2007), Plasmid system for the in-
tracellular production and purification of affinity-
tagged proteins in Bacillus megaterium, Biotech
Bioeng, 96(3):525-537.
148
Bacillus subtilis, super-
oxidizing strains; Thiol-
disulfde oxidoreductases
(TDORs); Thioredoxin A (TrxA)
depletion - disulfde bridge
optimization
University of Groningen - Licensor, primary
Description: Methods are provided for control-
ling the cytoplasmic redox control of a host cell
to maximize secretion of disulfide-bridge con-
taining proteins by Bacillus subtilis, along with
methods and strains for improved Bacillus subtilis
production of secreted disulphide bond-contain-
ing proteins. The expression levels of many of
the (potential) thiol-disulfide oxidoreductases
(TDORs) of B. subtilis are modulated and/or
TDORs from heterologous Gram-positive organ-
isms are introduced into vectors.
Three combined approaches were found to be
most successful: depletion of the major cytoplas-
mic reductase TrxA (a TDOR); introduction of the
heterologous oxidase DsbA from Staphylococcus
carnosus; and addition of redox-active compounds
to the growth medium. TrxA generally refers to
the capacity of TrxA to act as a redox control pro-
tein. As has been shown with the disulphide bond-
containing E. coli PhoA as a model protein, the
combined use of these three approaches allowed
for the secretion of ~3.5 times increased amounts
of active PhoA, as compared to the parental strain
B. subtilis 168. These findings suggest that super-
65
oxidizing Bacillus strains can be engineered for
the biotechnological production of heterologous
high-value proteins containing disulfide bonds.
Use with: Bacillus subtilis
Background: Thioredoxin, thioredoxin reductase
and NADPH, which together form the thiore-
doxin system, are ubiquitous from Archea to man.
Thioredoxins with a dithiol/disulphide active site
are major cellular protein disulphide reductases.
They can serve as electron donors for enzymes
such as ribonucleotide reductases, thioredoxin
peroxidases (peroxiredoxins) and methionine
sulfoxide reductases. Thioredoxin isoforms are
present in most organisms. Mitochondria have a
separate thioredoxin system. Thioredoxin (Trx)
and glutaredoxin (Grx) operate in thiol-disulfide
reactions via two vicinal (CXYC) active site
cysteine residues, which either form a disulfide
(oxidized form) or a dithiol (reduced form). The
thioredoxin superfamily includes a growing num-
ber of proteins all having the same basic folding
as thioredoxin and glutaredoxin with the active
site located at the C-terminal end of a B-strand
and followed by an alpha-helix. Trx is reduced
to the dithiol form by NADPH and thioredoxin
reductase (together called the thioredoxin system).
Patents: Exemplary patents include
WO/2004/056987, MODULATION OF THE
THIOREDOXIN PATHWAY, PRINS, A.,W.,
et al., assigned to the University of Groningen,
concerning host cells depleted of endogenous
thioredoxin A (TrxA)-activity.
Groningen regarding commercial use and licens-
ing.
149
Bacterial artifcial chromosome
(BAC) expression; pBAC
vectors - bacteria
University of Wisconsin - R&D
Wisconsin Alumni Research Foundation
(WARF) - Licensor, primary
Description: Bacterial artificial chromosome
(BAC) expression vectors (pBAC) with high copy
numbers are useful for producing large quantities
of recombinant proteins that are often be unstable
or toxic to the host if gene expression is not
tightly regulated. BAC (or pBAC) vectors typi-
cally accommodate inserts in the range of up to
300 kilobase pairs.
Although BACs are usually used for PCR
and other non-protein expression applications,
WARF claims The inventors have constructed
a controlled BAC expression system that can
produce large amounts of genomic polypeptide
upon induction. The pBAC system includes both
a conditionally amplifiable origin of replication
(oriV) with a broad host range, and a tightly regu-
lated, inducible transcriptional promoter linked
to a gene of interest. The conditional oriV and
the inducible promoter can be activated jointly or
separately by signals in the host cell. BAC copy
number is controlled by a single inducing protein,
TrfA, which induces replication from oriV. Gene
transcription is controlled by an arabinose-induc-
ible promoter, which can be induced to produce
many copies of the inserted gene. Thus, pBAC
includes all of the elements necessary for tran-
scribing large quantities of polynucleotides in a
controlled fashion.
Bacterial artificial chromosome (BAC) vec-
tors (and plasmid forms, pBAC) are single-copy
vectors used to maintain large genomic DNA
fragments, and have generally not been used as
expression vectors.
Use with: bacteria
Expression vector with dual control of replica-
tion and transcription, Szybalski, et al., Oct. 29,
2002, assigned to Wisconsin Alumni Research
Foundation, the patent agent for the Univ. of
Wisconsin.
66
150
Caulobacter crescentus
expression; PurePro
Caulobacter Expression
System - Caulobacter bacteria
hosts
Research Corporation Technologies Inc. (RCT)
- Licensor, primary
University of British Columbia (UBC) - As-
signee, patent
Description: The Caulobacter Expression System
is a protein secretion and display system using
Caulobacter crescentus, a harmless gram-nega-
tive, non-pathogenic, aerobic bacteria which
produces and secretes a single expressed protein
that assembles on its outer surface, forming a two-
dimensional paracrystalline array or S-layer. The
S-layer represents 10% of the total cellular protein
and is secreted by a flexible Type I secretion
mechanism. Management of this bacterial expres-
sion system results in the scalable, inexpensive se-
cretion of recombinant proteins (via fusion with a
C-terminal secretion signal) or display of peptides
or proteins on the cell surface (via insertion into
the complete S-layer gene sequence). The re-
combinant protein can then be easily isolated and
cleaved from the polypeptide carrier. The system
is best for small- to medium-sized (<450 amino
acids) recombinant proteins.
Although the Univ. of British Columbia reports
that Caulobacter has not yet been scaled-up for
commercial biopharmaceutical manufacture, it is
fully developed and validated. and it is expected
to be amenable to commercial-scale manufacture.
Use with: Caulobacter bacteria
Background: Caulobacters have a two-dimen-
sional crystalline array on their surface composed
of a single secreted protein. This surface network
(the S-layer) probably helps Caulobacters fend off
pathogenic bacteria, viruses and lytic enzymes.
The secretion capabilities of Caulobacters highly
expressed crystalline surface layer protein has
been modified so that these harmless bacteria can
secrete foreign proteins, such as subunit vaccines
and many other proteins of commercial relevance.
When specific sequences are inserted into the
S-layer, they can be expressed and displayed at
high density on the bacteriums surface or the iso-
lated S-layer protein can be assembled onto lipid
vesicles or an inert surface. This technology also
offers a system that fuses the recombinant protein
to a polypeptide carrier that is exported from the
bacteria through a type I secretion system. The
recombinant protein can then be easily isolated
and cleaved from the polypeptide carrier. Because
Caulobacters spontaneously attach to surfaces and
will form a dense monolayer of stalked cells, they
may be appropriate for fixed-cell bioreactors.
Benefits: Claimed benefits include:
Production is easily scalable using simple me-
dia/fermentation systems and inexpensive batch
culture that contains no animal or plant proteins.
Caulobacter secreted proteins can be retrieved
by conventional methods to concentrate protein
from the medium, especially as there are no prote-
ases and very low levels of other cellular proteins.
Alternatively, a slow shaking method results in an
aggregation of caulobacter S-layer fusion proteins
and is readily collected by simple filtration with
up to 95% purity.
Large quantities (10% of total cell protein) of a
wide range of secreted proteins can be produced;
a typical yield is 100 mg/ml for low density batch
cultures. However, yields of 500-700 mg/ml can
be obtained.
Proteins ranging from 2-70 KDa have been
successfully expressed, without post-translational
glycosylation.
Export system is capable of tolerating a wide
variety of foreign proteins and is stable in ex-
pressing repeated polymer sequences.
The endotoxin potential of the Caulobacter
LPS is approximately 100-1000 times lower than
E. coli.
Cloning vehicle is a single high copy number
vector that replicates in Caulobacter as well as E.
coli cloning strains.
67
Patents: Patents have issued in the United States,
Australia and Singapore; patent applications are
pending in Europe, Japan, Australia, Israel, Mex-
ico and New Zealand. Exemplary U.S. patents
assigned to the Univ. of British Columbia include
6,861,245; 6,210,948; 5,976,864; and 5,500,353.
Availability: CAULOBACTER S-LAYER
PROTEIN SECRETION KITs are available from
Invitrogen for non-commercial use.
Licensing information: Contact the University
of British Columbia regarding commercia use and
licensing.
Further info.:
Nomellini et al 2007. S-layer-mediated dis-
play of the immunoglobulin G-binding domain
of streptococcal protein G on the surface of
caulobacter crescentus: development of an im-
munoactive reagent. Appl Environ Microbiol.
May;73(10):3245-53
Bingle et al. 2000. Secretion of the Caulobacter
crescentus S-layer Protein: Further localization
of the C-terminal secretion signal and its use for
secretion of recombinant proteins. J. Bacteriol.
182: 3298-3301.
Duncan et al, 2005. Evaluating a new system
for the development of particulate enzymes based
on the surface (S)-layer protein (RsaA) of Caulo-
bacter crescentus; fusion with the -1,4-glycanase
(Cex) from the cellulolytic bacterium Cellulomo-
nas fimi yields a robust, catalytically active prod-
uct. Appl. Biochem. Biotechnol. 127: 95-110.
151
Clostridium Expression
System; NTNH promoter from
Clostridium botulinum
Description: Clostridium genus bacterial gene
constructions may be expressed in a Clostridium
host. A mobilizable transfer plasmid is provided
which permits the direct transfer of the plasmid
and genes carried on it from E. coli into Clos-
tridium species. A promoter is provided for use
in Clostridium species. Also, a useful host strain
is provided which is nontoxigenic and which per-
mits high levels of expression of clostridial genes
(e.g., botulinum toxins) or other genes using the
clostridial promoter.
Use with: Clostridium bacteria
Expression system for clostridium species,
Johnson, et al., Sept/ 21, 1999, assigned to Wis-
consin Alumni Research Foundation (WARF).
The first 5 of 15 claims state:
1. A conjugative transfer plasmid comprising: an
origin of replication effective in E. coli; an origin
of replication effective in Clostridium species; a
gene for an antibiotic resistance marker; and an
origin of conjugative transfer which is capable of
modulating the conjugative transfer of the plasmid
from E. coli into a Clostridium species.
2. The conjugative transfer plasmid of claim 1
wherein the origin of conjugative transfer is RP4
oriT.
3. The conjugative transfer plasmid of claim 1
further comprising a genetic construction effective
to express an antibiotic resistance in Clostridium
botulinum.
4. The plasmid pJIR1457.
5. The plasmid pJIR1456.
Other claims include the NTNH promoter from
Clostridium botulinum and use of C. botulinum
strain LNT01. One useful component for the sys-
tem is a wide host range shuttle vector, pJIR1457,
capable of transferring DNA constructs from E.
coli to clostridial species and to C. botulinum in
particular. Examples include expression of botuli-
num toxin (comparable to non-recombinant toxin,
e.g., used in BOTOX from Allergan).
Licensing information: Contact WARF, the
licensing agent for the University of Wisconsin.
68
152
Flavobacterium heparinum
expression - glycoproteins
BioMarin Pharmaceutical Inc. - Licensor,
primary
Description: The bacterium Flavobacterium
heparinum, naturally capable of expressing gly-
coproteins, is useful as host cells for recombinant
(glyco)protein expression. Vectors for transfor-
mation of F. heparinium, pIBFX1 and pIBFX2,
are provided. A method enables expression of
homologous and heterologous genes in F. hepari-
num and the coupling of expression to secretion
to produce a biologically active polypeptide or
protein. Vectors contain a functional origin of
replication (OriC) from F. heparinum or another
functional origin of replication, replication (rep)
genes which direct the replication of the vector, a
promoter derived from a gene endogenous to the
F. heparinum host; and also usually a chromosom-
al integration sequence. Vectors may also contain
selective markers and/or a regulated promoter for
the expression of desired gene sequences.
Use with: Flavobacterium heparinum
Background: The DNA sequences and methods
for the expression of homologous and heterolo-
gous proteins in Flavobacterium heparinum, a
Gram negative, non-pathogenic soil bacterium,
had previously not been investigated, despite the
microorganism naturally producing glycosyl-
ated proteins. In addition, F. heparinum naturally
produces low levels of the glycosaminoglycan de-
grading enzymes that act upon heparin and other
glycosaminoglycans.
Patents: Exemplary patents include U.S
.6,841,375, Flavobacterium heparinum expres-
sion system, Su, H., et al., Jan. 11, 2005, as-
signed to BioMarin Pharmaceuticals Inc. Claim
1 states, . A Flavobacterium heparinum host cell
transformed with a recombinant DNA expres-
sion vector selected from the group consisting of
pIBFX1 and pIBFX2.
Licensing information: Contact BioMarin.
Products made using this tech.: BioMarin
reports, The overexpression of homologous and
heterologous cloned genes in the F. heparinum
host cell, and the suitability of F. heparinum for
the expression of recombinant proteins is demon-
strated by the expression of the enzymes, hepari-
nase I, heparinase II, heparinase III, chondroitin-
ase AC, and chondroitinase B from F. heparinum
; dhfrII and beta-galactosidase encoding genes
from E. coli.
153
Lactococcus lactis htrA-
expression - protease depletion
GTP Technology S.A. - Licensor, primary
Institut National de la Recherche Agronomique
(INRA) - Assignee, patent
Sabatier University - Assignee, patent
Description: Lactococcus lactis provides a tightly
controlled expression system, both highly induc-
ible and well repressed, combining inducible
expression and protein stability for heterologous
protein production; providing an alternative to Ba-
cillus subtilis and E. coli. In the absence of HtrA,
the extra-cellular proteolysis of all tested proteins,
in particular heterologous ones, has been com-
pletely abolished, and the yield of intact protein
significantly increased.
The technology minimally consists of 2 ele-
ments: a tightly controlled expression system,
both highly inducible and well repressed and; a L.
Lactis htrA mutant strain, devoid of any related
surface protease (serine protease of the trypsin
type) to increase heterologous proteins yield. A
L. Lactis htrA mutant strain devoid of any surface
proteases has increase heterologous proteins yield,
and allows protein purification with a very safe,
GRAS (Generally Recognized as Safe) bacteria
commonly used in foods. This lactococcal expres-
sion/secretion system uses also both PZnzitR, an
expression cassette tightly controlled by envi-
ronmental zinc, and a consensus signal peptide,
69
SPExp4, for efficient protein production. HtrA
deletion or inactivation may also be used in other
bacteria, e.g., E. coli.
Inactivation of HtrA (or complementation of
the Sec machinery with B. subtilis SecDF ac-
cessory protein) results in the increase in heter-
ologous protein yield, as evidenced by protein
yields (protein amount/biomass) comparable to
those obtained using NICE (see related entry) or
P170 expression systems under similar laboratory
conditions.
The use of bacteria as cell factories to produce
heterologous exported proteins is often limited
by extra-cellular proteolysis. A Lactococcus lactis
mutant strain was developed in which recom-
binant or heterologous exported proteins are
stable. A previously unknown membrane prote-
ase belonging to the HtrA/DegP family (HtrA)
was first identified in L. lactis subsp. lactis strain
IL1403. Inactivation of the chromosomal gene
revealed that HtrA acts as a surface housekeeping
protease by elimination of abnormal and/or mis-
folded proteins, and that HtrA is also responsible
for the maturation of natural exported proteins,
such as the L. lactis bacteriolysin, AcmA. IThese
results suggest that HtrA is the sole extra-cellular
housekeeping protease in L. lactis, in agreement
with the analysis of the complete IL1403 genome
sequence. The mutant htrA strain is an efficient
tool to improve yields of heterologous exported
proteins in L. lactis.
Use with: Lactococcus lactis; bacteria
Background: Lactococcus lactis, the model
lactic acid bacterium (LAB), is a food grade and
well-characterized Gram positive bacterium. L.
lactis can also be used as a protein producer in
fermentors. Many heterologous proteins have
already been produced in L. lactis but only few
reports allow comparing production yields for a
given protein either produced intracellularly or
secreted in the medium. Available data show that
i) secretion is preferable to cytoplasmic produc-
tion; ii) secretion enhancement (by signal peptide
and propeptide optimization) results in increased
production yield; iii) protein conformation rather
than protein size can impair secretion and thus
alter production yields; and iv) fusion of a stable
protein can stabilize labile proteins.
Patents: INRA reports patent file nFR 9816462
entitled: Gram positive bacteria deprived of HtrA
proteasic activity and their uses, extended in
Europe, In the US and in Canada; and patent file
nFR0210805 entitled: Zinc regulated prokary-
otic expression cassettes, extended in Europe, In
the US and in Canada.
Exemplary U.S. applications include
20060228377, Poquet, I., et al., Oct. 12, 2006,
with its 11th claim (the first remaining after can-
cellation of the first 10) stating, A method of pro-
ducing a fermented product, comprising: culturing
a bacterial strain with a fermentation substrate
under conditions suitable to produce a fermented
product, wherein the bacterial strain does not
express a functional HtrA.
Benefits include:
A Safe system. - GRAS (Generaly Recog-
nized As Safe) and endotoxin free system.
Easy to use. Microbial host with rapid growth and
very simple metabolism.
Easy to scale-up. Growth in stirred fermentors
without aeration : scale up to thousands of liters in
one step only.
Easy dowstream process. Efficient secretion of
proteins and limited endogen proteins : the protein
of interested is often more than 50% pure in the
supernatant.
Licensing information: Contact GTP Technology
S.A. or INRA (mercier@paris.inra.fr).
Further info.:
Morello E, Bermdez-Humarn LG, Llull D,
Sol V, Miraglio N, Langella P and Poquet I.
2008 Lactococcus lactis, an efficient cell factory
for recombinant protein production and secretion.
J. Mol. Microbiol. Biotechnol.14:48-58.
Cortez-Perez N.G., Poquet I., Oliveira M., Grat-
adoux J.-J., Madsen S.M., Miyoshi A., Corthier
G., Azevedo V. & Langella P. 2006. Construction
and characterization of a Lactococcus strain defi-
cient in intracellular ClpP and extracellular HtrA
70
proteases. Microbiology, 152: 2611-2618.
Protein secretion in Lactococcus lactis : an ef-
ficient way to increase the overall heterologous
protein production, Microb Cell Fact., 2005; 4:2,
2005 January 4.
HtrA is the unique surface housekeeping protease
in Lactococcus lactis and is required for natural
protein processing. Mol Microbiol 35, 1042-51.
Controlled production of stable heterologous pro-
teins in Lactococcus lactis. Appl Environ Micro-
biol 68, 3141-6.
Heterologous protein production and delivery
systems for Lactococcus lactis. Genet Mol Res 2,
102-11.
154
Lactose (lac) promoters;
Isopropyl-beta-galactosidase
(IPTG) induction - bacteria
Description: Lactose (lac) promoters enables
regulation of expression by the lac repressor, with
induction of expression by isopropyl-beta-galacto-
sidase (IPTG). This has been and continues to be
widely used.
Use with: for E. coli and other bacteria
Patents: No relevant U.S. patents assigned to
the inventor, Yanisch-Perron, Rutgers University,
were retrieved. First reported in 1983, any related
patents have expired.
Availability: Related reagent available from
many vendors.
Further info.:
Yanisch-Perron et al. Gene 33: 103-109 {1985}.
155
NIsin-Controlled gene
Expression (NICE); Lactococcus
lactis expression; nisA
promoter - antibiotic selection
NIZO food research B.V. - Licensor, primary
Description: Thie nisin-controlled gene expres-
sion (NICE) system with antibiotic selection is
used in the gram-positive host, Lactococcus lactis,
and provides a well-characterized and established
inducible expression system. It is considered one
of the most successful and widely used Gram-pos-
itive gene expression systems. NICE has more
than 10-years track record in academic research
and is supported by some examples of large scale
production (e.g., the production of lysostaphin)
This system exploits the autoregulatory char-
acteristics of the production of the lantibiotic nisin
by this bacterium for controlled expression of
homologous and heterologous genes that can be
triggered by addition of nisin as an inducer. The
NICE system includes several nisRK-express-
ing production hosts and a variety of expression
vectors that allow translational or transcriptional
fusion of the gene of interest to the tightly con-
trolled nisA promoter.
Use of a dual plasmid-based cassette system
or derivatives of this system has shown that the
NICE system could also be functionally imple-
mented in a wide range of alternative gram-posi-
tive hosts, including B. subtilis
The NICE system has been used successfully
for the construction of nisin-controlled autolytic
lactococcal strains and several conditional mu-
tants, as well as in industrial-scale protein and
peptide production.
Use with: Lactococcus lactis
Background: Nisin is a 34-amino acid anti-mi-
crobial peptide (lantibiotic) with various unusual
amino acids and five ring structures.
Lactic acid bacteria producing lantibiotics similar
71
to nisin A; and 5,914,248, Method for controlling
the gene expression in lactic acid bacteria, both
assigned to Stichting Nederlands Instituut Voor
Zuivelonderzoek (NIZO).
Licensing information: Contact NIZO regarding
Further info.: Autoregulation of nisin biosynthe-
sis in Lactococcus lactis by signal transduction, J
Biol Chem. 1995 Nov 10;270(45):27299-304.
Trends Biotechnol., Controlled overproduc-
tion of proteins by lactic acid bacteria, 1997
Apr;15(4):135-40.
Optimization of the Lactococcus lactis nisin-con-
trolled gene
expression system NICE for industrial appli-
cations, Microbial Cell Factories 2005, 4:16
doi:10.1186/1475-2859-4-16.
156
P170 Expression
System;Lactococcus lactis
expression
Bioneer Corp. - Licensor, primary
Description: The P170 Expression System using
the P170 promoter for inducible expression in
Gram positive lactic acid bacteria, particularly
Lactococcus lactis, with induction of expression
when a certain threshold of lactate is reached in
the culture, with the protein of interest fused to a
secretion signal facilitating secretion of the gene
product into the culture supernatant. This type of
auto-induction eliminates the addition of exog-
enous components to induce gene expression. The
extracellular export of the protein product reduces
the number of steps needed for the subsequent
downstream processing, helping to reduce the
overall cost of the process. A fermentation me-
dium has been developed that is devoid of animal-
derived components.
Despite all of these advantages, the production
of lactic acid - the primary end product of glucose
metabolism - remains a limiting effect on biomass
production in L. lactis. Lactic acid inhibits growth
even when the acid is neutralized by addition
of base to keep pH constant. As a consequence,
the yield in biomass production is below that of
other expression systems, with cell densities of
approximately 20 OD600 units. With this limited
cell density, Bioneer has produced up to 200-300
mg/L of secreted protein, a level acceptable for
some high value proteins. Methods for higher pro-
duction levels are being developed. Lactococcus
lactis P170 expression system may be combined
with Reverse electro enhanced dialysis (REED)
for lactate control.
Use with: Lactococcus lactis
Benefits: The P170 Expression System is par-
ticularly well suited for industrial scale production
of pharmaceutical proteins. Lactic acid bacteria
do not produce endotoxins, which makes them at-
tractive protein production host organisms. In ad-
dition, several lactic acid bacterial strains includ-
ing L. lactis strains do not produce extracellular
proteases and are capable of secreting peptides,
polypeptides or proteins ensuring high gene prod-
uct stability facilitating subsequent purification.
7,358,067, Fermentation method for production
of heterologous gene products in lactic acid bac-
teria, Vrang, A., et al., April 15, 2008, assinged
to Bioneer A/S. Claim 1 states, 1. A method of
producing a heterologous peptide, polypeptide
or protein in a Lactococcus lactis bacterium, the
method comprising the steps of: (i) construct-
ing a recombinant Lactococcus lactis bacterium
comprising a nucleotide sequence coding for the
heterologous peptide, polypeptide or protein and
operably linked thereto, appropriate regulatory
nucleotide sequences to control the expression of
the coding sequence, (ii) cultivating said recom-
binant bacterium under fed-batch or continuous
cultivation conditions in a chemically defined
medium, to express the nucleotide sequence, and
(iii) harvesting the recombinant bacterium or
the peptide, polypeptide or protein, wherein the
concentration of glucose is kept at a pre-selected
concentration of at least about 0.5 g/L by con-
72
trolled feeding of glucose.
Licensing information: Contact Bioneer regard-
157
Pfenex Expression;
Pseudomonas fuorescens
biovar I (MB101) - E. coli
alternative
Dowpharma, Dow Chemical Co. - Licensor,
primary
Description: Pfenex is an expression system us-
ing a natural isolate of Pseudomonas fluorescens
bacterium (strain MB101) as host cells for recom-
binant protein manufacture. Pfenex has proven
to be a robust and cost effective platform for the
production of numerous classes of therapeutic
proteins, including various types of antibody
derivatives/fragments. High cell densities in the
fermentor along with high specific protein yields
result in high target protein titers, e.g., yields over
25 grams/L have been reported. Very high cell
densities are also achievable. An extensive tool
box of unique host strains has also been devel-
oped. These strains have phenotypes selected to
impact the amount of target protein produced and
its solubility and activity. High throughput meth-
ods allow dozens of different expression strategies
and host cell combinations to be rapidly tested
in parallel. Also, efficient periplasmic secretion
of proteins has been developed. This capability
allows the formation of disulfide bonds, critical to
the production of active antibody derivatives, and
also the development of simplified downstream
processes. The technology is easily employed in
traditional E. coli and other fermentation, recov-
ery and purification settings with no need for
additional, unique equipment. Pfenex may join or
replace E. coli as a first-line expression technol-
ogy, particularly as the complexity of new thera-
peutics continues to increase.
Pfenex can achieve soluble, correctly folded
proteins with a yield improvement that can be
over ten times greater than E. coli. With Pfenex,
the vast majority (75-80%) of proteins expressed
so far have folded completely the first time - there
are very few proteins that have not and even then
they have still come out partially folded.
Pfenex is able to express single chain antibod-
ies (scFVs) at high yield and in some cases as
soluble active protein . Active scFvs have been
expressed in both the cytoplasm and periplasm. P.
fluorescens expresses cytoplasmic, soluble, active
scFv at
3g/L.
Many high performance production strains
have been developed. In most cases, Dowpharma
can identify 10-20 different forms of Pfenex for
a given protein and deliver a result within eight
to twelve weeks, while with an insoluble protein
using E. coli this process can take up to a year,
Proteins are expressed in high yields with correct
disulfide bond formation, reduced proteolytic deg-
radation and enhanced solubility/activity. Dozens
of modified host strains can be tested in parallel
to identify expression strategy/host cell combina-
tions that result in improved target protein accu-
mulation and/or stability.
Pfenex Expression Technology has been
developed with regulatory requirements in mind,
specifically eliminating the need for antibiotics
for plasmid stability, while also avoiding ani-
mal-derived products using a defined medium.
In developing Pfenex, Dowpharma claims that
it continues to invest a significant amount of
research and development in identifying high-
performing strains of Pseudomonas fluorescens,
gene mapping these strains and improving them
by application of sophisticated genomic tools.
Dowpharma can quickly and easily modify the
strains to increase the expression of a particular
customers biotherapeutic.
Protein production in P. fluorescens can be
manipulated at the genetic level so that the organ-
ism can secrete proteins into the perisplasm (the
space between the inner and outer membranes of
the cell). Secretion can be of value since simpler
methods can be used to recover the protein (me-
73
chanical cell breakage is not required). Secretion
is typically mediated by a short amino acid leader
sequence on the amino terminus of the secreted
proteins allowing transport across the inner mem-
brane. This sequence is removed at the periplas-
mic face by a signal peptidase.
Use with: Pseudomonas fluorescens
Use to make: proteins; antibody fragments
Background: Pseudomonas fluorescens is an
abundant and non-infectious component of the
microbial flora of soil, water, and plants. This
obligate aerobe is nonpathogenic (BL-1). Deriva-
tives of P. fluorescens strain MB101 have been
used for a number of years to produce a range of
protein types, including industrial enzymes, one
of which has received GRAS notification from the
FDA for a food processing application. Pseu-
domonas fluorescens biovar I MB101 has been
safely used to commercially produce a variety
of proteins at scales up to thousands of liters, for
over 15 years.
Benefits: Money is saved using Pfenex primar-
ily because of the much greater yields it is able
to produce per fermentation batch compared to
E. coli. Because protein comes out in a soluble
form, it is easier to handle and allows a clearer
path through the purification process. Pfenex
technology also fits directly into customers
existing E. coli equipment, allowing for a direct
swap out, requiring no extra investment and it is
operationally very similar to the E. coli process.
A downside of Pfenex is that like all prokaryotes,
including E. coli, Pseudomonas cant glycosylate
proteins (although various glycosylation modifi-
cations may enable this; see related entries).
Claimed benefits include:
Increased protein expression
Secretes proteins to the periplasmic space
Maintains solubility
Increased downstream yield
Scale up is easily
Resolving of protease issues
Improve activity characteristics
Lower cost of goods
Genome fully sequenced by Dow
Advanced bioinformatics and functional ge-

nomics capability
Reliable strain and manufacturing process
development
No animal-derived products or antibiotics used
in manufacturing or strain development
Can be used with standard equipment
Enabling rapid development of a robust manufac-
turing process:
Rapid development of high yielding manufac-
turing programs
Fermentor yields exceeding 15 grams per liter
20050130160, 20050186666, 20060008877,
20060040352 and 20070238153. These, respect-
ably, include coverage for high level expression
of extremozymes at a total productivity of at least
1 g/L; an improved expression system for the
production of recombinant polypeptides utilizing
auxotrophic selectable markers; use of Sec-system
secretion signal peptides in Pseudomonas; im-
proved production of human proteins, particularly
in comparison with E. coli and; expression sys-
tems for producing recombinant disulfide bond-
containing proteins using Pseudomonas.
Licensing information: Contact DowPharma
regarding commercial uses and licensing. Pfenex
expression system includes two plasmids, several
promoters and ribozyme binding sites, numerous
chaperones and disulfide bond isomerases, over
30 protease deletion mutants and over 2000 open
reading frames targeted by transposon mutations.
All these designed and prepared with the goal
of rapid and reliable development of production
strains for biopharmaceutica production. Dow-
pharma offers extensive CRO/CMO services,
including custom manufacture using Pfenex.
Cambrex (now Lonza) has partnered with
Dowpharma and offers Pfenex CRO/CMO manu-
facturing services.
DowPharma has concluded a number of
licenses. This includes a multi-product license
with Merck in 2007 for commercial manufac-
ture. Dowpharma also has formed collaborations
and/or granted licensed Pfenex to Pfizer, Abbott,

74
VGX Pharmaceuticals and Viventia Biotech, now
Insmed (a biosimilars developer).
Products made using this tech.: Biovel Life
Sciences P Ltd. (India) is using Pfenex for manu-
facture of recombinant human growth hormone
(somatropin) expected to enter the Indian market
in mid 2009.
158
Plasmids stabilization
Benzon Pharma A/S - Former inv.
NycoMed Pharma A/S - Licensor, primary
Description: Stabilized plasmids are useful for
large-scale production of recombinant proteins,
with no particular bacterial strains or mutants
needed to secure plasmid maintenance. Also, it is
not necessary to employ specific composition of
nutrient media to prevent loss of the plasmid from
the bacterial population.
Use with: bacteria, gram negative
4,760,022, Molin, et al., July 26, 1988, assigned
to Alfred Benzon A/S (now part of Nycomed).
Claim 1 states: A plasmid which replicates in
gram negative bacteria and which has been stabi-
lized by the insertion of a DNA fragment consist-
ing essentially of par region A, par region B or
both par region A and par region B of plasmid
R1, said DNA fragment having a length under 19
kilobase pairs, the plasmid comprising a gene or
genes not naturally related to the plasmid.
Licensing information: Nycomed reports that
this patent has been licensed for commercial use
by several companies. With its related patents
now 20 years old, this technology is now likely in
the public domain.
159
Pseudoalteromonas
haloplanktis TAC125
(PhTAC125) - cold expression
University of Naples, Naples Italy - Licensor,
primary
Description: A cold gene-expression system
for the recombinant secretion of heterologous
proteins in psychrophilic (cold-loving) Pseudoal-
teromonas haloplanktis TAC125 (originating from
Antarctica; reclassified from Altermonas halo-
planktis) in which the gspE gene was knocked-
out, resulting in a remarkable reduction of the
extra-cellular protease secretion, is useful for
recombinant protein expression. The system in-
cludes use of the psychrophilic beta-amylase from
P. haloplanktis TAB23 as a secretion carrier, and
allows an effective extra-cellular addressing and
export of recombinant proteins. Chimeric fusion
proteins of psychrophilic beta-amylase fused to a
desired protein are translocated into the extra-cel-
lular medium with a secretion yield always higher
than 80%. The decrease in extra-cellular proteo-
lytic activity results in a substantial improvement
in the stability of the secreted proteins.
Low expression temperatures can facilitate
correct disulphide bond formation and folding of
difficult products. The use of P. haloplanktis
TAC125 has allowed the efficient production of
some intractable proteins in soluble and active
form at temperature as low as 4C
This system also avoids the presence of an
unwanted initial N- methionine on a protein that
does not normally contain it, as with E. coli.
This extra methionine can reduce the biological
activity and stability of the product, and require
additional steps for removal.
Use with: Pseudoalteromonas haloplanktis
Background: P. haloplanktis TAC125 was iso-
lated from Antarctic sea water. Pseudoalteromona-
les generally secrete a wide range of extra-cellular
proteases.
75
Benefits: The system efficiently combines the ob-
vious advantages of extra-cellular protein target-
ing with the positive effect of low temperature on
recombinant product solubility.
retrieved from simple searches.
Naples regarding commercial use and licensing.
Further info.:
Development of an improved Pseudoalteromonas
haloplanktis TAC125 strain for
recombinant protein secretion at low tempera-
ture, Parrilli, et al., Microbial Cell Factories
2008, 7:2 (online: doi:10.1186/1475-2859-7-2)
A novel genetic system for recombinant protein
secretion in the Antarctic Pseudoalteromonas
haloplanktis TAC125, Cusano, A.M., et al.,
Microbial Cell Factories 2006, 5:40 (online:
doi:10.1186/1475-2859-5-40),
C. Mdigue, E. Krin, G. Pascal, V. Barbe, A.
Bernsel, P. N. Bertin, F. Cheung, S. Cruveiller, S.
DAmico, A. Duilio, G. Fang, G. Feller, C. Ho, S.
Mangenot, G. Marino, J. Nilsson, E. Parrilli, E.
P.C. Rocha, Z. Rouy, A. Sekowska, M. L. Tutino,
D. Vallenet, G. von Heijne, and A. Danchin. 2005.
Coping with cold: the genome of the versatile
marine Antarctica bacterium Pseudoalteromo-
nas haloplanktis TAC125. Genome research
Oct;15(10):1325-35
Cusano AM, Parrilli E, Duilio A, Sannia G,
Marino G, and Tutino ML. (2006) Secretion of
psychrophilic alpha-amylase deletion mutants in
Pseudoalteromonas haloplanktis TAC125. FEMS
Microbiol Lett. 258:67-71.
Papa R, Rippa V, Sannia G, Marino G, Duilio A:
An effective cold inducible expression system
developed in Pseudoalteromonas haloplanktis
TAC125. J Biotechnol. 2007, 127:199-210.
160
Quasi-synthetic vectors;
Synthetic gene sequences in
vectors - bacteria
Description: Genentech received a patent in 1982
(now expired) that rather broadly covered com-
bined use of DNA from mRNA reverse transcrip-
tion with synthesized nucleotide sequences for
the expression of mammalian polypeptides in
microbial cloning systems. Methods and means
enable the construction and microbial expression
of quasi-synthetic genes arising from the combi-
nation of organic synthesis and enzymatic reverse
transcription from messenger RNA sequences
incomplete from the standpoint of the desired
protein product. Expressed proteins lack bio-inac-
tivating leader sequences common in eukaryotic
expression products but problematic with regard
to microbial cleavage to yield bioactive material.
Use with: bacteria; E. coli
Patents: Related patents include 4,704,362;
5,221,619; and 4,342,832. Claim 1 of U.S.
4,342,832, Method of constructing a replicable
cloning vehicle having quasi-synthetic genes,
Goeddel, et al., August 3, 1982, states:
In the method of constructing a replicable clon-
ing vehicle capable, in a microbial organism, of
expressing a particular polypeptide of known
amino acid sequence wherein a gene coding for
the polypeptide is inserted into a cloning vehicle
and placed under the control of an expression
promoter, the improvement which comprises: (a)
obtaining by reverse transcription from messen-
ger RNA a first gene fragment for an expression
product other than said polypeptide, which frag-
ment comprises at least a portion of the coding
sequence for said polypeptide;
(b) where the first fragment comprises protein-
encoding codons for amino acid sequences other
than those contained in said polypeptide, eliminat-
ing the same while retaining at least a substantial
76
portion of said coding sequence, the resulting
fragment nevertheless coding for an expression
product other than said polypeptide; the product
of step (a) or, where required, step (b) being a
fragment encoding less than all of the amino acid
sequence of said polypeptide;
(c) providing by organic synthesis one or more
synthetic non-reverse transcript-gene fragments
encoding the remainder of the amino acid se-
quence of said polypeptide, at least one of said
fragments coding for the amino-terminal portion
of the polypeptide; and
(d) deploying the synthetic gene fragment(s) of
step (c) and that produced in step (a) or (b), as
the case may be, in a replicable cloning vehicle
in proper reading phase relative to one another
and under the control of an expression promoter;
whereby a replicable cloning vehicle capable of
expressing the amino acid sequence of said poly-
peptide is formed.
Licensing information: Patents expired. Now in
the public domain.
In 1996, Genentech Inc. filed suit in U.S.
District Court against Amgen alleging infringe-
ment of its patents, including U.S. 4,704,362;
5,221,619; and 4,342,832, concerning bacterial
vectors and methods for expressing cloned genes
in relation to two Amgen products - Neupogen
and Neulasta. The court dismissed one patents
claims in May 1998, and ruled in Oct. 2000 that
Amgen had not infringed the other three pat-
ents. In April 2002, on appeal by Genentech, the
Court of Appeals for the Federal Circuit (CAFC)
reinstated the infringement suit against Amgen.
In Aug. 2003, Amgen and Genentech settled their
G-CSF-related patent disputes, with both parties
dismissing their lawsuits, and Amgen making an
unspecified one-time payment to Genentech, but
not taking any license to Genentech patents.
Products made using this tech.: This has prob-
ably been used in connection with manufacture
of multiple products. It is used by Amgne for
manufacture of Neupogen [Filgrastim - Granu-
lokine; Granulocyte Colony Stimulating Factor,
recombinant; G-CSF] and Neulasta [Pegfilgrastim
- Neupopeg; filgrastim, recombinant pegylated;
granulocyte-colony stimulating factor, N-L-me-
thionyl-, PEG-].
161
Ralstonia eutropha expression;
Alcaligenes eutrophus
expression
Dartmouth College - Licensor, primary
Description: A Ralstonia eutropha bacterial ex-
pression system overcomes some of the shortcom-
ings of traditional Escherichia coli-based protein
expression systems, particularly the propensity
of such systems to form inclusion bodies during
high-level expression. R. eutropha has been used
at a scale of several hundred thousand liters for
the production of polyhydroxyalkanoate (PHA),
a biodegradable polymer, by ICI/Zeneca and later
Monsanto. The system permits high-cell-density
culture in a defined minimal medium, does not
require the addition of antibiotics to maintain
plasmid stability, and does not require exogenous
addition of inducers such as IPTG to initiate pro-
tein expression.
This protein expression system is based on the
industrial fermentation organism Ralstonia eutro-
pha (formerly known as Alcaligenes eutrophus),
NCIMB 40124. The system has been used to
produce high levels (>10 g/liter) of soluble, active
organophosphohydrolase, a model enzyme prone
to inclusion body formation in E. coli.
Use with: Ralstonia eutropha (bacteria)
Background: The Ralstonia eutropha genome has
been sequenced and numerous high-cell-density
processes have been reported. Unlike E. coli and
other enterobacteria which preferentially metabo-
lize hexose sugars through the Embden-Meyer-
hof-Parnas pathway, R. eutropha preferentially
uses the Entner-Douderoff pathway to metabolize
hexoses. This results in a higher NADPH/NADH
ratio, which causes a higher degree of biosyn-
thetic as opposed to respiratory reducing equiva-
77
lents. This positively affects protein formation,
since R. eutropha has the ability to balance the
reducing equivalents generated from carbon
metabolism by using the production of polyhy-
droxybutyrate (PHB) as a sink for reducing power
(i.e., NADPH) in the absence of a final electron
acceptor such as oxygen. PHB is a high-molecu-
lar-weight polymer that causes no osmotic stress
on the cell and does not adversely affect overall
bacterial growth. High-cell-density fermentations
of R. eutropha (up to 230 g/liter) in large-scale
reactors have been described previously.
Patents: No as yet published patents or applica-
tions retrieved from several simple searches.
Licensing information: Contact Dartmouth Col-
lege regarding commercial use and licensing.
Further info.: High level recombinant pro-
tein expression in Ralstonia eutropha using T7
RNA polymerase based amplification, Gavin C.
Barnard, Grant E. Henderson, Sriram Srinivasan
and Tillman U. Gerngross, Protein Expression
and Purification, Volume 38, Issue 2, December
2004, Pages 264-271. The abstract states: We
report further development of a novel recom-
binant protein expression system based on the
Gram-negative bacterium, Ralstonia eutropha. In
this study, we were able to express soluble, active,
organophosphohydrolase (OPH), a protein that is
prone to inclusion body formation in Escherichia
coli, at titers greater than 10 g/L in high cell
density fermentation. This represents a titer that is
approximately 100-fold greater than titers previ-
ously reported in E. coli for this enzyme. R. eutro-
pha strains expressing OPH were generated in two
cloning steps. First, the T7 RNA polymerase gene
was placed under the control of the strong, induc-
ible phaP promoter and integrated into the phaP
locus of R. eutropha NCIMB 40124. Second, a
single copy of the oph gene under control of the
T7 promoter was randomly integrated into the
chromosome using a transposon cloning vector.
162
Rhodospirillum rubrum
(bacterial) expression -
membrane proteins
Description: The bacterium Rhodospirillum
rubrum host/vector system uniquely suited for the
expression of membrane proteins, taking advan-
tage of an unusual attribute of the Rhodospirillum
rubrum. R. rubrum forms an extensive intracyto-
plasmic membrane (ICM), which is non-essential
for growth, in response to membrane protein
synthesis. The host in this system is a mutant R.
rubrum strain that does not synthesize its own
major membrane proteins and, thus, does not
natively form ICM. However, the strain retains
the ability to make ICM in response to protein
synthesis, including the expression of foreign
proteins. Because the strain can produce ICM in
the absence of its own membrane proteins, it can
incorporate foreign and over-expressed membrane
proteins into this extra membrane. Gene expres-
sion in this system is regulated by oxygen, allow-
ing expression to be tightly controlled by a simple
means that does not require potentially toxic or
costly chemical inducers.
Use with: Rhodospirillum rubrum (bacteria)
Use to make: proteins (membrane proteins)
Background: Rhodospirillum rubrum is a facul-
tatively phototrophic purple nonsulfur bacterium.
Under reduced oxygen concentration, this organ-
ism forms an intracytoplasmic membrane (ICM)
that is the site of the photosynthetic apparatus. R.
rubrum may grow phototrophically under anaero-
bic light conditions or by respiration under aero-
bic or anaerobic conditions in the dark. R. rubrum
is capable of growth under conditions for which
the photosynthetic apparatus is not required.
* Because over-expressed membrane proteins
can be incorporated into the ICM, the technology
78
overcomes problems (e.g. host lethality) associat-
ed with membrane protein over-expression in cur-
rent commercially available expression systems
* Expression vector contains a promoter induced
by reduced oxygen concentration, a simple, low-
cost stimulus applicable to both laboratory and
production scales
* Host is easy to work with - R. rubrum is non-
pathogenic to humans and grows on simple media
* ICM is readily separated from other cellular
material, easing membrane protein purification
* System can facilitate analysis of putative mem-
brane proteins encoded by unknown genes identi-
fied in genome sequencing efforts
* Potential for efficient, large-scale production of
non-infectious vaccines
* Expression of a native and a foreign membrane
protein have been demonstrated in this system
Patents: Exemplary patents include U.s.
6,680,179, Host/vector system for expression of
membrane proteins, Perille-Collins, et al., Jan.
20, 2004, assigned to WiSYS Technology Foun-
dation, Inc. Also, U.S. application 20040086969,
Host/vector system for expression of membrane
proteins, Mary Lynne, C.P., et al., with its ab-
stract stating, A method of expressing proteins is
disclosed. In a preferable embodiment, the meth-
od comprises placing a DNA sequence encoding a
protein or peptide and expression vector contain-
ing a regulatable promoter expressible in Rho-
dospirillum rubrum and expressing the protein
within a bacterial host, wherein the host has extra
capacity for membrane formation and wherein the
host is a member of the genus Rhodospirillum
Licensing information: Contaact WARF (ref. no.
T00006US).
163
Staphylococcus carnosus
expresion
Boehringer Ingelheim Pharma KG - Licensor,
primary
PharmedArtis GmbH - Licensor, secondary
Description: Staphylococcus carnosus is useful
as a host for production of recombinant proteins.
Methods for manufacture of heterologous re-
combinant proteins in S. carnosus host cells are
provided.
The bacterium Staphylococcus carnosus has
Generally Recognized as Safe (GRAS) status
from FDA for food uses, and is used in the food
industry for the fermentation of meat and fish.
It is free from virulence factors and proteolytic ac-
tivities in the supernatant and is capable of secret-
ing large amounts of protein. Only small amounts
of host cell-coded proteins are secreted, which
makes it easier to purify the protein produced.
Bacterial enzymes have already been recombi-
nantly produced and expressed in S. carnosus.
One crucial disadvantage of numerous bacte-
rial systems is their use of rare codons, which
is very different from the codon preference in
human genes. The presence of rare codons in E.
coli leads to delayed and reduced expression of
recombinant genes. S carnosus overcomes this
disadvantage and provides a prokaryotic expres-
sion system with improved properties.
Use with: Staphylococcus carnosus
6,821,755, Preparation of a recombinant protein
in a prokaryotic host cell, Werner, et al., No 23,
2004, assigned to Boehringer Ingelheim Inter-
national GmbH, with claim 1, A method for
preparing a heterologous recombinant protein in
a S. carnosus host cell comprising: (a) transform-
ing said host cell with one or more nucleic acids
which code for tRNA or tRNAs specific for co-
dons with an average frequency of less than 15%
in said host cell; (b) transforming said host cell
of (a) with a nucleic acid coding for the heterolo-
gous recombinant protein; and (c) expressing said
nucleic acid in (b).
Licensing information: Contact Boehringer
Ingelheim Pharma KG (BI) regarding commercial
uses and licensing, and related CMO services.
PharmedArtis GmbH has also reported offering
and/or developing S. carnosus expression technol-
79
ogy, perhap, licensed or in collaboration with BI
or on its own.
164
Subtilin Regulated Gene
Expression; SURE competency
- B. subtilis
NIZO food research B.V. - Licensor, primary
Selexis - Licensor, secondary
University of Groningen - R&D
Description: The Subtilin Regulated Expression
(SURE) system was developed analogously to the
NICE system (for Nisin Controlled Gene Expres-
sion; see related entry). SURE is based on the
regulatory module involved in cell-density-depen-
dent control of the production of subtilin (in Ba-
cillus subtilis), with the cells highly transformable
(competent). SURE provides an integration vector
for introduction of the essential sensor-regulator
couple spaRK into the amyE locus of the B. sub-
tilis chromosome, and a B. subtilis 168-derived
production host in which the spaRK genes were
functionally introduced. SURE also provides ex-
pression plasmids harboring the subtilin-inducible
wild-type spaS promoter or a mutated derivative
of this promoter, which facilitates both transcrip-
tional and translational promoter-gene fusions.
Addition of subtilin, a lantibiotic bacteriocin, to a
growing culture leads to fast and strong induction
of gene expression. Also, the SURE system adds
the possibility to strictly control gene expression,
allowing the conditional production of otherwise
toxic gene products, or complete and strict separa-
tion of growth and production phases.
Despite the attractive characteristics of the
SURE system, most experience and examples
have been generated with the analogous nisin con-
trolled gene expression system (NICE) designed
for Lactococcus lactis. The development of the
SURE system is considered less advanced com-
pared to the NICE system. But the system holds
great promises for effective protein production in
Bacillus subtilis, could potentially be implement-
ed in other bacilli, and appears compatible with
large scale production applications.
Use with: B. subtilis
Benefits: The advantages of the SURE system
largely correlate with the those of Bacillus subtilis
as a protein-production host:
(1) food grade production host,
(2) production host does not produce endotoxins,
(3) production host grows to very high cell-densi-
ties under aerobic conditions,
(4) production host is renowned for its suitability
as a host for heterologous protein secretion.
Other benefits include:
Random integration at single site
Integration position sites known to be very stable
No chromosomal rearrangement
No chromosomal breaks
No multiple integration sites
No amplification required
The SURE technology platform from Strata-
gene includes Selexis MARtech technology (see
related entry) and reduces traditional cell line
development (using DHFR) from 12 months to 5
weeks (after transfection to isolating cell clones)
without any change in the scale-up process.
Patents: The system protected by the NICE
patent held by NIZO food research (lantibiotic
regulated gene expression systems:
Exemplary patents include 5,512,468;
5,707,841 and others, Process of producing
highly transformable bacterial cells and cells
produced thereby, Greener, include claims for
Gram- bacteria expressing alpha-amylase degrad-
ing enzymes rendering the bacteria very compe-
tent to transformation.
Availability: Stratagene markets SURE cells for
Licensing information: It is possible to obtain
both cloning vectors and production host sys-
tems for the use of the SURE system in academic
research. For commercial applications a license
needs to be negotiated with NIZO food research.
It is also possible to perform research projects
evaluating the SURE system for its potential for
80
the production of a particular protein in collabo-
ration with NIZO food research. These projects
are carried-out on contract basis and under strict
confidentiality.
Selexis has been involved with SURE and of-
fers related CRO/CMO services.
Diosynth, a CMO, has been reported to have
licensed this technology.
Products made using this tech.:
Further info.: Bongers, R. S., J. W. Veening, M.
Van Wieringen, O. P. Kuipers, and M. Kleerebe-
zem. 2005. Development and characterization of
a subtilin-regulated expression system in Bacil-
lus subtilis: strict control of gene expression by
addition of subtilin. Applied and Environmental
Microbiology 71:8818-8824.
165
E. coli expression/vectors
City of Hope National Medical Center - R&D
Description: Various early E. coli expression
vectors developed in the 1970s were developed
by Genentech in collaboration with the City of
Hope National Medical Center (COH). This
included use of the E. coli. lac operon and E. coli.
tryptophan operon as promoters in E. coli vector
constructs. Drs. Riggs and Itakua, City of Hope
National Medical Center (COH), developed basic
expression vector methods and constructs for
microbial polypeptide expression for which they
received U.S. patents 5,583,013 and 5,221,619,
both entitlted Method and means for microbial
polypeptide expression, and 4,704,362, Re-
combinant cloning vehicle microbial polypeptide
expression. These patents were exclusively
licensed to Genentech, and were widely used and
licensed by Genentech, including for essentially
all early Escherichia coli (E. coli)-expressed
products.
Use with: E. coli
Patents: U.S. 4,704,362, Recombinant clon-
ing vehicle microbial polypeptide expression,
Itakura, et al., Nov. 3, 1987, abstract states, The
specification discloses: 1. Recombinant microbial
cloning vehicles comprising heterologous DNA
coding for the expression of mammalian hor-
mone (e.g., somatostatin) and other polypeptides,
including plasmids suited for the transformation
of bacterial hosts. The latter incorporate a regu-
lon homologous to the host in its untransformed
state, in reading phase with the structural gene
for the heterologous DNA; 2. Cloning vehicles
coding for the microbial expression of a protein
variously comprising (a) a polypeptide hapten
and additional protein sufficient in size to confer
immunogenicity on the product of expression,
which may find use in raising antibodies to the
hapten for assay use or in the manufacture of vac-
cines; and (b) a desired polypeptide product and
additional protein from which the desired product
may be cleaved; and 3. Methods of preparing syn-
thetic structural genes coding for the expression
of mammalian polypeptides in microbial cloning
systems.
A dispute over these patents (i.e., royalties) is
still ongoing. Under Genentechs interpretation
of its 1976 development agreement with COH,
Genentech paid COH royalty payments on sales
of products made using methods from COH and
the sales of its (sub)licensees having explicitly
(sub)licensed these patents, with COH receiving
~$300 million from Genentech over 20 years (into
later 1990s). However, COH filed a contract dis-
pute suit against Genentech in 1999. In Oct. 2001,
the first trial resulted in a hung jury. Illustrat-
ing the importance of contract language, in June
2002, a jury retrial directed Genentech to pay
~$300 million in additional royalties, including
royalties on products for which Genentech itself
and its (sub)licensees had not used the patents
or Genentech received royalties, plus punitive
damages of $200 million (total, slightly over $500
million). In Oct. 2004, Genentech lost an ap-
peal of this ruling before the California Court of
Appeal. The court cited substantial evidence of
fraud and malice by Genentech, which failed to
81
pay royalties due on its own products and those of
its various licensees during the time period cov-
ered. This included Genentech allegedly hiding
nonpayment of licensing fees due from sales of
recombinant hepatitis B virus vaccines and other
products using COH inventions over a 15-year
period. Genentech is appealing to the California
Supreme Court. The company has set aside $600
million to cover court-ordered payments, if the
company loses its appeal.
main.
166
CASCADE expression; pALEX1
plasmids - E. coli
Biomedal, S.L. - Licensor, primary
Active Mofif - Retail seller
Description: The CASCADE expression sys-
tem uses two linked regulatory circuits to increase
gene expression in E. coli, with induction by
inexpensive salicylates (e.g,. acetyl-salicylate or
Aspirin) and lactose. This results in high protein
expression levels when induced and significantly
lower basal levels than traditional gene expression
systems. The system is composed of two regula-
tory genes. The nahR gene encodes for a positive
regulator of the Psal promoter, which controls ex-
pression of xylS2, a second regulator. The xylS2
gene positively regulates expression from the Pm
promoter, which is fused to the gene of interest.
The result is gene amplification 10- to 20-times
greater than systems using only one regulator.
The first portion of the Cascade system (nahR/
Psal:xylS2) is located in the chromosome of the
TAP expression strain. The second portion of
the system can be used for either plasmid-based,
multi-copy expression or chromosomal-based,
single copy expression. The lacZ gene was cloned
into a T7 expression vector, pCAS, pCAS-lacO
and into pCHROMO-pCAS-lacO, which was
integrated into the chromosome for single-copy
expression. The Cascade vectors are induced
by addition of salicylate and lactose to the cul-
ture medium. The pCHROMO transfer vector is
included and is used to transfer the desired gene
into the chromosome of the Cascade expression
strain. The delivery system of the pCHROMO
vector is based on a mini-Tn5 transposon.
The pALEX1a, b and c plasmids are highly
improved CASCADE expression vectors sold
by Biomedal (and Active Motif) to be used in E.
coli hosts strains bearing the salicylate-inducible
nahR/Psal::xylS2 regulatory module. pALEX vec-
tors are derivatives of pCAS vectors and contain
the 3 moiety of the Streptococcus pneumoniae
lytA gene (C-LYTAG protein) between Pm pro-
moter/lac operator and the multiple cloning site.
These vectors are high-copy number, ampicilin-
selectable plasmids that incorporate a mutant Pm
promoter with even lower basal transcriptional
activity, for tightly regulated and high capacity
protein expression. pALEX vectors also carry an
enterokinase recognition sequence that enables
removal of the C-LYTAG moiety from the fusion
protein. Among other features, pALEX1a, b and
c have improved, alternative, multicloning sites
for easy and flexible cloning, in-frame with an
ATG start codon located at an optimal distance
from a highly efficient ribosome binding site, and
strong transcriptional terminator sequences and
stop codons downstream of the MCS. In addition,
the expression cassette in pALEX1a, b and c is
flanked by rare cutter NotI sites, for conve-
nient subcloning into mini Tn5 delivery vectors
and construction of genetically stable expression
strains.
Use with: E. coli
Benefits: Claimed advantages include:
a) Cascades tightly regulated expression system
provides significantly reduced background levels
compared to T7 systems.
b) Very economical for large-scale protein produc-
tion. Induction with salicylate is approximately
1,000 fold less costly than IPTG.
c) With the chromosomal based system, there is
no need for antibiotic selection during expression,
82
which further reduces the cost of protein produc-
tion.
d) Low basal expression levels of toxic proteins
prevent selection for mutations.
e) The additional lacO control in the pCAS-lacO
Kit provides even lower background expression
levels
6,803,224, Regulated expression of cloned genes
using a cascade genetic circuit, assigned to
Counsejo, Supeslor De Investigaciones Cientifi-
cas (Spanish National Research Council), exclu-
sively licensed to Biomedal
ercial use from Biomedal
Licensing information: Contact Biomedal re-
garding commercial use and licensing.
Further info.: Microbial Cell Factories 2006,
5(Suppl 1):S35 (full text available online).
167
Choline-binding fusion affnity
tags - bacteria
Description: Choline-binding domain fusion
proteins with a desired protein are useful affinity
purification tags. Protein expression and purifica-
tion systems have been successfully tested out by
different independent scientists who have found
satisfactory results where other commercial sys-
tems had failed. This fusion tag is able to interact
specifically with simple immobilized choline
chromatographic supports. Recombinant hybrid
proteins can therefore be purified in a single-step
in these resins in a rapid, low-cost procedure. Use
as a bacterial protein expression system provides
tightly regulated (high-level) and cost-effective
protein expression, and allow scaling-up the pro-
duction of purified proteins. One-step purification
from crude lysate provides > 95% pure protein.
The base resin (unspecified) is simple, inexpen-
sive, reusable and compatible with virtually all
common buffers.
Use with: bacteria
Patents: No relevant patents or published applica-
tions were retrieved by the author.
Licensing information: Contact Biomedal, S.L.
regardmg commercial use and licensing.
168
Clean Genome E. coli;
Stripped-down E. coli
Scarab Genomics LLC - Licensor, primary
(WARF) - Assignee, patent
Description: Clean Genome E. coli involves
deletion of over 15% of the E. coli K-12 genome,
optimizing the E. coli for protein expression.
Elimination of recombinogenic or mobile DNA,
toxins and virulence genes and non-essential
genes provides increased genome and plasmid sta-
bility and improved metabolic efficiency. Without
having to spend excess energy on features that
arent necessary for its existence, the E. coli can
reproduce much faster. Clean Genome E. coli
is the strain of choice for a wide spectrum of ap-
plications ranging from routine cloning to pro-
duction of biopharmaceuticals. Clean Genome
E. coli lack mobile elements and yield insertion
sequence (IS)-free plasmids. The system provides
robust cell growth in minimal media; exceptional
transformation efficiency; eliminates cloning
artifacts due to IS transposition; increased plas-
mid stability; excellent plasmid DNA yield; and is
compatible with T7-based expression vectors (see
related entry).
While much of the excitement/publicity related
to this invention concerns gene therapy plasmid
production and vaccines, this technology is very
relevant to protein manufacture.
Use with: E. coli
Benefits: CLEAN GENOME E. coli benefits
include:
83
Superior genomic stability - Removal of mo-
bile insertion sequence (IS) elements, eliminates
the danger of undesirable IS transposition into
your clone and reduces the mutation rate of the
strain itself. Genome alterations due to IS trans-
location occur surprisingly frequently and many
commonly used laboratory strains have unrecog-
nized transposition mediated genome alterations.
High transformation efficiency - Among the
highest transformation efficiency of commercially
available electrocompetent E. coli strains.
Increased success cloning difficult or unclon-
able genes - Eliminates cloning artifacts due to
IS transposition and minimizes plasmid rearrange-
ment during propagation.
Improved metabolic efficiency - Elimination
of unnecessary genes, and its products, increases
metabolic efficiency so that energy is focused
on production of the target of choice, DNA or
protein.
EP1915445, REDUCED GENOME E. COLI,
Campbell, et al., assigned to Scarab Genomics
LLC, with its abstract stating, Reduced genome
strains of E. coli MGl 655 are described. In vari-
ous embodiments, the strains have one or more
of equal or improved growth rate, transformation
efficiency, protein expression, DNA produc-
tion, DNA yield and/or DNA quality compared
to the parental strain and commercially available
strains.
Licensing information: Contact Scarab Genom-
ics LLC. Commercial entities may conduct their
evaluation on annual research license terms. Com-
mercial use of the material requires a commercial
license. Scarab Genomics is interested in broadly
licensing its technology and will work with col-
laborators on custom development of strains for
particular applications. Several companies are
evaluating the system.
The Clean Genome E. coli is the result of Dr.
Fred Blattners, et al., research at the University
of Wisconsin, Madison. Scarab Genomics has
licensed the patented reduced genome technology
from Wisconsin Alumni Research Foundation on
an exclusive, worldwide basis.

Products made using this tech.: One product in
Phase I trials, and one is in Phase II.
Further information: Psfai, G., et al., 2006.
Emergent Properties of Reduced-Genome Esch-
erichia coli. Science 19 May 2006:_Vol. 312. no.
5776, pp. 1044 - 1046 (Published online April 27
2006)
Kolisnychenko, V., et al., 2002. Engineering a
reduced Escherichia coli genome. Genome Res.
12(4): 640-7.
169
GroEL,GroES chaperones;
Chaperonins - proper folding;
universal; E. coli
censor, primary
Hoffmann-La Roche AG - Parent co.
Description: E. coli GroEL,GroES and related
chaperone proteins (small, Hsp60, heat shock
proteins and isomerases), expressed along with
the desired protein are useful to assure proper pro-
tein folding, configurion and reduce aggregation.
These chaperones are often used in E. coli. These
co-expressed folding helper proteins can be used
with essentially all prokaroytic and eukaryotic
proteins. Chaperonin GroE is a protein complex
composed of GroEL (14 subunits, 57 kDa) and
GroES (7 subunits, 10 kDa) and supports pro-
teins in forming their tertiary structure upon (or
immediately after) translation. GroE is naturally
essential to assembly (and presumably reassembly
after denaturation) of protein complexes in vivo.
Addition of GroEL and GroES proteins can also
be used to facilitate proper folding, but co-expres-
sion is likely more efficient for larger-scale use.
Use with: E, coli; bacteria; eukaryotic cells
Background: The most-investigated chaperone
is GroEL which is a member of the Hsp60 family
from E. coli. Members of the Hsp60 families are
also referred to as chaperonins and are divided
84
into two groups. GroEL and its co-chaperone
GroES and their strongly homologous relatives
from other bacteria as well as mitochondria and
chloroplasts, form group I chaperonins (Sigler et
al., 1998; Fenton and Horwich, 1994). The Hsp60
proteins from the eukaryotic cytosol from Arche-
bacteria together form the group II chaperonins
(Gutsche et al., 1999). The Hsp60 proteins in both
groups have a similar oligomeric structure. In the
case of GroEL and the other group I chaperonins,
14 GroEL subunits associate to form a cylinder
composed of two heptamer rings whereas the
heptamer ring structure in the group II chapero-
nins from Archebacteria is usually composed of
two different subunits. Non-native proteins can be
intercalated and bound in the central cavity of this
cylinder. The co-chaperone GroES also forms a
heptameric ring and binds in this form to the poles
of the GroEL cylinder. However, this binding
of GroES results in a limitation of the substrate
binding depending on its size 10-55 kDa; (Ewalt
et al., 1997). The substrate binding is regulated by
ATP-binding and hydrolysis.
Method for the expression of proteins in in vitro
translation systems with coexpression of folding
helper proteins, Buchner, Aug. 16, 2005, as-
signed to Roche Diagnostics Operations, Inc. This
concerns a method for the expression of target
proteins in in vitro translation systems, charac-
terized in that folding helper proteins are co-ex-
pressed in this system. The co-expressed folding
helper proteins are selected from one or several
of the following protein classes: Hsp70, Hsp60,
Hsp90, Hsp100 protein family, the family of small
heat shock proteins and isomerases. This patent
could well cover many, most or all Hsp70, Hsp60,
Hsp90, Hsp100 chaperones.
Availability: Takara markets GroEL and GroES
for non-commercial uses.
tein Expression Group (RPEG) regarding com-
mercial uses and licensing.
170
Methylobacterium extorquens
(bacterium) expression
Biotechnology Research Institute, National Re-
search Council of Canada (NRC- BRI) - Licen-
sor, primary
Description: Genetically modified methylotro-
phic bacteria, particularly Methylobacterium
extorquens (ATCC 55366) are useful for protein
expression. The method involves introducing an
expression vector into the methylotrophic bacte-
rium, the expression vector comprising a polynu-
cleotide sequence, coding for a peptide or protein,
or allowing for production of a product under the
control of a regulated promoter.
The growth of this bacterium in a fed-batch
fermentation system has resulted in cultiva-
tion at very high cell densities using a relatively
cheap substrate, methanol, for the production of
poly-beta-hydroxybutyrate, a polyester plastic.
The ability to produce high biomass densities in
fermenters, combined with the newly acquired
genetic information obtained from the genome
sequencing of M. extorquens [Alper (1999) Sci-
ence 283:1625-1626], makes this microorganism
a potential expression system for recombinant
peptides or proteins and for the production of
industrially important bulk chemicals.
The modified methylotrophic bacterium may
be cultured in a minimal salts medium lacking
organic sugars and containing methanol. A metal
ion may be used for regulating the expression of
the polynucleotide sequence by the promoter.
Use with: Methylobacterium extorquens (bac-
terium)
Background: Methylotrophic bacteria are a group
of prokaryotic microorganisms that can utilize
one-carbon (C1) compounds more reduced than
carbon dioxide as a source of carbon and energy.
Formaldehyde, an intermediate in the oxidation of
reduced C1 compounds, is incorporated into cells
organic molecules via the serine pathway or via
85
other pathways, and/or can be further oxidized in
a series of reactions to CO2, generating energy
in the form of reducing equivalents. Methylo-
bacterium extorquens (ATCC 55366) is a pink
pigmented facultative methylotroph isolated from
a hydrocarbon-contaminated sandy soil.
Patents: Related patent applications include U.S.
20030104527, Methylotrophic bacterium for
the production of recombinant proteins and other
products, Figueira, M.M., et al., June 5, 2003
Licensing information: Contact NRC-BRI re-
garding tech. no. 11404.
171
CANGENUS; Streptomyces
(lividans and griseus)
expression
Cangene Corp. - Licensor, primary
Apotex Holding Ltd. - Parent co.
Description: CANGENUS technology using a
vectors containing a signal peptide of protease B
from Streptomyces griseus enables Streptomyces
bacteria, e.g., S. lividans 66, and S. griseus, to
secrete properly folded, bioactive proteins into the
extracellular medium. The Streptomycetes are a
relatively well-characterized group of nonpatho-
genic filamentous bacteria that have the capacity
to secrete large amounts of protein. In particular,
Streptomyces lividans has the ability to secrete
human proteins at a commercially viable level,
thanks to relatively well established plasmid-
based expression systems, a high-biomass fer-
mentation process and a low level of endogenous
protease activity.
Use with: Streptomyces lividans; Streptomyces
griseus
Background: Streptomyces are soil bacteria
known for the diversity of antibiotics produced
by them. Several Streptomyces secondary me-
tabolite (not recombinant protein) products are in
clinical use as antibacterials (e.g. tetracyclines),
antifungals (amphotericin B), in oncology (e.g.
doxorubicin), and as immunosuppressive agents
(tacrolimus)..
Among the Streptomyces spp., S. lividans has
been extensively utilized as a potential host for
both cytoplasmic and secreted protein production
because it lacks restriction systems that are gener-
ally present in other Streptomyces and prevent the
genetic manipulation of host cells. In addition,
S. lividans also exhibits a very low endogenous
extracellular proteolytic activity.
Expression system comprising DNA encoding
the signal peptide of protease B from Streptomy-
ces griseus, Garvin, et al., May 7, 1996, assigned
to Cangene, concerning an isolated Streptomyces
griseus signal peptide which directs the secretion,
via a fused intermediate, of a protein from the cell
within which the DNA signal sequence is ex-
pressed. Use of the signal sequence in expression
constructs results in the signal peptide correctly
directing the secretion of a mature protein of de-
sired structure, particularly from prokaryotes also
expressing protein disulphide oxidoreductase, EC
5.3.4.1, e.g., Streptomyces lividans 66.
Other related Cangene U.S. patents include
6,127,144, Method for expression of proteins
in bacterial host cells; 5,856,166, Streptomy-
ces proteases and methods for improved secre-
tion of recombinantly-expressed proteins; and
5,616,485, Streptomyces proteases and improved
streptomyces strains for expression of peptides
and polypeptides.
Licensing information: Contact Cangene regard-
Products made using this tech.: Cangene is
developing multiple biosimilar/follow-on proteins
using this technology. Among these, Accretropin
[Somatropin (rDNA origin) for Injection] is mar-
keted in the U.S. and Leucotropin [Sargramostim
- Granulocyte/Macrophage Colony Stimulat-
ing Factor, recombinant; GM-CSF] is marketed
in Canada. Both are also sold in various other
countries where lack of product-specific and other
patents allow this.
Further information: Heterologous biopharmaceu-
86
tical protein expression in Streptomyces, C Bin-
nie, J Douglas Cossar, DIH Stewart - Trends in
Biotechnology, Volume 15, Issue 8, August 1997,
Pages 315-320.
172
Saccharomyces cerevisiae
expression
Delta Biotechnology Ltd. - Licensor, primary
Novozymes Delta A/S - Parent co.
Description: Delta Biotechnology Ltd., Novo-
zymes has developed a proprietary Saccharomy-
ces cerevisiae-based expression system that is
the proven choice for high yielding, animal-free
protein production. A series of Saccharomyces
cerevisiae strains has been developed withi vari-
ous desirable traits including genetic stability,
high copy number expression plasmids, protease
deficient deficient mutants and strains deficient in
the enzymes involved in o-linked glycosylation.
The system is optimized for the production of
recombinant proteins where glycosylation does
not naturally occur without impacting product
efficacy.
Novozymes has reported the first successful
secretion and purification of a fully-folded recom-
binant transferrin from yeast. This yeast technol-
ogy overcomes many of the problems associated
with prokaryotic alternatives such as improper
disulfide bond assignment, risks of protease deg-
radation and inclusion body formation. Success
in producing difficult to express proteins such as
fully functional transferrin further has shown the
utility of our strains.
Use with: Saccharomyces cerevisiae
Patents: Apparently related applications include:
a) U.S. 20070275889, Gene Expression Tech-
nique, Sleep, D., et al., Nov. 29, 2007, assigned
to Delta Biotechnology, now Novozymes, con-
cerning 2m-family plasmids including chaper-
ones. Related patents include 5,637,504, Stable
yeast 2 m vector; 5,783,423, Yeast strains con-
cerning reduction (preferably elimination) of the
HSP150 protein in a yeast used to produce desired
foreign proteins, especially human albumin, by
facilitating purification of the protein
b) U.S..4,937,193, Process for the genetic modi-
fication of yeast, assigned to Delta Biotechnol-
ogy, concerning yeast is genetically modified by
transformation with an integration vector com-
prising two copies of a homologous 2m plasmid
DNA sequence in direct orientation relative to
one another and encompassing the said DNA se-
quence, and then isolating, from the transformed
yeast obtained, cells containing the endogenous 2
m plasmid modified by incorporation of the said
DNA sequence but not containing the said vector.
The resulting yeast can be maintained under non-
selective growth conditions.
3) 6,379,924, Protein expression strains, Sleep,
April 30, 2002, concerning reduction/elimination
of Ubc4p or Ubc5p activity in fungal cells.:
Licensing information: Contact Delta Biotech-
nology Ltd.
Products made using this tech.: Delta/Novo-
zymes, besides using this technology for its CMO
clients, manufactures recombinant transferrin,
used to supplement culture media, and Recombu-
min [human albumin, recombinant; rHA] used in
place of bovine serum albumin in the formulation
of marketed biopharmaceuticals.
173
Streptomyces stationary
phase expression; SPE
system; Streptomyces vectors;
Secreted Protease Production
(SPP) System
Plant Bioscience Ltd. - Licensor, primary
Description: Streptomyces stationary phase
expression is a plasmid-based technology that
enables the expression of any gene of interest in
stationary phase cultures of Streptomyces species.
The SPE system is a plasmid cassette that, in a
87
modified industrial Streptomyces lividans strain,
is capable of producing high levels of foreign
proteins and will do so in the absence of chemical
inducers (potentially requiring removal). The sys-
tem relies upon the initiation of a tightly regulated
promoter cascade which is only switched on once
the culture has attained stationary phase. Insertion
of a gene of interest into the plasmids expression
cassette permits its strong expression in a highly
regulated and efficient manner under high bio-
mass conditions when the main growth phase has
been completed.
Also included with the SPE system is the novel
stationary phase dependant Secreted Protease Pro-
duction (SPP) System. This system is regulated by
the promoter from a secreted protease which has
been identified through microarray analysis as be-
ing only switched on during stationary phase.
Use with: Streptomyces lividans
EP1244799, METHODS AND MATERIALS
RELATING TO GENE EXPRESSION, Chater,
K.F., et al., assigned to Plant Bioscience Ltd., with
claim 1, An expression cassette for the expres-
sion of a nucleic acid of interest, the expression
cassette including: a regulatory portion or portions
including: a first regulatory element which in-
cludes either an mmyR gene encoding an MmyR
polypeptide, or an mmfR gene encoding an MmfR
polypeptide, or both; a second regulatory element
which includes an mmfL gene encoding an MmfL
polypeptide; and a promoter (the repressible pro-
moter), the function of which is repressed by the
expression product of the first regulatory element,
that repression being alleviated or removed by a
product, the production of which is conferred by
the MmfL polypeptide, and a heterologous nucleic
acid of interest, in operative association with said
promoter, wherein; said mmyR polypeptide shows
at least 80% amino acid sequence identity with
the sequence of Figure 8a (optionally excluding
the first 6 listed amino acids); said mmfR poly-
peptide shows at least 80% amino acid sequence
identity with the sequence of Figure 8e; and said
mmfL polypeptide shows at least 80% amino acid
sequence identity with the sequence of Figure
8d.
Licensing information: Contact Plant Bioscience
Ltd.
174
Streptomycetes hyper-
inducible expression; PnitA-
NitR system; Caprolactam
induction - Streptomycetes
Tsukuba Industrial Liaison and Cooperative
Research Center (ILC) - Licensor, primary
Description: The PnitA-NitR system for regula-
tory gene expression has been adapted for use in
most Streptomycetes bacteria. The PnitA-NitR
system is based on the expression mechanism
of Rhodococcus rhodochrous J1 nitrilase, which
is highly induced by an inexpensive and safe
inducer, epsilon-caprolactam (6-aminohexanoic
acid). Heterologous protein expression studies
have shown that the system allows suppressed
basal expression and hyper-inducible expression,
yielding target protein levels of as high as ~40%
of all soluble protein. The system functions in
important streptomycete strains.
In the actinomycete Rhodococcus rhodochrous
J1, which is used for the industrial production
of acrylamide and nicotinamide (13), nitrilase is
strongly induced on the addition of e-caprolac-
tam to the culture medium. The induced nitrilase,
which catalyzes the hydrolysis of nitrile to acid
and ammonia, corresponds to 35% of all soluble
protein in R. rhodochrous J1. In this strain, the
expression of nitrilase encoded by nitA is regulat-
ed by a transcriptional positive regulator protein,
NitR. NitR forms a complex with e-caprolactam,
and binds to a specific site within the nitA pro-
moter region for activation of nitrilase expression.
This simple and strong induction mechanism was
used for the construction of a hyper-inducible
nitR-based expression system for streptomycetes.
88
Use with: Streptomycetes
Benefits: The induced expression levels with the
system are much higher than those with any other
streptomycete expression systems so far known.
Regulatory gene for the expression of nitrile hy-
dratase gene, assigned to Nitto Chemical Indus-
try Co., Ltd.
Licensing information: Contact University of
Tsukuba; Kitasato Institute for Life Sciences, Ki-
tasato University and/or; Nitto Chemical Industry
Co., Ltd.
Further info.: Hyper-inducible expression
system for streptomycetes, Sachio Herai, Yoshit-
eru Hashimoto, Hiroki Higashibata, et al., Proc
Natl Acad Sci U S A. 2004, September 28, 2004;
101(39): 14031-14035,
175
ApoLife Yeast Expression;
S. cerevisiae Twin Cassette
Plasmids - antibodies in yeast
ApoLife - Licensor, primary
Apex Bioscience, Inc. - R&D
Description: The ApoLife Yeast Expression sys-
tem provides proprietary plasmids, yeast strains,
media formulations and optimized production
conditions, with the system particularly capable of
expression and secretion of fully-formed, 2-chain
human-like antibodies. Simultaneous expression
of both antibody chains on a single plasmid favors
equimolar expression (See the New Cabilly entry,
which may need to be licensed). The proteins
expressed may include, but are not limited to, re-
combinant monoclonal antibodies, single antibody
chains, chimeric antibodies, immunotoxins, etc.
ApoLife uses a twin cassette system to produce
antibodies, which employs two promoters for ac-
tivating light-chain and heavy-chain genes in the
same plasmid. These plug-n-play vectors are eas-
ily manipulated, and the resulting functional IgG
secreted from stable yeast cell lines can be used
to screen antibodies for discovery, lead selection
and commercial manufacture. The system allows
rapid strain development for small- or large-scale
manufacture.
Use with: S. cerevisiae; yeasts
ies
Benefits: Advantages of the Apolife system
include:
Rapid, cost-effective production
Reduced capital investment
Enhanced flexibility to expand production
Efficient secretion of proteins, including full-
length antibodies and immunotoxins
Proprietary yeast strains and media formulations
for high level secretion
Expression of heterologous multi-domain
proteins in yeast, Motwani, et al., March 19,
2002. The first three claims state: 1. A vector
comprising a first and second expression cassette
wherein the first expression cassette includes a
nucleic acid encoding a heterologous recombinant
fusion protein which comprises an immunological
molecule and a toxin wherein the immunologi-
cal molecule is a first chain of an immunological
molecule and the toxin is glucose oxidase, and the
second expression cassette includes a nucleic acid
encoding a heterologous recombinant fusion pro-
tein which comprises an immunological molecule
and a toxin wherein the immunological molecule
is a second chain of an immunological molecule
and the toxin is horseradish peroxidase.
2. The vector of claim 1 wherein said first chain is
heavy chain and said second chain is light chain.
3. The vector of claim 1 wherein said first chain is
light chain and said second chain is heavy chain.
Other relevant Apolife patents include
6,924,125, August 2005 and U.S. application
0191726-A1, Sept. 2005
Patent sexclusively licensed by (and pre-
sumably included with licensing from) Apolife
include 5,827,693, Expression of recombinant
hemoglobin and hemoglobin variants in yeast;
and 6,172,039, Expression of recombinant hemo-
89
globin and hemoglobin variants in yeast, both
assigned to Apex Bioscience, Inc. (Durham, NC).
Licensing information: conct ApoLife regarding
176
Arxula adeninivorans
expression - alternative yeast
PharmedArtis GmbH - Licensor, primary
Rhein Biotech Ltd. - Assignee, patent
Dynavax Technologies Corp. - Parent co.
Institut fur Pflanzengenetik und Kulturpflan-
zenforschung - R&D
Description: The Arxula adeninivorans expres-
sion system is a yeast platform claimed to pro-
vide an excellent alternative and supplement to
H. polymorpha. This yeast is dimorphic and can
grow as budding yeast or as filamentous growth.
Strong promoter elements are available and new
ones are under development. New vector elements
have been developed that result in recombinant
strains of high copy number and high productiv-
ity, substantially improving the existing A. aden-
invorans-based platform. A simple and efficient
fermentation process has been designed. The ex-
pression system includes two host strains, namely
A. adeninivorans LS3, which forms budding cells
at 30 degrees C and mycelia at >42 degrees C,
and the strain A. adeninivorans 135, which forms
mycelia at temperatures as low as 30 degrees C.
The non-pathogenic, dimorphic, haploid,
ascomycetous yeast Arxula adeninivorans is able
to assimilate and ferment a range of compounds
as its sole carbon or nitrogen source. As such, it
efficiently utilizes n-alkanes and degrades starch
and purines. Features like thermo- and osmore-
sistance as well as its unusual growth and secre-
tion behavior add to its commercial potential.
Arxula adeninivorans is able to grow at cultiva-
tion temperatures of up to 48 C, even surviving
cultivation at 55 C for some hours. It maintains
growth in presence of strong ionic, osmotic and
water stresses. In addition, it exhibits a reversible
temperature-dependent dimorphism in growing
as budding cells below 42 C and as mycelia at
higher temperatures. The morphological status is
correlated to changes of secretion characteristics.
Mycelial cultures accumulate proteins in concen-
trations 2 fold higher than budding cells including
the secreted enzymes glucoamylase and invertase.
Use with: Arxula adeninivorans
a) attractive fermentation design
b) growing IP protection
c) strong inducible (under development) and con-
stitutive promoters
d) dimorphism-dependent modification of pro-
teins
Patents: Exemplary patent applications include
WO/2008/052797, EXPRESSION VECTORS
FOR MULTIPLE GENE INTEGRATION AND
OVEREXPRESSION OF HOMOLOGOUS AND
HETEROLOGOUS PROTEINS IN YEASTS OF
THE GENUS ARXULA, Zimmer, F.-J., assigned
to Institut fur Pflanzengenetik und Kulturpflan-
zenforschung, licensed to PharmedArtis; and
EP1280893, METHOD FOR THE PRODUC-
TION OF PROTEINS IN A YEAST OF THE
GENUS ARXULA AND SUITABLE PROMOT-
ERS, Kuntz, G., coassigned to Rhein Biotech Ltd.
(subsidiary of Dynavax) and Institut fur Pflanzen-
genetik und Kulturpflanzenforschung, licensed to
PharmedArtis.
Availability: Arxula adeninivorans may be
purchased for non-commercial uses from Centra-
albureau voor Schimmelcultures (CBS 8244) and
other culture collections.
Licensing information: Contact PharmedArtis
GmbH regarding commercial use and licensing.
Further info.: FEMS Yeast Res. High-level pro-
duction and secretion of recombinant proteins by
the dimorphic yeast Arxula adeninivorans, 2002
Aug;2(3):363-9.
J Ind Microbiol Biotechnol. A wide-range inte-
grative yeast expression vector system based on
Arxula adeninivorans-derived elements, 2004
Jun;31(5):223-8. Epub 2004 Jun 3.
90
Appl Microbiol Biotechnol. 2000, Genetic trans-
formation and biotechnological application of the
yeast Arxula adeninivorans, Nov;54(5):619-24.
177
Chrysosporium lucknowense
expression; C1 Express
Hyperproducing Protein
Expression System - fungi
Dyadic International, Inc. - Licensor, primary
Description: Strains of the fungal microorganism
Chrysosporium lucknowense (C1) are useful as
host cells for both rapid discovery and expres-
sion of both eukaryotic and prokaryotic proteins.
By utilizing a single organism for all steps of the
R&D process, this integrated platform eliminates
many of the bottlenecks inherent with other tech-
nologies currently used to bring gene products to
market. Dyadics novel technology platform con-
sists of two parts: The C1 Fungal High-Through-
put Robotic Screening System and the C1 Express
Hyperproducing Protein Expression System.
C1 is the only integrated system...equipped
to go from robotic gene discovery to commer-
cial manufacturing within the same organism,
thereby significantly increasing the chance of
commercializing newly discovered gene products
while dramatically shortening the time to com-
mercialization...Because the same C1 organism is
used for gene discovery and gene expression, the
probability of successfully increasing the level of
protein expression is very high.
C1 has been in commercial use since 1996, and
a number of processes have been readily scaled
up from the laboratory to 150,000 liters (40,000
gallons). Growth conditions of the isolated
Chrysosporium strain are very versatile. It can be
cultivated at temperatures from 25 to 43C and at
acidic to alkaline pH, allowing optimal cultivation
at human physiological conditions
Use with: Chrysosporium lucknowense (fungus)
Benefits: C1 has a number of claimed advantages
over current systems including:
a) Extremely efficient protein production and
secretion. The protein yields from C1 are unprec-
edented in the industry.
b) A variety of both inducible and constitutive
promoters. C1 has a variety of genetic switches
to choose from, thus increasing the likelihood of
successful gene expression.
c) Varying host genetic backgrounds. Host strains
with varying genetic backgrounds are currently
under development to meet a variety of expres-
sion needs. Whether the goal is to produce large
amounts of protein inexpensively, or to produce
a single protein in pure form, C1 offers a host
system well suited for either target.
d) Favorable growth conditions. C1 can grow
and express proteins over a wide range of pH and
temperature. This enables the genetic expression
of diverse targets, which often require varying
conditions that are highly specific to the desired
gene or protein. Additionally, C1 is superior to
other fungal expression systems due to its ability
to produce proteins at neutral pH, allowing for
the production of proteins of physiological and
biopharmaceutical interest.
C1 Express advantages/characteristics include:
Versatile fermentation properties
Broad pH / temperature range
Large scale fermentation developed
Gene expression system developed
Gene transfer system
Expression vectors
Efficient gene expression
6,573,086, Transformation system in the field of
filamentous fungal hosts, Emalfrab, et al., June
3, 2003, with claim 1, A mutant Chrysosporium
strain comprising a nucleic acid sequence encod-
ing a polypeptide of interest, said nucleic acid
sequence being operably linked to expression-reg-
ulating region selected from the group consisting
of a promoter sequence associated with cellulase
expression, xylanase expression, or gpdA expres-
sion; and optionally a secretion signal sequence,
said mutant strain expressing said polypeptide of
91
interest at a higher level than the corresponding
non-mutant strain under the same conditions.
Licensing information: Contact Dyadic, which
offers CMO services.
178
CoMed system; Universal Yeast
vectors; pCoMed vectors;
Arxula adeninivorans-derived
TEF1 promoter
PharmedArtis GmbH - Licensor, primary
Description: The CoMed system, including
Universal Yeast vectors, is a modular yeast strain/
vector system. Particular combinations of vector
elements result in a Universal Yeast vector that
can be addressed to a range of yeast platforms,
i.e., different yeast expression systems, includ-
ing in parallel for testing. A derivative CoMe-
dImmune system is under current development,
designed for the production of antibodies and
derivatives. Co-transformation and co-assessment
can be performed with several yeasts at the same
time, including in Hansenula polymorpha, Arxula
adeninivorans and other yeasts.
Since vector systems of different yeast spe-
cies are based on different basic vectors it has
been very difficult to exchange single cassettes
between the yeast systems. To reduce this dis-
advantage, the CoMed vector system was estab-
lished containing the pCoMed basic vector for
integration of ARS, selection markers, rDNA
sequences and expression cassettes. The single
modules are flanked by identical restriction sites
and are integrated in the same location of the ba-
sic vector. In this system, various modules can be
integrated. Due to high conservation in yeasts of
the included coding regions targeting of all yeast
species is feasible. If for instance the combination
of rDNA and the ALEU2 gene is chosen, a range
of yeasts with this auxotrophy can be targeted.
The same holds for the insertion of a dominant
selection marker like the E.coli-derived hph gene
conferring resistance to hygromycin B in all yeast
species tested so far. The expression cassette is
inserted in a final step as fragments derived from
pre-constructed plasmids. A range of such cassette
elements exists harbouring a promoter of choice,
among others the A. adeninivorans-derived TEF1
promoter mentioned before, and a S. cerevisiae
PHO5 terminator separated by a multiple cloning
site. This promoter was found to be functional in
all yeast species tested so far. A selection of ARS
sequences is available that will result in either epi-
somal (S. cerevisiae) or chromosomally integrated
plasmids (Hansenula polymorpha). However in-
clusion of such a sequence may reduce the range
of addressible hosts.
By easy exchange of modules, such a vector
can be converted into a plasmid that is optimal for
an individual platform, for instance by inserting
an expression cassette with a promoter element
that elicits efficient gene expression in methy-
lotrophic yeasts only. Variants of the CoMed
basic vector for the production of antibodies and
derivatives are under development. In yet another
design, it is possible to linearize the plasmids
in a way that leaves behind all bacterial DNA
sequence
Use with: yeasts
ies
Patents: IP exclusively licensed by PharmedArtis
GmbH includes EP05/004550.9, Recombinant
protein expression system, claiming a modu-
lar universal vector system for yeasts; and
WO01/385400, Vectors and methods for produc-
ing recombinant proteins in fungi, claiming a
method of rDNA integration and rDNA target-
ing element components of the universal vector
system
Licensing information: Contact PharmedArtis
regarding commercial use and licensing.
92
179
Fungal expression systems
Description: Fungi normally secrete high concen-
trations of proteins, making them candidates for
heterologous protein expression. One can gener-
ally culture fungus under conditions that induce
expression of target gene(s), extract mRMA and
construct cDNA libraries in E. coli; use libraries
in conjunction with a selectable marker/reporter to
transform yeast or other fungal host, and culture
in medium that allows selection of desired mutant
phenotypes.
As can be seen from the related entries, a vari-
ety of yeast and other commercially-viable fungal
expression systems are available.
Fungi, unlike bacteria, generally use promoters
from same species.
180
GlycoFi technology; Next
Generation Biotherapeutics
- Pichia pastoris; yeasts;
glycosylation; antibodies
Dartmouth College - R&D
GlycoFi, Inc. - Licensor, primary
Merck & Co., Inc. - Parent co.
Description: Various mannosidase-family genes
inserted into yeast provide proteins with mamma-
lian- or human-like glycosylation patterns, includ-
ing antibodies, with unprecedented control and
uniformity. Yeasts, particularly Pichia pastoris,
or other fungi are modified to produce N-glycans
such as Man5GlcNAc2 or other structures along
human glycosylation pathways. This technol-
ogy is useful to perform the final, most complex
step of human glycosylation and can produce a
broad variety of recombinant proteins, includ-
ing antibodies, with fully human (or human-like)
glycosylation structures. Expressing a protein in
multiple GlycoFi strains results in a library of gly-
coforms (protein-glycan combinations) that can
be analyzed through in vitro and in vivo meth-
ods to identify which glycans confer the desired
properties. Strains generating mixtures of specific
glycan structures are also possible, increasing the
power of this approach. For example, Pichia pas-
toris yeast was transformed with 14 heterologous
genes so that it would secrete human erythropoie-
tin (EPO) with fully complex, terminally sialyated
N-glycans.
GlycoFi has engineered a broad selection of
yeast strains, each capable of attaching a specific
and pre-determined glycan structure to a pro-
tein of interest. Each of these strains is ready to
receive a recombinant gene construct and is able
to produce a uniformly glycosylated recombinant
protein, including fully-assembled antibodies
and Fc-fusions. This process can be scaled from
96-well microtiter plates to large-scale fermentors
while maintaining the same product homogene-
ity. Typically expression levels of secreted protein
are in the 100-2600mg/l range (up to 2.6 g/L) in a
3-day process prior to optimization.
This technology is commonly portrayed (or
hyped) as potentially eliminating the need for pro-
ducing many therapeutic glycoproteins in mam-
malian systems. As validation of this technology,
Merck & Co. acquired GlycoFi, the systems
commercial developers, for over $400 million,
with this technology probably the companies
primary asset (vs. some early-stage therapeutics).
Next Generation Biotherapeutics was GlycoFis
pre-Merck acquisition trademark for this technol-
ogy.
Use with: Pichia pastoris; yeasts; fungi
ies
Benefits: Obviously, being able to manufacture
complex glycoproteins, including antibodies,
in yeast rather than mammalian systems saves
considerable money and time. Other advantages
of GlycoFis strains include demonstrated genetic
stability, robust scale-up within standard cGMP
infrastructures, and thoroughly documented strain
history, including a sequenced genome. GlycoFi
strains ensure rapid transition from lab bench
93
to clinical phase development with typical turn
around time of 3-6 months from gene to gram
quantities of secreted protein.
Patents: GlycoFi, now part of Merck & Co.,
owns or controls over 60 issued and pending
patents in the U.S. and abroad relating to glyco-
sylation engineering and the production of human
glycoproteins with proper glycosylation in yeast
and fungi. GlycoFi has also in-licensed comple-
mentary protein expression technology.
Methods for producing modified glycoproteins,
Gerngross, T.U., et al., April 18, 2006, with its
abstract stating [truncated], cell lines having
genetically modified glycosylation pathways that
allow them to carry out a sequence of enzymatic
reactions, which mimic the processing of glyco-
proteins in humans, have been developed. Re-
combinant proteins expressed in these engineered
hosts yield glycoproteins more similar, if not sub-
stantially identical, to their human counterparts.
The lower eukaryotes, which ordinarily produce
high-mannose containing N-glycans, including
unicellular and multicellular fungi are modified
to produce N-glycans such as Man5GlcNAc2
or other structures along human glycosylation
pathways. This is achieved using a combination of
engineering and/or selection of strains which: do
not express certain enzymes which create the un-
desirable complex structures characteristic of the
fungal glycoproteins, which express exogenous
enzymes selected either to have optimal activity
under the conditions present in the fungi where
activity is desired, or...
Availability:
Licensing information: GlycoFi strains are
available for out-licensing on a non-exclusive
basis. Terms of a strain license include an upfront
development and licensing fee, milestone pay-
ments, annual maintenance fees and royalties on
product sales. Specific economic terms are protein
and indication dependent.
GlycoFi offers CRO/CMO services, and works
collaboratively with its customers to identify the
most appropriate glycan structure(s) for a given
application.
181
Hansenula expression (yeast)
Rhein Biotech Ltd. - Licensor, primary
Description: The Hansenula Expression Platform,
including vectors and control sequences, has been
developed Rhein Biotech Ltd. and is being used
for manufacture of a marketed hepatitis B virus
surface antigen (HbsAg) vaccine. Hansenula (not
neccessarily this specific technology) holds the
record in yeast derived-protein production, with
a productivity of more than 13 g/L in phytase
production.
Use with: Hansenula yeasts
Patents: Exemplary patents include Recombi-
nant production of proteins in yeast, 5,741,674,
Schweden, et al., April 21, 1998, assigned to
Rhein Biotech. The claims of this patent state:
1. A process for the recombinant production of
proteins which are heterologous in the yeast Han-
senula, which comprises transforming Hansenula
with an expression cassette which comprises the
following structural elements encoded: where
L is a leader sequence, A is an adaptor with the
sequence SEQ ID NO:1, P is a processing signal
and GEN is a structural gene for the required
protein; growing Hansenula in a suitable growth
medium; and recovering said protein; wherein
said protein is correctly processed.
2. A process as defined in claim 1, wherein
the leader sequence of the glucoamylase from
Schwanniomyces occidentalis is used as leader
sequence L.
3. A process as defined in claim 1, wherein the
peptide sequence Lys-Arg is used one or more
times as processing signal P.
Another key patent is 5,389,525, DNA-mole-
cules coding for FMDH control regions and struc-
tured gene for a protein having FMDH- activity
and their uses. This has been cited as forming
the basis for HEPLISAV, a recombinant hepatitis
B vaccine that was developed at Rhein Biotech
94
GmbH in the early 1990s. This patents abstracts
states, The invention relates to DNA-molecules
comprising DNA-sequences encoding control
regions and the structural gene for a protein
having formate dehydrogenase (FMDH) activ-
ity. Said DNA-molecules may be combined with
DNA-sequences encoding foreign genes so as to
bring these genes under the stringent control of
the regulation of the FMDH regulatory sequences
and/or may be combined to DNA-sequences cod-
ing for secretory signals. The invention further
relates to recombinant vectors containing said
DNA-molecules and micro-organisms contain-
ing said vectors or DNA-molecules. Furthermore,
the invention relates to a process for producing a
useful substance by producing this substance by
culturing said micro-organisms and recovering the
substance.
U.S. 5672487, Recombinant production of
proteins in yeast, assigned to Rhein Biotech and
BASF, describes the use of a pr-pro-sequence
derived from a Carcinus maenas (v) protein that
is compatible to the commonly used MFa1 leader.
Fusions with this pre-pro fragment facilitate yeast,
including Hensensula, secretion of heterologous
proteins.
Licensing information: Contact Rhein Biotech.
Products made using this tech.: Rhein/Dy-
navax manufactures and markets (in countries not
subject to basic hepatitis B virus surface antigen
patents held by Chiron/Novartis) HEPLISAV
hepatitis B virus vaccine.
182
Hansenula polymorpha (yeast)
expression - E. coli alternative
Artes Biotechnology GmbH - Licensor, pri-
mary
Bio-Technical Resources (BTR) - Licensor,
primary
Description: Hansenula polymorpha offers a
cost-effective and commercially-viable expres-
sion system. Hansenula polymorpha is often an
alternative to E. coli for producing therapeutic
proteins or technical enzymes. Recombinant Han-
senula strains harbor many copies of the heterolo-
gous DNA, up to 150 copies per cell., resulting in
highly efficient productivity. Up to four different
genes/proteins may be expressed. Yields of up to
13.5 g/L have been obtained, with low levels of
impurities, high product stability and low protease
activity. Typical yields range from 0.5 to several
g/L. H. polymorpha is a very efficient secretor.
H. polymorpha can use alcohol as its sole carbon
source. The system offers high batch-to-batch
reproducibility.
The combined effect of promoter efficiency
and gene dosage results in Hansenula expression
yields superior to other microbial systems. Han-
senula polymorpha is considered an ideal system
for stable high gene dosage dependent expression
and for co-expression of multiple genes, with
high stability of strains (800 generations tested).
Simple, inexpensive media without methanol can
be used, with short fermentation times, e.g., 70-
120 hours.
Use with: Hansenula polymorpha
Background: Artes Biotechnology GmbH has
20+ year experience with contract R&D and
manufacture using Hensensula.
Patents: The Hansenula technology platform is
protected by a number of patents covering pro-
moters, signal sequences, chaperones as well as
auxotrophic markers and integration sites. As re-
ported by Artes, related patents licensed, some or
many presumably with exclusivity, or developed
by the company include:
a) MOX promoter: EP 173 378, US 5,240,838
(licensed from Rhein Biotech)
b) FMD promoter: EP 299 108, US 5,389,525
(licensed from Rhein Biotech)
c) TPS promoter: WO 00/65071 (licensed from
Rhein Biotech)
d) rDNA integration: WO 01/38510 (licensed
from Rhein Biotech)
e) ARO7 marker: WO 00/65071 (licensed from
Rhein Biotech)
95
f) CMK secretion: WO 00/68400 (licensed from
Rhein Biotech)
g) GAM-KEX2 leader: EP 716 705, US 5,741,674
(licensed from Rhein Biotech/BASF)
h) CHH-KEX2 leader: EP 725 822, US 5,672,487
(licensed from Rhein Biotech/BASF)
i) Glucose Fermentation Process: EP 0911416
(licensed from Hoffmann-La Roche)
j) Xylose reductase / Xylitol dehydrogenase (pat-
ented by ARTES)
k) Cell permeabilization: WO 01/38544 [licensed
from the Institute of Plant Genetics and Crop
Plant Research (IPK)].
Licensing information: Bio-Technical Resources
(BTR) is the exclusive representative for Artes
Biotechnology in North America. Otherwise, con-
tact the developer, Artes Biotechnology GmbH
Licensees include Proteo Biotech AG (2004).
183
Kluyveromyces lactis
expression; pKLAC1;
Acetamidase/Acetamide
selection; K. lactis GG799 -
yeast
DSM Biologics - Licensor, primary
New England Biolabs - Licensor, primary
Description: The yeast Kluyveromyces lactis is
useful as host cells for high yield recombinant
protein expression. K. lactis technology offers
high yield protein expression; rapid high cell den-
sity growth; includes glycosylation; can clone and
express genes toxic to E. coli; competent K. lactis
cells are available; no expensive antibiotics or
methanol required; and easy-to-use protocols for
those inexperienced with yeast systems. Acetami-
dase (amdS) expressed from the pKLAC1 vector
permits transformed cells to utilize acetamide as
a sole nitrogen source in defined medium, al-
lowing for simple selection of transformed cells.
Acetamide selection also promotes formation of
cells containing multiple integrations of pKLAC1
which results in higher yields of protein. The
technology includes the K. lactis GG799 strain, an
industrial isolate that has no auxotrophies, rapidly
grows to high cell density, and efficiently secretes
heterologous proteins; pKLAC1 Vector; and
pKLAC1-malE Control Plasmid.
Proteins may be produced intracellularly or be
secreted using the integrative expression vector
pKLAC1. To achieve protein secretion, a gene
of interest is cloned downstream of the K. lactis
alpha-mating factor secretion domain, which is
eventually processed in the Golgi resulting in
secretion of the desired protein.
Use with: Kluyveromyces lactis
Benefits: The K. lactis expression system offers
several advantages over other yeast and bacte-
rial protein expression systems. K. lactis has
been used to produce proteins at industrial scale
in the food industry for over a decade due to its
ability to rapidly achieve high culture densities
and abundantly produce recombinant proteins.
Yeast expression is driven by a variant of the
strong LAC4 promoter that has been modified
to lack background expression in E. coli. There-
fore, genes toxic to E. coli can be cloned into
pKLAC1 in bacteria prior to their expression in
yeast. Highly competent K. lactis cells make the
technology easy-to-use for those not accustomed
to working with yeast. Their high transformation
efficiency makes the system suitable for methods
that require large numbers of transformants, for
example, expression cloning using cDNA librar-
ies. Proteins expressed in K. lactis have access to
eukaryotic protein folding and glycosylation ma-
chinery that E. coli cells do not possess, making
it an important alternative to bacterial expression
systems.
Method for construction and use of Kluyvero-
myces lactis promoter variants in K. lactis that
substantially lack E. coli transcriptional capabil-
ity, Taron, C., et al., June 24, 2008, assigned to
New England Biolabs, Inc. The abstract states,
Methods and compositions are provided relating
96
to production of recombinant protein in yeast. A
modified PLAC4 is described where one or more
mutations may be introduced into the Pribnow
box-like sequences in the promoter. The modi-
fied promoter when placed upstream of a target
gene in a vector causes a significant reduction of
target gene expression in transformed bacteria but
produces efficient expression of the target gene in
yeast.
Availability: Related reagents are marketed or
non-commercial uses by New England Biolabs,
Inc.
Licensing information: A license to use this
system for manufacture of clinical grade material
or commercial purposes is available from New
England Biolabs, Inc. or DSM Biologics.
Further info.:
Kluyveromyces lactis LAC4 Promoter Variants
That Lack Function in Bacteria but Retain Full
Function in K. lactis, Paul A. Colussi and Chris-
topher H. Taron, Appl Environ Microbiol. 2005
November; 71(11): 7092-7098 (doi: 10.1128/
AEM.71.11.7092-7098.2005).
184
NeuBIOS expression;
Neurospora crassa expression;
NeuKARYON - flamentous
fungi; glycosylation; Cabilly-
Boss workaround
Neugenesis Corp - Licensor, primary
University of Hawaii - Assignee, patent
Description: NeuBIOS is a Neurospora crassa
(filamentous fungus)-based expression system
with the ability to fold and trim complex mam-
malian proteins correctly, to glycosylate proteins,
and to secrete them into inexpensive liquid media,
making downstream processing and purification
simple and inexpensive. The fungus-based system
also doesnt express toxins and the system has
no known viruses. Wild strains of Neurospora
typically produce more than a gram per liter of
secreted proteins when grown on a liquid minimal
medium for several days.
Neugenesis is developing a host cell line
capable of producing and secreting recombinant
products at levels measured in grams per liter and
at initial purities of more than 90%. Neugenesis
aims to use NeuBIOS to produce high value re-
combinant mammalian proteins, including human
biopharmaceuticals and proteins for research and
development use. To date, Neugenesis has been
able to produce cytokines, monoclonal antibodies,
a thrombolytic protein, and insulin-like proteins.
The NeuKARYON system for antibody manu-
facture, a version of Neugenesis CombiKARY-
ON technology, uses two individually transformed
strains of N. crassa, one producing high levels
of the light subunit and the other producing high
levels of the heavy subunit, which are fused to
form a multicellular, multinucleate strain (hetero-
karyon), which produces both subunits and pro-
cesses them into the intact monoclonal antibodies.
Variants of each subunit gene are generated by
standard techniques and then are inserted into host
cells to produce libraries of strains. NeuKARYON
has the ability to select for equal production of
each subunit before the cells are fused.
For recombinant monoclonal antibodies
manufacture, Neugenesis claims that using MAb
expression technology embodied in US Patent
5,643,745, we can offer the only known alterna-
tive technology to that owned by Genentech under
US Patent 6,331,415. Thus, this is proposed as
an alternative to the need to license the Cabilly-
Boss patent from Genentech (see related entry).
The expression strains are exceptionally stable
and are designed to secrete these proteins into in-
expensive liquid media, which contains no animal
serum or other potential product contaminants.
This makes downstream processing and purifica-
tion extraordinarily simple and inexpensive, re-
sulting in a low overall cost structure. The expres-
sion system has no known viruses, and produces
no toxins or harmful secondary metabolites.
In July 2007, Neugenesis received a multi-
million dollar Phase I contract from the Defense
97
Advanced Research Projects Agency (DARPA)
to develop expression technologies to acceler-
ate recombinant protein production for the rapid
manufacturing of vaccines and biopharmaceuti-
cals. The goal is to develop a commercially viable
system that can produce up to 3 million doses of a
vaccine or a monoclonal antibody therapeutic in a
12 week period.
Use with: Neurospora crassa (filamentous
fungus)
ies
Background: The conventional recombinant ap-
proach to the production and assembly of hetero-
meric proteins, such as monoclonal antibodies,
has been to transform the genes for both subunits
into a single host cell (e.g., see Cabilly-Boss en-
try). Transformed host cells are then selected for
production of the protein. Neugenesis expression
technology enables a new alternative. Two indi-
vidually transformed strains, one producing high
levels of the first subunit and one producing high
levels of the second subunit, are fused to form a
multicellular, multinucleate strain (heterokaryon),
which produces both subunits, and processes
them into the intact molecule. Important features
of Neugenesis protein expression technology
include the ability to fold and trim complex mam-
malian proteins correctly, resulting in bioactivity.
The systems can add sugar residues to secreted
proteins, thus producing N- and O-linked glyco-
proteins.
Neugenesis technology can produce small and
large proteins, including heteromeric proteins
such as human monoclonal antibodies, certain
hormones, and cell surface receptors. Relevant
compounds produced in Neugenesis systems
include: a human cytokine (rhM-CSF), a hu-
man blood protein (HSA), a mammalian food
processing enzyme (bovine chymosin), a mam-
malian hormone (porcine relaxin), a plant protein
(zeamatin), and mammalian plasminogen activa-
tor (DSPA).
Benefits: NeuBIOS is readily scalable and manu-
facturing requires significantly less infrastruc-
ture than traditional methods. Large amounts of
proteins, including glycosylated proteins, can be
rapidly manufactured. Compared to established
mammalian cell culture production systems,
fungal expression systems hold the potential for
significant cost savings. This cost reduction will
come from reduced capital investments, yield
benefits and lower operating costs in production.
Fungal fermentation holds the potential for yields
exceeding several grams/liter during a 10 day
batch cycle. Development times are significantly
reduced for fungal production hosts (typically
6-12 months compared with 18-24 months for
mammalian hosts).
For antibody producers, in particular, and bio-
pharmaceutical companies, in general, the attrac-
tiveness this technology provides
Proprietary intellectual property (IP) position
is free from dominating/competing IP
Reduced capital investments and production
cost through use of proven and efficient fungal
production systems
Scalability and flexibility - A stable and
robust production process that offers flexibility
in optimization and development of production
processes.
Patents: Exemplary patents assigned to the
University of Hawaii and exclusively licensed
to Neugenesis include 5,834,191 (glucoamylase
promoters and vectors); 5,643,745 (concerning
antibodies expression); and 5,695,965 (Neuros-
pora expression system)
Licensing information: Contact Neugenesis
regarding commercial use, licensing, custom ser-
vices and collaborations.
Neugenesis is working with Novozymes in
expression systems development. Novozymes
has further initiated a research collaboration with
a university partner for evaluation of technology
that will allow for in-vivo modification of fungal
glycosylation patterns, addressing questions con-
cerning post-translation modifications. The part-
nership is currently looking for additional partners
who have an interest in securing access to future
MAb manufacturing capacity and technology.

98
Products made using this tech.: Neugenesis
is developing its first commercial product - re-
combinant Neurospora crassa-expressed human
macrophage colony stimulating factor (M-CSF),
a biosimilar version of Leukine from Amgen.
Neugenesis expects to manufacture biogeneric/bi-
osimilar versions of other products.
Neugenesis reports working with multiple
biopharmaceutical companies on product manu-
facture.
In April 2009, Sepragen Corp. and Neugenesis
Corporation jointly announced a collaboration
to develop a means of high throughput, low cost
production of monoclonal antibodies and vaccines
for pandemic influenza.
185
Neurospora expression; cotA
promoter - fungi
Genencor International - Licensor, primary
Danisco A/S - Parent co.
Description: Genencor has developed a Neuros-
pora (filamentous fungus) expression system, in-
cluding the cotA promoter. Presumably, Genencor
uses this system in-house, e.g., for the enzymes it
manufactures.
Use with: Neurospora; other filamentous fungi
Patents: Exemplary U.S. patents assigned to
Genencor include 5,695,965 and 6,171,817. U.S.
6,171,817, Berka, et al., Jan. 9, 2001, has abstract
stating, Novel vectors are disclosed for express-
ing and secreting heterologous polypeptides
from filamentous fungi. Such vectors are used
in novel processes to express and secrete such
heterologous polypeptides. The vectors used for
transforming a filamentous fungus to express and
secrete a heterologous polypeptide include a DNA
sequence encoding a heterologous polypeptide
and a DNA sequence encoding a signal sequence
which is functional in a secretory system in a
given filamentous fungus and which is operably
linked to the sequence encoding the heterologous
polypeptide. Such signal sequences may be the
signal sequence normally associated with the
heterologous polypeptides or may be derived from
other sources. The vector may also contain DNA
sequences encoding a promoter sequence which is
functionally recognized by the filamentous fungus
and which is operably linked to the DNA se-
quence encoding the signal sequence. Preferably
functional polyadenylation sequences are opera-
bly linked to the 3 terminus of the DNA sequence
encoding the heterologous polypeptides. Each of
the above described vectors are used in novel pro-
cesses to transform a filamentous fungus wherein
the DNA sequences encoding the signal sequence
and heterologous polypeptide are expressed. The
thus synthesized polypeptide is thereafter secreted
from the filamentous fungus..
Licensing information: Contact Genencor.
186
Ophiostoma expression -
ascomycetes fungi
Macquarie University - Licensor, primary
University of Waikato-New Zealand - R&D
Description: The dimorphic ascomycetes fungus
Ophiostoma floccosum (and also Ophiostoma
piliferum) is useful for recombinant protein
expression. Due to the capability of these Ophios-
toma species to be fermented, they are particularly
suitable as hosts capable of excreting extracellular
recombinant proteins. A series of expression vec-
tors containing amylase regulatory sequences and
partial amyl gene were constructed. Extracellular
xylanases and lipases from these species have
post translational modifications. Promoters and
various transcriptional elements such as secre-
tion signals have been elucidated from selected
proteins using chromosome walking methods.
Use with: Ophiostoma floccosum (fungus)
Background: Ophiostoma spp. belong to the
Ophiostomataceae family, a large group of asco-
mycetes, which are the most frequent blue stain
99
fungi isolated from stained wood. Some albino
variants of O. floccosum and O. piliferum have
been used as biological control agents to prevent
blue staining.
Benefits: This whole organism approach plus the
added capability of extracellular protein secretion
makes Ophiostoma spp. attractive for industrial
applications. In addition, Ophiostoma produces
only a small range of abundantly secreted proteins
in liquid culture, which can facilitate downstream
purification of a recombinant product. Genes
encoding efficiently secreted proteins provide a
potential souce for strong promoters for high-level
gene expression. These characteristics provide
an excellent starting point for the development of
novel expression systems.
WO/2005/028637, FUNGAL HOSTS
FOR EXPRESSION OF RECOMBINANT
PRODUCT,NEVALAINEN, H., et al., assigned
to MACQUARIE UNIVERSITY. The abstract
states, An isolated mutant Ophiostoma species
having enhanced protein excretion capability as
compared with its parent strain cultured under
similar conditions.
Licensing information: Contact Macquarie
University.
Further info.: Developing Ophiostoma flocco-
sum as a novel expression system (thesis), Wu,
Caiyan, Macquarie University. Dept. of Chemistry
and Biomolecular Sciences (full text online).
187
Yeast expression systems
Washington Research Foundation (WRF) - Li-
censor, primary
Genentech, Inc. - Assignee, patent
University of Washington - Assignee, patent
Description: A variety of yeast-based expression
systems offer utilization of cheaper substrates,
increased protein secretion efficiency and/or
allow manipulation of post-translational events,
e.g,. glycosylation. A gene for a desired protein,
usually cDNA to avoid problems from introns, is
often flanked by a yeast promoter and transcrip-
tional terminator. Human proteins expressed in
yeast are generally correctly folded and disulfide
bridged, but have inherently different glycosyl-
ation. A variety of technologies address this non-
mammallian/non-human glycosylation.
Yeasts require a signal sequence, usually
a short (e.g, 15-30 aa, such as poly-arginine)
peptide sequence, for protein secretion. Cleavage
of the signal sequences is carried out upon entry
of expressed protein into the cells endoplasmic
reticulum (the start of the secretory process in
eukaryotes). The cleavage site is not sequence-
specific, but is controlled by size and charge
distribution of the signal sequence (usually a
positively-charged N-terminal region with a hy-
drophobic core). It is common to include a short
pro-sequence after the signal sequence and
before the N-terminus of the target protein, e.g., to
aid secretion from cell. The endopeptidase KEX2
is the main pro-sequence used in S. cerevisiae.
Most S. cerevisiea and other yeast plasmids are
shuttle vectors that can be propagated in E. coli or
S. cerevisiea. Transformed DNA integrates into
yeast genome via homologous recombination, al-
lowing genes to be knocked out and replaced. The
complete S. cerevisiae genome sequence has been
available since 1996.
Excessive or hyperglycosylation (e.g., mannose
chains over 40 units) can occur with yeasts with
N-linked carbohydrate chains often much longer
and containing high levels of mannose (uncharac-
teristic of human glycoproteins). As can be seen
from the related entries, there are a number of
technologies available to better control this.
Yeasts can express intracellular accumulated
proteins at titers in the g/L range. Secreted pro-
teins levels are generally lower, e.g., up to 100
mg/L or more. Many yeasts are classed by FDA as
GRAS (generally regarded as safe) food additives,
and present no human pathogenic or biosafety
hazards.
Use with: yeasts
100
Use to make: proteins; glycoproteins Benefits:
Saccharomyces cerevisiae, the most commonly
used yeast and most commonly used for commer-
cial recombinant glycoprotein protein manufac-
ture, is classed by FDA (as a food ingredient) as
a generally recognized as safe (GRAS) organism.
S. cerevisiae and other yeasts employ stable high
copy number expression plasmids. Recombinant
heterologous proteins can be produced at high
levels, e.g., up to 30% of total protein.
Secretion of proteins by yeasts into media
reduces downstream processing costs (e.g., com-
pared to E. coli inclusion bodies). Additionally,
yeast expression generally involves:
No serum/animal products (defined medium)
No mammalian prions or viruses
No endotoxins
No oncoproteins
Availability: Yeast expression system compo-
nents and reagents are available for non-commer-
cial use from many vendors.
Patents: Genentech and the researchers at the
Univ. of California and Univ. of Washington
were among the first to develop commercially-vi-
able yeast, particularly S. cerevisiae, expresion
systems. The Washington Research Foundation
(WRF), acting for the Univ. of California and
Univ. of Washington, was involved in a two-de-
cade patent interference (first to invent) dispute
with Genentech over yeast expression systems,
first used commercially for manufacture of re-
combinant hepatitis B virus S-protein (HBsAg;
hepatiitis B virus vaccines). The Court of Appeals
for the Federal Circuit (CAFC), the highest court
for most patent disputes, in April 2001 supported
lower court rulings that the Univ. of California
(Rutter, Valenzuela, Goodman, et al.), along with
the Univ. of Washington, was the first to invent
yeast-expression for HBsAg useful for vaccines
in 1979, beating Genentech (Hitzeman, Levinson,
et al.) by just three weeks. Related royalties re-
ceived by the Univ. of California from Genentech
totalled over $225 million in 2001, and royalties
received by the Univ. of Washington totalled over
$56 million.
Licensing information: The Washington Re-
search Foundation (WRF) is now the exclusive
licensing agent for the Univ. of California/Univ.
of Washington/Genentech family of yeast expres-
sion patents. WRF asserts that essentially all com-
mercial uses of yeast, particularly Sacharomyces
cervisieae, expression systems require taking a
license (or at least clearing your particular appli-
cation). The issued patents, particularly the more
recently issued patents, broadly claim processes
and materials for expression of proteins in yeast.
Patents include U.S. 5,854,018 and 5,856,123
(continuations of 5,618,676) concerning recom-
binant production of proteins in yeast expression
systems including Saccharomyces, Kluyveromy-
ces, Pichia and Hansenula. All three U.S. patents
will expire in 2014. The new patents are broader,
as they claim processes and materials for expres-
sion of proteins in recombinant yeast systems
generally. Other related patents include also U.S.
4,769,238 and 5,196,194.
As can be seen from the related entries, there
are some dominant or frequently used commercial
yeast expression systems, e.g,. Saccharomyces
cerevisiae and Pichia. With each of these systems,
broad use and equivalent foreign patents cover
basic aspects of these systems, e.g., it would be
hard to use either of these systems without taking
a license from the Washington Research Founda-
tion (WRF; S. cerevisiae) or RCT (Pichia). Use of
Pichia may require a license WRF, besides RCT
(although there seems to be disagreement on com-
plexities on this point).
188
Zygosaccharomyces bailii
expression; Zbleu2 strain -
yeast
University of Milano-Bicocca - Licensor,
primary
Description: The yeast Zygosaccharomyces
bailii, including a Z. bailii auxotrophic mutant
(Zbleu2 strain), is useful for economic, large-scale
101
recombinant (glyco)protein manufacture. The
yeast secretes expressed protein into the culture
medium, simplifying the isolation process; and
may be cultivated in chemically defined medium.
Direct and indirect approaches allow for the im-
provement of heterologous proteins and secretion
levels in Zygosaccharomyces bailii. Zbleu2 strain
has shown a great improvement in recombinant
protein production, mainly related to higher plas-
mid stability, compared with the wild type strain
transformed with a similar plasmid but selecting
for an antibiotic resistance.
Use with: Zygosaccharomyces bailii (yeast)
Background: The yeast Zygosaccharomyces
bailii belongs to the so-called group of non-con-
ventional yeasts, poorly studied in the past. Only
recently has this yeast attracted attention due to its
characteristics of stress resistance, particularly to
acidic environments.
Benefits: Major advantages of Z. bailii as a host
organism for production of heterologous proteins
include a naturally favorable codon usage and the
comparatively low demands on the culture condi-
tions. This is in particular due to a high acid and
temperature tolerance as well as a weak Crabtree
effect allowing the cultivation with a high sugar
concentration from the beginning (i.e. batch
instead of fed-batch cultivation), and the omission
of sophisticated regulation of the culture condi-
tions such as temperature or pH. This provides a
cost effective production of proteins at industrial
scale yielding proteins of high purity.
Process for expression and secretion of proteins
by the non-conventional yeast zygosaccharomy-
ces bailii, U.S. 20070065905, Branduardi, et al.
Milano-Bicocca.
Further info.: The first auxotrophic mutant
of Zygosaccharomyces bailii for recombinant
productions: a road to practical applications,
Microbial Cell Factories 2006, 5(Suppl 1):
P67doi:10.1186/1475-2859-5-S1-P67 (available
online).
189
EASYEAST; Saccharomyces
cerevisiae strains - easy
protein release
Description: EASYEAST strains of Saccharo-
myces cerevisiae readily release of recombinant
proteins with no need for enzymatic or chemical
treatment or specialized equipment. The EAS-
YEAST strain series have mutations that allow
a conditionally fragile cell wall. Intracellular
proteins can be released in an easy and efficient
way by growth in low osmotic media at moderate
temperature (37C). High density cultures can be
achieved. This cell technology avoids the require-
ment for chemical, enzymatic or mechanical
means to break yeast cell envelopes. EASYEAST
can be used for large-scale production of cytoplas-
mic proteins in yeasts; heteromultimeric protein
production; bioassays in yeast for drug discovery;
and high-throughput protein expression/purifica-
tion with purification tags.
Availabiilty: Marketed for non-commercial uses
by Biomedal, S.L.
regarding commercial uses and licensing.
190
Yeast cell lines and vectors;
cell lines - proper folding and
glycosylation
Yeast Protein Sciences, Inc. - Licensor, primary
Pennsylvania State University - R&D
Description: Vectors and yeast cell lines, part-
cularly Saccharomyces cerevisiae, have been
developed with inhibiton of O-glycosylation. The
quality control mechanism employed by fungi
102
which returns misfolded proteins to the cyto-
sol for degradation is manipulated so that these
proteins are instead secreted. This promotes the
proper expression and secretion of heterologous
secretory proteins in yeast by overcoming the pre-
vious problems associated with the yeast expres-
sion systems where many heterologous proteins
fail to fold properly. In addition, this invention
improves the yields and activity of proteins where
yeast expression has shown some success. This
allows the production of heterologous proteins in
yeast to be more similar (if not identical) to the
proteins synthesized in the original host organism,
including glycoproteins with more human-like
folding and activity. In a preferred embodiment,
recipient yeast cells are manipulated so that an
enzyme associated with O-glycosylation or the
Bypass of Sec Thirteen families are inhibited. As
a part of quality control, proteins with yeast spe-
cific modifications are eliminated. Inhibition of
O-glycosylation prevents improper yeast specific
modification thereby avoiding the yeast internal
quality control mechanism.
Use with: yeasts; S. cerevisiae
Patents: Exemplary pending applications include
U.S. 20020068325, Methods and compositions
for highly efficient production of heterologous
proteins in yeast,. Ng, D.T.W., et al., June 6,
2002, assigned to Pennsylvania State University,
and exclusively licensed to Yeast Protein Sci-
ences.
Licensing information: Contact Yeast Protein
Sciences.
191
Pichia pastoris expression
- Licensor, primary
Phillips Petroleum - R&D, of tech.
Description: The yeast Pichia pastoris has be-
come a major platform for recombinant protein
manufacture. P. pastoris can be readily manipulat-
ed and cultured in high density. Pichia species can
perform N-glycosylation and can be engineered
to produce homogenous and customized glyco-
sylations. The very potent AOX1 promoter is
often used with Pichia (see related entry). Another
advantage of P. pastoris as a production host is its
secretion of product into fermentation medium at
titers of up to 3 g/L. Primary recovery and product
purification are less laborious and time-consum-
ing for this system than for bacterial ones, result-
ing in more cost-efficient purification processes.
P. pastoris grows on a simple mineral media and
does not secrete high amounts of endogenous pro-
tein. Therefore, the heterologous protein secreted
into the culture is relatively pure and purification
is easier to accomplish. Pichia is similar to S.
cerevisiae in practice, but it can give 10 -100 fold
higher levels of expression for a foreign gene.
Pichia offers complex, single or multicopy
integration of plasmids; high mitotic stability;
clonal variation (unlike E. coli); product release
(secretion) into the fermentation medium; high
productivity (intracellular compared with secreted
and released), e.g., >10 g/L; sometimes unwanted
product variants; N-glycosylation; O-glycosyl-
ation; and process development time (e.g., gene to
the first mg of purified product) of 5-7 months.
Pichia is a methylotrophic yeast that can use
methanol as its sole carbon source. It does this
by formation of formaldehyde and hydrogen
peroxide inside peroxisomes. The enzyme that
carries out this reaction is alcohol oxidase. This
enzyme is made in large amounts in peroxisomes.
Pichia expression vectors use the generally use
the AOX1 promoter (see related entry) to drive
expression of foreign genes. This gene can be
induced by methanol, while glucose represses the
AOX1 gene.
Pichia tends to be more like higher eukary-
otes in its glycosylations, but is different from S.
cerevisiae. S. cerevisiae-expressed proteins tend
to be more antigenic. 8-14 mannose are added per
chain compared to 50-150 in S. cerevisiae, and
the terminal linkages in S. cerevisiae are alpha 1,3
glycan linkages.
103
Use with: Pichia pastoris
Background: Pichia pastoris and also Pichia
augusta (formerly Hansenula polymorpha) are
widely used commercially. Both utilize (metabo-
lize) methanol and possess the strong methanol-
inducible promoter from the methanol oxidase
gene (AOX1, see related entry), which is used to
drive expression of cloned gene.
P. pastoris was initially developed and used by
Phillips Petroleum Company for large-scale pro-
duction of single-cell protein for animal feed,
Benefits: Development times - from gene to the
first milligram of purified product - are about the
same (five to six months) for both E. coli and P.
pastoris. However, in P. pastoris all expression
constructs must be integrated into the chromo-
some. Although autonomous multicopy plasmids
exist for this yeast, chromosomal integration has
been more successful. This more complex integra-
tion process is driven by homologous recombi-
nation, which may result in clonal variation. To
identify the most productive clones, more labori-
ous screening procedures have been developed
and optimized. Sometimes thousands of P. pasto-
ris transformants must be analyzed to identify a
valuable production clone. Clonal variation may
require automated high-throughput screening.
P. pastoris has an additional advantage of
achieving very high cell densities in fed-batch
culture while wasting a lower fraction of sub-
strate than does E. coli, for example. The main-
tenance energy of P. pastoris is lower than that of
other microorganisms . Nevertheless, heat evolu-
tion does set clear limits for this yeast as well.
Benefits/characteristics include:
Fast growth up to very high cell densities
Regulatory status: P. pastoris is a generally rec-
ommended as safe (GRAS) organism
Availability of several signal sequences for
protein secretion and concomitantly simplified
downstream processing
Integrative expression plasmids are available
and single- and multiple-copy integrants with high
mitotic stability can be obtained.
Possible development of customized homog-

enous glycosylation
Widely used with P. pastoris , the AOX pro-
moter is one of the strongest promoters known.
Other even stronger promoters can be identified
and developed for P. pastoris.
Readily controllable process
Generations of stability and durability
Product processing similar to mammalian cells
P. pastoris processes sometimes exhibit N- and
C-terminal product variants that usually repre-
sent the major product impurities in fermentation
supernatant. Because of their nearly identical
physicochemical properties, sophisticated pu-
rification is needed to eliminate them. Product
variants result from incomplete or incorrect
N-terminal processing and C-terminal truncation
by proteolytic cleavage. Generation of product
variants and undesired O-glycosylation must
be tightly controlled by strain development and
specific fermentation protocols. Moreover, the
need for high-cell-density fermentation using
methanol as an inducer typically causes excessive
heat production. Despite these drawbacks, Pichia
has great potential as a manufacturing organism
for proteins.
Patents: The Phillips and other patents covering
the Pichia system broadly cover components and
methods.
Exemplary patents include 4,882,279, Site
selective genomic modification of yeast of the
genus pichia, Cregg, J.M., Nov. 21, 1989, as-
signed to Phillips Petroleum Co., concerning site-
directed mutation of yeast. The abstract states:
Method for the site specific genomic modifica-
tion of yeasts of the genus Pichia and novel DNA
sequences useful therefor are provided. Pichia is
transformed with a serially arranged linear DNA
fragment comprising first and second insertable
DNA fragments, which flank a marker gene. The
insertable DNA sequences are homologous to de-
fined portions of the Pichia genome and integrate
by recombination at such defined sites. The result-
ing transformed organism contains the marker
gene and any additional DNA sequences which

104
are positioned between the insertable DNA frag-
ments. In addition, the resulting transformed or-
ganism has either a disrupted or deleted sequence
at the site of the genomic modification. Enhanced
production of heterologous gene products is
observed when using as the host for expression
strains in which the alcohol oxidase gene has been
disrupted. Upon disruption of the primary alcohol
oxidase gene of Pichia, the existence of a second
alcohol oxidase gene in Pichia was also discov-
ered.
U.S. 4,414,329, assigned to assigned to Phil-
lips Petroleum Co., concerns growth of yeast such
as Pichia pastoris to ultra-high cell densities, i.e.,
cell densities in excess of 100 g/L.
U.S. Pichia transformation, Cregg, J.M., et
al., May 29, 1990, concerns methods for mak-
ing whole cells of Pichia pastoris competent for
transformation.
Licensing information: Contact RCT. RCT has
licensed the system to more than 120 companies.
RCT is one of the few companies to publish
information about licensing rates/terms (available
at its Web site). This includes three different types
of nonexclusive licenses, termed Class A, Class B
and Class C. Proteins excluded from production in
Pichia (due to previous licensing commitments),
bulk product terms and legal aspects are identi-
cal among all three license classes. All classes of
license agreements have similar legal language
but different financial arrangements. Commercial
license issue fees (simply stated) range from $20-
75,000 (for Class A), with annual mimimum roy-
alty from $50-75 K (A); a royalty rates (initial 2
products) of 3% (A), 4% (B), 5% (C). A research
license involves a $50,000 license issue fee, with
no royalties.
Patent coverage from some of the earlier com-
ponent Pichia technologies are or will soon begin
to expire, but RCT holds a large portfolio that will
likely extend this for further years.
Products made using this tech.: More than 500
products have been reportedly expressed in P. pas-
toris. Most of those are developmental products.
Dyax Corp. has developed DX-88 (kallikrein
inhhibitor protein, recombinant) for treatment of
hereditary angioedema (HAE), with a BLA pend-
ing in the U.S. Nuvelo Inc. (and CMO, Avecia)
manufacture Alfimeprase [3-203-fibrolase (3-ser-
ine) (Agkistrodon contortrix contortrix recombi-
nant)] for marketing by Bayer Schering, with a
BLA pending in the U.S.
AquaVac IPN salmon vaccines for infectious
pancreatic necrosis prevention in salmon from
Schering Plough Animal Health is manufactured
in Pichia
Other products manufactured using Pichia tech-
nology from RCT include: Albrec (Recombinant
human serum albumin, or rHSA) from Mitsubishi
Pharma Corp.; DX-890 from Dyax Corporation,
in trials; recombinant collagen from Fibrogen,
Inc.; recombinant allergens (house dust mite, cat,
mouse and cockroach) from INDOOR Biotech-
nologies are available for research use; Superior
Stock Nitrate Reductase, enzyme for water treat-
ment and testing from The Nitrate Elimination
Co., is on the market; and recombinant endotoxin
neutralizing protein from Associates of Cape Cod,
Inc., East Falmouth, is marketed for non-drug-re-
lated endotoxin assays.
See the extensive list/bibliography, Heterologous
Proteins Expressed in Pichia pastoris, at http://
faculty.kgi.edu/cregg/Pichia2004.htm, by Dr.
Cregg, primary inventor of Pichia technology.
ACE Expression System - MAb-expressing CHO
cells lines
192
VelociMab; EESYR expression
system; FASTR cell lines -
CHO expression optimization;
antibodies
Regeneron Pharmaceuticals, Inc. - Licensor,
primary
Description: FASTR (CHO) cell lines are use-
ful for recombinant (glyo)protein, particularly
antibody, manufacture (along with high-through-
put analysis, sorting and other services provided
105
by Regeneron). Use of the EESYR cell line
progresses seamlessly through an optimized
bioprocess resulting in consistent, predictable,
high-titer yields of the desired protein therapeutic.
This turnkey, high-throughput process enables
significantly more therapeutic antibody candi-
dates to be evaluated using purified, clinical-grade
protein expressed from the actual production cell
lines. The EESYR cell line platform can be used
to efficiently express any biologic and protein
reagent...The Regeneron flow cytometry-based
autologous secretion trap (FASTR) technology is
a novel method for the high-throughput analysis
and sorting of live cells based on expression level
of the protein to be manufactured. This enables
the creation of production cell lines specifically
tailored to the desired bioprocess parameters for
maximum bioreactor yields and favorable cost of
goods.
Use with: CHO cells
ies
Patents: Related patents include 6,919,183, Iso-
lating cells expressing secreted proteins, Fandl,
et al., July 19, 2005, with abstract, A method
for identifying and isolating cells which produce
secreted proteins. This method is based upon a
specific characteristic or the expression level of
the secreted protein by transiently capturing the
secreted protein on the surface of an individual
cell, allowing selection of rare cell clones from a
heterogeneous population. Also provided is the
use of this method to generate cells which pro-
duce a desired level of secreted protein or se-
creted protein of a particular characteristic(s), and
organisms which possess such cells. In particular,
the method allows rapid isolation of high expres-
sion recombinant antibody-producing cell lines,
or may be applied directly to rapid isolation of
specific hybridomas, or to the isolation of anti-
body-producing transgenic animals. This method
is applicable for any cell which secretes protein.
Licensing information: Contact Regeneron
regarding related services and licensing opportu-
nities.
Plants
193
Plastid Transformation;
Translation-based vectors
(TBV) - plants
Icon Genetics AG - Licensor, primary
Bayer Corp. - Parent co.
Description: Translation-based vectors (TBV)
provide high levels of recombinant protein pro-
duction in the plastids of plants (tobacco and to
date four other species). Plastid transformation
provides high precision gene technology with
pronounced biological safety due to lack of pollen
transmission of transgenes. Pigmentation is used
as a visible marker for plastid transformation, and
plastid mutants with a clearly detectable pheno-
type are generated. Subsequently, the transgene of
interest is introduced and photosynthetic capacity
is restored. Transformed tissue is readily identi-
fied and genetically uniform lines are rapidly
produced. A selection of specialized receptor lines
is available.
Use with: plants; tobacco
Patents: Related patents include U.s. 7,193,131,
Processes and vectors for plastid transforma-
tion of higher plants, Eibl, C., March 20, 2007,
assigned to Icon Genetics AG. The abstract states,
A process for producing multicellular plants,
plant organs or plant tissues transformed on their
plastome by the following steps is provided: (a)
altering or disrupting the function of a gene in
a plastid genome for producing a selectable or
recognizable phenotype; (b) separating or select-
ing plants or cells having plastids expressing said
phenotype; (c) transforming said plastid genome
of said separated or selected plant, plant organ or
plant tissue with at least one transformation vector
having a restoring sequence capable of restoring
said function; and (d) separating or selecting said
transformed plant, plant organ or plant tissue hav-
ing plastids expressing said restored function.
106
Licensing information: Contact ICON Genetics .
194
Chlamydomonas reinhardtii
chloroplast expression;
promoter - algae, single-cell
Scripps Research Institute, The - Licensor,
primary
Description: A Chlamydomonas reinhardtii
chloroplast expression system using a deleted/
non-functional C. reinhardtii psb1 gene, which
encodes oxygen evolving enhancer protein num-
ber one (OEE1), and use of the apsbD promoter
provides enhanced chloroplast translation and
protein expression. Vectors are provided, and ex-
pression may be modulated by exposure to light.
Direct replacement of chloroplast Photosystem
II (PSII) reaction center protein coding regions
achieves expression of recombinant protein above
5% of total protein. The algal expressed protein
is produced predominantly as a soluble protein
with functional activity intact. As the host algae
is edible, biologics may be manufactured in this
organism for oral delivery of proteins/peptides,
especially gut active proteins, e.g., oral vaccines.
C. reinhardtii nuclear mutant FuD44 lacks the
ability to express OEE1 and is therefore com-
pletely deficient in photosynthetic oxygen evolu-
tion. C. reinhardtii cells deficient in OEE1 are
incapable of photoautrotrophic growth, i.e., the
OEE1 protein is absolutely required for photosyn-
thesis. In FuD44, a 5 kilobase (kb) DNA insertion
into the 5 min region of the psbl gene results in
the complete absence of OEE1 mRNA and pro-
tein. Methods for transformation are a key aspect
of the system.
Use with: Chlamydomonas reinhardtii
Use to make: proteins; vaccines, edible
Background: See the related Entelechon C. rein-
hardtii entry.
WO/2007/133558, ROBUST EXPRESSION
OF A BIOACTIVE MAMMALIAN PROTEIN
IN CHLAMYDOMONAS CHLOROPLAST,
published Nov. 22, 2007, Mayfield, Stephen
P., assigned to Scripps Research Institute; and
EP0342824, Methods and systems for transform-
ing chlamydomonas reinhardtii., Mayfield,
Stephen P., assigned to Scripps Research Institute,
published in 1989. Claim 1 of the recent filing
states, A method of expressing a gene compris-
ing: transforming an algae cell by replacing an
endogenous chloroplast gene via integration of a
chimeric construct having a heterologous coding
sequence, a promoter sequence, and at least one
UTR, wherein the promoter is cognate or non-
cognate to the endogenous chloroplast gene; and
cultivating the transformed algae cell under condi-
tions to allow for expression of the transgene.
Licensing information: Contact The Scripps
Research Institute.
Further info.: PROC. NATL. ACAD. SCI. USA,
vol. 84, February 1987, pages 749-753; S.P.
MAYFIELD et al.: Expression of the nuclear
gene coding oxygen-evolving enhancer protein 2
is required for high levels of photosynthetic oxy-
gen evolution in Chlamydomonas reinhardtii
195
Chlamydomonas reinhardtii
expression - algae, single-cell
Entelechon GmbH - Licensor, primary
Regensburg University - R&D
ASA Spezialenzyme Ltd. - R&D
Description: The single-cell algae-based Chlam-
ydomonas reinhardtii expression system offers a
range of advantages including easy cultivation, a
glycolization pattern similar to mammalian cells,
targeted protein transport and directed excretion
to different cell compartments, besides a remark-
able biocompatibility for clinical applications.
Industrial fermentation can be perfomed under
easy conditions and provides high expression
yields. The alga is non-toxic and non-pathogenic.
It can be easily grown and contained. As carbon
source, both CO2 and acetate can be used. This
107
reduces the cost of fermentation and makes con-
tamination less likely. Simple disposable plastic
reactors enable the efficient assembly of large
production volumes. This technology is ideally
suited to rapidly establish gene expression that
would take much longer in higher plant systems.
Chlamydomonas displays a glycosylation
pattern very similar to mammalia. It is thus a
valuable alternative for yeasts and other systems
which may not provide suitable glycosylation
modifications.
Complete control over the localization of ex-
pressed proteins can be achieved. Proteins can be
directed into cell compartments such as mito-
chondria, chloroplasts, extraceullar medium or the
membrane.
The cells are significantly more biocompatible
with any application in a clinical context than E.
coli. In particular the cells are free of endotoxin
and avoid the expensive and tedious endotoxin
removal.
Fermentation can be performed under a batch-,
fed-batch- or flowthrough regime. Chlamydomo-
nas can be fermented in simple disposable plastic
reactors, and can be grown heterotrophically or
photoautotrophically. Typical yields in the ex-
ponential growth phase yields are currently 230
mg/l of dry biomass of protein. Typical expres-
sion rates, as seen in similar green algae, are 1.4
mg/(l*h) to 8.75 mg/(l*h). The latter has been
achieved in Chlamydomonas with added acetate,
under a 24h light/dark regime.
As a proof of principle, Entelechon has suc-
cessfully expressed a bleomycine/luciferase
fusion protein, and has directed the product
alternatively into the cytosol or the extracellular
medium. A bleomycine/GFP fusion protein has
been directed into the nucleus, whereas a GFP-
APH VIII fusion protein has been directed into
the cell membrane. The GFP has been displayed
extracellularily, whereas APH VIII was located
on the cytosolic side, both linked by a transmem-
brane domain.
Benefits: Cheap, simple, quick, safe with basic
glycosylation.
Background: Chlamydomonas reinhardtii is a
single-cell alga which has been used as a model
organism for several decades. Its generation peri-
od is 8-24 hours and enables the rapid generation
of clones and crossbreeds. The alga can be grown
both heterotrophically and photoautotrophically.
C. reinhardtii employs the same photosynthetic
scheme as higher plants, and contains photosyn-
thetic complexes that are identical to those of
higher plants both in function and protein com-
ponents. However, in contrast to higher plants,
C. reinhardtii has a rapid (6-24 hour) replication
schedule, contains a genome that is a fraction of
the size of most plant genomes, and is well char-
acterized genetically.
Although haploid by default, the alga can dif-
ferentiate into two mating types which can form
diploid zygospores. This enables crossbreeding
and easy combination of genetic features. Cell
wall-deficient mutants which have significantly
reduced cell wall protein content facilitate the
export of transgenic proteins into the medium.
The 100 Mbp genome of Chlamydomonas - com-
prised of 17 chromosomes - has been completely
sequenced, with 8-fold coverage. The alga easily
forms colonies on agar plates, and can be easily
transformed. Entechelon has developed tools to
rapidly and efficiently create transgenic clones for
the expression of arbitrary proteins.
Patents: Entelechon reports not yet filing pat-
ent applications for this technology, and that this
Chlamydomonas reinhardtii expression system
and the related one from Scripps (see related en-
try) do not have conflicting or overlapping aspects
and were developed independently.
Availability: Entechelon is commercially de-
veloping this technology, and offers CRO/CMO
services.
Licensing information: Entechelon states, We
are ready to grant non-exclusive licenses for
research and manufacture of proteins with the
help of this expression system to industry clients.
The exact terms of such liences depends strongly
on the scope of the intended application, and is
subject to negotiation. As with many candidate
108
alternative expression systems, Entechelon re-
ports, that the expression system has not yet been
used on an industrial scale. We are currently in the
process of upscaling several pilot projects from a
1 litre and 10 Litre scale to higher volumes, but
this still needs some time. Much of the reseach
on this trechnology has been supported by a grant
from BioChance program of the German govern-
ment.
Further info.: Chlamydomonas reinhardtii: A
protein expression system for pharmaceutical and
biotechnological proteins, Molecular Biotechnolo-
gy, Volume 34, Number 2 / October, 2006 (review
article).
196
Drosophila Expression System
(DES) - insect cell culture
Invitrogen, Life Technologies, Inc. - Licensor,
primary
GlaxoSmithKline Inc. (GSK) - licensor, pri-
mary
SmithKline Beecham Corp. (SKB) - R&D
Description: The Drosophila Expression System
(DES) enables protein expression in Drosophila
(mosquito) cell culture. DES is claimed to com-
bine the best features of mammalian and insect
expression systems for simple, efficient produc-
tion of recombinant protein, e.g., using Drosophi-
la S2 cells. DES provides straightforward genera-
tion of insect cell lines that stably express high
levels desired protein. Vectors with an inducible
promoter allow for expression of toxic proteins.
DES offers a variety of easy-to-use vectors with
features such as inducible promoters, secretion
signals, and tags. DES can include use of the
Drosophila metallothionein (MT) promoter for
high-level, inducible expression of your gene of
interest from vectors. The MT promoter is tightly
regulated and is easily induced by the addition of
copper sulfate.
See also the entry for Drosophila melanogaster
S2 cells.
Use with: Drosophila cells.
Use to make: proteins, glycoproteins
Background: DES was originally developed by
SmithKlineBeecham (SKB), now GlaxoSmith-
Kine (GSK), primarily for vaccines.
Benefits: Drosophila Expression offers: higher
protein yields than most mammalian systems;
easy high-density cell culture; and non-lytic ex-
pression for reduced degradation. Drosophila S2
cells are claimed to be perfectly suited for high-
level, low-cost production of eukaryotic proteins.
S2 cells are cost-effective and easy to use because
they:
Grow to high densities without supplementary
CO2 in serum-free medium, reducing costs
Produce proteins with eukaryotic posttransla-
tional modifications
Integrate multiple copies of expression plas-
mids to allow isolation of high-producing poly-
clonal stable cell lines
Also, endogenous Drosophila proteins generally
do not interact with mammalian proteins, al-
though they may be immunogenic.
Patents: Drosophila Expression System (DES)
and its use are the subject of U.S. patents
5,550,043; 5,681,713; 5,705,359; 6,046,025, each
titled, Expression of heterologous proteins in
drosophila cells, and other pending patents as-
signed GlaxoSmithKine and licensed exclusively
to Invitrogen. Claim 1 of 5,550,043 states: 1. A
method for altering the copy number of a heter-
ologous gene integrated into a Drosophila cells
genome which comprises: selecting cultured Dro-
sophila cells as a host for transfection; cotrans-
fecting said host cell with (i) a first vector com-
prising a gene expression unit having a promoter
of Drosophila origin and a DNA sequence encod-
ing a heterologous gene product and; (ii) a second
vector comprising a promoter of Drosophila
origin and a DNA sequence encoding a selection
marker, wherein the ratio of said cotransfected
vectors is altered such that the first vector is pres-
ent in the ratio of from about 1 to 100 times that
of the second vector; and culturing the transfected
Drosophila cells.
109
Availability: Invitrogen markets DES and com-
ponents for non-commercial use.
Licensing information: Invitrogen states, Pur-
chase of DES products from Invitogen grants you
a limited, non-exclusive license to use the product
for research purposes only. For more
information, contact Invitrogen... It is unstated
whether Invitrogen or GSK handles commercial
licensing.
197
Streptomyces lividans
Centro Informtico Cientfico de Andaluca
(Spanish Center on Biotechnology) - Licensor,
primary
Description: Streptomyces lividans bacteria, e.g,
Y62 strain, with extracellular overproduction of
desired protein and decreased signal petidases
type I (protease) activity, are useful for protein
manufacture. Genes coding for signal petidases
type I are used to obtain Streptomyces deficient in
extracellular protease activity. Secreted proteins
remain more stable extracellularly over time.
Use with: Streptomyces lividans
Benefits: Streptomyces lividans is easy to culti-
vate.
WO/2004/016730, BACTERIA THAT CAN
OVERPRODUCE AND SECRETE PROTEINS,
MELLADO, et. al., assigned to CONSEJO SUPE-
RIOR DE INVESTIGACIONES CIENTFICAS.
Licensing information: Centro Informtico
Cientfico de Andaluca. Collaborators and licens-
ees are sought.
Further info.: FEMS Microbiology Letters, An
inducible expression system of histidine-tagged
proteins in Streptomyces lividans for one-step
purification by Ni2+ affinity chromatography,
Volume 137 Issue 2-3 Page 135 - April 1996.
Eukaryotes
198
Antibiotic inducible promoters
- eukaryotes
Cistronics Cell Technology GmbH - Licensor,
primary
Institute for Chemical and Bio-Engineering,
ETH - Licensor, secondary
Description: Actinomycetes antibiotic resistance
promoters in expressed fusion proteins are useful
for regulated expression in eukaryotic cells, and
provide a new system for antibiotic-regulated
gene expression in eukaryotic cells. Proteins are
provided that can be used to activate or repress
transcription from a desired nucleotide sequence
operatively linked to a Pabr sequence. This sys-
tem is based on sequences from an Actinomycetes
antibiotic responsive operon (resistance pro-
moter), the Pabr sequence, operatively linked to a
second polypeptide which activates or represses
transcription in eukaryotic cells, with activation
or repression using polypeptides that bind to Pabr
(without use of antibiotics). Vectors contain the
Pabr-bind sequence along with the desired protein
for expression of fusion proteins which regulate
transcription.
6,287,813, Antibiotic-based gene regulation
system, Fussenegger, et al., Sept. 11, 2001, as-
signed to Cistronics Cell Technology GmbH. The
abstract states: The invention relates to a novel
system for gene regulation in eukaryotic cells, and
methods of using the same for protein production,
tissue engineering and gene therapy. In particular,
the invention provides a new system for antibi-
otic-regulated gene expression in eukaryotic cells
based on sequences from Actinomycetes antibiot-
ic resistance promoters, polypeptides that bind to
the same in an antibiotic responsive manner, and
nucleotides encoding such polypeptides. Further,
110
the invention provides novel and sensitive meth-
ods of screening for candidate antibiotics.
Licensing information: Contact Cistronics
Cell Technology GmbH, which has licensed this
technology from the Institute for Chemical and
Bio-Engineering, ETH.
Further info.: Animal Cell Technology Meets
Genomics, Proceedings of the 18th ESACT
Meeting Granada, Spain, May 11-14, 2003.
199
AttSite recombinases - precise
gene insertion; eukaryotes
RheoGene, Inc. - Licensor, primary
University of Pittsburgh Medical Center
(UPMC) - Assignee, patent
Description: AttSite recombinase enzyme-
based technology is useful to guide gene inser-
tion into specific hot spots within the genome
to ease the exchange of DNA and development
of new genetic sequences and eukaryotic cell
lines (plants, and animals). Each AttSite enzyme
recognizes a different DNA sequence. Different
enzyme combinations can be used to reproducibly
introduce multiple genes into the same site in the
genome, or into different sites in the same cell.
Only RheoGenes AttSite platform has this level
of precision and flexibility.
The methods involve providing a eukaryotic
cell that has a first recombination attachment
site and a second recombination attachment site;
contacting the first and second recombination
attachment sites with prokaryotic recombinase
polypeptide, resulting in recombination between
the recombination attachment sites, in which the
recombinase polypeptide can mediate recombi-
nation between the first and second recombina-
tion attachment sites. The first recombination
attachment site is a phage genomic recombina-
tion attachment site (attP) or a bacterial genomic
recombination attachment site (attB), the second
recombination site is attB or attP, and the recom-
binase is selected from Listeria monocytogenes
phage recombinase, a Streptococcus pyogenes
phage recombinase, a Bacillus subtilis phage re-
combinase, a Mycobacterium tuberculosis phage
recombinase and a Mycobacterium smegmatis
phage recombinase,
20060172377, Site-specific serine recombinases
and methods of their use,Padidam, M., assigned
to RheoGene
Licensing information: Nonexclusive research
and commercial licenses for AttSite(TM) Recom-
binases are available from RheoGen.
Reported licensees have been reported to include
Centocor/J&J and Symphogen.
200
Bovine growth hormone (bGH)
polyadenylation sequence
- eukaryotes; expression
enhancement
- Licensor, primary
Upjohn Co. - Assignee, patent
Pfizer, Inc. - Parent co.
Description: The bovine growth hormone poly-
adenylation (bgh-PolyA) signal from the gene for
bovine growth hormone is a specialized termina-
tion sequence for protein expression in eukaryotic
cells. This DNA sequence is useful for producing
proteins and peptides in culture, and increasing
gene expression in gene therapy and protein pro-
duction in transgenic animals.
Use with: eukaryotes
Patents: The bovine growth hormone (bgh)
polyadenylation signal is patented under U.S.
5,122,458, European Patent 0 173 552, and Japa-
nese Patent 6-83669. U.S. 5,122,458, Use of a
bGH gDNA polyadenylation signal in expression
of non-bGH polypeptides in higher eukaryotic
cells, Post, et al., June 16, 1992, concerns use of
111
the polyadenylation signal to achieve a high level
of expression of peptides in eukaryotic cells. This
is assigned to Upjohn (subsequently acquired by
Pfizer).
Licensing information: RCT is now the licens-
ing agent for this technology, probably on behalf
of Pfizer, unless it fully acquired the technology
itself.
Products made using this tech.: As reported
in Biopharmaceutical Products in the U.S. and
European Markets, a bovine polyadenylation
signal is used in the expression vector for CHO
cell manufacture of Aldurazyme [Laronidase -I2S;
alpha-L-iduronidase] by BioMarin, marketed by
Genzyme.
201
Expression enhancers; Copy
number increase - eukaryotic
cells
RepliGen Corp. - Licensor, primary
Abbott Laboratories, Inc. - Licensor, secondary
Damon Biotech, Inc. - R&D
Massachusetts Institute of Technology (MIT)
- Assignee, patent
Description: Vectors with an a cellular enhancer
element achieve higher expression levels in
eukaryotic cells. The cellular enhancer element
increases the level of transcription of genes on its
3 and 5 sides. A blocking element is interposed
between the enhancer element and the marker
gene which shields the promoter of the marker
gene from the transcription-stimulating function
of the enhancer, limiting the effect of the enhancer
to transcriptions of the DNA encoding the protein
product of interest. Use of the vectors permits
isolation of viable clones characterized by a very
high level of expression of the protein of interest.
The resulting transformants express the exons
of the transcription unit at high levels as the
enhancer element increases the copy number of
mRNA. The enhancer element operates to in-
crease transcription independent of its orientation
and position provided it is located within an active
region on the DNA, generally between about 1-10
kilobases (kb) from the 3 or 5 end of the tran-
scription unit.
4,663,281, Enhanced production of proteinaceous
materials in eucaryotic cells, Gillies, et al., May
5, 1987, assigned to MIT, originally exclusively
licensed to Damon Biotech, then to Repligen,
which acquired certain Damon Biotech assets.
U.S., 5,665,578, Vector and method for
achieving high level of expression in eukaryotic
cells, Gillies, et al., Sept. 9, 1997, with similar
claims, is assigned to Abbott Labs., with Repligen
having sublicensing rights (apparently as a result
of a cross-licensing settlement between the two
companies).
On May 4, 2004, RepliGen filed a suit against
ImClone alleging use of a cell line infringing
U.S. 4,663,281 in connection with manufacture
of Erbitux (see below). In Sept. 2007, the dispute
was setted, with ImClone paying a total of $65.0
million in cash for full and final settlement of the
claims against ImClone in the matter, as well as
for a royalty-free, irrevocable worldwide subli-
cense to technology patented under U.S. Patent
No. 4,663,281. Repligen paid MIT with a portion
of the settlement payment. Repligen had original-
ly approached ImClone about licensing its patent
in 1996, seeking $200,000 plus 1% royalties, but
was denied.
Repligen also granted to ImClone a royalty-
free, irrevocable worldwide sublicense for the
future use of other patented technology, includ-
ing U.S. 5,665,578, which is owned by Abbott
Laboratories, but to which Repligen has the power
to sublicense under an agreement between Abbott
Laboratories and Repligen. Abbott had filed suit
aganst ImClone alleging infringement of U.S. Pat-
ent No. 5,665,578 in early 2007.
Licensing information: Contact Repligen regard-
Products made using this tech.: A transformed
murine cell line (M225) using this enhancer is
112
used by ImClone for manufacture of Erbitux
[Cetuximab - IMC-C225; epidermal growth fac-
tor receptor monoclonal antibody, recombinant]
marketed by Bristol-Myers Squibb.
202
Homologous Recombination;
Knock-out/knock-in animals
and cells
Institut Pasteur - Assignee, patent
Description: Homologous Recombination
enables specific replacement of a copy of a gene
present in the genome of a recipient eukaryotic or-
ganism by the integration of a gene different from
the inactivated gene. This is useful for the genera-
tion of gene knock-out/knock-in animals and cells
as models for screening and protein production.
Preferably, the recipient gene is present in at least
2 copies in the transfected host cell. The recipient
gene is defined as the gene where the insertion of
the different gene is made.
Patents: Related patents assigned to Institute
Pasteur and exclusively licensed to Cellectis
include U.S. 6,528,313 and 6,528,314, both with
the title, Procedure for specific replacement of a
copy of a gene present in the recipient genome by
the integration of a gene different from that where
the integration is made. These broadly claim the
insertion by homologous recombination in mam-
malian cells of a coding sequence and a selection
gene and a promoter with a coding sequence and
a selection gene and the conditions to obtain such
recombined cells.
Licensing information: Contact Cellectis. Li-
censees include GlaxoSmithKline.
203
Internal Ribosome Entry
Sequences (IRES) RNA
translation enhancers; pCITE
vectors; Cardiovirus 2A IRES;
pIRES - eukaryotes
Description: The cardiovirus internal ribosomal
entry site (IRES), particularly encephalomyocar-
ditis (EMCV IRES), or fragments are useful for
enhancing RNA translation (RNA to protein) and
protein expression, making these broadly useful in
DNA-based vectors. The EMCV IRES is a popu-
lar RNA element used widely in experimental and
biopharmaceutical applications to express proteins
in eukaryotic cells and cell-free extracts. Inclu-
sion of the wild-type element in monocistronic or
bicistronic messenger RNAs (mRNAs) confers a
high level of cap-independent translation activity
to appropriately configured cistrons. The literature
now describes thousands of cDNA constructions
utilizing IRES technology. The encephalomyo-
carditis translational enhancer coding region is
included in a vector deposited as ATCC 67525.
In 1986, shortly after EMCV-R was sequenced,
the region responsible for IRES activity was local-
ized to a genome fragment about 430 bases long,
immediately 5 to the AUG, which begins the viral
polyprotein open reading frame (ORF). When
the enhancer element was excised and linked to
other portions of the virus ORF, the resulting T7
transcripts were highly active messenger RNAs
(mRNAs) in the absence of 5 capping reactions.
A related vector was commercialized in 1990 by
Novagen as pCITE-1. pCITE-1 provides high lev-
els of enhancer-conferred protein expression. This
vector allows easy linkage of exogenous cistrons
onto the cap-independent ranslation enhancer for
transcription of hybrid mRNAs and facile protein
expression in cell-free extracts. pCITE-2, a de-
rivative vector, was released by Novagen in 1990.
113
In 1995, Clontech released a new vector
(pIRES) that could be delivered directly into
mammalian cells as cDNA. Transfection with
IRES induced nuclear transcription of capped bi-
cistronic mRNAs is driven by a cytomegalovirus
(CMV) promoter (see related entry). Translation
of any upstream gene A
inserted into the 5-most A multiple cloning site
is (MCS) cap-dependent. Linked in tandem is a
modified EMCV IRES, a second MCS (B), and
a poly(A) signal sequence, protein synthesis for
gene B inserted at MCS-B was IRES-depen-
dent.
WARF is now offering a new IRES based on
EMCV 2A protein, particularly vectors encoding
the cardiovirus 2A polypeptide operably linked
to a promoter. When expressed in mammalian
cells, in the absence of any other viral proteins or
genes, protein 2A localizes to the nucleolus, binds
to ribosomes, and inhibits cap-dependent mRNA
translation, effectively preventing the translation
of anything that is not a viral protein. Because
cardiovirus protein 2A does not inhibit cap-in-
dependent IRES-driven mRNA translation, the
result is a net increase in IRES-dependent pro-
tein expression relative to cap-dependent protein
expression.
These vectors have a DNA transcriptional
promoter and a DNA enhancer coding sequence
capable of coding for an RNA translational
enhancer. The enhancer (IRES) has the charac-
teristics of a cardiovirus RNA enhancer sequence
that is located 5 of a cardiovirus AUG sequence.
The DNA enhancer sequence is positioned on the
vector so as to be subject to the transcriptional
promoter. There is also foreign DNA gene for
a protein of interest which is positioned on the
vector so as to be subject to the transcriptional
promoter. After transcription of the foreign DNA
gene and the DNA enhancer coding sequence to
their RNA variants, translation of the RNA variant
of the foreign DNA gene is subject to the con-
trol of the RNA variant of the enhancer coding
sequence (IRES).
Background: Translation of the EMCV-driven
cistron is independent of the upstream gene, an
activity that could not be attributed to leaky scan-
ning or ribosome readthrough mechanisms. The
name, internal ribosome entry site or IRES was
used and has stuck with this element. IRES has
since been applied to many other virus or cellular
RNA fragments that also direct eukaryotic transla-
tion in a cap-independent manner.
Benefits: Claimed benefits for WARFs new
IRES include:
Introducing 2A into a cell doubles the ribo-
some content of that cell
May be used to enhance the yield of proteins
in cap-independent translation via the IRES
Acts independently-does not require other viral
proteins for its function
Can regulate cap-dependent or IRES-depen-
dent mRNA translation
Inhibits cellular mRNA transcription, but not
rRNA transcription, when combined with another
cardiovirus protein
Able to inhibit cellular mRNA transcription in
a virally-infected cell
Highly toxic-eventually kills every cell it
enters, and may be used therapeutically to selec-
tively kill cells, such as tumor cells
Patents: The primary exemplary U.S. patent
covering early IRESs is 4,937,190, Palmenberg,
et al., June 26, 1990, assigned to the Wisconsin
Alumni Research Foundation (Univ. of Wiscon-
sin). This likely has or will soon expire.
An exemplary application covering the new
WARF IRES is U.S. 20050019808, Composi-
tions and methods for regulating mRNA tran-
scription and translation, Palmenberg, A.C., et
al., Jan. 27, 2005. Claim 1 states: A nucleic acid
construct comprising: at least one DNA depen-
dent RNA polymerase promoter; a 2A cardiovirus
polynucleotide sequence encoding a 2A polypep-
tide, wherein the 2A polynucleotide is positioned
on the construct so as to be operably linked to at
least one promoter; and at least one foreign poly-
nucleotide coding for a detectable polypeptide,

114
wherein the at least one foreign polynucleotide is
positioned immediately downstream of and oper-
ably linked to the 2A polynucleotide.
Availability: Marketed by Clontech, Invitrogen
and other reagent vendors and also available from
ATCC, for non-commercial uses only.
Licensing information: Contact the Wisconsin
Alumni Research Foundation.
Further information: Translational efficiency of
EMCV IRES in bicistronic vectors is dependent
upon IRES sequence and gene location, Yury A.
Bochkov and Ann C. Palmenberg, BioTechniques
41:283-292 (September 2006)
204
Light-switchable promoters
Description: Light-induced promoters allow an
increase protein expression by over 1000-fold
after a single pulse of red light (wavelength peak
maximum of 665 nm). The system has a very
robust degree of inducibility over a large dynamic
range, and is appears to be univerally useful in
eukaryotic cells. The red light induced increase in
expression can be completely halted by a subse-
quent far-red pulse (wavelength peak maximum
of 730-760 nm), with expression switched off
completely, back down to the background control
levels. The level of induction is quite respect-
able in absolute terms compared to a very strong
activator. The promoter system is very tight in
the uninduced state.
A molecule of the plant photoreceptor phyto-
chrome is targeted to the specific DNA binding
site in the promoter by a protein domain that
is fused to the phytochrome and that specifi-
cally recognizes this binding site. This bound
phytochrome, upon activation by light, recruits
a second fusion protein consisting of a protein
that binds to phytochrome only upon light activa-
tion and a transcriptional activation domain that
activates expression of the gene downstream of
the promoter.
Benefits: Advantages of such an inducible system
include low background activity (negligible basal
expression in the absence of the inducer), high
inducibility, noninvasive inducibility, switchable,
inducible at a similar level in any tissue or organ-
ism; rapid, reversible, dose-dependent, gene-spe-
cific induction of expression upon administration
of light; inexpensive; non-toxic; accurately dos-
able, lacking pleiotropic and unintended effects,
simple to administer, and combinable with cell or
tissue specific expression.
6,858,429, Universal light-switchable gene
promoter system, Quail, P.H., et al., February 22,
2005, assigned to the Univ. of California.
Licensing information: Contact the Univ. of
California.
205
Tetracycline (Tc; Tet)
Expression Systems -
eukaryotes
TET Systems Holding GmbH & Co KG. - Li-
censor, primary
BASF AG - Parent co.
Abbott Laboratories, Inc. - Assignee, patent
Description: Tetracycline (Tc; Tet) Expression
(promoter) Systems allow inducible control of ex-
pression using the antibiotic tetracycline. ET gene
control elements allow genes to be turned on or
off, either in cultured cells or in whole organisms
such as yeast, plants and mammals, by exposure
to tetracycline. The Tet Systemuses prokaryotic
core components - the Tet repressor (TetR) and its
cognate binding site, the tet operator (tetO), which
makes the system essentially inert in eukaryotes.
See also the tetracycline promoter system used in
prokaryotes.
The Tet Technology provides efficient, precise
and reversible control over timing and level of
gene expression in eukaryotic cells. Tet Systems
115
claims, Its unusual specificity and the well
studied chemical and physiological properties of
the inducing agents have made the Tet System the
most widely applied inducible gene expression
system as demonstrated by the extensive litera-
ture available [although this primarily involves
research, not commercial, use].
Tet System comprises two complementary
control circuits, initially described as the tTA de-
pendent and rtTA dependent expression systems.
They are now commonly referred to as the Tet-Off
System (i.e., tTA dependent) and the Tet-On Sys-
tem (i.e., rtTA dependent). In each system, a syn-
thetic tetracycline-controlled transcription factor
(tTA or rtTA) interacts with a tTA/rtTA responsive
promoter, Ptet, to drive expression of a gene of
interest. Expression is regulated from outside by
the effector substance, tetracycline, or one of its
derivatives. Tetracyclines act at the level of DNA
binding of tTA and rtTA transcription factors. rtTA
requires a tetracycline ligand for DNA binding,
whereas the interaction between tTA and DNA is
prevented by tetracycline. Thus, the two versions
of the Tet System respond to tetracyclines in
opposite manner, making them complementary,
as each system has its unique characteristics and
strengths.
Doxycycline, a tetracycline derivative, is cur-
rently considered the most suitable effector sub-
stance for both the Tet-On and the Tet-Off Sys-
tems. It binds with high affinity to tTA as well as
to rtTA and, thus, is fully effective in the Tet-Off
system at such low concentrations as 1-2 ng/ml in
the case of tTA and in the Tet-On systems effec-
tive down to concentrations as low as 80 ng/ml in
the case of rtTA2-syn1.
Although the Tet System in its original ver-
sion has been and still is in wide use, a number of
modifications and refinements have significantly
improved its use. These modifications concern
the tTA/rtTA responsive promoters, Ptet, as well
as the transcription activators tTA and rtTA. In
addition, a new class of components has been
developed: tetracycline controlled transcriptional
silencers, tTS.
Use with: eukaryotes. The systems have been
used in cultured cells from mammals, plants,
amphibians, and insects as well as in whole
organisms including yeast, Drosophila, plants,
mice, and rats.
Benefits: The systems provide high induced
expression levels combined with low basal back-
ground, resulting in a wide dynamic range for
both expression systems, allowing regulation of
gene expression over many orders of magnitude
without interference with host cell physiology.
Patents: In Jan. 2004, Tet Systems exclusively
licensed core Tet expression-related patents from
Abbott Labs. Patents originally assigned to Abbott
include 6,783,756, Methods for regulating gene
expression, Bujard, et al., Aug 31, 2004. Other
patents include 5,464,758, Tight control of gene
expression in eucaryotic cells by tetracycline-re-
sponsive promoters, Gossen, et al., Nov. 7, 1995;
6,136,954; 6252136; and 5,814,618, now assigned
to Tet/BASF.
Availability: The components of the Tet System
are available through a number of authorized
reagent distributors.
Licensing information: Contact TET Systems.
The company provides technical support to licens-
ees of the TET Technology to help promote suc-
cessful new product development.
Tight control of gene expression in mamma-
lian cells by tetracycline-responsive promoters,
PNAS June 15, 1992 vol. 89 no. 12 5547-5551.
206
Zinc fnger DNA-binding
proteins (ZFPs); ZFP
Transcription Factors - gene
modifcation; mammalian cells;
plant cells
Sangamo - Licensor, primary
Description: Zinc Finger DNA-binding Proteins
(ZFPs) are transcription factors (TFs) useful for
116
gene modification; targeted insertion and targeted
gene knock-outs, providing a basis for efficient
site-specific genomic manipulation in mamma-
lian somatic cells and rapid cell line develop-
ment. Genes can be selectively added, modified
or knocked-out. This includes selection marker
genes (e.g., dhfr, GS); glycosylation enzymes; si-
alylation enzymes (e.g.,sialidases); pro-apoptotic
genes; proteases; and enhancers and repressors of
transcription/translation. Sangamo claims it can
design ZFPs to bind/target any endogenous DNA
sequence.
Homologous recombination can be stimulated
at the site of a DNA cut. The cell can be tricked
into using added DNA to repair the cut, resulting
in the added DNA being used to repair the break.
This can be used to target integration of an ex-
pression construct to a specific genomic location
(hot spot); can introduce changes into endogenous
genes; can optimize or de-optimize codons and
can make genes more human like. The modi-
fication approach can also be used to disrupt (or
knockout) a specific gene by simply not adding a
donor fragment.
Another application of the technology com-
bines the gene targeting function of an engineered
ZFP and the functional domain of a restriction
endonuclease, an enzyme that cuts DNA. The
resulting protein, a ZFP nuclease or ZFN may be
used to modify a specific DNA sequence within a
gene enabling corrrection or disruption of a gene
or the addition of a new DNA sequence.
Transcription factors typically consist of two
principal components: a DNA-binding domain,
which recognizes and binds to a specific DNA
sequence within or near a particular gene, and
a functional domain that causes the target gene
to be activated (turned on) or repressed (turned
off). The recognition domain is composed of
two or more zinc fingers; each zinc-complexed
finger recognizing and binding to a three base
pair sequence of DNA and multiple fingers can be
linked together to more precisely recognize longer
stretches of DNA. By modifying those portions
or the critical amino acid contacts of a ZFP that
interact with DNA, novel ZFPs can be engineered
capable of recognizing defined DNA sequences in
any gene. Engineered ZFPs with a variety of dif-
ferent functional domains to generate proteins can
activate or repress gene expression.
Use with: mammalian cells; plant cells
nal antibodies
Benefits: Benefits include:
No need for sustained presence of an inhibitor
(e.g., siRNA)
Both heterozygous and homozygous knockouts
are possible
ZFPs can be designed to recognize unique DNA
sequences within a large complex genome.
ZFP TFs can both activate or repress genes, en-
hancing their versatility.
ZFP TFs can be used to regulate the genes of hu-
mans, animals, plants, microbes and viruses.
ZFP TFs have proven effectiveness from cell
models through to the corresponding animal
models.
ZFP TFs can themselves be regulated, allowing
conditional and reversible regulation of a gene.
ZFP TFs can be used to regulate an endogenous
cellular gene rather than a transgene, providing a
workaround solution for genes whose cDNAs are
patented.
7,163,824, Regulation of endogenous gene ex-
pression in cells using zinc finger proteins, Cox,
et al., Jan. 16, 2007
Availability; Marketed by Sangamo for non-com-
mercial uses.
Licensing information: Contact Sangamo re-
In April 2007, Genentech reported licensing
Sangamos ZFP technology. Dow AgroSciences
has licensed it for use with plant expression
systems. Medarex is using the technology for
development of monoclonal antibodies, including
several currently in early trials. Sigma-Aldrich
has licensed it for production cell line develop-
ment.
117
Protozoa
207
Ciliate Performance Expression
System; CIPEX system;
Tetrahymena (protozoa)
expression
Cilian AG - Licensor, primary
Description: A Tetrahymena thermophila expres-
sion system is provided. Heat shock proteins
(HSPs) are used as heat-inducible (temperature-
controllable) promoters for the expression of
homologous and/or heterologous proteins in the
ciliate Tetrahymena thermophila. Tetrahymena
is a unicellular, apathogenic eukaryotic micro-
organism, and a very common organism in fresh
water like ponds and lakes. Ciliates of the genus
Tetrahymena naturally secrete large quantities of
enzymes, so-called lysosomal acid hydrolases,
into the surrounding culture medium. Ciliates
appear ideally suited in many respects for the
large-scale production of proteins. The organism
is already used commercially, e.g., for enzymes
manufacture.
During fermentation, expression can be
stopped easily by just lowering the temperature:
cells will recover and the biosynthesis can be
re-induced again. Tetrahymena thermophila offers
higher yields than other systems. As continuous
fermentation is a well established method for
ciliates and as thermal stress can be taken away
from the culture easily, the production cycle can
be repeated over and over again. These properties
of the invention lead to a new, efficient production
system.
Use with: Tetrahymena thermophila (protozoa)
Background: Protozoa represent an alterna-
tive for the recombinant production of heterolo-
gous proteins, however few protozoa have been
characterized to the extent necessary for routine
heterologous protein expression. Well-character-
ized pathogenic protozoa that have been geneti-
cally engineered to express heterologous proteins
include Trypanosoma cruzi, Trypanosoma brucei,
and Leishmania spp.
Tetrahymena is a ciliated eukaryotic unicellular
organism belonging to the regnum of Protozoa
and bearing two nuclei, a transcriptionally si-
lent, diploid germline micronucleus (MIC) and a
transcriptionally active, polyploid somatic mac-
ronucleus (MAC). In 1923, when Nobel Laureate
Andre Lwoff succeeded in growing Tetrahymena
in pure culture, the basis for exploiting this Al-
veolate as a model organism was laid. Milestone
discoveries made in Tetrahymena are the discov-
ery of dynein motors, telomeres, RNA-mediated
catalysis, telomerase and the function of histone
acety transferases in transcription regulation.
Within the last decades, molecular biological
techniques have been developed to alter Tetrahy-
menas genome and proteome. DNA transfection
methods range from microinjection into the MAC,
over electroporation into the MAC to biolis-
tic bombardment of MIC and MAC. Episomal
plasmids based on a rDNA-replicon are avail-
able, as well as knock-out/-in techniques based
on homologous recombination. Heterologous
expression of related species has been performed
and also endogenous proteins were silenced by a
novel antisense-ribosome-technique. Mammalian
and protozoan signal peptides function in T. cruzi
to target a heterologous protein to different cel-
lular compartments. The biotechnological poten-
tial of Tetrahymena has been proven in numerous
publications, demonstrating fast growth, high
biomass, fermentation in ordinary bacterial/yeast
equipment, up-scalability, existence of cheap and
chemical defined media. Background level of
transcriptional activity is low.
Protozoa represent an alternative for the re-
combinant production of heterologous proteins.
However few protozoa have been characterized
to the extent necessary for routine heterologous
protein expression. Well-characterized pathogenic
protozoa that have been genetically engineered
to express heterologous proteins are available
(see related entries). Protozoa are characterized
118
by a cell membrane glycosylphosphatidylinosi-
tol (GPI) anchoring system that allows targeted
surface expression, or display, of various
endogenous proteins, providing novel methods for
vaccine delivery.
Patents: WO/2007/006812, TETRAHYMENA
HEAT INDUCIBLE PROMOTERS AND THEIR
USE, HARTMANN, M., Jan. 18, 2007, assigned
to Cilian AG.
Licensing information: Contact Cilian AG.
The company offers CRO/CMO services, and
is developing its own T. thermophiia-expressed
protein (enzyme) for the treatment of pancreatic
insufficiency.
Further info.:
Weide T, Bockau U, Rave A, Herrmann L,
Hartmann MWW (2007), A recombinase system
facilitates cloning of expression cassettes in the
ciliate Tetrahymena thermophila, BMC Microbi-
ology 2007, 7:21
Herrmann L, Bockau U, Tiedtke A, Hartmann
MWW, Weide T (2006), The bifunctional dihy-
drofolate reductase thymidylate synthase of Tetra-
hymena thermophila provides a tool for molecular
and biotechnology applications, BMC Biotechnol-
ogy 2006, 6:21
208
LEXSY; Leishmania tarentolae
expression system - protozoa
Jena Bioscience - Licensor, primary
Description: Leishmania tarentolae protozoa
has been developed as an inducible expression
system. The L. tarentolae parasite natural host is
Tarentolae annularis (lizard). The systems offers
post-translational mammalian-like glycosylation
with full eukaryotic protein folding capabilities.
A wide range of proteins has been expressed us-
ing LEXSY. High level transcription of protein
coding genes can be mediated by a repressor-con-
trolled T7 RNA polymerase. LEXSY is particu-
larly useful for the expression of proteins that are
expressed at low yields or inactive in established
systems.
Jena Bioscience claims. The system repre-
sents a eukaryotic analogue of the most successful
bacterial expression architecture, and has dem-
onstrated that the system can inducibly over-
express intracellular proteins with a yield of over
300mg protein per liter of culture. Cell densities
in suspension cultures reach >10.sup.8 cells/ml.
The system is suitable for production of secreted
mammalian-like glycosylated proteins as shown
by the example human erythropoietin (EPO).
The N-glycosylation of EPO was exceptionally
homogenous, with a mammalian-type bianten-
nary oligosaccharide and the Man3GlcNAc2 core
structure accounting for >90% of the glycans
present. L. tarentolae is thus the first described
biotechnologically useful unicellular
eukaryotic organism producing biantennary fully
galactosylated, core-a-1,6-fucosylated N-gly-
cans.
Once established, the recombinant strains are
stable for hundreds of generations, which quali-
fies them for large scale fermentation. L. tarento-
lae handling procedures are amenable to automa-
tion.
Use with: Leishmania tarentolae (protozoan)
Patents: Relevant patents include WO0132896,
PROTEIN EXPRESSION SYSTEMS FOR
NON-PATHOGENIC KINETOPLASTIDAE,
KIRILL and Mathias, published 2001-05-10,
assigned to Jena Bioscience. The abstract states
The object of the present invention is a method
of recombinant protein production in vitro with
cultivated non pathogenic Kinetoplastidae para-
sites (Leishmania tarentolae, Crithidia fasciculata,
Wallaceina inconstans) (former Proteomonas
inconstans), (Leptomonas collos, Leptomonas
sp. Cfm, Leptomonas sp. Nfm or Leptomonas
seymouri). This includes description of cultiva-
tion conditions for several species that allow large
scale growth of these organisms on cheap media.
The method of the invention includes strategies
and construct description for generation of stably
119
transfected organisms capable of expression of
recombinant proteins.
Availability: Available for purchase for non-com-
mercial use from Jena Bioscience.
Licensing information: Contact Jena Bioscience
209
Perkinsus marinus expression -
protozoa express large proteins
University of Maryland Biotechnology Institute
- Licensor, primary
Description: The marine protozoa Perkinsus ma-
rinus is useful as recombinant host cells, particu-
larly for manufacture of large proteins, e.g., over
800 a.a. The organism offers scalable growth in a
full-defined cell free medium. This single-celled
eukaryotic organism is a natural pathogen of oys-
ters, with extensive genomic studies available, but
is non-pathogenic to humans and other species.
Use with: Perkinsus marinus (protozoan)
Patents: A PCT patent application was reported
as pending, but apparently has not published
yet. The primary inventor is Dr. J.A. Fernndez-
Robledo.
Licensing information: Contact University of
Maryland Biotechnology Institute (UMBI Refer-
ence: Robledo, 07-05; 07-006)
Products made using this tech.: This is noted as
useful for expression of large protozoan antigens,
e.g., for malaria vaccines.
Further info.:
1. Fernndez-Robledo JA, Lin Z, Vasta GR. 2008.
Transfection of the protozoan parasite Perkinsus
marinus. Mol Biochem Parasitol. 157(1):44-53.
2. Robledo JA, Courville P, Cellier MF, Vasta GR.
2004. Gene organization and expression of the
divalent cation transporter Nramp in the protistan
parasite Perkinsus marinus. J Parasitol. 90(5):
1004-14.
3. Gauthier, J.D., Feig, B. and Vasta, G.R. 1995.
Effect of fetal bovine serum glycoproteins on the
in vitro proliferation of the oyster parasite Per-
kinsus marinus: Development of a fully defined
medium. J. Eukaryot Microbiol. 42: 307-13.
4. Gauthier, J.D. and Vasta, G.R. 1995. In vitro
culture of the eastern oyster parasite Perkinsus
marinus: Optimization of the methodology. J.
Invertebr Pathol. 66: 156-68.
210
TetraExpress; Tetrahymena
(protozoan) expression
Tetragenetics, Inc. - Licensor, primary
University of Georgia - R&D
Description: The TetraExpress expression system
provides vectors bearing a beta-tubulin gene
enabling selection of transformed Tetrahymena
protozoa cells based on sensitivity to paclitaxel,
i.e., transgenic protozoan host cells are select-
able by negative selection using paclitaxel. The
beta-tubulin allele confers paclitaxel sensitivity on
cells. The technology is particularly well suited
to the production of eukaryotic membrane and
secretory proteins that are difficult to express in
conventional systems, including vaccine antigens
and monoclonal antibodies. Methods for rans-
formation and inducible high-level expression of
heterologous genes in the Tetrahymena system
have allowed Tetragenetics to produce foreign
membrane proteins in these cells at theoretical
maximal yields on the order of grams/liter of
culture.
Optionally, as with other Tetrahymena sys-
tems, expressed polypeptide can be displayed
with a GPI anchor on the plasma membrane of
the transgenic protozoan host cell and cleaved to
release the polypeptide (providing a novel vaccine
delivery method). Tetragenetics, Inc. is develop-
ing a Ichthyophthirius multifiliis antigen-bearing
Tetrahymena-expressed vaccine for use in fish
based on this.
Use with: Tetrahymena (protozoa) cells
ies
120
Background: See the CIPEX entry.
Benefits: Characteristics of the system include:
GPI-anchored membrane proteins from patho-
genic protozoa to levels of ~ 300mg/l
High-level expression of foreign genes under
the control of a strong inducible promoter using
a new rDNA-based vector that replicates to 9,000
copies per cell (18,000 copies of transgene)
Fully sequenced macronuclear genome (27K
genes with many encoding ion channels, ABC
transporters, GPCRs, Polo, Aurora and NIMA
kinases, etc.)
Successfully expressed human and other verte-
brate proteins with no codon optimization
Claimed advantages include:
Cells can be readily transformed with foreign
DNA using electroporation or biolistic bombard-
ment
Stably transformed cell lines are readily es-
tablished using artificial chromosomes or stable
integration via homologous recombination into
the host genome
Expressed membrane proteins are appropri-
ately modified, disulfide-bonded and targeted to
the cell surface
Protein glycosylation patterns have a simple
N-glycoside structure lacking non-mammalian
types of modifications such as mannose-rich
residues often found in yeast, or xylose residues
found in plants
Cells are amenable to high-throughput screen-
ing based on growth assays with fluorescent
(constitutive GFP-producing) or colorimetric (pig-
ment-producing) strains
6,846,481, Recombinant expression of heter-
ologous nucleic acids in protozoa, Gaertig, et
al., Jan. 25, 2005, assigned to the University of
Georgia.
Availability: Tetragenetics markets kits/reagents
Licensing information: Contact Tetragenetics
Products made using this tech.: IchVax is a
recombinant Ichthyophthirius multifiliis subunit
vaccine in development by Tetragenetics for the
prevention of white-spot disease in freshwater

fish.
Further info.:
Lin YK, Cheng G, Wang XT, Clark TG. 2002.
The use of synthetic genes for the expression of
ciliate proteins in heterologous systems. GENE
288 (1-2): 85-94.
Gaertig J, Gao Y, Tishgarten T, Clark TG, Dick-
erson HW. 1999. Surface display of a parasite
antigen in the ciliate Tetrahymena thermophila.
NATURE BIOTECHNOLOGY 17 (5): 462-465.
211
Tetrahymena thermophila
(protozoan) expression
Nutrinova Nutrition Specialties & Food Ingre-
dients GmbH - Licensor, primary
Celanese - Parent co.
Description: A Tetrahymena thermophila expres-
sion system has been developed, and is apparently
currently used for large-scale manufacture of
nutritional products. U.S. patent claims granted
to Nutrinova Nutrition Specialties & Food Ingre-
dients GmbH, Celanese AG, appear particularly
broad and may cover all T. thermophilia expres-
sion systems; and describe the first successful
expression of recombinant human serum albumin
(HSA) in Tetrahymena.
Use with: Tetrahymena thermophila; ciliate
protozoa cells
Patents: U.S. 6,962,800, Expression of recom-
binant human proteins in Tetrahymena, Kiy,
T., et al., Nov. 8, 2005, asisgned to Nutrinova
Nutrition Specialties & Food Ingredients GmbH.
The exemplary claim (no. 1) states, Method for
production of recombinant human proteins in Tet-
rahymena, comprising the steps: a) transforming
said Tetrahymena cells with recombinant DNA
containing at least one functional gene that codes
for said recombinant human protein, said gene
comprises a DNA sequence encoding a human
leader sequence; b) culturing said transformed
121
Tetrahymena cells and expressing said gene; and
c) isolation said human protein. The other claims
are comparably broad.
Licensing information: Contact Nutrinova re-
Animals, Misc.
212
Milk protein promoter -
proteins in milk of transgenic
animals
Advanced Cell Technology, Inc. - Licensor,
primary
Genzyme Transgenics Corp. (GTC) - Assignee,
patent
Genzyme Corp. - Parent co.
Description: The milk protein promoter is broad-
ly useful for expression of heterologous recom-
binant (glyco)proteins in the milk of transgenic
animals at high yields. The method of producing
a recombinant protein in transgenic nonhuman
mammals involves inserting into an embryo of
a mammal a DNA construct with a gene for a
desired protein under transcriptional control of
a milk protein promoter sequence that does not
naturally control transcription of the gene along
with a gene for a signal peptide enabling secre-
tion of the protein into the milk of the transformed
mammal; transferring the embryo to a female
mammal of the same species; allowing the em-
bryo to develop into an adult nonhuman mam-
mal whose genome includes this DNA construct;
inducing lactation in the mammal or its transgenic
progeny of the mammal; and collecting milk from
the animal and purifying the desired protein.
This permits the production of any desired
protein in an easily maintained stable, portable
culture system, i.e., a living domesticated mam-
mal, which is capable not only of producing the
desired protein, but also passing on the ability to
do so to its female offspring as well. Secretion of
the protein into the host mammals milk facilitates
purification and obviates removal of blood prod-
ucts and culture media additives.
Use with: mammals, non-human
ies
Transgenic animals secreting proteins into milk,
Gordon, et al., May 16, 2006, assigned to GTC
Biotherapeutics, Inc.
Licensing information: Am exclusive hicense
has been granted to Advanced Cell Technology.
Contact ACT and/or GTC, both of likely hold and
may offer other transgenic animal technologies.
Pharming B.V. and, presumably, other lead-
ers in transgenic animal development for protein
manufacture have licensed this technology.
213
Shrimp expression; Penaeus
stylirostris expression; Taura
Syndrome Virus (TSV) IRES
vectors - glycoproteins;
antibodies
Advanced BioNutrition Corp. (ABN) - Licen-
sor, primary
Description: Marine shrimp (Penaeus styliros-
tris) and a Taura Syndrome Virus (TSV) internal
ribosomal entry site (TSV IRES)-based shrimp
expression vector are useful for rapid scale-up
and manufacture of recombinant (glyco)proteins,
including vaccine antigens and monoclonal anti-
bodies.
Advanced BioNutrition Corp. (ABN) is
validating the use of crustaceans as biofactories
for the production of vaccines and other proteins
of pharmaceutical interest using its proprietary
transfection technology. The Company is also de-
veloping a high-density GMP-capable production
system for shrimp cultivation as well as meth-
ods for the isolation and purification of the final
products. ABNs goal (associated with its DARPA
122
contract, see below) is to demonstrate the capabil-
ity of production of three million doses of vaccine
or immunological-based therapeutic within 12
weeks of the first recognition of a new pathogen.
Use with: shrimp
ies
Patents: Related applications include EP1756276,
CRUSTACEAN EXPRESSION VECTOR,
Dhar, H.K., et al., 02/28/2007, assigned to Ad-
vanced BioNutrition Corp. (ABN).
Licensing information: Contact Advanced Bio-
Nutrition Corp. (ABN), which primarily develops
and markets nutrional supplies for shrimp aqua-
culture.
In April 2007, Advanced BioNutrition Corp
received a $1.7 million contract from the Defense
Advanced Research Projects Agency (DARPA),
Dept. of Defense (DOD), to develop new shrimp-
based methods for biopharmaceutical production,
with abstract, Methods and constructs for genetic
manipulation of one or more of shrimp, shellfish,
mollusks, and fish are disclosed. The nucleic
acid construct includes a promoter and an inter-
nal ribosome entry site of an insect picornavirus,
such as a cricket paralysis-like picornavirus. One
or more open reading frames can be operably as-
sociated with one or both of the promoter and the
internal ribosome entry site, and one or more pro-
teins or protein subunits can be expressed upon
introduction of the construct into a host cell, such
as into a shrimp. Method for producing immortal-
ized crustacean cell lines using enhancer elements
derived from shrimp and/or shrimp viruses are
also described.
214
Transgenic rabbits - humanized
polyclonal antibodies
Hoffmann-La Roche AG - Parent co.
Therapeutic Human Polyclonals Inc. - As-
signee, patent
Description: Vectors and methods enable manu-
facture of polyclonal antibodies in transgenic
rabbits. The animals contain one or more human-
ized immunoglobulin loci capable of undergoing
gene rearrangement and gene conversion in the
transgenic animals to produce diversified human-
ized immunoglobulins. The humanized antibodies
have minimal immunogenicity to humans.
Use with: rabbits
Use to make: antibodies, polyclonal
Production of humanized antibodies in trans-
genic animals, Buelow, et al., Oct. 31, 2006,
with claim 1, A transgenic vector comprising a
humanized Immunoglobulin (Ig) locus, wherein
(a) said humanized Ig locus comprises multiple
Ig gene segments, including multiple variable (V)
gene segments, multiple J gene segments, and
one or more constant region gene segments, (b) at
least one of said gene segments is a functional V
gene segment encoding a human V region amino
acid sequence, (c) said V gene segments are sepa-
rated only by non-coding, non-human sequences
derived from a non-human animal that generates
antibody diversity primarily through gene conver-
sion and/or hypermutation, (d) at least one of said
functional V gene segments encoding a human V
region amino acid sequence is placed downstream
of the other V gene segments, and (e) said Ig gene
segments are juxtaposed in an unrearranged, par-
tially rearranged or fully rearranged configuration,
and wherein, as a result of structural features (a)-
(e), said humanized Ig locus is capable of under-
going gene conversion and producing a repertoire
of humanized immunoglobulins with V region
amino acid sequences encoded by segments of
more than one V region gene, in said non-human
animal.
Licensing information: Contact for Therapeutic
Human Polyclonals Inc. (and/or Hoffmann-La
Roche).
Largely validating this technology, in April
2007 Hoffmann-La Roche paid $56.5 million to
acquire Therapeutic Human Polyclonals (THP;
founded by RCT and Sangstat). THP was the first
company to create transgenic rabbits with a hu-
123
man antibody repertoire for producing both mono-
clonal and polyclonal antibody therapeutics.
Mammalian
215
AmProtein vectors - stongest
mammalian vector set;
universal
AmProtein, Inc. - Licensor, primary
Description: AmProtein has developed vectors
enabling extremely high protein and antibody
expression, e.g., 60-120pg/cell/day in 3 weeks,
in fast-growing CHO-KS cells and apparently
broadly useful in eukaryotic cells. AmProtein
claims it has revolutionized protein manufacture
through use of two important proprietary systems:
1. The strongest mammalian gene expression
vector set known to date. 2. Single-use plastic
bioreactors with novel O2 transfer method; and
has discovered an universal mechanism govern-
ing mammalian gene expression. A universal
vector set works in most cell types, reaching 30-
60pg/cell/day in 96-well plate by a single stable
gene transfection. Also, AmProtein Expression
technology reduces conventional cell line devel-
opment with DHFR from 12 months to 1 month.
AmProtein has rapidly generated more than 10
CHO-KS cell lines, which produced more than
10 different proteins and antibodies at levels of
60-120pg/cell/day. All of these were achieved
in three weeks in fast-growing CHO-KS cells
after stable gene transfections. These results
have strongly suggested that this technology has
revolutionized the protein production industry
and made mammalian protein production rapid,
affordable and dominant over other transgenic
production methods. Currently, AmProtein is
working on the more detailed mechanism and is
applying this discovery to plant, yeast and insect
gene expression.
ies
Patents: No yet published patent applications
or patents assigned to Amprotein were retreived
from simple searches.
Availability: AmProtein offers development of
mammalian cell lines and manufacturing under
cGMP conditions at its cGMP production facility
in Hongzhou, China.
Licensing information: AmProtein is interested
in transfer and out-licensing of these technologies.
AmProtein claims it owns the most advanced
and innovative technologies for mammalian cell
line development used for recombinant proteins
and monoclonal antibodies production. AmPro-
tein is also developing innovative recombinant
therapeutics using its technology. While its public
pronouncements may seem boastful, the company
was founded by former Amgen scientists and their
claims may well be justified.
216
Anti-apoptosis expression
system; BCL-xL or BCL-2
expressing cell lines - CHO,
NS0, BHK, SP2/0-Ag14
primary
Description: Hamster or murine myeloma cell
lines, e.g., NS0, modified by introduction of
nucleic acid sequences that encode for an anti-
apoptosis gene (member of the Bcl-2 gene super-
family, BCL-xL or BCL-2), a selectable amplifi-
able marker gene, and at least one gene of interest
are useful for recombinant protein and antibody
manufacture. This substantially increases the level
of anti-apoptosis active genes within host cells,
and host cells have enhanced cell viability (live
longer) by delaying/inhibiting programmed cell
death naturally occurring in these cells. Anti-
apoptosis genes are provided that are suitable for
preparing host cells showing an enhanced cell
viability by delaying/inhibiting programmed cell
death.
Use with: myeloma cells; NS0 cells
124
ies
U.S. 20030219871, Host cells having improved
cell survival properties and methods to generate
such cells, Enenkel, B., et al., Nov. 27, 2003, as-
signed to Boehringer Ingelheim Pharma KG.
Ingelheim Pharma KG.
217
Autocatalytic cleavage sites;
Mengo virus vectors; Scission
cassettes - mammalian cells
Description: Autocatalytic cleavage sites (scis-
sion cassettes) found in picornaviruses, e.g.,
Mengo virus, are useful for protein expression.
When a fusion protein is expressed containing
these sequences, the autocatalytic cleavage sites
automatically release the foreign protein. A scis-
sion site is an RNA or DNA sequence encoding
a protein capable of undergoing non-molecular,
autocatalytic self-cleavage. Gene constructs from
Mengo virus (a replication competent picornavi-
rus) are provided that contain at least two such
sites. In a much preferred embodiment of the in-
vention, the construct is preferably a Mengo virus
vector with a shortened poly(C) region with no C
residues. WARF cites uses including vaccination,
with protein antigens expressed in hosts, but this
seems also relevant to more conventional rDNA
protein manufacture, including use in affinity fu-
sion protein purification schemes.
Also, the scission sites need not be identi-
cal - that is, they may come from two different
picornaviruses, e.g. one from Mengo and one
from encephalomycarditis (EMC) virus. The
method involves placing a foreign protein coding
sequence between the two autocatalytic cleavage
sites.
Use with: mammalian cells
Autocatalytic cleavage site and use thereof in a
protein expression vector, Palmenberg, et al.,
June 15, 1999, assigned to WARF.
Licensing information: Contact WARF.
218
Cell adhesion optimization;
cdkl3, siat7e, and lama4 genes
- antibody-expressing cells
primary
Description: Compositions and methods are
useful for improving the growth characteristics
of cells , including HEK-293 and CHO cells,
engineered to express antibodies or glycosylated
proteins. The expression of various genes (e.g.,
cdkl3, siat7e, or lama4) modulates growth char-
acteristics, such as adhesion properties, of the
cell lines, allowing increased recombinant protein
yields and reducing product production costs.
This technology provides a method for identifying
genes whose expression modulates cellular adhe-
sion characteristics.
Use with: HEK-293 cells; CHO cells
Patents: U.S. Provisional Application No.
60/840,381 filed 24 Aug 2006 (HHS Reference
No. E-149-2006/0-US-01)
Availability:
Licensing information: The technologys status
is described as Late Stage - Ready for Produc-
tion. Contact NIH. Available for exclusive or
non-exclusive licensing. The primary inventors
are: Joseph Shiloach and Pratik Jaluria (NIDDK,
NIH).
125
219
CMV (human) promoter;
Cytomegalovirus promoter;
Complete Control Inducible
Mammalian Expression System
- mammalian cells
University of Iowa Research Foundation
(UIRF) - Licensor, primary
Description: The human cytomegalovirus (CMV;
a member of the herpesvirus family) immedi-
ate-early regulatory DNA sequence, termed the
CMV promoter, is probably the most widely-used
promoter for manufacture of most mammalian
cell-culture manufactured recombinant proteins.
Upon infection of human or other susceptible
cells with CMV, the CMV promoter drives virus
protein expression and replication. This promoter
has been shown to be capable of significantly in-
creasing the expression of a wide variety of genes.
These genes cover a broad eukaryotic host range,
making the CMV promoter particularly useful for
recombinant protein production in diverse expres-
sion vectors/systems.
Benefits: HIghly effective; usable in most any
expression vector/system; well-studied; very
familiar to regulatory agencies
Patents: Use of the CMV promoter is covered
under U.S 5,168,062 and 5,385,839, assigned
to the University of Iowa Research Foundation
(UIRF). U.S. 5,168,062, Transfer vectors and
microorganisms containing human cytomegalo-
virus immediate-early promoter-regulatory DNA
sequence, has abstract, The cloning of a eucary-
otic promoter-regulatory region that functions
preferentially in human cells is disclosed. The in-
vention is exemplified by the cloning of a section
of the human cytomegalovirus genome compris-
ing a DNA sequence with regulatory and promot-
er signals and an initiation site for RNA synthesis.
The fragment, termed the human cytomegalovirus
(HCMV) promoter-regulatory sequence, was
obtained from purified HCMV DNA.
Availability: Available from many vendors for
non-commercial use.
Licensing information: Available for nonexclu-
sive commercial use licensing from the University
of Iowa Research Foundation.
Products made using this tech.: Marketed bio-
pharmaceuticals known to be manufactured using
the CMV promoter include:
1) Erythropoietin (epoetin delta; Dynepo; Gene-
Activated Erythropoietin; GA-EPO; EPO, gene
activated, recombinant) expressed in vivo in hu-
man cells; developed by Transkaryotic Therapies,
Inc., a subsidiary of Shire Pharmaceuticals Group
plc, which markets the product outside the U.S.
(blocked by Amgen EPO patents), with contract
manufacture by Lonza.
2) Myozyme [Alglucosidase alfa - Pompase;
alpha glucosidase; glucosidase alpha (rhGAA)]
manufactured and marketed by Genzyme Corp.
3) Aldurazyme [Laronidase - I2S; alpha-L-iduron-
idase, recombinant] manufactured by BioMarin
and marketed by Genzyme.
Note, there are probably many other biophar-
maceuticals manufactured using the CMV pro-
moter.
220
Cumate gene-switch; Q-
mate Inducible Expression -
mammalian cells
sor, primary
Description: The prokaryotic-based cumate-in-
ducible expression system allows induction of
expression in mammalian cells by addition of
p-cumate, and allows turning on the expression
of a given protein only when needed. Regulatory
mechanisms of bacterial operons (cmt and cym)
are used to regulate gene expression in mam-
malian cells using three different strategies. This
system is especially important for proteins that
126
would slow down or even prevent the growth of
the very cells that are used to produce the protein.
Since prokaryotic inducers usually do not have
any target sites in mammalian cells, the possibility
of interference with cellular processes is reduced.
The cumate-inducible expression system has
three configurations: the Repressor, the Transac-
tivator and the Reverse Transactivator. In the first
configuration, the repressor molecule, CymR, is
used to repress transcription from a mammalian
promoter (CMV5) by binding the CymR bind-
ing element, the cumate operator (CuO) placed
downstream of the initiation site. Addition of the
inducer (cumate), at concentrations that are not
toxic to mammalian cells causes a change in the
configuration of CymR such that it can no lon-
ger bind the DNA sequence of the operator, thus
relieving repression and allowing production of
the desired protein. In the second configuration, a
chimeric transactivator (cTA) is generated by the
fusion of CymR, as a DNA binding domain, with
the activation domain of VP16. A chimeric pro-
moter, CR5, is also created by inserting 6 copies
of the cumate operator sequences upstream of the
CMV minimal promoter. This scheme allows ac-
tivation of the CR5 promoter only in the absence
of cumate. Finally, a reverse CymR molecule was
generated by mutagenesis, such that it binds to the
CuO in the presence rather than in the absence of
cumate. The transactivator formed by the fusion
of the reverse CymR mutant with VP16, gives
rise to the reverse transactivator, which activates
expression from the CR5 promoter in the presence
of cumate. The ability to regulate target gene ex-
pression by any one of these three configurations
makes the system more versatile and thus suitable
for a large range of applications.
The system uses the cym operon, which in
Pseudomonas putida F1 controls the expression
of genes intervening in the transformation of p-
cymene (found in volatile oils from several plant
species) to p-cumate. The cmt operon, located
downstream of the cym operon, controls the
further degradation of the cumate molecule. Up-
stream of the cym operon lies a regulatory gene,
cymR, encoding a 28-kDa repressor molecule
(CymR), which monitors expression of both the
cym and cmt operons and is induced by p-cumate.
Qbiogene has combined Q-mate-based vectors
with the Q-mate CymR System, involving repres-
sion of gene expression is mediated by the cumate
repressor protein CymR bound to operator sites
in the absence of the inducer molecule cumate.
With cumate present, CymR binds to cumate and
undergoes a conformational change resulting in
its release from the operator sites. This permits
transgene expression. The reporter construct con-
sists of three components: a strong promoter, the
CymR binding sites (cumate operator sequences,
CuO), and a reporter or a transgene.
Benefits: This is based on a prokaryotic regula-
tory system adapted to construct inducible sys-
tems that function in mammalian cells. Since
prokaryotic inducer molecules usually do not have
any target sites in mammalian cells, the possibility
of interference with cellular processes is reduced.
Benefits include:
Controllable gene expression
Tight regulation of transgenes
Possibility to co-express GFP
Possibility to modulate levels of transgene
expression
Rapid response after induction
Patents: Related patent applications include
WO/2006/037215, EXPRESSION SYSTEM,
COMPONENTS THEREOF AND METHODS
OF USE, Yan, Y., et al., assigned to National Re-
search Council of Canada. Claim 1 (of 52) states,
An isolated polypeptide comprising a sequence
variant of CymR that exhibits a higher affinity
for a CymR response element when in a presence
rather than an absence of an effector molecule.
Availability: Kits/reagents are available from
Qbiogene for non-commercial uses.
Licensing information: Contact the Biotechnol-
ogy Research Institute, National Research Council
of Canada.
Further info.: :The cumate gene-switch: a system
127
for regulated expression in mammalian cells,
Mullick,A., et al., BMC Biotechnology 2006,
6:43.
221
Dihydrofolate reductase (DHFR)
System - selectable marker/
amplifcation; CHO and NS0
cells
Description: The Dihydrofolate Reductase
(DHFR) System is a selectable marker/amplifica-
tion widely used in CHO and NS0 cells and other
expression systems involving cells with inherent
low levels of DHFR expression. Use of the DHFR
as a selectable marker (see related entry) allows
selection of properly transformed cells that can
grow in absence of thymidine, glycine and hypo-
xanthine with methotrexate (MTX) positive selec-
tion/amplification, with the DHFR gene allowing
transformed cells to express DHFR, allowing
production of tetrahydrofolate and survival in the
presence of otherwise toxic methotrexate. Without
dhfr gene expression, cells are unable to produce
tetrahydrofolate, an essenatial metabolic cofactor.
Also, Dhfr- cells are only able to grow in media
containing thyrmidine, glycine and hypoxanthine,
which allow cells to overcome tetrahydrofolate
deficiency. Stable, DHFR+ transfected cells can
grown in unsupplemented media.
See also the entries for DHFR- CHO cell lines.
One of the disadvantages, compared to some
newer technologies, is that while the DHFR am-
plification system takes a longer time to develop
high-expressing clones through multiple rounds of
selection and amplification.
Use with: CHO cells; NS0 cells
Use to make: proteins; glcyoproteins
Background: Dihydrofolate reductase (DHFR;
5,6,7,8-tetrahydrofolate: NADP + oxidoreductase;
EC 1.5.1.3) catalyzes the NADPH-dependent
reduction of dihydrofolate to tetrahydrofolate, an
essential carrier of one-carbon units in the biosyn-
thesis of thymidylate, purine nucleotides, serine
and methyl compounds. It is an essential enzyme
in both eukaryotes and prokaryotes.
In selecting a preferred host cell for trans-
fection by DHFR vectors, it is appropriate to
select the host according to the type of DHFR
protein employed. If wild type DHFR protein
is employed, it is preferable to select a host cell
deficient in DHFR, permiting the use of the
DHFR coding sequence as a marker for success-
ful transfection in selective medium which lacks
hypoxanthine, glycine, and thymidine (e.g., see
related CHO cell line entries). On the other hand,
if DHFR protein with low binding affinity for
MTX is used as the controlling sequence, it is not
necesary to use DHFR resistant cells. Because
the mutant DHFR is resistant to methotrexate,
MTX containing media can be used as a means of
selection, provided that the host cells are them-
selves are methotrexate sensitive. Most eukaryotic
cells which are capable of absorbing MTX are
methotrexate sensitive, e.g., CHO-K1 (see related
entry).
Patents: Originally commercially developed by
Genentech, related patents have now expired and
the classic system is now in the public domain.
Exemplary patents include U.S. 4,713,339, Poly-
cistronic expression vector construction, Levin-
son, et al., Dec. 15, 1987, assigned to Genentech.
The abstract states: A polycistronic construction
of xenogeneic sequences expressible in eukary-
otic cells is described. DHFR encoding sequences
under control of the same promoter with another
foreign gene can be amplified with methotrex-
ate, and thus cause coamplification of the foreign
gene. Application of DHFR to the commercial
manufacture of Activase, recombinant tissue plas-
minogen activator (tPA), is claimed in 5,010,002
assigned to Genentech.
Licensing information: Now in public domain.
Products made using this tech.: Despite the
widespread promotion and use of the GS System
(see related entry), DHFR is still used for com-
mercial biopharmaceutical manufacture. For
example, products known to use the system (as
128
reported in Biopharmaceutical Products in the
U.S. and European Markets; see www.biopharma.
com) include
a) Myozyme [alglucosidase alfa; Pompase; alpha
glucosidase; glucosidase alpha (rhGAA) [recom-
binant] from Genzyme.
b) Activase, recombinant tissue plasminogen acti-
vator (tPA) from Genentech.
c) Kogenate FS [Antihemophilic Factor (Recom-
binant), Formulated with
Sucrose; Factor VIII, recombinant] from Bayer;
and relabeled as Advate by Baxter
d) Epogen (Erythropoietin, recombinant; EPO)
from Amgen and equivalent - Procrit from Ortho/
J&J.
e) Cerezyme (Imiglucerase -- beta-Glucocerebro-
sidase, recombinant) from Genzyme
f) Rebif (Interferon beta-1a) from Merck Serono
g) Enbrel (Etanercept - tumor necrosis factor
receptor2-immune globlulin G1 Fc fusion protein,
recombinant) from Wyeth
h) TNKase (Tenecteplase - TNK-tPA; tissue
plasminogen activator, TNK-, recombinant) from
Genentech
i) Luveris (Lutropin alfa - human luteinizing hor-
mone, recombinant) from Merck Serono.
222
Flp-In expression system; FLP-
Mediated Gene Modifcation
in Mammalian Cells; FLP
recombinase - mammalian
cells
Salk Institute for Biological Studies - Licensor,
primary
Life Technologies, Inc. - Retail seller
Description: A gene activation/inactivation and
site-specific integration system has been de-
veloped for mammalian cells. This is based on
recombination of transfected sequences by FLP,
a recombinase derived from Saccharomyces. In
several cell lines, FLP has been shown to rapidly
and precisely recombine copies of its specific tar-
get sequence. A chromosomally integrated, silent
reporter gene can be activated for expression by
FLP-mediated removal of intervening sequences
to generate clones of marked cells. Or, FLP can be
used to target transfected DNA to specific chro-
mosomal sites.
Thje Flp-In system provides efficient targeted
gene integration and saves time when generating
stable cell lines by eliminating the need to isolate
single clones. The Flp-In System integrates a
single copy of the gene of interest at a particular
genomic locus in every transfected cell. The time-
consuming process of isolation
and characterization of single cell clones is no
longer necessary, as a single Flp-In transfection
will yield an isogenic, homogenous population of
stably expressing cells. This integration of a gene
of interest into a single site limits chromosomal
position effects on gene expression, and allows
for reproducible analysis of multiple
genes (or variants of a single gene) without the
variability introduced by multiple integrated
copies or single integration events at different
chromosomal locations.
The Flp-In System, as sold by Invitrogen,
consists of three plasmids: the target site vector
(pFRT/lacZeo or pFRT/lacZeo2), an expression
vector (pEF5/FRT/V5-TOPO, pcDNA5/FRT/V5-
His or pSecTag/FRT/V5-His-TOPO), and the
vector that transiently expresses Flp recombinase
(pOG44). In a straightforward two-step process,
the gene of interest is inserted into the same
genetic locus in every cell (Figure 2). Since every
transfected cell is the same, one can
quickly select stable cells as a population rather
than isolating single clones.
FLP-mediated gene modification in mammalian
cells, and compositions and cells useful therefor,
Wahl, et al., August 5, 1997; and 5,677,177.
Availability: Invitrogen markets the FLP system
for research use only.
129
Licensing information: Contact the Salk Institute
for Biological Studies.
223
Gene-Activated (GA)
expression, in vivo -
mammalian cells
Transkaryotic Therapies, Inc. (TKT) - Licensor,
primary
Shire Pharmaceuticals Group plc - Parent co.
Cell Genesys, Inc. - R&D
Description: Methods and vectors are provided
enabling specific activation of expression of a
human gene within human cells, i.e., the genome
is altered to significantly increase expression of
the desired preexisting human gene. This method
is used by Transkaryotic Therapies, Inc. (TKT),
Shire Pharmaceuticals Group plc, for manufac-
ture of Dynepo (Gene-Activated Erythropoietin).
Exogenous DNA is stably integrated into the cell
genome or is expressed in the cells episomally.
The method may be used with primary and sec-
ondary somatic cells, such as fibroblasts, keratino-
cytes, epithelial cells, endothelial cells, glial cells,
neural cells, formed elements of the blood, muscle
cells, other somatic cells which can be cultured
and somatic cell precursors, which have already
been transfected with exogenous DNA encoding a
desired protein.
Patents: U.S. 5,994,127, In vivo production and
delivery of erythropoietin or insulinotropin for
gene therapy, Selden, et al., Nov. 30, 199, as-
signed to Transkaryotic Therapies, Inc. describes
Gene-Activated (GA) production of EPO and in-
sulinotropin. The abstract states, The present in-
vention relates to transfected primary and second-
ary somatic cells of vertebrate origin, particularly
mammalian origin, transfected with exogenous
genetic material (DNA) which encodes erythro-
poietin or an insulinotropin [e.g., derivatives of
glucagon-like peptide 1(GLP-1)], methods by
which primary and secondary cells are transfected
to include exogenous genetic material encod-
ing erythropoietin or an insulinotropin, methods
of producing clonal cell strains or heterogenous
cell strains which express eruthropoietin or an
insulinotropin, methods of gene therapy in which
the transfected primary or secondary cells are
used, and methods of producing antibodies using
the transfected primary or secondary cells. The
present invention includes primary and secondary
somatic cells, such as fibroblasts, keratinocytes,
epithelial cells, endothelial cells, glial cells, neural
cells, formed elements of the blood, muscle cells,
other somatic cells which can be cultured and
somatic cell precursors, which have been trans-
fected with exogenous DNA encoding EPO or
an insulinotropin, which is stably integrated into
their genomes or is expressed in the cells episom-
ally.
Licensing information: Contact TKT/Shire re-
Cell Genesys (now Abgenix, Inc.) originally
developed gene activation methods for recom-
binant expression of human therapeutic proteins.
In June 2002, Cell Genesys exclusively licensed
(divested) this technology for certain therapeutic
proteins to Transkaryotic Therapies, Inc. (TKT).
Cell Genesys received an up-front license fee of
$26 million, and was eligible to receive payments
up to $17 million upon the completion of certain
patent-related milestones. The agreement did not
include any royalty payments.
In Aug. 2004, Serono S.A. (now Merck Serono
S.A.) filed suit in U.S. District Court alleging that
TKT and Cell Genesys Inc. were infringing its
U.S. patent 5,272,071, assigned to Applied Re-
search Systems Ars Holding N.V., a subsidiary of
Serono S.A., concerning gene activation. The U.S.
patent office had previously declared an interfer-
ence brought by Cell Genesys between 5,272,071
(U.S. equivalent of EP 0505500) and Cell Gene-
sys gene activation patent application to which
TKT had a license. TKT reported that the patent
office had determined that both Serono and Cell
Genesys (TKT) were entitled to some claims in
130
their respective patent and application, and both
parties appealed this decision. [Final resolution of
this dispute was not retrieved, but TKT continues
to manufacture its GA-EPO product for marketing
where lack of Amgen patents covering EPO allow
this. In April 2005, Shire Pharmaceuticals Group
plc initiated acquisition of TKT for ~$1.6 billion.
Products made using this tech.: TKT uses this
method for manufacture of Dynepo [Erythropoi-
etin - epoetin delta; Gene-Activated Erythropoie-
tin; GA-EPO; EPO, gene activated, recombinant].
224
Glutamine synthetase
(GS) System - NS0, CHO,
mammalian cells
Celltech Biologics plc - R&D
Lonza Biologics plc - Licensor, primary
Description: The GS system allows selection and
amplification of successfully transformed cells.
Glutamine synthetase (GS) catalyzes formation
of glutamine from glutamate and ammonium
ion. This enzymatic reaction provides the only
pathway for glutamine formation in a mamma-
lian cells. Mammalian cells deficient in the GS
gene/protein require glutamine supplementation
to grow and survive. With these GS- cell lines, a
transfected GS gene can function as a selectable
marker by permitting growth in a glutamine-free
medium. The GS gene is used in recombinant vec-
tors as a selectable marker along with the desired
gene(s) for expression in cells deficient in GS,
e.g., NS0. Only successfully transformed cells,
i.e., carrying both the GS and desired gene(s), are
capable of producing their own GS enzyme and
surviving in glutamine-deficient culture media,
with this allowing easy, reliable selection of
transformed cells. Other cell lines, such as CHO,
express sufficient GS to survive without exog-
enous glutamine. With these cells, the specific GS
inhibitor, methionine sulphoximine (MSX), can
be used to inhibit endogenous GS activity such
that only transfectants with additional GS activity
can survive. This can be used to amplify the target
gene (also amplifying the GS gene, so success-
fully transformed cells overproduces glutamine).
Use of a weak promoter on the GS gene and
strong promoter on product gene selects for rare
integration into transcriptionally efficient sites
in the cells genome. Using appropriate screen-
ing techniques, amplification is often not found
to be necessary. Particularly with constitutively
GS-minus cell types, high yielding cell lines can
often be identified on first selection. However,
yield improvements may be made by the addition
of MSX, particularly for GS-plus cell lines such
as CHO. Hybridoma cells expressing GS produce
less ammonia/ammonium byproducts, can be cul-
tured in media with glutamine, increasing produc-
tion of monoclonal antibodies.
In practice, the most commonly used cell
lines are reported to be NS0 and CHO. The GS-
NS0 cell line has been used most commonly for
antibody production. GS-CHO has been used to
express a large range of other protein types.
Maximum expression levels attainable depend
on the product, but cell lines have been created
that are capable of producing up to 3.6g/L of
recombinant antibody, with specific production
rates of 15-65pg/cell/day.
Use with: mammalian cells; CHO cells; NS0
cells
ies
speed (DNA to IND in 12 months possible)
ease of use (support package available to licens-
ees)
high yielding cell lines (>5g/L for GS-CHO, in
chemically-defined, protein-free media)
regulatory acceptance, with multiple approved
products
predictable scale Up (1L to 5000L or more)
Patents: The glutamine synthetase (GS) gene
amplification/expression system used to select
and amplify transformed cells is based on patents
issued to Celltech Biologics plc, now assigned to
Lonza Group plc, subsidiary of Alusuise-Lonza
Group. Related patents include U.S. 5,770,359
131
and 5,747,308 (coassigned to the University
of Glasgow). These patents describe dominant
selectable markers for use in co-amplification of
non-selected genes and transformation of host cell
lines to glutamine independence. The GS gene
is used in recombinant vectors as a selectable
marker along with (an)other gene(s) for expres-
sion in cells deficient in GS, e.g., NS0 cells. Only
successfully transformed cells are capable of
producing their own GS enzyme and surviving
in glutamine-deficient culture media, with this
allowing easy, reliable selection and amplification
of transformed cells.
Licensing information: The system including
vectors, GMP host cells, and technical manuals
can be supplied under Research Evaluation or
License Agreements. Contact Lonza.
Products made using this tech.: Lonza reports,
Over 70 biotechnology and pharmaceutical
companies have successfully used this system
[presumably, licensed], the GS gene expression
system, worldwide. Lonza Biologics has itself
created over 250 cell lines using the GS System.
Marketed products reported by Lonza as manu-
factured using the GS Gene Expression System
include:
a) Zenapax [Daclizumab - interleukin-2 alpha re-
ceptor monoclonal antibody, recombinant], manu-
factured and marketed by Hoffmann-La Roche
Inc., originally developed by Protein Design Labs.
(Protein Biosciences).
b) Synagis [Palivizumab - MEDI-493; respira-
tory syncytial virus (RSV) monoclonal antibody,
recombinant] developed, manufactured and mar-
keted by Medimmune, now part of AstraZeneca.
Other marketed products, as reported by Bio-
pharmaceutical Products in the U.S. and European
Markets (www.biopharma.com), include:
a) ReoPro [Abciximab - Platelet monoclonal anti-
body, recombinant chimeric] from Centocor/J&J
b) Saizen; Serostim; Zorbtive (multiple trade
names for Somatropin (rDNA origin) from EMD
Serono, Inc., Merck Serono S.A.
c) Zenapax [Daclizumab - Interleukin-2 alpha
receptor monoclonal antibody, recombinant] from
Hoffmann-La Roche AG, with Lonza a contract
manufacturer
d) Remicade [Infliximab - cA2; Avakine; tumor
necrosis factor monoclonal antibody, recombi-
nant] from Centocor/J&J
e) Campath [Alemtuzumab - MabCampath; CD52
monoclonal antibody, recombinant humanized rat]
from Genzyme Corp. and Schering-Plough
f) Mylotarg [Gemtuzumab Ozogamicin - CMA-
676; recombinant humanized CD33 monoclonal
antibody-calicheamicin cytotoxin conjugate (im-
munotoxin)] from Wyeth
g) Erbitux [Cetuximab - IMC-C225; epidermal
growth factor receptor monoclonal antibody,
recombinant] from Bristol-Myers Squibb and
ImClone
h) Xigris [Drotrecogin alfa (activated) - recombi-
nant human activated protein C; rhAPC] from Eli
Lilly, and
i) Rituxan [Rituximab - IDEC-C2B8; MabThera;
CD20 monoclonal antibody, recombinant] from
Hoffmann-La Roche AG.
225
GPEx Gene Product Expression
Technology - mammalian; CHO
Catalent Pharma Solutions - Licensor, primary
Blackstone Group - Parent co.
Cardinal Health, Inc. (see Catalent Pharma
Solutions) - Former inv.
Gala Biotech - Assignee, patent
Description: GPEx retrovector technology is
based on a pseudotyped high-titer vector, which
ensures that stable transductions occur in virtually
100% of target cells (any mammalian cell line,
but usually CHO cells). Virtually any cDNA, in-
cluding monoclonal antiibodies, can be packaged
into the GPEx retrovector and used to transduce
diverse mammalian cell lines. Catalent claims,
The GPEx gene insertion technology produces
mammalian cell lines - typically CHO cells - that
deliver higher initial yields of proteins than any
conventional system based on transfection or elec-
troporation.
132
This technology typically produces a stable
clonal cell line in about four months for proof of
concept studies. High producing cell lines suitable
for Master Cell Banking (MCB) and subsequent
cGMP production are typically obtained in less
than five months after receipt of client cDNA.
Selected clones have consistent productivity from
25 to over 50 picograms per cell, per day. Process
development with minimal inputs can consistently
reach several gm/L in six months in batch-fed
bioreactor systems.
One reason for higher yields is that the GPEx
retrovector targets high-expressing chromosomal
matrix attachment sites (MARS) in the target cell
genome, leading to markedly higher expression of
the gene in transduced cells compared with other
systems. Another important reason is that the
optimization of GPEx cell lines employs an itera-
tive insertion process that drives up the inserted
gene copy number and proportionally increases
protein expression levels. The optimized cell line
may therefore have a dozen or more copies of the
desired gene, all stably inserted and expressing
the target protein.
Cardinal manufactured its first Mab for clinical
use (for Ludwig Institute for Cancer Research)
using GPex in 2005.
Use with: mammalian cells; CHO cells; BHK
cells
ies
Benefits: GPEx eliminates the need for selectable
markers, saving time and cost. In comparison,
conventional transfection or electroporation meth-
ods can produce an insertion frequency as low as
1 out of every 100,000 cells, necessitating addi-
tional selection steps and use of selectable marker
genes. Other methods carry the risk of gene exci-
sion or silencing, making them relatively unstable.
Using conventional gene insertion and expres-
sion techniques, developing a stable production
cell line for a target protein can take as long as
18 months. GPEx overcomes the inefficiencies of
conventional systems and delivers a stable pro-
duction cell line for your target protein in as little
as 4.5 months.
Monoclonal antibody heavy- and light-chain
coexpression
Receptors, coexpressed with a secreted sur-
rogate
Inactive proteins, coexpressed with associated
processing enzymes
Sequential transduction can be used to intro-
duce required genes without antibiotic selection
Expressions can be enhanced by clonal selec-
tion or additional rounds of transduction without
antibiotic selection
Cell lines are developed in serum-free culture
media
No antibiotic selection requirement
Host cells containing multiple integrating vec-
tors, Bremel, R.D. ,et al., Feb. 19, 2008, assigned
to Gala Design, Inc., and licensed to Chronos.
The abstract states, The present invention relates
to the production of proteins in host cells, and
more particularly to host cells containing multiple
integrated copies of an integrating vector. Suitable
integrating vectors for use in the present inven-
tion include retrovirus vectors, lentivirus vectors,
transposon vectors, and adeno-associated virus
vectors. Methods are provided in which the host
cells are prepared by using the integrating vectors
at a high multiplicity of infection. The host cells
are useful for producing pharmaceutical proteins,
variants of proteins for use in screening assays,
and for direct use in high throughput screening.
Availability: GPEx technology has been used to
generated over 150 cell lines for production of
recombinant proteins and antibodies. Cardinal/
Catalent has concluded agreements with multiple
companies for development of GPex cell lines,
including with Centocor/J&J and Trubion. The
company is developing a cell line for expression
of a prrostate specific membrane antigen (PSMA)
Mab for PSMA Development Company LLC
(PDC).
Licensing information: Cardinal/Catalent does
not actually licence GPEx to any firm, although,
its would consider doing so if we were presented
with the right size deal from a large company.
133
Rather, it offers in-house R&D and contract man-
ufacturing services, including strain development
and optimization, and full commercial manufac-
turing capabilities. Usually at the conclusion of a
feasibility study, the client can out-license the cell
line for production at the facility of your choice,
or continue to work with Catalent.
Cardinal (before it became Catalent in sp-
ing 2006) said it was facing a robust market
in terms of interest in its GPEx technology and
already had 30 various deals in place.
Products made using this tech.: Cardinal re-
ported that a biogeneric product is manufactured
using GPex.
226
MARtech; Matrix Attachment
Regions; Scaffold Attachment
Regions; SARs - mammalian
cells
Selexis - Licensor, primary
Ecole Polytechnique Fdrale de Lausanne
(EPFL) - R&D
Description: The MARtech expression system
provides increased recombinant protein expres-
sion in mammalian cells in suspension and in
serum-free media by more than 20 fold. MARtech
is based on human Matrix Attachment Regions
(MARs) genetic elements which control the
dynamic organization of chromatin. MARs func-
tion by insulating nearby genes from the effect of
surrounding chromatin, thereby increasing copy
number dependent, position-independent, expres-
sion of genes. MARtech increases the number
of independently transformed cells that express
the desired protein and promotes higher amounts
of recombinant protein produced by these cells.
MAR-binding proteins may also recruit histone
acetyltransferases and ATP-dependent chromatin
remodeling complexes, and thereby increase gene
expression.
MARs (Matrix Attachment Regions), also
called SARs (Scaffold Attachment Regions), are
300-3000 bp long DNA elements proposed to play
a role in nuclear and chromosomal architecture.
They attach chromatin to proteins of the nuclear
matrix and thereby partition the eukaryotic ge-
nomes into independent chromatin loops. Because
of their co-localization with transcription units
and regulatory elements in genomes, MARs have
been implicated in the regulation of mammalian
gene expression.
Benefits: Gene transfer in eukaryotic cells and
organisms suffers from epigenetic effects that
result in low or unstable transgene expression and
high clonal variability. Use of epigenetic regula-
tors such as matrix attachment regions (MARs) is
an approach to alleviate these unwanted effects.
Advantages over traditional approaches in-
clude:
High Yield : Predictable high expression levels at
rates typically ranging from 50 to 80 p/c/d/
Speed : High productivity clones approximately 5
weeks
Expression : High levels and long-term expression
of therapeutic proteins
Stability : Not associated with chromosomal rear-
rangements nor chromosomal breaks
Random integration a single site
Integration position sites known to be very stable
(sub-telomeric)
No chromosomal breaks
No multiple integration sites
Effective : Very effective in a variety of cell lines
Regulatory : Eeasily analyzed for regulatory ap-
proval.
7,129,062, Matrix attachment regions and
methods for use thereof, Mermod, et al., Oct. 31,
2006, assigned to Selexis S.A.
Licensing information: Contact Selexis regard-
Further info.:
Use of scaffold/matrix-attachment regions for pro-
tein production, Florian M. Wurm, CHAPTER 10,
in Makrides (Ed.) Gene Transfer and Expression
134
in Mammalian Ce!!s, by Wiley, 2008.
227
RheoSwitch Mammalian
Inducible Expression System;
RheoSwitch Ligand RSL1
promoter ; Ecdysone receptor
induction - mammalian cells;
adjustable expression
RheoGene, Inc. - Licensor, primary
New England Biolabs - Retail seller
Description: The RheoSwitch Mammalian
Inducible Expression System permits maxi-
mum control of gene expression in mammalian
cells. Analogous to the operation of a rheostat,
RheoSwitch technology allows induction and
adjustable control of gene expression. Vectors and
steroid hormone 20-OH ecdysone (beta-ecdysone;
EcR) control elements from Drosophila enable
control of expression using a synthetic ligand of
an insect steroid receptor superfamily. The steroid
hormone 20-OH ecdysone controls timing of
development in many insects. Precise regulation
of gene expression is achieved through the highly
specific interaction of a synthetic inducer, Rheo-
Switch Ligand RSL1, with a chimeric bipartite
nuclear receptor. This receptor is activated in the
presence of RSL1 ligand, and the level of gene
expression can be regulated by adjusting the con-
centration of RSL1 ligand in the culture media.
The system is built on a two-hybrid switch
format that activates transcription in the presence
of inducer and represses gene expression in its
absence. The synthetic receptor is composed of
two proteins, RheoReceptor-1 and RheoActiva-
tor, that dimerize to make a holoreceptor. Both are
expressed from strong constitutive promoters on
plasmid pNEBR-R1. The RheoReceptor-1 protein
is a highly engineered ligand-binding domain
(LBD) of an insect EcR nuclear receptor fused to
the yeast GAL4 DNA binding domain. The Rheo-
Activator protein is an insect/mammalian RXR
hybrid LBD fused to the viral activation domain
VP16. RSL1 ligand is synthetic diacylhydrazine,
[N-(2-ethyl-3-methoxybenzoyl)-N-(3,5-dimeth-
ylbenzoyl)-N-tert-butylhydrazine]. It is one of a
family of compounds that have been found to act
as non-steroidal ecdysone agonists and can func-
tion as gene inducer.
The gene to be expressed is cloned into the
pNEBR-X1 plasmid under control of five tandem
repeats of the GAL4 response element (5XRE). In
the absence of RSL1 ligand, the receptor represses
transcription by binding to the GAL4 elements in
a transcriptionally inactive conformation. Upon
induction, the RSL1 ligand tightly binds and
changes the conformation of the RheoRecep-
tor-1 protein which stabilizes the holoreceptor
heterodimer on the 5XRE. The activated holore-
ceptor and the VP16 activation domain bind and
recruit to the promoter transcriptional coactivators
along with basal machinery, resulting in a highly
induced transcriptional state. This high induction
of transcription coupled with extremely low basal
expression results in extremely high induction
levels (greater than 10,000 fold induction), with a
degree of control that is superior to other systems.
Precise regulation of expression levels
Negligible basal expression
Synthetic inducer and engineered receptor elimi-
nate non-specific side effects
Greater than 1,000 fold induction levels routinely
obtained
No special culture and media requirements
The RheoSwitchs precise control is unrivaled
among mammalian expression systems, giv-
ing negligible levels of basal expression in the
absence of inducer and greater than 10,000 fold
induction when RSL1 ligand is present. Unlike
other systems, which rely on steroids or other
drugs, the synthetic ligand RSL1 shows no pleio-
tropic effects in mammalian cells and exhibits no
cross talk with endogenous transcription factors.
The RheoSwitch System has no special culture
media requirements.
135
Ecdysone receptor-based inducible gene ex-
pression system, Palli, et al., August 15, 2006,
assigned to RheoGene, Inc.
RHeoGene holds an exclusive license to Stan-
ford University patents U.S. 5,514,578, 6,245,531
and EP Patent 0517805 that cover certain ecdy-
sone-based products.
Availability: The system is available from Invit-
rogen, New England Biolabs and, perhaps, other
vendors for non-commercial uses.
Licensing information: Contact RheoGene re-
228
Selexis Genetic Elements
(SGEs) - mammalian cell lines
Selexis - Licensor, primary
Description: Selexis Genetic Elements (SGEs)
enable the rapid development of high performance
mammalian cell lines. SGEs can boost the number
of cells that actively produce recombinant pro-
tein after transfection up to 20-fold in serum-free
media. Selexis Genetic Elements (from 1.6Kb to
4Kb in length) are used to control the promoter
accessibility of the expression cassette. SGEs are
based on human DNA sequences which were dis-
covered using a novel bioinformatics tool and are
now applied to control the dynamic organization
of transgenes in chromatin. SGEs function by in-
sulating nearby genes from the effect of surround-
ing chromatin, thereby increasing copy number
dependent, position-independent expression of
genes. For this reason, SGEs increase the number
of independently transformed cells that express
desired protein and promote higher amounts of
recombinant protein produced by these cells.
Selexis Genetic Elements can reduce tradi-
tional cell line development from 12 months to ~5
weeks. The increased number of clones that have
integrated the transgene cassette (post transfec-
tion) allows a fast isolation of high-yield express-
ing cell clones.
Selexis Genetic Elements has been tested in a
variety of cell types including: CHO, HEK 293,
BHK, C2C12, and a B cell derivative. Selexis
with its partners have tested over 30 cell lines.
These include cell lines expressing green fluores-
cent protein (GFP), various monoclonal antibod-
ies, a clotting factor, the EGF receptor, EPO as
well as membrane bound receptors. Selexis has
tested different versions of the CMV promoter as
well as the ubiquitin promoter and the SV40 early
promoter.
Over 10 cell lines have been scaled up and
tested in bioreactors. The Selexis Genetic Ele-
ments cell lines were compared with cell lines
made the traditional manner. The cell lines made
with Selexis Genetic Elements were found to
maintain the high level of expression and yield
stable production at this high level during optimi-
zation in the bioreactor. The yield improvement
seen during the creation of the cell lines translated
into increased yields in the bioreactor.
Use with: mammalian cells; CHO; HEK-293;
BHK
ies
Background: It is believed SGEs function by
enabling the formation of highly transcribed loops
within the chromatin structure. When combined
with optimal cell line development procedure (the
Selexis SURE process; see related entry) a higher
number of high performance cells are generated.
Benefits: Aadvantages over traditional approach-
es include:
High Yield : Predictable high expression levels at
rates typically ranging from 50 to 80 p/c/d/
Speed : High productivity clones approximately 5
weeks
Expression : High levels and long-term expression
of therapeutic proteins
Stability : Not associated with chromosomal rear-
rangements nor chromosomal breaks; Random
integration a single site; Integration position sites
known to be very stable (sub-telomeric); No chro-
mosomal breaks; No multiple integration sites
Effective : Very effective in a variety of cell lines.
136
Regulatory : Selexis Genetic Elements cell lines
can be easily analyzed for regulatory approval.
Selexis provides a step-by-step description of
the assembly of all gene fragments present in its
vectors, including the Selexis genetic elements,
to form the final gene constructs. The entire
sequences of the expression vectors, including
Selexis genetic elements, are defined, satisfying
the requirements of the points to consider docu-
ment from the FDA.
Patents: No specific yet published patents/ap-
plications covering this technology were retrieved
from simple searching.
Licensing information: Contact Selexis.
As of the end of 2007, two companies had
taken cell lines developed with the Selexis tech-
nologies forward to produce cGMP material for
use in human clinical trials.
229
STabilizing Anti-Repression;
STAR elements - expression
enhancement; mamalian cells
Chromagenics BV - Former inv.
Crucell Holland B.V. - Licensor, primary
Description: STAR technology involves genetic
elements, called STAR elements, that enable
stable and high-yield gene expression important
to recombinant antibody and protein production in
mammalian cells. The technology has the poten-
tial to increase production yields, thereby reduc-
ing production costs. STAR is effective for pro-
duction of antibodies and proteins in mammalian
cell lines, such as Crucells PER.C6 human cell
technology and the widely used Chinese hamster
ovary (CHO) cell lines.
STAR sequences are nucleic acid sequences
that comprise a capacity to influence transcription
of genes in cis. Typically, although not necessar-
ily, the STAR sequences do not code by them-
selves for a functional protein. STAR technology
was discovered by Dr. Arie Otte (Nature Biotech-
nology 2003 May, 21 (5)) who founded Chroma-
genics B.V., a spin-off company of the University
of Amsterdam acquired by Crucell in March 2004.
Use with: CHO cells; PER.C6 cells; HEK-293
cells; NS0 cells; mammalian cells
ies
Means and methods for regulating gene ex-
pression, Otte, et al., Sept. 11, 2007 assigned
to Chromagenics, with claim 1, A method for
producing a proteinaceous molecule in a cell
comprising: providing a cell selected from the
group consisting of a cell having an adenovirus
Early Region 1 (E1) sequence, a HuNS-1 my-
eloma cell, a 293 cell, a CHO cell, a Vero cell,
a WERI-Rb-1retinoblastoma cell, a BHK cell,
a non-secreting mouse myeloma Sp2/0-Ag 14
cell, a non-secreting mouse myeloma NSO cell,
and an NCI-H295R adrenal gland carcinoma
cell; wherein said cell comprises an anti-repres-
sor activity sequence operably linked to a nucleic
acid sequence encoding a proteinaceous molecule
of interest, wherein said anti-repressor activity
sequence comprises SEQ ID NO:7; expressing the
proteinaceous molecule in said cell; and isolating
said proteinaceous molecule.
Licensing information: Contact Crucell.
Genentech and Medarex have been reported to
be testing STAR.
230
Ubiquitous chromatin opening
element (UCOE) expression -
mammalian cells
Celliance, Serologicals Ltd., now Millipore
- Licensor, primary
Innovata plc - Assignee, patent
ML Laboratories - R&D
Kings College - R&D
Description: The Ubiquitous chromatin opening
element (UCOE) system is an efficient expression
system for the rapid production of recombinant
proteins in mammalian cells, with rapid and high-
137
yielding production of correctly folded and gly-
cosylated proteins. Over 50% of UCOE-derived
clones have higher expression than the best non-
UCOE-derived clones, UCOE-derived vectors
produce substantially more expressing clones than
non-UCOE-derived vectors. High-yielding cell
lines can be derived in less than 60 days, without
amplification. When selected clones are grown
in bioreactors, yields of 1.5-2.0g/L can easily be
achieved in non-optimized conditions.
UCOE gives major improvements in gene
expression in stably-transfected mammalian cells
through effects on the structure of chromatin.
UCOE prevents transgene silencing and gives
consistent, stable and high-level gene expres-
sion irrespective of the chromosomal integration
site. UCOE expression elements are small DNA
elements (isolated from the area around house-
keeping genes, which need to be active most of
the time) that create a transcriptionally active,
open chromatin environment around an integrated
transgene, maximizing its potential to be tran-
scribed into protein, irrespective of the position
of the transgene in the chromosome. This has
potential advantages over transient transfection,
minimizing the quantities of DNA and costly
transfection agent, as well as allowing aliquots of
the pool to be stored frozen for future use if the
protein is required at a later stage.
UCOE improves the yield, consistency and
stability of protein production in cultured mam-
malian cells, allowing simpler and quicker
generation of stable, highly productive cell lines
suitable for larger-scale manufacture of protein
therapeutics. Ubiquitous Chromatin Opening Ele-
ments (UCOEs) have been isolated which ensure
efficient expression in a wide range of cell types
including CHO-K1, CHO-S, HeLa, and NS0 cells.
UCOE containing vectors have been shown to
markedly enhance a wide variety of intracellular,
and secreted proteins. Inclusion of a UCOE in
an expression vector permits efficient expression
in the vast majority of stable clones, while with
conventional vectors, only a minor proportion of
transfectants show high-level expression. There is
no need for gene amplification and as the vectors
integrate at low copy number, they are stable ge-
netically and expression has been demonstrated to
be maintained over 130 generations. The size of
the latest generation, optimized UCOEs has been
reduced to >2kb.
UCOEs allows the rapid isolation of high-
yielding, stably expressing clones and have
been evaluated with numerous therapeutic genes
including erythroprotein (EPO) and antibodies.
Typically a screen of 96 antibody expressing
clones gives 5-10 clones yielding greater than
1g/L in shake flasks within 8 weeks of transfec-
tion. Expression of antibody has been shown to be
stable for at least 50 generations.
Use with: mammalian cells; CHO cells; NS0
cells
ies
Background: When transfected genes integrate
into mammalian chromosomes, the structure
of the chromatin at the site of integration has a
profound effect on expression of the transgene.
Consequently, only relatively rare transfected
clones where the expression vector has integrated
into open chromatin show efficient expression
of the transgene. The successful integration of a
transgene into mammalian cells is largely de-
pendent upon the chromatin structure around the
integration site. Most mammalian chromatin ex-
ists in the closed (heterochromatin) form, which is
transcriptionally silent. With conventional vectors,
most integrations suffer from position effects and
chromatin shutdown, which leads to gene-silenc-
ing and unproductive integration events. As a
result, pools of stably-transfected cells generally
show low productivity and stability, as only a few
cells might be good producers, and gene silencing
and overgrowth of non-producers rapidly dimin-
ish the overall productivity of the pool. This also
makes it very difficult and time consuming to find
high-expressing stable clones needed for large-
scale manufacturing.
Benefits: UCOE benefits include:
Rapid production of gram quantities of pro-
138
teins using stable pools
Ensures high-level expression, independent of
the site of chromosomal integration
Evaluated for recombinant antibodies, secreted
and intracellular proteins
Rapid isolation of stably transfected clones
Unique mechanism that prevents gene silenc-
ing through effects on chromatin structure
Majority of clones show high-level expression
No extensive cell line screening
Cell lines are stable over 130 generations
Small (4-8kb) DNA elements are capable
of making two protein chains per plasmid, e.g.,
antibodies
Another advantage of this system is the abil-
ity to produce frozen stable pools, which can be
used in subsequent fermentations. The technology
represents a significant cost saving both in the
production of research grade quantities of product
and in the selection of high expressing clones for
large-scale manufacturing.
Polynucleotide, Antoniou, et al. (Kings Col-
lege, London), Feb. 10, 2004, assigned to ML
Labs., with its abstract stating, The present
invention relates to a polynucleotide comprising
a ubiquitous chromatin opening element (UCOE)
which is not derived from an LCR. The present
invention also relates to a vector comprising the
polynucleotide sequence, a host cell comprising
the vector, use of the polynucleotide, vector or
host cell in therapy and in an assay, and a method
of identifying UCOEs. The UCOE opens chro-
matin or maintains chromatin in an open state and
facilitates reproducible expression of an operably-
linked gene in cells of at least two different tissue
types.
Other UCOE patents assigned to ML Labs.
include U.S. 6,881,556 and 6,689,606
Licensing information: Contact Celliance/Mil-
lipore. UCOE was originally commercially
developed by Innovata plc (formerly ML Labs.
and Quadrant), and has been exclusively licensed
in 2005 to Celliance Corp., now part of Millipore
Corp.
Products made using this tech.: This technology

has been licensed and is used by contract manu-
facturer (CMO) Cobra Biomanufacturing plc.
The technology has been licensed by more
than 50 companies in North America, Europe and
Japan. including Bayer HealthCare AG (in Feb.
2008), Medarex and Maxygen. The full terms
of Bayers licensing agreement have not been
disclosed, but the agreement included an upfront
fee of $5 million to cover both R&D and protein
production throughout the company.
231
Whey acidic protein (WAP) milk
promoters - mammals
primary
Genzyme Corp. - Assignee, patent
Description: Whey acidic protein (WAP) milk
promoters are useful to drive expression of de-
sired proteins in the milk of transgenic animals,
mice, sheep, pigs, goats and cows, with animals
passing on their genome to offspring. This WAP
promoter platform technology provides a viable
alternative to other milk protein promoters for
generation of transgenic animals expressing pro-
teins in their milk.
Use with: mammals; mice, sheep, pigs, goats
and cows
Patents: U.S. 6,727,405, Transgenic animals
secreting desired proteins into milk, Gordon,
K., et al., has been issued (expiry date 2021) to
NIH scientists and their collaborators (Genzyme).
Claim 1 states, A non-human mammal whose
genome comprises a DNA construct comprising a
whey acidic protein promoter operably linked to a
DNA sequence encoding a heterologous protein,
wherein said construct further comprises a DNA
sequence encoding a secretory peptide operatively
linked to said DNA sequence encoding a heter-
ologous protein, wherein said mammal is selected
from the group consisting of mouse, sheep, pig,
139
goat and cow, and wherein said heterologous pro-
tein is expressed in the milk of the mammal.
Licensing information: Available for non-exclu-
sive licensing from NIH.
Chinese hamster ovary (CHO)
cells
232
ACE Expression System - MAb-
expressing CHO cells lines
Chromos Molecular Systems - Licensor, pri-
mary
Hungarian Academy of Sciences - Assignee,
patent
Description: The ACE System has been devel-
oped to rapidly generate stable high MAb ex-
pressing CHO cell lines. The system is a platform
technology based on a pre-engineered artificial
chromosome with multiple recombination accep-
tor sites that allows for the targeted transfer of
single or multiple copies of genes into cells. The
pre-engineered artificial
chromosome contains 50-70 recombination ac-
ceptor sites for targeted integration of single or
multiple copies of
product genes. The ACE System provides a rapid
and efficient process for generating high-express-
ing cell lines for commercial recombinant protein
manufacture. The ACE System is currently being
used to engineer cell lines. Mab-expressing cell
lines can be generated in less than 2 months from
transfection to produce research material and in
less than 3.5 months to produce candidate single
cell subclones that express industry-relevant lev-
els of antibody. The candidate cell lines cultured
in non-optimized, non-fed batch shake flasks,
express MAb titres of > 1 g/L.
Use with: CHO cells
ies
Patents: The core ACE System technology is
covered by a broad, worldwide patent portfolio.
by Invitrogen and Sigma-Aldrich, including cus-
tom cell lines services..
Licensing information: Non-exclusive licenses
are available for research and commercial applica-
tions. Chromos states, licensees obtain a license
to a complete engineering system that includes
the core technology along with license rights to a
number of third party proprietary regulatory ele-
ments that have been evaluated by Chromos for
optimal product gene expression when combined
with the ACE System. Our business model for
commercializing the ACE System is to gener-
ate near-term and longer-term revenues through
a combination of (i) non-exclusive research and
commercial licenses and (ii) fee-for-service to
engineer cell lines for customers.
Cambridge Antibody Technology, now part
of MedImmune, AstraZeneca; Pfizer; and Pain
Therapeutics have licensing the system.
Chromos used its proprietary ACE System to
engineer cell lines for CHR-1103, currently in
clinical trials.
233
Boehringer Ingelheim High
Expression System (BI HEX);
CHO-DG44
Boehringer Ingelheim Pharma KG - Uses for
manufacturing
Laureate Pharma, Inc. - Uses for manufacturing
Description: BI HEX technology from Boeh-
ringer Ingelheim (and available to its contract
manufacturing clients) uses serum-free transfec-
tion, suspension adapted CHO-D44 cells (see
related entry) cultured in chemically defined
media for high yield and large-scale recombinant
(glyco)protein manufacture. Several cell line de-
velopment projects have delivered titers above 1
g/L with minimal process development after clone
screening. Specific productivities above 50 pg per
cell and day for monoclonal antibodies have been
140
achieved and successfully translated into product
titers of > 4 g/L in an 11- day fed batch process
without compromising product quality, activity
and characteristics.
BI HEX combines several technology plat-
forms: Efficient vector systems with novel genetic
elements for high-level of product expression and
enrichment of high producers; Serum-free trans-
fection, selection and cultivation of suspension-
adapted CHO-DG44 cells cultivated in chemi-
cally defined media eliminating long adaptation
processes; Serum-free FACS-based automated
single cell cloning; Sophisticated design of high
throughput screening platforms, including plat-
form media and well-characterized down-scale
models, giving highly productive, robust clones
that grow to high densities; Fed batch process
platform with well characterized chemically
defined media and feeds; cGMP cell banking
of master and working cell banks in chemically
defined media. BI HEX platform provides shorter
development times to first use of a new biologic
entity in humans and it follows the paradigm do
it right the first time. Early focus on commercial
scale potential, process performance and product
quality, safety and comparability avoids unneces-
sary costs and delays later.
Starting with product gene cloning into BI
HEX vectors, material for toxicological studies
from monoclonal cell lines can be supplied in
just 15 months. BI HEX processes can be used
directly to produce Phase I clinical material. Early
parallel development of the upstream, down-
stream, and formulation processes, including care-
ful product characterization, is key to success.
Use with: CHO cells
nal antibodies
Licensing information: The BI HEX system is
royalty-free for Boehringer Ingelheim s contract
manufacturing customers. For third party manu-
facture, use of the system is royalty-bearing. BI
HEX has apparently been licensed by Biogen
Idec.
In Feb. 2008, Laureate, a CMO, formed a col-
laboration with Boehringer Ingelheim for access
to Boehringer Ingelheim s technology platforms
for biophamaceutical manufacture, including
BI-HEX. Laureate and Boehringer Ingelheim will
harmonize bioprocessing methods to ensure seam-
less transfer of manufacturing activities from Lau-
reate to Boehringer Ingelheim. Laureate now offer
its clients bioprocessing services that include:
creation and development of production cell lines
using the BI HEX expression system
European Markets, CHO-expressed biopharma-
ceuticals manufactured by Boehringer Ingelheim
include:
a) Enbrel [Etanercept - tumor necrosis factor
receptor2-immune globlulin G1 Fc fusion protein,
recombinant] developed by Immunex (now Am-
gen) and marketed by Amgen, and
b) TNKase [Tenecteplase - TNK-tPA; tissue
plasminogen activator, TNK-, recombinant]
developed and marketed by Genentech, also with
U.S marketing by Schering-Plough Corp. and Cor
Therapeutics Inc.
234
CHO cell line (Puck); CHO-K1
cell line
University of Colorado - Licensor, primary
Description: The original, parental CHO cell
line was initiated from a biopsy of an ovary of
an adult Chinese hamster (Cricetulus griseus)
(Puck, Univ. of Colorado, 1958). Since then, the
cells have been well characterized with regard to
the karyotype, chromosome structure, gene map,
general culture conditions, and cell physiology.
Also, multiple derivative cells lines with desirable
characteristics have been developed (see related
entries).
In the late 1960s the first nutritional mutant
(CHO-K1) was isolated (Kao and Puck 1968). It
requires proline in the medium in order to grow.
It was later adapted to DHFR- phenotype (see
related entry).
141
Dr. Puck was an early pioneer of somatic cell
genetics and single-cell plating ( i.e., traditional
cell cloning) He developed the CHO cell line
from chinese hamster ovarian cells for this work.
Use with: CHO cells
Licensing information: Initially reported 50
years ago, the original cell line is now in the
public domain. However, biopharmaceutical
manufacturers may want to obtain cells directly
from Dr. Puck or others retaining/maintaining
the original cell line, or from a source capable of
providing regulatory-related testing and documen-
tation.
Products made using this tech.: Various bio-
pharmaceutical developers have used the original
cell line. Product manufactured using CHO cells
explicitly reported as obtained from Dr. Puck
include Xolair [Omalizumab - rhuMab-E25; im-
munoglobulin E25 monoclonal antibody, recom-
binant; IgE Mab, rDNA] from Genentech
Further info.: Puck, T. (1958). J. Exp. Med. 108,
945.
235
CHO DG44 cells, DHFR-;
CHO-DG44; DUK-XB11; CHO
K1 DUX B11 (DHFR-) cells;
Dihydrofolate reductase
selection/amplifcation, CHO
cells
Chasin, Columbia University - Licensor, pri-
mary
Life Technologies, Inc. - Retail seller; Licensor,
secondary
Description: The CHO DG44 cell line (dhfr-)
developed by Dr. Chasin and co-workers, Co-
lumbia University, is one of the original dihy-
drofolate reductase-negative (deleted) CHO cell
lines. DG44 cells are routinely used to establish
cell lines for production of recombinant proteins.
They were originally developed using gamma
rays to eliminate the entire dhfr locus. In non-mu-
tated cells, dhfr is an essential enzyme in CHO
cells for de novo synthesis of glycine, purines,
and thymidylate. This allows dhfr to be used as a
dominant selectable marker and a gene amplifier
for the expression of proteins in dhfr cell lines.
The dhfr mutation in DG44 cells is stable and
irreversible, which makes it a suitable mammalian
cell line for production of recombinant proteins.
The cells can be cultured in animal origin com-
ponent-free, chemically defined media containing
hypoxanthine and thymidine.
Benefits: established cell line, familiar to regula-
tory agencies
Patents: Although first reported in 1980, a related
U.S. patent was granted in 1989, with this now
presumably expired. U.S. 4,818,679, Chasin, et
al., April 4, 1989, Method for recovering mutant
cells covers a broad method for recovering mutant
cells which produce a substance [e.g., DHFR] in a
quantifiably reduced amount relative to wild type
cells. The abstract states, This invention con-
cerns a method for recovering mutant cells which
produce a substance in a quantifiably reduced
amount relative to wild type cells, and the mutant
cells so recovered. The method involves contact-
ing the mutant cells under suitable conditions with
a suitable amount of an appropriate detectable
compound capable of binding to the substance
when it is present in the cells so as to permit the
detectable compound to bind to the substance.
The amount of the detectable compound bound to
the substance in the cells is then detected, and the
cells which produce the substance in the quantifi-
ably reduced amount are thereby detected. Such
cells which produce the quantifiably reduced
amount of the substance are then recovered. This
invention is applicable to a wide variety of cell
types of various genetic characteristics and should
therefore be useful in providing a wide range of
useful mutant cells.
Availability: CHO DG44 cells are available from
a number of reagent suppliers and other sources,
e.g., as ATCC CRL 9096 and ECACC 94060607.
However, a company planning to manufacture
142
a human biopharmaceutical would probably do
best, if only to better satisfy FDA, by getting cells
from their original source, Dr. Chasin.
Licensing information: In correspondance, Dr.
Chasin reported, The dihydrofolate reductase
negative CHO cell lines DXB11 and DG44 can
be licensed from me for a one-time fee for all
products a company manufactures. No license is
required for research purposes, including research
and development. A license is required only for
commercial use (selling a product made in these
cells). Companies should contact me for the cur-
rent fee at lac2@columbia.edu.
These cell lines may be obtained directly
from me. I do not vouch for cells obtained from
the ATCC, which we did not deposit. Derivatives
of these cells can also be obtained from Invitro-
gen. The cells may also be licensed from Invitro-
gen.
The full text of a SAMPLE LICENSE
AGREEMENT follows: In consideration for the
payment of $25,000, receipt of which is hereby
acknowledged, Lawrence Chasin and Gail Urlaub
Chasin grant to XXX a paid-up, non-exclusive
right and license throughout the world to make
and use the Chinese hamster ovary (CHO) cell
dihydrofolate reductase-deficient mutant cell
lines DG44 and DXB11 (also known as DUK-
XB11and DUKX) that we have isolated and
described.
In connection with the grant of this license, we
represent and warrant that:
1.) We are the persons responsible for the induc-
tion and isolation of these cell lines; 2.) No au-
thorization by any person is required to permit the
granting of this license or the exercise of rights
thereunder, except authorizations from Columbia
University and the National Institutes of Health,
which authorizations have been obtained; 3.) We
have the right and power to grant the license;
and 4.) The granting of this license conveys no
rights under any valid patent owned by any other
person.
Products made using this tech.: Dr. Chasin
noted, I do not have any official information
about what products are being manufactured in
these cell lines. My impression is that it is about
half the currently marketed recombinant proteins.
We have licensed most of the major biotechnol-
ogy and pharmaceutical companies making pro-
teins in mammalian cells as well as many small
companies.
Further info.: See Urlaub, G. and L.A. Cha-
sin (1980). Isolation of Chinese Hamster Cell
Mutants Deficient in Dihydrofolate Reductase
Activity, Proceedings of the National Academy of
Science 77, 4216-4220.
236
CHO SSF cell lines - adherent
CHO cells; protein-free media
Swiss Federal Institute of Technology, Zurich
- R&D
Description: CHO SSF3 cells that grow in sus-
pension culture with adaptation to protein-free
media, have been further modified to grow in ad-
herent culture. CHO SSF3 X9 strain conditionally
expresses an attachment factor leading to induc-
ible adhesion to surfaces, while CHO SSF3 KX1
is a constitutive CHO cell mutant that only grows
as adherent cells. These lines offer the benefits
of serum-free growth with all the advantages of
immobilized cell bioreactor systems for produc-
tion of heterologous proteins. A medium was also
developed which allows these cells to grow in the
absence of any exogenously added growth factors.
In vivo, the secretory component (SC) inserted
into the parent cell line is synthesized as the extra-
cellular part of an integral membrane glycoprotein
of specific cells responsible for the transport of
polymeric immunoglobin (Ig) to external mucosal
surfaces.
Use with: CHO cells
ies
Patents: Related patents include
WO/1996/018734, PRODUCTION OF RE-
143
COMBINANT SECRETORY COMPONENT,
assigned to Ciba-Geigy AG, now Novartis AG.
The Swiss Federal Institute of Technology, Zurich
assisted in R&D.
Availability: Marketed by QIAGEN GmbH for
Licensing information: Contact Novartis AG.
Further info.: Production of Recombinant
Proteins in Chinese Hamster Ovary Cells Using A
Protein-Free Cell Culture Medium, Nature Bio/
Technology 13, 389 - 392 (1995). This concerns
a recombinant secretory component (rSC) obtain-
able from a recombinant Chinese Hamster Ovary
(CHO) cell line.
237
CHO Supercell; Targeted
transfection; CHO DG44 cell
line
Xcellerex - Licensor, primary
Description: Xcellerex, a CRO/CMO, offers
cell line development services using Supercell its
CHO DG44 parental cell line along with a propri-
etary vector. A gene of interest can be integrated
into a tagged hotspot in a CHO DG44 cell line
and contains sequences specific for the hotspot,
allowing the gene to be targeted to this site. This
targeted transfection eliminates randomness and
minimized time required for traditonal transfec-
tion
Use with: CHO cells
Licensing information: Contact Xcellerex
regarding these custom services and access to its
technologies.
238
CHOZn CHO DG44 cell lines -
antibodies
Sangamo BioSciences, Inc. - Licensor, primary
Description: A stable CHO cell line, CHOZn,
constitutively expressing Zinc Finger DNA-bind-
ing Protein (ZFP; see related entry) specific for
a 9bp sequence fused to the VP16 activation
domain can be used as a recipient for any host
vector(s), and is particularly useful for its high-
level expression of monoclonal antibodies. The
cell line was derived from CHO DG44 (see re-
lated entry). CHOZn shows good growth charac-
teristics and functional activity. Bioreactor studies
have confirmed utility of the cell line.
Use with: CHO cells
nal antibodies
Licensing information: CHOZn is available for
evaluation under MTA (6-9 months). Nonexclu-
sive research and commercial licenses are also
available.
239
GS-CHO Protein Free System;
CHOK1SV cell lines - protein-
free media
Description: Lonza has developed highly produc-
tive CHO cell lines adapated to serum and pro-
tein-free media and readily adapted to suspension
culture using its GS expression technology (see
related entry). Due to CHOK1SV being pre-
adapted to the desired culture conditions, adapta-
tion to suspension culture is faster and easier.
Use with: CHO cells
Use to make: proteins; glycoproeins; antibodies
Benefits: The pre-adapted CHOK1SV host cell
line results in a substantial timeline reduction for
production cell line development. High yielding
144
GS-CHO cell lines adapted to chemically-defined
medium can be created within six months.
Availability: The cell line is available from Chro-
mos Molecular Systems for non-commercial uses.
Licensing information: A GMP cell bank of
CHOK1SV is now available. The cell line is
offered at no extra charge for GS System licens-
ees; and presumably is available for licensing to
others.
In 2004, Chromos acquired non-exclusive
rights to use Lonzas CHOK1SV cell line in com-
bination with Chromos proprietary ACE System
(see related entry) for cellular protein production
applications.

240
Sympress expression; Human
polyclonal antibodies (rpAB) -
CHO cells
Symphogen A/S - Licensor, primary
Description: Sympress expression uses CHO
cells with site-specific integration technology
using the FRT/Flp-In recombinase system to
generate antibody-producing cell lines enabling
consistent production of target-specific recombi-
nant human(-like) polyclonal antibodies (rpAb).
Sympress involves stable, site-specific integration
of antibody genes in all transfected producer cells,
ensuring that the manufacturing process yields a
consistent and reproducible mixture of polyclonal
antibodies. Sympress makes it possible to main-
tain diversity and obtain batch-to-batch consis-
tency when producing a polyclonal mixture.
The process involves culturing a mixture of
transformed CHO cell lines, each of which ex-
presses one distinct antibody. The expressed rpAb
constitutes a large diversity of individual antibody
members produced in the same bioreactor, with
diversity maintained from batch to batch. Sym-
phogen has demonstrated proof-of-concept for the
effective utilization of the Sympress manufactur-
ing platform for multiple internally developed
products.
Use with: CHO cells
Use to make: human antibodies, polyclonal
Benefits: Sympogen claims to be the only com-
pany with a viable platform for producing recom-
binant polyclonal proteins such as antibodies.
Patents: European Patent number 1,583,830,
Haurum, et al., titled Method for manufacturing
recombinant polyclonal proteins. broadly covers
Sympress technology, including pAb manufactur-
ing cell lines and Symphogens polyclonal anti-
body expression libraries. The exemplary claim
(2) states, A method for generating a collection
of cells suitable as a recombinant polyclonal man-
ufacturing cell line, said method comprising: a)
providing a library of vectors comprising a popu-
lation of variant nucleic acid sequences, wherein
each of said vectors comprises 1) one single copy
of a distinct nucleic acid sequence encoding a
distinct member of a polyclonal protein compris-
ing distinct members that bind a particular antigen
and 2) one or more recombinase recognition
sequences; b) introducing said library of vectors
into a host cell line, wherein the genome of each
individual cell of said host cell line comprises re-
combinase recognition sequences, matching those
of the vector, at a single specific site in its ge-
nome; c) ensuring the presence in said cells of one
or more recombinases so that the variant nucleic
acid sequences of step (a) are integrated site-spe-
cifically in the cells of the host cell line, where
said one or more recombinases is/are either i) ex-
pressed by said cells into which said nucleic acid
sequence is introduced; ii) operatively encoded by
the vectors of step a; iii) provided through expres-
sion from a second vector; or iv) provided to the
cell as a protein; and d) selecting cells comprising
an integrated copy from said library of variant
nucleic acid sequences.
Licensing information: Contact Symphogen
Further info.: Production of target-specific
recombinant human polyclonal antibodies in
mammalian cells, Wiberg FC, Rasmussen SK,
Frandsen TP, Rasmussen LK, Tengbjerg K, Coljee
VW, Sharon J, Yang CY, Bregenholt S, Nielsen
LS, Haurum JS, Tolstrup AB, Biotechnol Bioeng.
145
2006 Jun 5;94(2):396-405 [An anti-Rhesus D
recombinant polyclonal antibody, Sym001, com-
prised of 25 unique human IgG1 antibodies, was
produced by Sympress expression technology].
.
241
Baby hamster kidney (BHK) 21
cells; ATCC CCL 10
Description: BHK 21 cells are one of the few
mammalian cell lines that can be grown both in
suspension as well as anchorage. As described be-
low, the cell line is used for manufacture of Factor
VIII, generally cited as the largest (and also dif-
ficult to manufacture in decent yield) recombinant
(glyco)protein. Syrian hamster kidney BHK-21
is a subclone (clone 13) of a parental line estab-
lished from the kidneys of five unsexed, one-day-
old hamsters in 1961.
Use with: BHK cells
Availability: Readily available from culture
collections, e.g., as ATCC CCL 10; ECACC
85011433; DSMZ ACC 61; and from vendors,
e.g., Sigma-Aldrich (SIAL) (SAFC).
Licensing information: First reported in 1962
(Virology 1962;16:147-151), BHK 21 cells are
now in the public domain.
Products made using this tech.: NovoSeven
[Factor VIIa, rDNA; Coagulation Factor VIIa)
from Novo Nordisk may be manufactured us-
ing this cell line. The gene for human Factor VII
was cloned and is expressed in a transformed
baby hamster kidney (BHK) cell line (apparently,
ATCC CRL 1632 or ATCC CCL 10). The cells are
cultured on microcarrier beads in conventional
stirred tanks with media replaced/harvested daily
and purified.
Hybridomas
242
Human MORPHODOMA;
Morphogenics; Hypermutation;
PMS2 gene screening,
modifcation - human
monoclonal antibodies from
hybridomas
Morphotek Inc. - Licensor, primary
Eisai Co., Ltd. - Parent co.
Johns Hopkins University (JHU) - R&D
Description: Human MORPHODOMA provides
a platform for generation and screening of host
cells lines and manufacture of human monoclo-
nal antibodies from selected stable hybridomas.
Hybridoma cells have proven suitable for genetic
optimization using the Morphogenics process and
have shown potential for large-scale manufactur-
ing.
Morphogenics is a broad-based technology that
allows for the selective regulation of the DNA
spell check function of a host cell line. Insertion
of a truncated mutant of a dominant negative
allele of a mismatch repair gene, PMS2, induces
hypermutation in hybridomas. Transient regula-
tion of cellular DNA mismatch repair processes
is used to enhance traits (e.g., affinity and titers)
of mAb-producing hybridomas and improve
suboptimal hybridoma cells generated by means
of ex vivo immunization and immortalization of
antigens-pecific human B cells for therapeutic
Mab development. This provides a rapid means
to create genetic diversity within a cell line and
leads to variant sib cells with novel characteristics
as well as gene products with enhanced biological
activities for therapy. Human MORPHODOMA
applies this to hybridomas derived from in vitro
immunization or B-cells from disease patients
to generate fully human antibodies from stable
hybridomas.
Human MORPHODOMA technology works by
immunizing human B-cells with a disease antigen
146
to yield B-cells producing target-specific antibod-
ies or B-cells derived from patients with autoim-
mune disease or cancer. Positive B-cells are fused
to an optimized myeloma cell line to generate
stable hybridomas. Antibodies are analyzed for
affinity, specificity and biological activity. Leads
with low-affinity are further optimized using
Morphogenics to genetically evolve parental
cells to derive sibs with structurally altered vari-
able (V-region) domains for increased affinity or
altered antibodies with enhanced immune-effector
function. In addition, hybridoma clones produc-
ing optimized antibodies are then further evolved
to yield high-titer cell lines suitable for scaleable
manufacturing.
The technology creates genome-wide hyper-
mutation within antibody-producing cells and
enables the discovery of variant antibody prod-
ucts with increased activities as well as host cells
with enhanced titers for scaleable manufacturing.
MORPHODOMAs perpetual mutagenesis allows
for maximum diversity in cells and gene products.
Libraries of diverse sib cells are directly screened
using automated high throughput screening to
identify clones exhibiting a phenotype of interest.
Lead cell lines are then genetically stabilized by
restoring the spell check system. Lead antibod-
ies generated from the immunization process
are further optimized for binding affinity and/or
increased immune effector-function via MORPH-
ODOMA technology.
Use with: human hybridomas
Benefits: This provides the ability to generate
stable high-titer hybridomas allows for the com-
mercial development of fully human antibodies
without the need for licenses to other 3rd party
enabling technologies (avoid royalty stacking
commonly associated with other antibody discov-
ery platforms).
Methods for generating genetically altered anti-
body producing cell lines with improved antibody
characteristics, Nicolaides, et al., Oct. 26, 2004,
assigned to Morphotek, Inc., a subsidiary of Eisai
Co., Ltd. Another related patent is 7,319,036,
Method for generating hypermutable organ-
isms, Nicolaides, et al., Jan. 15, 2008, coassigned
to Morphotek and Johns Hopkins University.
Morphotek has an exclusive license from Johns
Hopkins University for application of Morpho-
genics.
Licensing information: Contact Morphotek
regarding commercial uses and licensing. Mor-
photek markets its Human MORPHODOMA plat-
form in collaboration with third parties to yield
high quality human antibodies and attendant pro-
duction lines that alleviate the burdensome royalty
mandated by other antibody technologies.
Products made using this tech.: Morphotek has
two antibodies in clinical development. MORAb-
003 is in a multi-institutional phase 2 clinical trial
for the treatment of ovarian cancer and MORAb-
009 in a multi-institutional phase 1 clinical trial
for the treatment of pancreatic and lung cancers.
The company reports, Morphogenics technol-
ogy has been validated though research collabora-
tions with premier corporate partners including
Abgenix, Amgen, Centocor, Novo Nordisk, PDL,
GSK, Tanox, and Wyeth, as well as though its
application to internal development candidates
MORAb-003 and MORAb-009 which were
optimized for affinity and titers, respectively.
Morphotek has also entered into R&D collabora-
tions with other companies including GlaxoS-
mithKline.
243
Cell-free system -
glycoproteins; hybridoma
RIKEN - Licensor, primary
Description: A highly efficient cell-free trans-
lation system has been developed based on a
hybridoma cell lines with very strong protein
translation/glycosylation systems. The system
offers ease of use, low-cost, and well-developed
protein translation/glycosylation. Immortaliza-
tion of B-cells as hybridomas allows for easy and
147
cost-effective growth in the laboratory, while their
mammalian source confers glycosylation.
A major advantage of this system is that the
source hybridoma cell line is quite easy to main-
tain and grow. It can also be adapted to various
bioreactors for scale-up. Since the hybridoma cell
line is clonal in nature, there is little batch-to-
batch variability that plagues other animal-based
cell free systems. The system is quite robust, so
many different types of glycoproteins could be
synthesized successfully and in quantity. Human
choriogonadotropin1 protein from this cell-free
system has been shown to have biological activity.
Use with: hybridomas
ies
Patents: No relevant yet published applications or
patents were retrieved from simple searching.
Licensing information: Contact RIKEN.
Further info.:
A hybridoma-based in vitro translation system
that efficiently synthesizes glycoproteins, Ko-
bayashi, T., et al., Journal of Virological Methods,
Volume 142, Issues 1-2, June 2007, Pages 182-
188.
244
Ex-Cell EBx expression; EBx
cells; Chicken embryonic stem
cells; Chicken EBx cells; Duck
EBx cells
Vivalis - Licensor, primary
Description: The Ex-Cell EBx platform involves
use of immortal, stable chicken embryonic stem
(ES) cells as host cells for recombinant protein
expression and culture of viruses, e.g,. for vac-
cines. A Duck EBx platform application of the
EBx platform for the production of therapeutic
proteins has also been developed.
Telomerase expression constitutes a key feature
of these cells, and is responsible for the genetic
stability of the diploid EBx cells and their con-
tinuous cell growth (immortalization). These two
characteristics render EBx cells unique, because
cell line immortality is usually associated with
genetic instability like that in cancer cells or in
continuous cell lines that are transformed.
EBx cell lines display unique biological and
industrial properties: (1) they grow in suspension
in serum-free medium; (2) they reach high cell
densities (over 10.sup.7 cells/ml) in batch and
fed-batch bioreactors; (3) they are highly suscep-
tible to most viruses currently cultured in chicken
eggs or fibroblasts, including human and avian
influenza viruses or modern recombinant viruses
such as poxviruses; and (4) they are immortal, yet
genetically stable.
EBx cell lines is claimed to constitute an
innovative, safe and cost-effective production
platform that has the potential to rapidly evolve
as an industry standard for the manufacture of
human and animal vaccines currently produced
on the old-fashioned and cumbersome embryo-
nated egg production platform. Avian EBx cells
have been demonstrated to be amenable to easy
genetic modifications and to produce monoclonal
antibodies with favorable glycosylation profiles
and enhanced therapeutic profiles. Antibodies
produced using EBx cell lines show remarkably
low fucose content and increased immunogenic-
ity, with reduced fucose content associated with
improved antibody-dependent cell cytotoxicity
(ADCC) activity, particularly important in treating
cancerous cells.
Use with: chicken and duck embryonic stem
(ES) cells.
ies
Benefits: EBx cell lines have the potential to
replace the embryonated eggs or Chicken Embryo
Fibroblasts (CEF) for the industrial manufacture
of various human and animal vaccines systems
(e.g, human and avian influenza viruses, poxvi-
ruses...). In addition to being a simpler, faster,
cleaner and more robust system, the EBx platform
allows a very significant decrease in costs of
production.
148
U.S. 20040058441, Avian cell lines useful for
the production of substances of interest, Pain, B.,
March 25, 2004, concerning methods for genera-
tion of avian cell lines (EBx) cells so that cells
become adherent or nonadherent cells and are
capable of proliferating indefinitely in a basic cul-
ture medium, by progressive adaptation to culture
media.
Licensing information: The Vivalis EBx plat-
form is available for vaccine and protein produc-
tion under both research and commercial licenses.
Vivalis also offers extensive CRO/CMO services
using EBx cells, including full manufacturing
process development to its clients for the pro-
duction of their protein of interest, including the
production of preclinical and clinical batches.
Commercial licenses include an upfront payment
on signing followed by milestone payments at
defined points in the development and registration
process, and finally, payments of a percentage
royalty on future sales of product. The research
license can be transformed into non-exclusive or
exclusive commercial licenses that include:
- rights to Vivalis worldwide family of EBx pat-
ents;
- transfer of an EBx GMP premaster cell bank;
- access to the Biological Master File for the US
market or its equivalent file for other commercial
region;
- a complete know-how transfer.
Vivalis report that it has been for over 7 years
conducting research into the development of well
documented and high quality substrate cell lines
derived from ES cells. Vivalis proprietary EBx
platform is available for research and commercial
licensing on a non-exclusive basis.
More than 20 licenses have already been
signed with most leaders in vaccine industry (e.g.,
Sanofi, GSK, Novartis, CSL, ...). 27 partnerships
with 22 clients as of October 18th 2007 included:
- 8 Commercial licenses on EBx platform
Companies License type
Sanofi-Pasteur Recombinant vaccines for HIV
and cancers
Sanofi-Pasteur Undisclosed
GSK Influenza vaccine (seasonal and pandemic)
CSL Influenza vaccine (seasonal and pandemic)
Nobilon (Schering-Plough) Influenza vaccine
(seasonal and pandemic)
Merial Canarypox based vaccines for pets dis-
eases
Virbac Biologicals
SAFC Bio. (Sigma-Aldrich) Cell culture media
- 14 research licenses
Novartis Vaccines Kaketsuken (2 distinct licenc-
es)
Bavarian Nordic CEVA
Geovax Fort Dodge (Wyeth)
Intervet (Schering-Plough) Sanofi-Aventis
4 human undisclosed 1 veterinary undisclosed
Through a collaboration of Vivalis with
Sigma-Aldrich Biosciences (SAFC Biosciences),
SAFC now markets Ex-Cell EBx viral growth and
production media designed exclusively to support
EBx cells.
Vivalis has been working in collaboration with
Mat Biopharma to evaluate its EBx cell lines for
the production of Mat Biopharmas monoclonal
antibodies. The companies demonstrated that as
well as the reduced fucose content, the glycosyl-
ation profile of monoclonal antibody of IgG1 sub-
type produced in the avian EBx cells was similar
to human antibody glycosylation profiles.
Chicken/Poultry
245
Chicken egg expression;
Transgenic animals
TranXenoGen, Inc. - Licensor, primary
Description: TranXenoGen has developed mul-
tiple technologies for the production of high-vol-
ume recombinant therapeutic proteins in the white
of transgenic chicken eggs, and related technolo-
gies also useful with other animal species. These
include:
Direct-Egg Transfection - Non-viral method for
the transfection of large transgenes into chicken
149
eggs, such that transgenic chickens express the
recombinant protein in the albumin fraction of
their eggs.
PGC/CEC Stem Cell Technology - Primordial
germ cells (PGC) or chicken embryonic cells
(CEC) are harvested from developing embryos,
grown in culture to allow transfection with the
selected gene and then the cells are transferred
to a recipient egg to produce a chimeric chicken.
Potentially allows for efficient gene insertion or
deletion.
And multi-species transgenic technologies:
Gene-Testes Transfection - This technology is
designed to be applicable in many species. The
Gene-Testes Transfection technology targets the
regenerating sperm cells of the testes so that the
male produces transgenic sperm. Consequently,
the male can be bred with the objective of gener-
ating transgenic animals.
Cloning - TranXenoGen currently holds a world-
wide exclusive license to several patents relating
to cloning and sperm-mediated transgenesis. The
patent portfolio, with a filing date of February 3,
1993, covers techniques for the reprogramming
of somatic cell nuclei for the generation of cloned
and/or transgenic animals.
Use with: chickens; animals
Benefits: Chicken egg expression benefits in-
clude:
Rapid gestation and development times - Chick-
ens mature and lay eggs 6 months after hatching
Minimal development costs - Inexpensive spe-
cific pathogen free SPF animals/eggs; simple
husbandry
Prolific protein production - On average, one egg
per day; 21 per month; 252 per year from a single
hen
Post-translation processing - Similarity with hu-
man, ability to glycosylate and phosphorylate
Total containment/flock assurance - Chickens can
be housed in controlled health screened facilities
Existing regulatory experience - SPF eggs used
for vaccine production
Patents: The platform technologies are protected
by in-licensed, owned and pending patents.
Licensing information: Contact TranXenoGen,
Inc.
246
Chicken rimordial germ cells
(PGCs) - Human proteins/
monoclonal antibodies in
chicken eggs
Origen Therapeutics - Licensor, primary
Description: A system has been developed for
protein, including monoclonal antibody, expres-
sion in chicken (hen) eggs. The system uses
primordial germ cells (PGCs; stem cells), the very
earliest cells that normally mature into sperm
and eggs in the chicken embryo, harvested from
the chicken embryo. These cells can be isolated,
transformed and cultured indefinitely in the labo-
ratory, while remaining committed to their germ
(reproductive) cell lineage. Once inserted into a
recipient chicken embryo at the appropriate devel-
opmental stage, the modified PGCs will develop
normally into sperm and eggs, thus passing on the
introduced traits to the next generation of chick-
ens. Origen claims, This is the best system avail-
able to date for producing transgenic chickens.
Antibodies produced by this method have very
similar physical and biological characteristics to
those produced in CHO cells, including nearly
identical binding curves, similar affinities, and
an equal ability to be internalized by antigen
on prostate cancer cells. Th chicken-produced
antibodies lacked the sugar residue, fucose, which
greatly increases their antibody-dependent cellular
cytotoxicity (ADCC) /cellular immunity (by up to
100-fold) activity compared to CHO-produced an-
tibodies. Origen has designed vectors so that the
sequence for a MAbs variable domain (V genes)
can be easily inserted.
Unlike other transgenic animal systems, the
time from protein/antibody identification to
production in eggs can be as short as 8 months
versus 18 months to 3 years for goats or cattle.
150
The eggs are sterile and stable, providing a good
starting material for isolation and purification of
the protein of interest. Moreover, conditions for
good manufacturing practices have been long-es-
tablished for vaccines using chicken eggs.
To date, chimeric chickens have been made by
injecting blastodermal cells or embryonic stem
cells into the stage X embryo (recipient; nuclear
transformation), but the frequency and level of
chimerism is variable and unpredictable. Ori-
gen has developed a universal recipient cell that
contributes minimally, so that the contribution
of donor derived cells to the resulting chimera is
constantly high and predictable. These high-grade
chimeras can be used to produce biopharmaceuti-
cal proteins in chicken eggs as soon as the chime-
ras commence laying and before they transmit the
gene encoding the biopharmaceutical protein to
the next generation. In addition, these high-grade
chimeras allow the production of desired proteins
even if the transgene interferes with their ability
to reproduce. This provides a robust system to
produce high-grade chimeras in a highly predict-
able way. Using this system biopharmaceutical
proteins can be produced quickly, in a scalable
system and at minimal cost.
In Aug. 2005, Origen Therapeutics reported the
firrt fully functional, human sequence monoclo-
nal antibodies (mAbs) produced in chickens. The
antibodies were expressed solely in the chicken
oviduct and deposited into egg white in concen-
trations of 1-3 milligrams per egg. Antibodies pro-
duced in this manner demonstrated 10-100 fold
greater cell-killing ability (ADCC) compared to
therapeutic antibodies produced by conventional
cell culture methods.
Use with: chicken primordial germ cells (stem
cells); chickens
ies
Patents: Apparently, initially developed by
University of California, Davis, researchers and
licensed/assigned to Origen Therapeutics. Ex-
emplary patents assigned to Origen Therapeutics
include 6,861,572, Production of proteins in
eggs, Etches, et al., March 1, 2005; 7,145,057,
Chimeric bird from embryonic stem cells, and
7,323,618, Tissue specific expression of exog-
enous proteins in transgenic chickens, Jan. 29,
2008.
Licensing information: Contact Origen Thera-
peutics.
247
OVA System; Avian Transgenic
Biomanufacturing - chicken
eggs expresssion
Roslin Institute - Licensor, primary
Oxford BioMedica plc - R&D
Viragen - Former inv.
Description: The OVA System enables recombi-
nant oviduct-specific protein expression in the egg
white of eggs laid by transgenic chickens. The
OVA System is being developed as a large-scale
biomanufacturing platform alternative capable
of cost-effectively expressing many types of
therapeutic proteins, including glycoproteins and
antibodies. Protein expression can be maintained
through several generations, leading to a flock
of hundreds of the transgenic birds expressing a
desired protein(s). Equine infectious anemia virus
(EIAV) and other lentiviral vectors may be used
to transform chickens. Transgenic mammals are
capable of producing yields of several grams of
protein product per Liter equivalent, and the short
generation time for birds allows a rapid scale up
of production.
Ovalbumin normally constitutes more than half
of the protein in the white of a laid egg, and ex-
pression of the ovalbumin gene is restricted to the
tubular gland cells of the avian oviduct. Lentiviral
vectors deliver transgene constructs comprising
regulatory sequences from the ovalbumin gene
designed to direct expression of associated thera-
peutic proteins to the oviduct. Functional recom-
binant protein are expressed in a tightly regulated
tissue-specific manner, without any evidence of
transgene silencing after germ-line transmission.
151
Protein manufacture in chicken eggs has
several advantages as compared to mammalian
cell culture, or the use of transgenic mammalian
systems. Chickens have a short generation time
(24 weeks), which permits transgenic flocks to be
established rapidly. Secondly, the capital outlays
for a transgenic animal production facility are far
lower than that for cell culture. Extra processing
equipment required to facilitate transgenic protein
production is minimal in comparison to that re-
quired for cell culture. These lower capital outlays
result in the production cost per unit of transgenic
therapeutic being lower than that produced by cell
culture. In addition, transgenic systems provide
significantly greater flexibility regarding purifica-
tion batch size and frequency. This flexibility may
lead to further reductions in capital and operating
costs in purification through batch size optimisa-
tion.
Use with: chickens, transgenic
ies
Benefits: Avian transgenic production is expected
to offer significant economic and technological
advantages over traditional methods of protein
production including: ease of scale-up; low capital
risk; deferred capital investment; and competitive
costs.
Chicken proteins, unlike those of other trans-
genic expression animals, have sugars and glyco-
sylation similar to humans. This may offer distinct
advantages for patients who develop neutralizing
and binding antibodies to foreign sugar epitopes
on traditionally manufactured biotherapeutics
which, in turn, may negate some or all of the ben-
eficial effect of the protein drug in the patient.
The chicken egg, as natures bioreactor, offers
a far more preferable drug manufacturing vehicle
compared to present equipment or other trans-
genic production methods, such as with mammals.
Chicken eggs have been used in the manufac-
ture of non-recombinant, e.g., influenza, vaccines
for more than 30 years.
Patents: Related applications include U.S.
2008120732; and WO2006024867, METHOD
FOR IMPROVED TRANSGENE EXPRES-
SION, Elliot, E., WO filing published March 9,
2006, assigned to Viragen. The abstract states:
The present invention provides an improved
method for achieving efficient transcription and
translation of modified transgene constructs in
vector systems. The vector may be a lentiviral
vector. Such a method facilitates the production
of viral vector genomes with intact functional
transgene sequences allowing stable integration of
a transgene-containing viral vector genome into
the germline of an animal such as a transgenic
avian. The subsequent expression of the transgene
results in a recombinant protein product being
produced, which, in the case of a transgenic avian
can result in the targeted production of the protein
into the egg of the transgenic bird.
U.S. application 20050273872, Protein
production in transgenic avians, Sang, H., et al.,
December 8, 2005, coasisgned to Oxford Bio-
medica and Viragen, relates to the generation of
transgenic avians and the production of recom-
binant proteins. More particularly, the invention
relates to the enhanced transduction of avian cells
by exogenous genetic material so that the genetic
material is incorporated into an avian genome in
such a way that the modification becomes inte-
grated into the germline and results in expression
of the encoded protein within the avian egg.
Licensing information: Viragen formerly long
held the worldwide exclusive license to com-
mercialize the OVA System (Avian Transgenic
Biomanufacturing) as granted by the Roslin
Institute in consideration for royalty payments to
Roslin in the amounts 3.5% of sales of products
developed under the agreement and 17.5% in
connection with the sales or transfers of certain
intellectual property [licensing]. The LentiVector
gene delivery system, i.e., lentiviral vectors, was
licensed by Viragen from Oxford BioMedica plc.
Viragen had been developing the OVA System in
collaboration with the Roslin Institute.
In June 2007, Viragen halted its development
of the OVA System to concentrate on its own
therapeutics development, with rights returned to
152
the Roslin Institute and Oxford BioMedica plc
Avigenic has challenged OVA System-related
patents, but without success ot date.
Products made using this tech.: As proof-
of-principle, recombinant inteferon beta-1a (a
biogeneric version of Betaseron) was expressed in
the system in 2006. Other products expressed us-
ing the system include VG101, a humanized GD3
antibody from Viragen; and interferon alfa-2b (a
biogeneric version of Intron A).
Further info.:
Rapp, J.C., Harvey, A.J., Speksnijder, G.L., Hu,
W. and Ivarie, R. (2003) Biologically active
human interferon alpha-2b produced in the egg
white of transgenic hens. Transgenic Research,
12, 569-75.
Harvey, A.J., Speksnijder, G., Baugh, L.R.,
Morris, J.A. & Ivarie, R. (2002) Expression of
exogenous protein in the egg white of transgenic
chickens. Nature Biotechnology 20: 396-9.
248
Transgenic poultry; avian
transgenesis and nuclear
transfer - proteins in chicken
eggs
Avigenics, Inc. - Licensor, primary
Geron Corp. - Assignee, patent
University of Georgia - Assignee, patent
Description: Avigenics has developed and as-
sembled a large patent portfolio concerning
nuclear transfer in avians, and manufacture of
recombinant proteins in chicken eggs. In some or
many respects, Avigenics technology is similar
to that of Roslin/Viragen (see OVA System), and
the companies have been involved in related pat-
ent disputes. Basic nuclear transfer technology to
create cloned poultry was licensed in from Geron
Corp. Poultry, including chickens and quail, are
able to produce large amounts of protein in their
eggs at relatively low cost. This is being used for
manufacture of proteins, including humanized
monoclonal antibodies, in large amounts.
Use with: chickens, transgenic
ies
Patents: Exemplary U.S. patent applications
include: 20060185024, Avians that produce eggs
containing exogenous proteins, with its abstract
stating, This invention provides for proteins
which are expressed in the avian oviduct, pack-
aged into eggs laid by the avian, then isolated;
U.S. 20040158882, Novel vectors in avian
transgenesis, concerning vectors for chicken
transformation; 20060075513, Avian transgen-
esis and exogenous proteins; and 20040019922,
Exogenous proteins expressed in avians and
their eggs; all assigned the University of Georgia
and/or Avigenics (a univ. spinoff), and exclusively
licensed/assigned to Avigenics.
Licensing information: Contact Avigenics re-
garding CRO/CMO services and licensing.
Avigenics has licensed in multiple technologies
to assemble its chicken egg production technol-
ogy. Much of the core technology comes from the
University of Georgia.
Other licensed inventions or collaborations
include Nature Technology Corp., avian vec-
tors (see related entry); Salk Institute, ribozyme
Knockouts; Univ. of Maryland Accelerated Roost-
er Fertility; Stem Cell Sciences, IRES Vectors
(see related entry); Chromos Molecular Systems,
artificial chromosomes (see related entry) and;
Univ. of Cincinnati, gut-specific promoters.
Products made using this tech.: AviGenics
reports having successfully produced six bioactive
therapeutic proteins, including several tested in
early clinical trials.
249
Windowing Technology -
transgenics avians/chickens
Avigenics, Inc. - Licensor, primary
Description: A method is useful to create an aper-
ture or window through an avian eggs shell for
introducing foreign genetic material into the egg,
153
facilitating the generation of transgenic poultry.
Combined with other AviGenics other proprietary
technology and know-how, this forms the basis
for its methods for creating transgenic chickens.
Use with: birds; chickens
ies
Licensing information: Contact Avigenics.
Human
250
CEVEC Amniocyte Production
(CAP) expression; Human
amniocytes, immortalized
CEVEC Pharmaceuticals GmbH - Licensor,
primary
Description: The CAP expression system, based
on immortalized human amniocytes as host cells
using the E1A and E1B regions of adenovirus, is
useful for large scale manufacturing of biophar-
maceuticals and offers significant advantages over
existing production technologies. Amniocytes are
derived from amniotic fluid cells obtained by am-
niocentesis (from pregnant women). Primary hu-
man amniocytes are immortalized by adenoviral
genetic E1/pIX functions and selected for highly
efficient CAP (CEVECs Amniocyte Production)
Cell Lines. CAP cell Lines contain adenovirus
type 5 nucleotide sequence 505-4079 including
the E1 genes and the entire pIX sequence. Using
human cells, it should be possible to manufacture
essentially fully human glycoproteins and mono-
clonal antibodies
Use with: human amniocytes
Use to make: glycoproteins; monoclonal anti-
bodies
Background: Amniocentesis is a clinical rou-
tine procedure performed during week 16-20 of
gestation for prenatal diagnosis. It is an ethically
approved and accepted procedure. CEVEC, using
amniotic fluid volumes not required for clinical
diagnosis, is done in accordance with all legal and
ethical requirements.
Benefits: Advantages of CAP Cell Lines for the
production of biopharmaceuticals include: stable
and high expression of human proteins; human-
like post-translational modifications; growth in
suspension; high cell density; no spontaneous
transformation; non-tumor origin, immortalized
by a function not oncogenic in humans; easily
accessible, ethically accepted source of origin;
generation of new custom-made cell lines possible
on a routine basis; and complete cell source and
cell line development documentation.
METHOD FOR THE PRODUCTION OF
PERMANENT HUMAN CELL LINEAGES,
SCHIEDNER, et al., assigned to CEVEC Phar-
maceuticals GmbH; and WO0136615 and
equivalents, Permanent amniocyte cell line, the
production thereof and its use for producing gene
transfer vectors, KOCHANEK, et al.,
CEVEC has been successful in challenging
Crucell PER.C6 cell line patents concerning PER.
C6 cell modification to contain certain adenoviral
gene functions.
Licensing information: CEVEC is open to
establish partnerships in order to broaden the use
of CAP technology and via license agreements.
Contact CEVEC. CEVEC has concluded a global
marketing agreement with PharmaCell, combin-
ing CEVECs CAP technology and CAP cell line
development expertise with PharmaCells GMP
cell culturing facility and CMO services.
251
bcl-2 (p21) overexpressing NS0
cell lines - NS06A1(100)3 cell
line - antagonize apoptosis;
Mabs
University of Birmingham - licensor, primary
Description: Apoptosis (programmed cell death)-
resistant NS0 clones suitable for long-term
continuous perfusion culture have been developed
by Dr. M. Al-Rubeai and co-workers, University
154
of Birmingham. These NS0 cells overexpress a
bcl-2 gene, particularly p21. Expression of this
cell cycle inhibitor is induced at high cell den-
sities in perfusion reactor systems. Perfusion
bioreactors allow for extended culture periods
due to a constant supply of fresh medium passing
through a culture compartment. Product protein
commonly is constantly removed, though this not
a particularly limiting factor. High density culture
in a perfusion reactor system is accompanied by
co-expression of recombinant bcl-2 or another
apoptosis inhibitor protein. bcl-2can be any
naturally occuring eukaryotic, preferably mamma-
lian, bcl-2 protein that can act as a suppressor of
apoptosis (see Tsujimoto, Y. et al., Analysis of the
structure, transcripts and protein products of bcl-
2, the gene involved in human follicular lympho-
ma, PNAS 83,5214-5218,1986; Hockenbery, D.
et al., bcl-2 is an inner mitochondrial membrane
protein that blocks programmed cell death, Nature
348, 6299,1990). This includes anti-apoptotically
functional, natural isoforms of bcl-2 (e. g. bcl-XL)
as well as variants or muteins.
Benefits: These bcl-2 overexpressing NS0 cell
lines may be adapted to state-of-the-art antibody
manufacturing process without any major ad-
justments (i.e., bioprocessing using these cells
is much the same as with other NS0 and similar
mammalian cells), and with their longer life, may
dramatically improve the productivity of mamma-
lian cell culture processes.
WO/2002/099100, METHOD OF PRODUC-
TION OF A PROTEIN IN CELLS WHICH
INDUCIBLY EXPRESS THE CELL CYCLE
INHIBITOR PROTEIN, P21, assigned to Lonza
Licensing information: Contact Lonza or the
University of Birmingham.
Further information: See Metabolic en-
gineering of apoptosis in cultured animal
cells: implications for the biotechnology in-
dustry, online at http://www.sciencedirect.
com/science?_ob=ArticleURL&_udi=B6WN3-
48YVF7H-3&_user=10&_rdoc=1&_fmt=&_
orig=search&_sort=d&view=c&_acct=
C000050221&_version=1&_urlVersion=0&_us
erid=10&md5=b08e856ebc6834d2e99b29c29fb
56f44. This review is focused especially in the
encouraging initial results obtained with the over-
expression of cloned anti-apoptosis genes.
CHO DG44 cells, DHFR-; CHO-DG44; DUK-
XB11; CHO K1 DUX B11 (DHFR-) cells; Dihy-
drofolate reductase selection/amplification, CHO
cells
252
Cell fusion, NS0 cells -
antibodies
Abgenix, Inc. - Licensor, primary
Japan Tobacco Inc. - Assignee, patent
Description: A method has been developed for
producing multimeric proteins, e.g., antibodies,
from hybrid cells, e.g., NS0 cells, formed from
the fusion of two or more cells, with each cell
engineered to express one component of the mul-
timeric protein, e.g., an antibody chain; as well as
a method for screening for successful fusion of
the cells to produce a desired hybrid cell capable
of fully-formed antibody expression. The methods
are widely applicable to the production of multi-
meric proteins having two or more components.
Any mammalian cell line capable of express-
ing the desired multimeric protein and amenable
to fusion may be used. For example, where the
desired protein is an antibody, the cell line is any
mammalian cell capable of expressing a func-
tional antibody. A particularly preferred host cell
is a non-secreting (NS) myeloma cell, e.g., NS0
cell line (see related entries). Other myeloma cells
that may be used include mouse derived P3/X63-
Ag8.653, P3/NS1/1-Ag4-1(NS-1), P3/X63Ag8.
U1 (P3U1), SP2/O-Ag14 (Sp2/O, Sp2), PAI, F0,
and BW5147; rat derived 210RCY3-Ag.2.3; and
human derived U-266AR1, GM1500-6TG-A1-2,
UC729, CEM-AGR, DIR11, and CEM-T15.
Use with: NS0; myeloma cells
Use to make: monclonal antibodies
155
5,916,771; Production of a multimeric protein by
cell fusion method, Hori, et al., June 29, 1999,
assigned to Abgenix, Inc. and Japan Tobacco Inc.
Licensing information: Contact Abgenix.
253
Cholesterol/3-ketosteroid
reductase (p3-KSR) expression
- cholesterol selection/
induction of NS0 cells
BioFactura, Inc. - Licensor, primary
Description: The Cholesterol/3-ketosteroid re-
ductase (p3-KSR) expression system uses lack of
available cholesterol for selection of transformed
cells, particularly in inherently cholesterol-requir-
ing (auxotrophic) NS0 host cell lines (see related
entry), making NS0 cells more useful, including
for antibody manufacture. 3-ketosteroid reduc-
tase (3-KSR) is a member of a large family of
beta-hydroxysteroid dehydrogenases encoded by
this specific gene. The 3-KSR protein catalyzes
the conversion of zymosterone into zymosterol,
a precursor of cholesterol, with 3-KSR-contain-
ing vectors transforming cells so they do not need
cholesterol supplementation (make their own).
Vectors and methods are provided for obtain-
ing NS0 cells that have the ability to survive in a
medium lacking cholesterol, and which express
heterologous protein upon addition of (exposure
to) cholesterol in the culture medium. NS0 or
similar eukaryotic cells that are auxotrophic for
cholesterol are tranformed with a vector contain-
ing an enzyme, 3-KSR, and at least one poly-
nucleotide that encodes a heterologous protein;
followed by selectiion of cells that have the ability
to survive in medium lacking cholesterol. The
medium may be protein- and serum-free.
Use with: NS0 cells
ies
Benefits: Cholesterol is relatively cheap and non-
toxic, compared to many other substances used
for induction of expression.
Adding exogenous cholesterol to NS0 cell
culture media, as is required with most NS0 cells,
poses two problems: a) cholesterol is generally
animal-derived, and 2) media are difficult to
solubilize. In addition to the safety-related issues
surrounding animal-derived cholesterol-contain-
ing media, there are further complications with
regard to consistency, delivery, and solubility of
this media component.
Patents: Related patent applications include
EP1910421 (WO2006125126), COMPOSI-
TIONS AND METHODS FOR METABOLIC
SELECTION OF TRANSFECTED CELLS,
Branco and Sampey, assigned to BioFactura.
Availability: BioFactura sells kits that include
system components and NS0 cells
Licensing information: Contact BioFactura
254
GlycoExpress human cell lines;
NM-F9 cell line - glycolysis
GLYCOTOPE GmbH - Licensor, primary
Description: GlycoExpress human cell lines, usu-
ally human cell line NM-F9 of myeloid leukemia
origin, are useful for expression of glyco-opti-
mised, i.e, fully human, glycoproteins and chime-
ric and humanized antibodies. GlycoExpress cell
lines enable controlled optimization of glycosyl-
ation, such as sialylation, fucosylation, galactosyl-
ation and antennarity. Human post-translational
modifications include N- and O-glycosylation.
GlycoExpress generates fully human glycosylated
proteins with optimised sialylation degrees from
0-100% in order to improve bioactivity, serum
half-life and/or stability, or to avoid immunoge-
nicity. In case studies, it was possible to improve
the quality of biotherapeutics just by optimizing
the sialylation degree. GlycoExpress is based on
glycoengineered human cell lines with optimal
biotechnological properties, including suspension
culture, 15-24 h doubling time, easy cell cloning
and molecular biological modification, produc-
156
tion of high amounts of secreted glycoproteins,
including soluble fragments of membrane proteins
serum free and virus free, with origin and trans-
formation events known, and full documentation.
GlycoExpress has been used to improve the
bioactivity of hGM-CSF in vitro by 500-fold
compared to hGM-CSF from E.coli and yeast.
Due to fully human glycosylation with a high
sialylation degree, this is expected to provide
further advantages for the serum-half life and
antigenicity/immunogenicity.
Use with: human NM-F9 cell line
ies
GOLETZ, et al., Use of human cells of myeloid
leukaemia origin for expression of antibodies,
published April 16, 2008, assigned to GLYCO-
TOPE GmbH; and WO2008028686, FULLY
HUMAN HIGH YIELD PRODUCTION SYS-
TEM FOR IMPROVED ANTIBODIES AND
PROTEINS, GOLETZ, et al., published March
13, 2008, assigned to GLYCOTOPE GmbH.
Licensing information: Glytope offer CRO/
CMO services. As marketed by Glycotope, Gly-
coExpress is, An Integrated System involving
cloning of target protein into own expression vec-
tors; expression of various fully human glycosyl-
ated forms of the target protein with different
sialylation degrees; functional Screening of the
sialylation forms; and selection and characteriza-
tion of sialylation form with highest biological
activity; generation of stable high expression
clones; generation & purification of desired
sialylation form of target protein; up-scaling; and
production. GlycoExpress may be available for
out-licensing.
Further info.:
Novel Human Expression System Glycoengi-
neered for Optimal Glycosylation of Biotherapeu-
tics, Hans Baumeister, Ute Schber, Renate Stahn,
Heiner Pettan, and Steffen Goletz, BioProcess In-
ternational MAY 2006 SUPPLEMENT, pl. 36-41.
255
Human primary preB
lymphocytes; HupreB cells -
human monoclonal antibodies
4-Antibody AG - Licensor, primary
Description: Methods allowing the genetic
modification of long-term proliferating human
primary preB lymphocytes, forming human anti-
body producing cells (HupreB) cells, are useful
for expression of human antibodies. This technol-
ogy uses the natural immune system to generate
in-vivo high affinity fully human antibodies. In
combination with genetic engineering of preB
lymphocytes, forming HupreB cells, fully human
therapeutic antibodies can be generated within
only a few months. 4-ABs technology covers
two complementary approaches to either gener-
ate completely novel, fully human antibodies, or
to optimize the affinity of existing therapeutic
antibodies.
The genetic modification of murine preB cells
is rapid and flexible and allows the modifica-
tion of any aspect of the primary structure of the
expressed antibodies.
The genetically modified preB cells can be
subjected to T-cell dependent stimuli inducing
somatic hypermutation in the human antibody
constructs. Affinity matured antibodies can be
generated allowing the development of thera-
peutic antibodies with the best pharmacological
efficacies. This method can be applied for the
optimization of existing therapeutic antibody can-
didates or for the development of novel therapeu-
tic antibodies specific for any desired target.
4-Antibodys proprietary technology platform
is based on a defined long-term in vitro tissue cul-
ture system for modified primary mouse precursor
(preB) lymphocytes. Novel, fully human antibod-
ies can be developed if preB cell populations are
used, which express a diverse repertoire (library)
of human antibodies. High affinity antibody
producing cells can then be generated by standard
immunizations of mice transplanted with HupreB
157
cell libraries. In addition, the affinity of existing
therapeutic antibodies can be optimized if only a
limited repertoire (or at the extreme, a monoclonal
specificity) with defined antigen-reactivity is ex-
pressed in HupreB cells, and transferred into mice
for in-vivo affinity maturation.
Use with: human primary preB lymphocytes
Use to make: monoclonal antibodies, human
U.S. 20060052585, Method for the generation of
genetically modified vertebrate precursor lym-
phocytes and use thereof for the production of
heterologous binding proteins, Grawunde, et al.,
assigned to 4-Antibody AG.
Licensing information: Contact 4-Antibody AG.
256
NS0 murine myeloma cell line
Description: [See the other more specific NS0-re-
lated entries]. The NSO mouse myeloma cell line
was derived from a mouse plasma cell line (NS1)
that originally produced IgA antibodies. NS0 is
generally more productive than CHO cells and
suited for manufacture of recombinant monoclo-
nal antibodies. NS0 is one of the more popular
systems in large-scale heterologous protein and
antibody expression. Although all derivatives of
the original source murine tumor were high-level
secretors of IgG1, sequential cloning has incre-
mentally silenced that capacity. In its long his-
tory at many locations, NS0 has been subcloned
and otherwise selectively adapted and modified,
resulting in availability of a number of distinct
NS0 lines. NS0 transfectoma can be constructed
by direct cloning methods that promote high and
stable expression of a fully human MAb or other
recombinant protein under the control of multiple/
inducible promoters. Production with NS0 cells
has been scaled-up by multiple companies to 10k
liter scale, including using animal-derived compo-
nent- and protein-free medium.
NS0 cells are inherently cholesterol auxotrophs
and have an essential requirement of cholesterol
in culture medium (see the related cholesterol-
negative cell lines and cholesterol induction
entries).
Two expression systems for the production of
therapeutic proteins are widely used with most
NS0 large-scale production -- the dihydrofolate
reductase (DHFR) system and the glutamine syn-
thetase (GS) system (see related entries). In com-
parison to the DHFR system (now off-patent), the
GS system (requires licensing) is especially used
with nonsecreting (NS0) myeloma cells, offers a
significant time advantage during development,
and requires fewer copies of the recombinant gene
per cell. NS0 cells, unlike CHO cells, are pheno-
typically GS-deficient, with this allowing easy
selection of successful transfectants using the GS
system. The GS system for selection/amplification
is used for most large-scale NS0-based manufac-
ture.
NSO produces predominantly fucosylated G0
IgG. However, in addition to a capacity to add
galactose in (1-4) linkage to N-acetylglucosamine,
NSO may also add a further galactose-linked
(1-3) to the primary galactose. Mammals, with
the exception of higher primates and humans,
express the transferase for adding the galactose
(1-3) galactose structure. In humans, that structure
is an immunogenic epitope. Thus, it is estimated
that 1% of circulating IgG is a specific antibody
directed against the galactose (1-3) galactose-epi-
tope. Infusion of recombinant antibody bearing
that epitope would likely lead to the formation
of immune complexes and provoke a systemic
inflammatory response.
In addition to adding sialic acid at the (2-3)
position, NSO adds a variant N-glycolyl form of
sialic acid at the (2-3) position. NSO cells lack the
capacity to add bisecting N-acetylglucosamine.
Use with: NS0 cells
ies
Background: Cell line ECACC 85110503, the
original NS0, was originally deposited in the
ECACC culture collection in 1981, deposited by
Dr. J. Jarvis, MRC Laboratory of Molecular Biol-
ogy, Cambridge [73B(3) Methods in Enzymology
(1981)].
158
Benefits: Factors that have contributed to the
wide use of NSO include NS0s capacity to sta-
bly and productively incorporate foreign DNA,
protein processing capability, lack of endogenous
antibody production, extensive regulatory pedi-
gree, culture as unclumped single-cell dispersions
in suspension culture, robust growth and high
levels of production in a variety of media, and
adaptability to a variety of derivitization, selection
and production environments.
Licensing information: Multiple cell lines with
no current patent protection are available from
multiple sources, e.g., ATCC and other culture
collection and reagent vendors. Others, particu-
larly with desireable traits, such as adaption to
protein-free media or loss of need for cholesterol
supplementation, are more likely to be patented
and require licenses for commercial use.
Products made using this tech.: NS0-derived
cell lines have been used to manufacture three
FDA-approved recombinant antibodies for thera-
peutic applications in humans
a) Synagis [respiratory syncytial virus monoclonal
antibody, recombinant) from MedImmune, now
part of AstraZeneca.
b) Mylotarg, [Gemtuzumab Ozogamicin - CMA-
676; recombinant humanized CD33 monoclonal
antibody-calicheamicin cytotoxin conjugate (im-
munotoxin)] from Wyeth; and
c) Zenapax [Daclizumab - Interleukin-2 alpha
receptor monoclonal antibody, recombinant]
manufactured and marketed by Hoffmann-La
Roche Inc.
257
NS0-PFCF cells; NS0 cells,
protein- and cholesterol-free -
antibodies
Protein Biopharma, Inc. (PDL) - R&D
Description: PDL Biopharma has reported [Bio-
technol Bioeng. 2007 Feb 1;96(2):294-306] devel-
opment of a recombinant antibody production
platform based on fed-batch culture of non-GS
NS0 cells in protein-free and cholesterol-free me-
dia. In contrast to the commonly used NS0 host
cell lines, which require serum and cholesterol
for growth, and the commonly used expression
vector systems, which use a proprietary glutamine
synthetase selection marker (GS-NS0; see GS
system entries), these NS0 cells are cholesterol-
independent, grow well in a protein-free medium,
use a non-proprietary selection marker, and do not
require gene amplification for productivity im-
provement. See also the Cholesterol/3-ketosteroid
reductase (p3-KSR) expression system entry.
NS0 host cells (ECACC 85110503; see related
entry) were first adapted to grow in a protein-
free, cholesterol-free medium. The resulting host
cell line was designated NS0-PFCF (protein-free,
cholesterol-free).
PDL has reported that five production cell
lines were generated using a common protocol
consisting of transfection by electroporation and
subcloning. The NS0-PFCF host cell lines were
transfected using a single expression vector con-
taining the E. coli xanthine-guanine phosphoribo-
syl transferase gene (gpt), and the antibody heavy
and light chain genes driven by the CMV promot-
er. Each of the five cell lines had a single stably
integrated copy of the antibody expression vector
and antibody genes were correctly expressed.
Using a fed-batch bioreactor process, an average
specific productivity of 20-60 pg/cell-day, and a
volumetric productivity exceeding 120 mg/L-day
was consistently achieved. .
Use with: NS0 cells
Patents: No as yet published patent applications
were retrieved.
Licensing information: Contact the correspond-
ing author, taymar.hartman@pdl.com, and/or
PDL.
159
258
PER.C6 expression; Extreme
density (XD) Technology -
glycoproteins; antibodies
Crucell Holland B.V. - Licensor, primary
DSM Biologics - Licensor, secondary
PERCIVA PER.C6 Development Center - R&D
Description: The human PER.C6 cell line is
becoming a major host cell line and platform tech-
nology. The cell line was designed from the start
for recombinant glycoprotein, including antibod-
ies, manufacture at high yields, with human-like
glycosylation. The cells harbor and express
constitutively the adenovirus E1B gene, with ad-
enovirus E1B a well-known inhibitor of apoptosis
(programmed cell death). Associated XD technol-
ogy involves a proprietary process to achieve cell
densities of up to 150 million cells/mL in com-
bination with the PER.C6 cell line. The high cell
number directly accounts for record high yields of
antibody expression achieved with the cell line.
In June 2008, DSM Biologics and Crucell N.V.
announced a breakthrough -- the production of
IgG antibodies using the PER.C6 cell line (and
XD technology) to produce a record yield of an
unspecified regular (presumably human or
humanized) antibody at over 27 grams/Liter (with
over 95% viability and unchanged product qual-
ity). In March 2008, acheivement of 15 grams per
liter had been reported. This new record surpasses
all other mammalian cell recombinant protein
production systems. Also, the production scale
potential of the cell line has been demonstrated
in an unprecedented successful bioreactor run of
20,000 liters.
PER.C6 technology can be used in a variety
of culture formats including roller bottles, spin-
ner flasks, wavebags and stirred tank bioreactors.
Also, the PER.C6 technology has been made
suitable to perform well in a variety of production
processes including batch, fed-batch and perfu-
sion. Most of the other technologies cited in this
reference as relevant to or useful with CHO cells,
NS0 cells, other human and mammalian host cell
lines are useful with PER.C6 cells.
PER.C6 cells were generated by transfection
of primary human embryonic retina cells using a
plasmid that contained the adenovirus serotype
5 (Ad5) E1A- and E1B-coding sequences (Ad5
nucleotides 459-3510) under the control of the hu-
man phosphoglycerate kinase (PGK) promoter.
PER.C6 cells harbor and express constitutively
the adenovirus E1B gene, a well-known inhibitor
of programmed cell death, or apoptosis.
Although no products using PER.C6 have been
approved, many PER.C6 produced products have
been in the clinic, with several in or heading for
Phase III trials.
Originally starting in the gene therapy market,
which is governed by some of the highest safety
standards, PER.C6 produced products have had
no adverse reactions reported to date.
The PER.C6 cell line is documented like no
other cell line, including turmorigencity and as-
says that go beyond required international guide-
lines. Licensees to PER.C6 cells can review and
refer to the FDA BMF in their regulatory submis-
sions, there by potentially saving time and cost for
regulatory approvals. The Biologics Master File,
initially filed with FDA in 1999, is updated on an
annual basis in collaboration with Merck & Co.,
one of Crucells PER.C6 licensees.
Use with: PER.C6 cells
ies
Benefits: Claimed benefits include excellent
safety profile, scalability and productivity under
serum-free culture conditions. Other benefits of
the PER.C6 cell line include:
- Rapid development - Crucell, DSM and their
vendor network have worked closely together to
offer customers a serum-free, suspension adapted
PER.C6 cell line in three months, and move from
clone to Phase I material in less than 12 months.
- Regulatory acceptance & safety in the clinic
- Many PER.C6 produced products have been
tested in clinical trials, with several heading
towards Phase III trials. In the clinic, not a single
160
adverse event has been documented due to the
use of PER.C6 produced materials. The complete
history of the cell line is documented in an FDA-
filed Biologics Master File (BMF), in greater
detail than any other cell line.
- Human-like glycosylation - PER.C6 cells pro-
vide human-like glycosylation for monoclonal an-
tibodies. Products produced in rodent-derived cell
lines, such as NS/0 and CHO, do not have human
glycosylation profiles like the PER.C6 cell line.
- Scalable and high yields - The PER.C6 cell line
has successfully performed up to a 20,000L scale.
For IgG1 monoclonal antibodies, production lev-
els of 3.5g/L (and up to 40pg/cell/day) have been
achieved with PER.C6 cells..
Patents: PER.C6 was deposited under ECACC
no. 96022940. Crucell has obtained multiple pat-
ents/applications related to PER.C6 cells and their
uses, including manufacturing methods.
U.S. 6,855,544 (earliest U.S. patent), Re-
combinant protein production in a human cell ,
Hateboer, et al., Feb. 15, 2005, assignd to Crucell
Holland B.V., broadly claims, 1. A method for
producing at least one proteinaceous substance
in a eukaryotic cell, said method comprising:
providing a eukaryotic cell having a nucleic acid
sequence in the eukaryotic cells genome, said
nucleic acid sequence encoding adenoviral E1A
and E1B proteins, which eukaryotic cell further
does not comprise a sequence encoding a struc-
tural adenoviral protein in its genome; introduc-
ing a gene encoding a recombinant proteinaceous
substance into the eukaryotic cell; culturing said
eukaryotic cell in a suitable medium; and harvest-
ing at least one proteinaceous substance from said
eukaryotic cell, said suitable medium, or both said
eukaryotic cell and said medium.
U.S. 7,132,280, Recombinant protein produc-
tion in a human cell, Bout, et al., Nov. 7, 2006,
has an abstract stating, Methods and composi-
tions for the production of recombinant proteins
in a human cell line. The methods and composi-
tions are particularly useful for generating stable
expression of human recombinant proteins of
interest that are modified post-translationally, for
example, by glycosylation. Such proteins may
have advantageous properties in comparison with
their counterparts produced in non-human sys-
tems such as Chinese hamster ovary cells.
Availability: Invitrogen markets PER.C6 for non-
commercial uses.
Licensing: DSM and Crucell have formed a col-
laboration, PERCIVIA, for further research and
development using PER.C6 cells. DSM and/or
Crucell should be contacted regarding licens-
ing (and CRO/CMO services). Crucell gener-
ally licenses PER.C6 for all purposes, including
proteins, Mabs, gene therapy, etc. DSM generally
licenses it for proteins only..
Flexible license structures are available for
pure evaluation up through commercial rights.
Licensing terms and costs are customer spe-
cific. The PER.C6 technology platform (license)
includes requisite assistance/information concern-
ing cell line generation technology, cell culture
media development, upstream and downstream
processes, equipment selection, scale-up, technol-
ogy transfer, and regulatory documentation.
In June 2008, DSM Biologics and Crucell N.V.
entered into an agreement with Avid Bioservices,
Inc. of Tustin, California to join their Vendor
Network, with Avid becoming a pre-approved
contract manufacturer (CMO) for licensees of the
PER.C6 cell line located in the western U.S. Avid
is the first U.S.-based contract manufacturer to be
awarded this status.
Licensees are known to include Vaxin, Merck
& Co., GSK, MorphoSys, Sanofi Pasteur, AME/
Lilly Bio A&D, Tibotec (J&J) Medarex, Gen-
eMax, Wyethk Synergenics, Vascular Biogenics,
Acambis, Medimmune, Gene Medicine Japan,
Novartis, Transgene, Sartorius Biotech, Merial
Products made using this tech.: Merck & Cos
HIV vaccine candidate (development recently
abandoned), V520, was produced using PER.C6
technology.
161
259
Retrotransposon vectors -
transgenic avian/chicken cell
culture
Nature Technology Corp. - Licensor, primary
Description: Mammalian retrotransposon vec-
tors are broadly useful for transformation of avian
species, e.g., chickens. Retrotransposon particles
transduced into the new host cell are reverse
transcribed into DNA, and are introduced sta-
bly into the new host genome, from which RNA
transcripts can again be generated from either
the retrotransposon transcriptional unit or from
inserted internal transcriptional units, directing the
expression of useful genes. The introduced genes
are unable to replicate further in the absence of
helper functions.
This technology is useful for gene therapy,
gene insertion and protein expression in cell
culture, for the generation of transgenic animals,
and in other applications where traditional RNA
tumor virus vectors may be considered unsafe or
undesirable.
Use with: birds; chickens
ies
Mammalian Retrotransposons, Hodgson, C.P.;
and 6,027,722, Vectors for gene transfer, Hodg-
son, C.P., both assigned to Nature Technology
Corp.
Licensing information: Contact Nature Technol-
ogy concerning commercial use and licensing.
This technology has been reported as licensed
by Nature Tech. to Avigenics.
260
Sp2/0-Ag14 cells - protein-free
media; antibodies
Description: The Sp2/0-Ag14 myeloma cell line
P3X63Ag8.653 has been adapted to suspension
culture in chemically defined protein- and pep-
tide-free culture media. This a non-Ig-secreting
cell line is suitable for use as fusion parents for
the serum-free culturable hybridomas, and as host
cells for recombinant protein manufacture. One
vendor, Cell Culture Technologies LLC, claims
that The hybridoma Sp2/0-Ag14 cell line is be-
coming the host of choice for the expression and
production of several recombinant glycoproteins.
Sp2/0-Ag14 is a non-Ig-secreting or synthesizing
line derived from a cell line created by fusing a
BALB/c mouse spleen cell and the mouse my-
eloma P3X63Ag8. Th cell line is comparable to
other myeloma lines used for fusions relating to
growth properties, fusion frequency and stability
of resulting hybrids.
Use with: Sp2/0-Ag14 myeloma cells; hy-
bidomas
ies
Benefits: For industrial applications, lines of
evidence suggest that the newly selected cell lines
described here may positively affect part of the
steps involved in a typical process for producing
recombinant proteins, monoclonal antibodies, and
viral particles. The use of a chemically defined
minimal medium decreases the risk of variability
in cell growth characteristics, genetic arrange-
ment, product synthesis rate, and product integ-
rity. The absence of complex additives, proteins,
peptones and peptides in the medium and the high
cell viability achieved during cultivation contrib-
ute to lower the amount of released proteins in the
culture supernatant. The very low protein back-
ground in the supernatant enables the achievement
of products in an almost-purified form by lower
levels of cell biomass, or by cell lines that pro-
duce proteins at low concentrations. Last, but not
least in importance, the elimination of animal-de-
162
rived components dramatically decreases the risk
of virus and mycoplasma contamination, thereby
favoring any industrial process scale-up.
Patents: First reported in 1979 [J Immunol
1979;123:1548], like many other hybridoma-re-
lated technologies, this was not patented, and is
now clearly in the public domain.
Availability: The European Collection of Cell
Cultures (ECACC) and other culture collec-
tions distribute the cell lines (e.g., ECACC
85072401;ATCC CRL 1580) for non-commercial
use
Licensing information: Contact Cell Culture
Technologies LLC.
261
HEK 293 cell line, protein- and
peptide-free (Hektor G) media;
human embryonic kidney (HEK
293) cell line -
Cell Culture Technologies LLC - Licensor,
primary
European Collection of Cell Cultures (ECACC)
- Retail seller
Description: See the Main entry for HEK-293
cells. In 2005, Cell Culture Technologies an-
nounced the successful selection, routine main-
tenance and banking of the human embryonic
kidney HEK 293 cell line in the protein- and
peptide-free Hektor G minimal medium. The
Hektor G medium exclusively consists of non-
animal derived molecules of pharmaceutical grade
quality.
The newly selected cell line directly derives
from the original serum-dependent HEK 293 cell
line provided to Cell Culture Technologies by the
European Collection of Cell Cultures (ECACC).
After selection, the cell lines were transferred
back to ECACC in order to perform Quality Con-
trol testing, including DNA profiling.
Use with: HEK-293 cells
Availability: The European Collection of Cell
Cultures (ECACC), Porton Down, UK, distrib-
utes the cell line (ECAAC HEK 23, Prod. No.
85120602) together with the protein- and peptide-
free Hektor G medium and the basic culture pro-
tocols as a complete starter kit, for non-commer-
cial uses. The newly selected HEK 293 cell line
is released from the ECACC with a Certificate of
Analysis mentioning its suitability for cell culture
work as well as its human origin.
Licensing information: Contact Cell Culture
Technologies LLC regarding commercial use and
licensing.
262
HEK 293 expression
Biotechnology Research Institute, National
Research Council of Canada (NRC- BRI) - Licen-
sor, primary
Description: See the HEK-293 main entry. NRC-
BRI reports having developed a new process
for the production of recombinant proteins, by
transient transfection of suspension-grown hu-
man embryonic kidney cells (293 cell line and its
genetic variants) with an expression vector, using
polyethylenimine (PEl) as a transfection reagent
The vectors, medium and cell line were optimized
to allow highest expression levels in a short
period of time. Recovery of secreted proteins is
easily achieved as the process uses serum-free
medium. The process uses 293E cells expressing
the Epstein-Barr virus (EBV) EBNA 1 protein, in
combination with an oriP-based episomal expres-
sion vector having an improved cytomegalovirus
expression cassette comprising the CMV5 pro-
moter. See the related entries for a HEK-293 cell
lines from NRC-BRI adapted to protein/peptide-
free media.
Transient protein expression is achieved using
the small size pTT5 vector (4.4 kb), a family of
expression vectors that contains the EBV oriP and
an improved cytomegalovirus- based expression
cassette. Use of the pTT vector in HEK293E cells
provided a 2-3 fold increase in transgene expres-
163
sion compared with the pCEP4 vector and a 10-
fold increase compared to pcDNA3.1 vector (both
from Invitrogen).
The system provides fast and scalable transfec-
tion. A robust transient transfection procedure has
been developed that can be carried out at multi-
liters scale. The single step procedure is fast and
easy to perform as there is no need to change the
culture medium.
Optimization of the culture conditions has led
to the formulation of a serum-free growth medium
enriched with selected peptones. This medium
reduces the costs associated with protein purifica-
tion and allows higher transient gene expression
in the absence of serum. Protein expression levels
obtained with 293SFE cells grown (see related
entry) in the optimized serum-free medium were
nearly identical to those obtained with HEK293E
cells cultured in a serum-supplemented medium.
The system has been validated by the success-
ful production and purification (> 95% purity fol-
lowing a single IMAC purification step) of over
thirty recombinant proteins at scales ranging from
1 to 14-liters, with expression yields ranged from
20 to 60 mg/L for non- toxic proteins.
Use with: Hek-293 cells
Benefits: NRC-BRI claims, The process com-
bines in a single step the cell growth, transfection
and protein expression, is carried out without
changing the culture medium, and allows to
achieve high expression levels in a short period
of time. The process may be carried out in a
serum-free, low-protein culture medium, is easily
scalable, compatible with continuous production
processes, and fully adapted to high-throughput
production of milligram quantities of recombinant
proteins.
Transient gene expression is often preferable
to the establishment of stable transfectants as this
latter approach is time consuming and requires
that the expressed protein not adversely affect
the growth of the cells. HEK293SFE provides a
robust large-scale transient transfection process
for fast production of milligram to gram amounts
of proteins. This easily scalable technology, ame-
nable to high throughput production. was obtained
by combining optimized parameters in four key
aspects namely the cell line, the expression vector,
the transfection vehicle and the culture medium.
Patents: Applications include US 10/477148,
filed 2003-11- 07. European, Canadian and Singa-
pore patents are also pending.
Licensing information: Contact NRC-BRI
regarding invention no. 11266. Available for non-
exclusive licensing.
263
HEK 293 expression
Biotechnology Research Institute, National
Research Council of Canada (NRC- BRI) - Licen-
sor, primary
Description: See the HEK-293 main entry. NRC-
BRI reports having developed a new process
for the production of recombinant proteins, by
transient transfection of suspension-grown hu-
man embryonic kidney cells (293 cell line and its
genetic variants) with an expression vector, using
polyethylenimine (PEl) as a transfection reagent
The vectors, medium and cell line were optimized
to allow highest expression levels in a short
period of time. Recovery of secreted proteins is
easily achieved as the process uses serum-free
medium. The process uses 293E cells expressing
the Epstein-Barr virus (EBV) EBNA 1 protein, in
combination with an oriP-based episomal expres-
sion vector having an improved cytomegalovirus
expression cassette comprising the CMV5 pro-
moter. See the related entries for a HEK-293 cell
lines from NRC-BRI adapted to protein/peptide-
free media.
Transient protein expression is achieved using
the small size pTT5 vector (4.4 kb), a family of
expression vectors that contains the EBV oriP and
an improved cytomegalovirus- based expression
cassette. Use of the pTT vector in HEK293E cells
provided a 2-3 fold increase in transgene expres-
sion compared with the pCEP4 vector and a 10-
164
fold increase compared to pcDNA3.1 vector (both
from Invitrogen).
The system provides fast and scalable transfec-
tion. A robust transient transfection procedure has
been developed that can be carried out at multi-
liters scale. The single step procedure is fast and
easy to perform as there is no need to change the
culture medium.
Optimization of the culture conditions has led
to the formulation of a serum-free growth medium
enriched with selected peptones. This medium
reduces the costs associated with protein purifica-
tion and allows higher transient gene expression
in the absence of serum. Protein expression levels
obtained with 293SFE cells grown (see related
entry) in the optimized serum-free medium were
nearly identical to those obtained with HEK293E
cells cultured in a serum-supplemented medium.
The system has been validated by the success-
ful production and purification (> 95% purity fol-
lowing a single IMAC purification step) of over
thirty recombinant proteins at scales ranging from
1 to 14-liters, with expression yields ranged from
20 to 60 mg/L for non- toxic proteins.
Use with: Hek-293 cells
Benefits: NRC-BRI claims, The process com-
bines in a single step the cell growth, transfection
and protein expression, is carried out without
changing the culture medium, and allows to
achieve high expression levels in a short period
of time. The process may be carried out in a
serum-free, low-protein culture medium, is easily
scalable, compatible with continuous production
processes, and fully adapted to high-throughput
production of milligram quantities of recombinant
proteins.
Transient gene expression is often preferable
to the establishment of stable transfectants as this
latter approach is time consuming and requires
that the expressed protein not adversely affect
the growth of the cells. HEK293SFE provides a
robust large-scale transient transfection process
for fast production of milligram to gram amounts
of proteins. This easily scalable technology, ame-
nable to high throughput production. was obtained
by combining optimized parameters in four key
aspects namely the cell line, the expression vector,
the transfection vehicle and the culture medium.
Patents: Applications include US 10/477148,
filed 2003-11- 07. European, Canadian and Singa-
pore patents are also pending.
regarding invention no. 11266. Available for non-
264
HEK293 cell lines
Microbix Biosystems, Inc. - Licensor, primary
McMaster University - R&D
Description: HEK293 (HEK-293) human em-
bryonic kidney cells are a commonly-used hu-
man host cell line used for antibody and other
(glyco)protein manufacture. There never having
been patented has contribututed to their popular-
ity.
HEK-293 is a stable ell line generated by
transformation of human embryonic kidney cell
cultures (HEK) with sheared adenovirus 5 DNA,
and was first described in 1977 (Graham et al.,
J. Gen Virol 1977 Jul;36(1):59-74). The cells
have been adapted (or are adaptable) to grown in
serum-free media. The E1A adenovirus gene is
expressed in these cells and participates in trans-
activation of some viral promoters, allowing these
cells to produce very high levels of protein. HEK-
293 cells also have superior transfection efficien-
cies. For example, the 293-F line from Invitrogen
is characterized by above-average growth rates,
and is easily converted to monolayer culture for
adherent applications.
ies
Benefits: High expression levels; well-studied
and familar to regulatory agencies
Patents: The HEK293 cell line itself is not
patented or subjected to any onerous intellectual
165
property restrictions. For many years Dr. Gra-
ham, McMaster University, its developer, sent it
out without strings attached to anyone having a
legitimate use.
Availability: The best source of HEK293 cells
appears to be Microbix, which has the earliest
passage cells which they got as a result of an
agreement with Dr. Frank Graham, the origina-
tor of the cell line. Most people seem to use the
standard 293 line for protein expression, though it
often does not express high amounts of protein.
The cell line (at different passages) is also
available from many reagent suppliers, and from
ATCC (ATCC CRL-1573).
Licensing information: No license needed; pub-
lic domain
Products made using this tech.: Marketed re-
combinant proteins are manufactured using HEK-
293 cells include:
1) Protein C, activated [Drotrecogin alfa (activat-
ed) - Xigris; recombinant human activated protein
C] from Eli Lilly & Co.; and
2) Gendicine [Genkaxin; Ad-p53, recombinant;
adenovirus vector p53 gene therapy] from Shen-
zhen SiBiono Gene Technologies Co., currently
marketed in Japan.
265
HEK293SFE cell line
sor, primary
Description: See the entry for NRC-BRIs
HEK293 Expression systems and other HEK293
cell line entries. A genetic variant of the HEK293
line has been develooped that expresses the Ep-
stein Barr nuclear antigen (HEK293E) and allows
active propagation of plasmids carrying the appro-
priate origin of replication, resulting in augmented
levels of protein expression. HEK293SFE pro-
vides an improved process for the production of
recombinant proteins by the transient transfection
of suspension-growing cells. Transient expres-
sion can currently be used to provide up to gram
amounts in a relatively short time.
293SFE was established by stably transfecting
serum-free adapted HEK293SF-3F6 cells with
an EBNA1 (Epstein-Barr virus (EBV) Nuclear
Antigen 1) expression plasmid. Although the
readily available HEK293E cell line also allows
the episomal persistence of vectors containing the
EBV origin of replication oriP, the 293SFE cell
line offers the added advantage of being capable
of growing in a serum-free medium.
ies
Benefits: The process is carried out in a single
step, is easily scalable and achieves high expres-
sion levels in a very short period of time.
Licensing information: Contact NRC-BRI re-
garding nonexclusive licensing.
266
HKB-11 (HKB11) expression;
Hybrid of kidney and B cells -
HEK-293 alternative
Bayer Corp. - Licensor, primary
Description: HKB-11 (HKB11) cells, a hybrid
human embryonic kidney 293S (HEK-293S) cells
and
Burkitts lymphoma cells (B-cells), expressing
a(2-6) sialyltransferase [a(2,6)ST] are useful for
recombinant protein expression, providing an al-
ternative to HEK-293 and other mammalian host
cells. The HKB11 system is well suited for both
transient and long-term production of recombi-
nant therapeutic proteins including monoclonal
antibodies.. Clones of hybridized HKB-11 are
easily transfected by electroporation or cationic
liposome and easily adapted to growth in suspen-
sion culture. HKB11 is delivers biopharmaceu-
ticals that are structurally identical to the natural
product. The host/vector system supports the
production of gram quantities of proteins in a
large-scale transient transfection format as well as
166
the development of stable cell lines.
The primary goal of establishing this hybrid
cell line starting with HEK 293S and 2B8 cells
was to resolve the aggregation problem associated
with the adaptation of 293S cells into serum-free
suspension culture. G rowth adaptation studies
have shown that HKB clones grow readily as
serum-free suspension cultures without major
adaptation
efforts. In addition, HKB cells formes smaller and
looser aggregates, while 293S cells form larger
and tighter aggregates under serum-free suspen-
sion conditions.
One clone designated as HKB11 supports high
level expression of heterologous proteins. These
cells grow as small and loose aggregates under
serum-free suspension conditions, a characteristic
that is highly desirable for high cell density cul-
tures in bioreactors. This clone expresses a human
specific glycosylation enzyme, a(2-6) sialyltrans-
ferase [a(2,6)ST]. In addition, these cells do not
express any endogenous immunogloblins, mak-
ing these cells suitable for recombinant antibody
production. The reproducibility of this system has
been demonstrated at lab. scale with recombinant
antibodies, growth factors, and other heterologous
proteins. In one study, stable clones were derived
from HKB11 cells transfected with an improved
dhfr-expression vector. These
HKB11 clones secreted high levels of a tissue-de-
rived factor (40-50 pg/c/d) under serum-free
conditions from which kilogram-quantity of the
protein could be produced. CHO clones derived
under similar conditions produced 10-fold less
protein.
Use with: HKB-11 (HKB11) cells
nal antibodies
6,136,599, Human hybrid host cell for mam-
malian gene expression, Cho, M.-S., Oct. 24,
2000, assigned to Bayer Corp. The patent includes
example expression of recombinant human Factor
VIII, often cited as the largest and a very difficult
glycoprotein to manufacture due to low yields.
Licensing information: MorphoSys uses HKB
11 for its production of HuCAL antibodies
Further info.: Establishment of a Human So-
matic Hybrid Cell Line for Recombinant Protein
Production,
M.S. Cho, H. Yee, S. Chan, J. Biomed Sci., Vol. 9,
No. 6, 2002.
Plants
267
Agrobacterium tumefaciens; Ti
plasmids - transgenic plants
Washington University - Licensor, primary
Description: Agrobacterium bacteria, which
naturally carry the Ti plasmid, have the ability to
transfer genes to plants and fungi, and have been
the most commonly used method for generating
transgenic plants. Agrobacterium tumefaciens
contains Ti plasmids having the unique natural
ability to transform cells of susceptible host plants
by the insertion of an 8 to 23 kilobase (kb) sector
of plasmid DNA into host chromosomal DNA
(Chilton et al., Cell 11:263-71 (1977); Chilton
et al., Proc. Natl. Acad. Sci. USA, 77:2693-97
(1980). Dr. Mary-Dell Chilton, Washington Uni-
versity, and colleagues demonstrated that a single
gene (cytokinin autonomy gene) responsible for
pathogenicity (to plants) could be inactivated
without adversely affecting the gene-transfer pro-
cess or harming the plant cell. They also showed
that new genes from other organisms could be
placed into the agrobacterium DNA, and that
these genes would be incorporated into the plants
chromosomes and pass specific traits to the plant.
Genes to be introduced into a plant are cloned
into a plant transformation vector that contains
the T-DNA (transfer DNA) region of the disarmed
plasmid (pathogenicity-related gene deleted), to-
gether with a selectable marker (such as antibiotic
resistance) to enable selection for plants that have
been successfully transformed. Plants are grown
167
on media containing antibiotic (or other selec-
tive factor) following transformation, and those
that do not have the T-DNA integrated into their
genome die. Other methods for plasmid transfer
are available. When plants are grown from these
transgenic cells, they are fertile and pass the new
genes through seed to future generations of plants.
Transformation with Agrobacterium can be
achieved in two ways. Protoplasts or leaf-discs
can be incubated with the Agrobacterium and
whole plants regenerated using plant tissue
culture. A common transformation protocol for
Arabidopsis is the floral-dip method: the flow-
ers are dipped in an Agrobacterium culture, and
the bacterium transforms the germline cells that
make the female gametes. The seeds can then
be screened for antibiotic resistance (or another
marker of interest), and plants that have not inte-
grated the plasmid DNA die.
Agrobacterium does not infect all plant spe-
cies. There are several other effective techniques
for plant transformation including the gene gun.
Use with: plants; fungi
Background: Agrobacterium is a genus of
Gram-negative bacteria that uses horizontal gene
transfer to cause tumors in plants. Agrobacterium
tumefaciens is the most commonly studied species
in this genus. Agrobacterium is well known for its
ability to transfer DNA between itself and plants,
and for this reason it has become an important
tool for plant improvement by genetic engineer-
ing. A. tumefaciens naturally causes crown-gall
disease in plants. The disease is characterised
by a tumour-like growth or gall on the infected
plant, often at the junction between the root and
the shoot. Tumors are incited by the conjuga-
tive transfer of a DNA segment (T-DNA) from
the bacterial tumour-inducing (Ti) plasmid. The
plasmid T-DNA is integrated semi-randomly into
the genome of the host cell.
Patents: A patent application was filed in 1983 by
Washington University and the patent was granted
in April of 2000. U.S. 6,051,757, Regenera-
tion of plants containing genetically engineered
T-DNA, Barton, was issued on April 18, 2000.
The exemplary claim (no. 1) states, A method of
transforming a dicotyledonous plant susceptible
to transformation by Agrobacterium, comprising:
contacting the plant with an Agrobacterium tume-
faciens bacterium comprising a gene vector, the
vector comprising DNA foreign to the Agrobacte-
rium, and the vector not comprising a functional
cytokinin autonomy gene.
Licensing information: Contact Washington
University.
Products made using this tech.: The majority of
genetically modified plants used in commercial
agriculture have likely been developed using this
technology.
Further info.:
Agrobacterium Gene Transfer: Progress on a
Poor Mans Vector for Maize, Mary-Dell
Chilton, Proceedings of the National Academy of
Sciences of the United States of America, Vol. 90,
No. 8 (Apr. 15, 1993), pp. 3119-3120
Schell J, Van Montagu M., The Ti-plasmid of
Agrobacterium tumefaciens, a natural vector for
the introduction of nif genes in plants, Basic Life
Sci. 1977;9:159-79
Joos H, Timmerman B, Van Montagu M, Schell
J, Genetic analysis of transfer and stabilization
of Agrobacterium DNA in plant cells, EMBO J.
1983; 2(12): 2151-2160.
268
Antibiotic resistance markers
- plants
Description: Monsanto Co. holds patent rights
that reportedly effectively covering the use of any
antibiotic resistance gene as a selectable marker
for plant transformation.
Use with: plants; plant cells
Patents: Monsantos broad rights have been re-
ported to apply only in the United States and are
covered by three granted Patents: US 5034322;
US 6174724; US 6255560; Later two with issue
dates in 2001, with 17 year patent term...Users
168
in all other countries are free to employ in any
way the products claimed in the patents. The only
restriction would come when such users seek to
import products, i.e. plants containing chimeric
constructs claimed in the patents, into the United
States.
U.S. 5,034,322, Chimeric genes suitable for
expression in plant cells, Rogers, S.D., et al.,
July 23, 1991, has claim 1, A chimeric gene
capable of expressing a polypeptide in plant com-
prising
in sequence: a) a promoter region from a gene
selected from the group consisting of an Agro-
bacterium tumefaciens opine synthase gene and
a ribulose-1.5-bis-phosphate carboxylase small
subunit gene; b) a structural DNA sequence
encoding a polypeptide that permits the selection
of transformed plant cells containing said chime-
ric gene by rendering said plant cells resistant to
an amount of an antibiotic that would be toxic to
non-transformed plant cells, said structural DNA
sequence being heterologous with respect to the
promoter region; and c) a 3 non-translated region
of a gene naturally expressed in plants, said
region encoding a signal sequence for polyadenyl-
ation of mRNA.
Licensing information: Contact Monsanto re-
269
Chloroplast expression - plants
University of Central Florida - Licensor, pri-
mary
Description: The University of Central Florida
(Dr. H. Daniell, et al.) has developed chloroplast
expression technologies, which are currently be-
ing applied to the development of oral vaccines.
Use with: plant cells; plants; tobacco
Patents: Related patents/applications include
U.S. 7,294,506, Daniell, et al., Nov. 13, 2007,
with its abstract stating, Tobacco chloroplast
transformation vectors comprising a multi-gene
operon encoding a biopharmaceutical protein
and a chaperonin and; CA2608671 (and equiv.),
CHLOROPLASTS ENGINEERED TO EX-
PRESS PHARMACEUTICAL PROTEINS,
with its abstract stating, Vaccines for conferring
immunity in mammals to infective pathogens are
provided, as well as vectors and methods for plas-
tid transformation of plants to produce protective
antigens and vaccines for oral delivery. The vac-
cines are operative by parenteral administration
as well. The invention also extends to the trans-
formed plants, plant parts, and seeds and progeny
thereof. The invention is applicable to monocot
and dicot plants.
Central Florida
H. Daniell, A. Dhingra and L. Allison, (2002)
Chloroplast transformation: from basic molecular
biology to biotechnology. Reviews in Plant Bio-
chemistry & Biotechnology 1: 1-20.
Chloroplast Genetic Engineering, Henry Daniell,
Biotechnology Journal, Volume 1 Issue 1, Pages
31 - 33
H. Daniell, M.S. Khan and L. Allison (2002)
Milestones in chloroplast genetic engineering: an
environmentally friendly era in biotechnology.
Trends in Plant Science, 7:84-91.
270
Chloroplast Transformation
Technology (CTT) - plant cells
Dowpharma, Dow Chemical Co. - Licensor,
primary
Kentucky BioProcessing, LLC (KBP) - Licen-
sor, secondary
Chlorogen, Inc - Licensor, secondary
Auburn University - R&D.
University of Central Florida - R&D, of tech.
Description: Chloroplast transformation technol-
ogy (CTT) facilitates hyper-expression of recom-
binant proteins in tobacco chloroplasts. CTT is
based on the inherent nature of chloroplasts to
169
hyper-express genes during plant leaf develop-
ment. Plants with transformed chloroplasts have
potential to provide low-cost, stable and quality
proteins.
Each plant cell contains approximately 100
chloroplasts, each of which can have up to 100
copies of the total plant genome (all the genes
within the plant and the proteins they produce).
Therefore, a single cell can produce up to 10,000
copies of a specific protein, as opposed to only
one or two in the single plant cell nucleus. The
chloroplast has the capability to assemble com-
plex proteins such as monoclonal antibodies and
human cell regulators. Therefore, CTT has po-
tential to be a most efficient method of producing
important therapeutic proteins in plants.
CTT vector DNA is coated onto gold particles
and delivered to the chloroplasts using biolistic
mediated transfection. Once the transgene cassette
has been integrated into a copy of the chloroplast
genome and 20-25 cell divisions occur under
selective conditions, then all chloroplasts within
each cell will contain the transgene. Chloroplast
genomes are maternally inherited, so all seeds or
cloned tissue from a single plant will contain the
transgene. Therefore, the CTT system can pro-
duce master seed stock in a single generation.
Once the genetic manipulation of tobacco has
occurred and levels of protein expression are
sufficient for commercial application, the protein
must be extracted from plant material and purified
to meet FDA requirements. Compared with tech-
nologies such as mammalian cell culture, which
are capital and operation intensive, the manufac-
ture of tobacco produced proteins can be very cost
effective due to the low cost of field production
and upstream processing.
Use with: plants; tobacco
ies
Background: Plant cells contain several compart-
ments, including the nucleus, the mitochondria
and the chloroplasts, where photosynthesis is
conducted. Any and all of these components can
be penetrated via the most widely used method
of transforming cells, the Biolistics gene gun.
The gene gun, which Chlorogen and many other
companies use to introduce new genes into plants,
is not precise. It bombards a section of plant mate-
rial with tiny metal particles coated with DNA.
The process is just as likely to shoot new genetic
material into the chloroplast as the nucleus or
mitochondria. However, with proper genetic
instructions, the introduced genes function only
inside the chloroplast. Chlorogen has invented
and patented genetic sequences or regulatory sig-
nals that tell foreign genes to function within the
chloroplasts and only the chloroplasts. Research
by Chlorogen and other groups has demonstrated
successful chloroplast transformation in several
crops.
Patents:
Availability:
Licensing information: LBSC, the original
commercial developers, divested this technology
to Chlorogen. To facilitate scale-up and cGMP
production of proteins, Chlorogen has established
a strategic manufacturing alliance with Kentucky
Bioprocessing, LLC (KBP), a contract research
and manufacturing company, which purchased the
Owensboro, Kentucky-based assets of the former
Large Scale Biology Corporation (LSBC; which
has apparently developed/licensed much of CTT
with/from Auburn University and the University
of Central Florida.), the original developer, patent
assignee, etc. of this technology. KPB welcomes
offering CTT and other plant-based CRO/CMO
services.
In Sept. 2007, Dow AgroSciences LLC
secured exclusive rights to Chloroplast Transfor-
mation Technology (CTT) from Chlorogen, Inc.
(which obtained its rights from LBSC).
271
Coupled regeneration/
transformation, plants
Hi-Genomics, LLC - Licensor, primary
University of Toledo - R&D
Description: Coupled regeneration/transforma-
170
tion couples high output, rapid, genotype indepen-
dent regeneration to high frequency DNA transfer
using either Agrobacterium or biolistic (gene gun)
transformation. This allows for faster and more
robust breeding, genetic transformation, research
and development, production and market release
than any competing technology. The methods
are universal in their application to monocots and
dicots; and chloroplast transformation systems
for corn, soybeans and alfalfa. It can be used to
transfer any value-added genes and bring product
to market years ahead of the competing technolo-
gies. Because of its high rate of T-DNA transfer,
use of patented super-virulent Agrobacterium
strains is not needed.
The system offers a contained, compact, safe
growing protocol for those seeking to extract
products directly from seeds or flowers.
Benefits: What makes this unique is its coupling
of high output, rapid, genotype independent
regeneration technologies to high frequency DNA
transfer into crops for incorporating value-added
traits.
Patents: Apparently related patents include U.S.
5,376,543, Agrobacterium mediated transfor-
mation of germinating plant seeds, Chee, P.P., et
al., Dec. 27, 1994, assigned to the University of
Toledo, with its abstract stating, A non-tissue
culture process using Agrobacterium-mediated
vectors to produce transgenic plants from seeds of
such plants as the common bean and soybean.
Licensing information: Hi-Genomics is exclu-
sive licensee of this technology from the Univer-
sity of Toledo in the areas of plant tissue culture
and genetic engineering of monocots and dicots.
Exclusive licenses in other areas are available
from the University of Toledo in the areas of plant
tissue culture and genetic engineering of mono-
cots and dicots.
272
GENEWARE expression;
Tobacco mosaic virus (TMV)
vectors - tobacco; plants
Kentucky BioProcessing, LLC (KBP) - Licen-
sor, primary
Large Scale Biology Corp. (LBSC) - Assignee,
patent
Description: GENEWARE expression, generally
used in tobacco plants, provides a high-yield fac-
tory for manufacturing any protein, either human,
animal or plant, in bulk, with expression of for-
eign genes transiently, with no permanent genetic
modification of the host plant. GENEWARE
provides transient, plant viral-vector-based gene
expression utilizing a modified Tobacco Mosaic
Virus (TMV), or similar RNA viral replicon,
carrying its normal housekeeping genes plus an
introduced new RNA promoter and structural
gene to encode a protein of interest. LSBCs dual
subgenomic promoter system is designed for the
coordinated expression of a wide range of soluble
(free) protein products with high efficiency.
Kilogram amounts of a wide array of complex
biopharmaceuticals can be produced rapidly with
low capital investment relative to conventional
biotechnology manufacturing operations.
GENEWARE is an inherently safe technology,
expressly designed not to cause permanent genetic
modification. The viral vectors used to install
genes are well maintained within the tobacco
plant and do not spread or infect other plants or
organisms. These vectors are partially disabled
and non-competitive with wild-type virus, a
property that adds an additional measure of safety.
Whereas other viral vectors insert foreign DNA
into the genome - the basis of genetic modifica-
tion - GENEWARE does not. Instead, it merely
causes large quantities of messenger RNA - a
disposable, short-lived copy of the gene - to be
manufactured in the cytoplasm of each plant cell.
By targeting the accumulation of desired protein
products in various parts of the plant for easier
171
recovery, LSBC can efficiently manufacture and
purify a wide range of important molecules.
Use with: tobacco; other plants
Background: GENEWAREs basic principle is
to use a safe vector modified from a virus to place
any gene (or a large number of genes) within a
test organism. The organism then manufactures
the genes protein product, which can be collected
and purified efficiently in pilot and commercial
bioprocessing facilities.
Benefits: Cheap; simple; quick; no genetic modi-
fication (biosafety or containment concerns)
While genetic modification may remain a
controversial field, GENEWARE enables the
expression of foreign genes transiently, with no
permanent genetic modification of the host plant.
Coupled with the use of disabled viral vectors
and the fact that tobacco is not a food crop, this
ensures that GENEWARE is inherently safe.
Patents: LBSC, now KBP, has received a number
of related patents.
Availability: Contact KBP, which offers related
CRO/CMO services.
Licensing information: In June 2008, Kentucky
BioProcessing completed acquisition of the GE-
NEWARE intellectual property from LBSC. The
company stated, While there are a number of
technologies enabling the expression of proteins
in plants, most systems are held on a proprietary
basis with limited commercial availability for
those outside of its immediate ownership. In an-
nouncing the acquisition, KBP signaled its intent
to use the technology as a tool in business devel-
opment. It also seeks to enhance the reputation of
the Owensboro area in the plant-made pharma-
ceutical and plant-made industrial protein industry
by offering broad commercial licensing oppor-
tunities to the GENEWARE system...We believe
that combining the availability of this enabling
technology with our processing and commercial
scale production capabilities enables us to offer
researchers, collaborators and other product de-
velopers a clearly defined commercialization path
and competitive advantage.
KPB also stated, Tens of millions of dollars have
been invested in the development of Geneware
[by LBSC, primarily]. KBP offers access to this
expression system with a clear path to a commer-
cial license. If an upfront arrangement is made for
KBP to conduct the bioprocessing of the target
protein, special licensing arrangements can be
made.
Products made using this tech.: LSBC/KBP has
successfully made a number of products through
its GENEWARE biomanufacturing technology,
including r-Aprotinin, rh-Alpha Galactosidase, rh-
Interferon Alpha 2a and 2b, and personalized non-
Hodgkins lymphoma (NHL) cancer vaccines.
Sigma-Aldrich Fine Chemicals entered into an
agreement with LSBC (presumably now KBP) for
manufacture Apronexin NP, animal-free, recombi-
nant, plant-made, bovine-sequence aprotinin using
GENEWARE expression technology.
273
Glyco-Engineered Moss;
Physcomitrella patens
expression; moss expression
promoting regions (MEPRs) -
glycosylation; antibodies
Greenovation Biotech GmbH - Licensor, pri-
mary
Universithy of Freiburg - R&D
Description: Glyco-Engineered Moss, using the
moss Physcomitrella patens modified to glycosyl-
ate proteins in human-like fashion, is useful for
glycoprotein, including antibody, manufacture.
Cultures are easily scaled to several thousand
liters. Since proteins are excreted into the me-
dium, downstream purification is easier than with
mammalian cell cultures. Mosses are the only
known plants which show a high frequency of
homologous recombination, allowing targeted
gene knock-outs and production strain design.
Transgenic strains are generated without the use
of any marker and consequently no antibiotics are
172
used during the entire production process. The
fast and flexible transient expression system al-
lows analysis of optimal expression tools prior to
the generation of transgenic strains.
By knocking out the genes coding for relevant
transferases and by simultaneous introduction of
the human 1-4 galactosyltransferase-gene, hu-
manisation of N-glycans in mosses was achieved.
Cultivation in suspension allows up-scaling of the
photobioreactors up to several 1000 L. Through
phototrophic (using light/photosynthesis) cultiva-
tion, the system requires a simple medium and
together with secretion of the heterologous protein
greatly facilitates downstream processing. In
October, 2005, greenovation Biotech introduced
a polyethylene glycol-mediated transient gene
expression system for gene expression in moss
with or without the use of viral or Agrobacterial
vectors.
Transgenic strains of the moss Physcomitrella
patens were created in which the alpha(1,3)-fu-
cosyltransferase and beta(1,2)-xylosyltransferase
genes were knocked out by targeted insertion of
the human beta(1,4)-galactosyltransferase coding
sequence in both of the plant genes (knockin).
The transgenics lacked alpha(1,3)-fucose and
beta(1,2)-xylose residues, whereas beta(1,4)-ga-
lactose residues appeared on protein N-glycans.
Despite these significant biochemical changes,
the plants do not differ from wild type with regard
to overall morphology under standard cultivation
conditions. This combined knockout/knockin ap-
proach provides the safe and flexible production
of correctly processed pharmaceutical proteins
with human-like N-glycosylation profiles.
Time-to-market is comparable to traditional
expression systems. The transient expression sys-
tem allows feasibility studies and stable produc-
tion strain development does not require crossing
steps or regeneration of whole plants.
Antibodies which were synthesized and folded
in genetically modified moss strains have shown
40-fold higher activity than comparable antibod-
ies produced in mammalian cells. Variations of
the C-terminal part of the heavy chain of antibod-
ies normally detected on recombinantly produced
antibodies are not present on moss-derived
antibodies, resulting not only in highly reduced
heterogeneity but also providing a peptide struc-
ture which is more similar to the situation found
on human IgG molecules. Glyco-Engineered
Moss-expressed antibodies are assembled cor-
rectly, have shown the identical molecular weight
and the same specificity in comparison to its
parent antibody and also have shown the expected
glyco-optimized N-linked oligosaccharide pattern
lacking core fucosylation.
A more than 30-fold increased ADCC activity
of such a fucose-deficient therapeutic IgG1 anti-
body recognizing the carbohydrate antigen Lewis
Y (over-expressed on epithelial cancers) has been
be achieved independently of the FcgRIII geno-
type of the patient, i.e., expressed IgG1 antibodies
participate in cellular immunity/cytotoxic T-lym-
phocyte-based immunity. This improved ADCC
effector function is not impaired by normal human
serum up to 40%, in contrast to the ADCC activity
mediated by the parental antibody.
Use with: Physcomitrella patens (moss)
ies
20080141387, Protein Production, Reski, R.,
et al., June 12, 2008, with its abstract stating,
Bryophyte plants and bryophyte plant cells com-
prising dysfunctional fucT and xylT genes and an
introduced glycosyl transferase gene, methods for
the production of glycosylated proteins therewith,
vectors and uses thereof. U.S. 20060236432,
Moss expressing promoting regions, concerns
wild type moss nucleus-derived moss expression
promoting regions (MEPRs).
Licensing information: Contact greenovation
Biotech GmbH regarding commercial use, licens-
ing and CRO/CMO services.
Bayer Innovation GmbH hasconcluded a
licensing and service agreement with and greeno-
vation Biotech GmbH.
Further info.: Glyco-engineering of moss
lacking plant-specific sugar residues, Huether
173
CM, Lienhart O, Baur A, Stemmer C, Gorr G,
Reski R, Decker EL, Plant Biol (Stuttg). 2005
May;7(3):292-9.
274
iBioLaunch expression; Launch
vectors - proteins and Mabs in
plants
InB:Biotechnologies, Inc. - Licensor, primary
Description: iBioLaunch and related Launch
vectors carried by an Agrobacterium (see related
entry) are useful for transient high level expresion
of proteins, including antibodies, in transformed
plants. This unique accelerated technology plat-
form, iBioLaunch, has the potential to revolution-
ize the manufacture of biopharmaceuticals.
A launch vector is constructed of a cDNA se-
quence that encodes the target protein cloned into
a plant viral gene expression vector. The launch
vectors are introduced into Agrobacteria (see
related entry) where they multiply to very large
copy numbers. The Agrobacteria, carrying their
payload of launch vectors, are introduced into all
aerial parts of non-genetically-modified plants
by vacuum infiltration. This process provides
for near complete leaf coverage, increasing the
efficiency and speed of protein production. Once
the plants have been infiltrated with Agrobacteria,
the viral sequences of the launch vectors along
with the cloned target sequences are massively
amplified through virus replication. Translation
of these recombinant viral vector mRNAs results
in accumulation of hundred milligram quanti-
ties of target protein per kilogram of fresh plant
tissue in less than a week. The plants accumulate
these high levels of target protein in their leaf and
stem tissue. This is termed transient expression
because the target gene sequence is not incorpo-
rated into the plant chromosome and thus does
not result in the creation of transgenic plants. The
plant biomass is harvested, ground and clarified to
produce an extract containing the protein of inter-
est. Proteins are further purified as required using
well-established separation and chromatography
steps.
Use with: plants
ies
Benefits: Compared to other expession systems,
In:B claims very low overall cost, short produc-
tion timescale, very high scale-up capacity, high
product quality, only minor differences in glyco-
sylation, low rist of contamination, and inexpen-
sive storage. The iBioLaunch platform is capable
of:
- Rapid & Efficient Production-transient nature of
the gene expression technology and short growing
cycle of non-genetically-modified plants create
time-efficient production capabilities.
- High Production Capacity & Scalability-non-
transgenic plants can be easily scaled-up in con-
tained growth facilities to provide large amounts
of biomass and expressed protein in a short period
of time.
- Increased Safety-plants are not infected with
pathogens hazardous to humans or animals and
the system eliminates the use of live infectious
agents or tissues and materials of animal origin.
- Low Cost-facilities and source material provide
economical product manufacturing capabilities.
- Flexibility-capable of rapid modification to pro-
duce enhanced vaccines in response to emerging
or mutating virus strains
Patents: Dr. Eric B. Kmiec, formerly with the
Univ. of Delaware, now with Thomas Jefferson
University, is the primary inventor of iBioLaunch.
Related patent applications include U.S
20030163849, Plant gene targeting using oligo-
nucleotides, May, G.D., et al., with its abstract
stating, Methods and compositions are presented
for the generation of targeted alterations in a
plant genome using double-stranded homoge-
neous oligonucleotides containing a single type of
nucleotide. These methods can be used to correct
mutations, introduce mutations and/or alter gene
activity in a plant cell. A cell-free assay system for
monitoring genetic alteration using the oligonu-
cleotides of the invention is also presented; and
174
20030236208, Kmiec, E.G., Targeted chromo-
somal genomic alterations in plants using modi-
fied single stranded oligonucleotides, et al., Dec.
25, 2003, with its abstract stating, Presented are
methods and compositions for targeted chromo-
somal genomic alterations with modified single-
stranded oligonucleotides. The oligonucleotides
of the invention have modified nuclease-resistant
termini comprising LNA, phosphorothioate link-
ages or 2-O-Me base analogues or combinations
of such modifications.
Licensing information: In:B, as a CMO, primar-
ily offers the iBioLaunch technology platform to
biotechnology and pharmaceutical companies for
production of vaccines, antibodies and therapeutic
proteins.
Products made using this tech.: In April 2008, it
was reported that an influenza vaccine manufac-
tured by InB provided complete protection against
infection in the ferret challenge model and proved
highly immunogenic in a mouse model. A number
of other plant-expressed vaccines and infectious
disease monoclonal antibodies are in early devel-
opment.
The InB:Bio technology platform has demon-
strated utility for the production of a broad range
of proteins including vaccine antigens, antibodies
and other therapeutic proteins.
275
LEX System; Lemna
(duckweed) expression - algae,
whole plants
Biolex Therapeutics - Licensor, primary
Description: The LEX System uses vector-
transformed Lemna genus whole alage, small,
free-floating, fresh-water plants for expresssion
of recombinant proteins. Lemna can secrete
the target protein directly into inorganic media,
considerably simplifying purification. The aquatic
plants secrete the protein of interest into a simple
inorganic growth media comprised of water and
minerals. The level of protein production per liter
of medium in duckweed is on the same order
of magnitude as yeast gene expression systems.
Plants demonstrate post-translational processing
that is similar to mammalian cells.
The LEX System can produce antibodies with
a fully human glycosylation structure without
fucose. The technology also has the ability to pro-
duce antibodies that have a fully G0 glycosylation
structure, lacking galactose. This can provide
antibodies with greater efficacy and potency, less
side effects, and reduced infusion times.
Lemna does not support human or zoonotic
pathogens and, because all other components of
the system are defined and synthetic, viral inacti-
vation steps in purification are unnecessary. Cost
advantages result from simple media requirements
for plant growth, inexpensive facilities, and sim-
plified down-stream processing.
The Lex System grows Lemna plants in con-
tained and closed systems. Transgenic Lemna
are grown in aseptically sealed vessels, housed
in growth rooms with artificial lighting, and kept
in a controlled and contained facility. The plants
grow on aqueous media consisting of water and
inorganic nutrients. CO2 in the air is the only
carbon source. The completely artificial and
enclosed environment, combined with lack of
flowering and seed production, make the Lemna
System uniquely contained and controlled when
compared to other plant and animal transgenic
systems.
Duckweed plant or duckweed nodule cultures
can be efficiently transformed with an expres-
sion cassette containing a nucleotide sequence
of interest by any one of a number of methods
including Agrobacterium-mediated gene transfer
(see related entry), ballistic bombardment, or elec-
troporation.
Use with: Lemna acquatic plants
ies
Background: The duckweeds are the sole mem-
bers of the monocotyledonous family Lemnaceae.
The four genera and 34 species are all small, free-
floating, fresh-water plants whose geographical
175
range spans the entire globe. Although the most
morphologically reduced plants known, most
duckweed species have all the tissues and organs
of much larger plants, including roots, stems,
flowers, seeds and fronds. The growth habit of the
duckweeds is ideal for microbial culturing meth-
ods. The plant rapidly proliferates through veg-
etative budding of new fronds, in a macroscopic
manner analogous to asexual propagation in yeast.
This proliferation occurs by vegetative budding
from meristematic cells. The meristematic region
is small and is found on the ventral surface of
the frond. Meristematic cells lie in two pockets,
one on each side of the frond midvein. The small
midvein region is also the site from which the
root originates and the stem arises that connects
each frond to its mother frond. The meristematic
pocket is protected by a tissue flap. Fronds bud al-
ternately from these pockets. Doubling times vary
by species and are as short as 20-24 hours.
Sexual reproduction in duckweed is controlled
by medium components and culturing conditions,
including photoperiod and culture density. Flower
induction is a routine laboratory procedure with
some species. Plants normally self-pollinate, and
selfing can be accomplished in the laboratory by
gently shaking cultures. By this method, inbred
lines of Lemna gibba have been developed.
Benefits: Human and zoonotic pathogens do not
infect the plants, which grow in inorganic media
(water and salts) with light and CO2.
Both FDA and the UKs Medicine and Health-
care products Regulatory Agency (MHRA) have
said that approval of proteins expressed in Bio-
lexs Lex System will not require dedicated viral
inactivation and removal processes.
Other benefits/characteristics include:
Efficient production of complex proteins
Protein refolding not needed
Novel protein production method
Broad freedom to operate
Low capital requirements
Low operating expenses
Shorter lead times
Patents: BioLex acquired LemnaGene SA and
combined the two companies patent estates cov-
ering two members of the Lemnacaea family.
Exemplary U.S. paents include 6,815,184,
Expression of biologically active polypeptide in
duckweed, Stomp, A.-M., et al., Nov. 9, 2004,
assigned to BioLex, with its abstract stating:
Methods, nucleic acid sequences, and trans-
formed duckweed plant or duckweed nodule
cultures for the expression and the secretion of
biologically active polypeptides from genetically
engineered duckweed are provided. Expression of
recombinant polypeptides in duckweed is im-
proved by modifying the nucleotide sequence of
the expression cassette encoding the polypeptide
for improved expression in duckweed. Recovery
of biologically active polypeptides from duck-
weed is improved by linking the biologically
active polypeptide to a signal peptide that directs
the secretion of the polypeptide into the culture
medium.
Licensing information: Contact Biolex regarding
CRO/CMO services, commercial use and licens-
ing.
Products made using this tech.: The LEX
System has successfully produced every protein
(>35) attempted. Biolex has produced proteins for
Centocor/J&J and other clients.
BioLex is developing Locteron, a biosimilar/
follow-on biologic version of interferon alfa-
2b (similar to Intron A). The company has the
capability to produce one million doses/year in its
current manufacturing facility. Positive Phase IIA
trial results were reported in April 2008.
LEX System Biolex, in conjunction with Me-
darex, has produced monoclonal antibodies which
appear to be superior in vitro when compared to
those produced by traditional CHO cell methods.
176
276
Nuclear transfer Cultured
inner cell mass cells (CICM)
- transgenic animals; cloning
from somatic cells
stART Licensing, Inc. - Licensor, primary
University of Massachusetts (UMass) - As-
signee, patent
Advanced Cell Technology, Inc. - Assignee, pat-
ent
Description: Improved methods for nuclear
transfer involving the transplantation and culture
of donor differentiated cell nuclei into enucle-
ated oocytes of the same species as the donor cell
are provided. The nuclei of the transfected cells
are injected into matured oocytes from which the
original DNA has been removed. The transgenic
embryos are then transferred to foster mothers for
development into transgenic calves. The resultant
nuclear transfer units are useful for multiplication
of genotypes and transgenic genotypes by the pro-
duction of fetuses and offspring, and for produc-
tion of isogenic cultured inner cell mass (CICM)
cells, including human isogenic embryonic or
stem cells. Production of genetically engineered
or transgenic mammalian embryos, fetuses and
offspring is facilitated, since the differentiated
cell source of the donor nuclei can be genetically
modified and clonally propagated.
A single transgenci dairy cow can produce up
to 10,000L/year of milk. With expression levels of
recombinant proteins in the milk of transgenic an-
imals in the range of 1-10g/L, only a limited num-
ber of cows is enough to meet worldwide market
demands for many recombinant (glyco)proteins
This method also substantially reduces the time
needed for product development, as production
herds are established directly, bypassing the con-
ventional breeding process. As little as 15 months
after establishing the first pregnancies, signifi-
cant quantities of milk can be obtained through
lactation (see relate milk protein promoter-related
entries) induction to initiate preclinical product
assessment. Large-scale production of calving-in-
duced milk can begin after about 33 months.
Use with: human cells; animal cells; transgenic
animals
ies
Background: Whereas Herman, the worlds
first transgenic bull, was generated through mi-
croinjection, nuclear transfer involves importing
transgenic DNA into female bovine cells. .
Cloning using donor nuclei from proliferating
somatic cells, August 31, 1999, Slice, S.L., et al.,
with claim no. 1 broadly claiming, An improved
method of cloning a non-human mammal by
nuclear transfer comprising the introduction of a
non-human mammalian donor cell or a non-hu-
man mammalian donor cell nucleus into a non-hu-
man mammalian enucleated oocyte of the same
species as the donor cell or donor cell nucleus
to form a nuclear transfer (NT) unit, implanta-
tion of the NT unit into the uterus of a surrogate
mother of said species, and permitting the NT unit
to develop into the cloned mammal, wherein the
improvement comprises using as the donor cell
or donor cell nucleus a proliferating somatic cell
that has been expanded in culture, or a nucleus
isolated from said somatic cell. U.S. 6,234,970
also covers the technology.
Licensing information: Contact stART, which
has a strong patent portfolio concerning nuclear
transfer to generate transgenic animals, including
technology developed by the University of Mas-
sachusetts and Advanced Cell Technology, Inc.
In 2006, stART Licensing, Inc. settled a patent
dispute with Advanced Cell Technology, Inc. and
the University of Massachusetts (University), with
stART receiving licensing rights.
Pharming B.V. and, presumably, other leaders
using nuclear transfer for transgenic animal de-
velopment for protein manufacture have licensed
this technology. Pharming uses transgenic cattle
for manufacture of lactoferrin, and is seeking
Generally Recognized As Safe (GRAS) food
177
additive status in the U.S. Pharming developed
Rhucin [C1INH; C1-INH; human complement C1
esterase inhibitor, re-combinant] expressed from
transgenic rabbits apparently using this technol-
ogy. Although approval was recently denied in
the EU for treatment of hereditary angioedema
(HAE), this was due to clinical efficacy, not prod-
uct-related, issues. A BLA for Rhucin has been
filed with FDA.
277
Nuclear Transformation Suite,
plants
Icon Genetics - Licensor, primary
Description: A suite or collection of various
technologies for generation of transgenic plants
includes nuclear transformation systems based on
translational (promoter-less), translation-fusion,
polycistronic vectors and vectors that utilize vec-
tor assembly in planta or gene fragment trans-
splicing, along with non-leaky gene switches
based on proteins. This provides biosafety, speed,
precision, expression control and freedom-to-op-
erate.
This suite of technologies offers:
Efficient translational vectors
Translation-fusion vectors
Bi- and polycistronic vectors
Vectors utilizing intein-based trans-splicing
Gene locks/switches
New binary vector system
Artificial plastid transit peptide
IRES elements for eukaryotes
T-DNA assembly in planta
Rapid line conversion
Use with: plant cells; plants
Patents: The suite of technology is covered by 11
or more patents.
Licensing information: Icon Genetics provides
related training, technology transfer and contract
research and manufacturing services.
278
Plant expression - glycosylation
Washington State University Research Founda-
tion (WSURF)
Description: Methods based on preventing
acquisition of golgi-mediated modifications to
asparagine-linked glycans (preventing undesirable
glycosylation of proteins in plants) by retaining
proteins in the endoplastic reticulum (ER) of plant
cells enable expression of pharmaceutically active
glycoproteins with human-like glycosylation in
plants.
Background: Most proteins have to be put into
the secretory pathway before they acquire the
proper structure to be functionally active. A
complication is that these proteins almost always
have sugar chains attached to them when they are
first expressed and put into the lumen of the ER.
At this stage, the sugar chains of plant-expressed
(glyco)proteins are not different from those on
human proteins. It is only when the newly syn-
thesized protein moves to the golgi apparatus
that plant-specific changes which are very im-
munogenic in certain animals occur. By trapping
the desired protein in the ER, the changes can
be prevented. For many reasons, this goal has
been quite difficult to achieve. The discovery of a
unique function of a protein by Washington State
University researchers appears to accomplish this
goal and be useful in plant biotechnology applica-
tions where it is important to prevent acquisition
of golgi-mediated modifications to recombinant
proteins. Directing a recombinant protein into the
ER to PAC vesicle pathway where it can accu-
mulate without undergoing glycan modifications
or proteolytic cleavage seems to accomplish the
goal.
Patents: Application(s) reported filed, but no
published applications retrieved from several
simple searches.
178
State University Research Foundation (WSURF).
279
pPIPRA vectors - plants; public
domain
Public Intellectual Property Resource for Agri-
culture (PIPRA) - Licensor, primary
Description: The Public Intellectual Property Re-
source for Agriculture (PIPRA) is facilitating the
design, construction, and testing of plant transfor-
mation vectors with maximal freedom-to-operate
(no or minimal patent licensing). PIPRA staff, a
working group of leading plant transformation
scientists, and PIPRAs pro bono attorneys are
working together to create vectors with as many
components as possible from the public domain or
owned by PIPRA members with known licensing
terms. The vectors will be distributed on a roy-
alty-free basis for humanitarian uses.
The pPIPRA plant transformation vector
system will incorporate technically proven, plant-
derived components and marker-free strategies.
PIPRA will design, develop and test the transfor-
mation system in model monocot and dicot plant
systems before making the transformation tools
available. The pPIPRA transformation system is
funded by a $600,000 grant from the Rockefeller
Foundation.
Use with: plants
Licensing information: Contact PIPRA.
280
ProCellEx Plant Expression -
plant cells; glycosylation
Protalix BioTherapeutics, Inc - Licensor, pri-
mary
Description: ProCellEx is an expression system
based on plant cell, such as carrot and tobacco
cells, culture for the development, expression and
manufacture of recombinant proteins, including
expression of enzymes with human-like glyco-
sylation. The system facilitates the creation and
selection of high expressing, genetically stable
plant cell lines capable of expressing recombinant
proteins. Large-scale production, takes place in
flexible, sterile, polyethylene bioreactors which
are confined to a clean-room environment. Plant
cell cultures are grown on aqueous media consist-
ing of highly purified water and defined inorganic
nutrients in a completely closed and controlled
environment using simple polyethylene bioreac-
tors that are able to be maintained at the room
temperature of the clean-room in which they are
placed. Agrobacterium tumefaciens is cell used
for transfection (see relate entry).
ProCellEx produces enzymes which have uni-
form glycosylation patterns and therefore do not
require the lengthy and expensive post-expression
modifications that are required for certain proteins
produced by mammalian cell-based systems, in-
cluding the proteins for the treatment of Gaucher
disease (e.g., Cerezyme or recombinant gluco-
cerebrosidase manufactured and marketed by
Genzyme, which is glycosylated using enzymes
after expression of the protein). Such post-expres-
sion modifications in mammalian cell-produced
proteins are made in order to expose the terminal
mannose sugar residues. In the production of Cer-
ezyme, exposing these terminal mannose sugar
residues involves a multitude of highly technical
steps using enzymes which add time and cost to
the production process. In addition, these steps do
not guarantee the exposure of all of the required
terminal mannose sugar residues, resulting in
potentially lower effective yields and inconsis-
tency in potency from batch to batch. ProCellEx
protein expression system, by contrast, produces
glucocerebrosidase in a ready to use form that
does not require additional glycosylation or other
modifications to make it suitable for use in en-
zyme replacement therapy for Gaucher disease.
Use with: plants cells
Patents: Exemplary applications include U.S.
179
20080038232, Production of high mannose pro-
teins in plant culture, Shaaltiel, Y., et al., Feb. 14,
2008, assigned to Protalix Ltd., with its abstact
stating, A device, system and method for produc-
ing glycosylated proteins in plant culture, particu-
larly proteins having a high mannose glycosyl-
ation, while targeting such proteins with an ER
signal and/or by-passing the Golgi. The invention
further relates to vectors and methods for expres-
sion and production of enzymatically active high
mannose lysosomal enzymes using transgenic
plant root, particularly carrot cells. More particu-
larly, the invention relates to host cells, particular-
ly transgenic suspended carrot cells, vectors and
methods for high yield expression and production
of biologically active high mannose Glucocer-
ebrosidase (GCD). The invention further provides
for compositions and methods for the treatment of
lysosomal storage diseases.
Licensing information: Contact Protalix.
Products made using this tech.: Protalix is con-
ducting clinical trials with carrot cell-expressed
glucocerebrosidase. A Phase III U.S. trial started
in late 2007.
281
Profcia expression - transient
expression, plants
Medicago, Inc. - Licensor, primary
Description: The Proficia expression system for
transient expression enables recombinant protein
manufacture in dicotyledonous (higher) plants. A
gene of interest under the transcriptional con-
trol of promoting sequences is regulated by the
presence of nitrogen sources. Very high level of
expression can be achieved (about 10X more than
transgenic plants) with production starting in five
days. This technology may be used with Lemna
(duckweed; see related entry).
In a preferred embodiment, the method in-
volves: a) introducing the vector into a suitable
Agrobacterium tumefaciens strain; b) using the
Agrobacterium strain to transfer T-DNA into a
plant cell; c) selecting for transgenicity of plant
cells on a suitable medium; d) regenerating
embryos or plantlets from transgenic cells; and e)
growing mature plants from regenerated embryos.
Use with: plants
7,125,978, Promoter for regulating expression
of foreign genes, Vezina, et al., Oct. 24, 2006,
including a promoter with expression induced
by light; and 6,420,548, Method for regulating
transcription of foreign genes, both assigned to
Medicago.
Licensing information: Contact Medicago.
Acambis plc (becoming part of Sanofi Pasteur
S.A.) has been reported to have evaluated this
technology.
282
RNA-dependent RNA
polymerase (RdRP) - universal
Description: RNA-dependent RNA polymerase
(RdRP; EC 2.7.7.48) gene sequences encode
polypeptides having the enzymatic activity of an
RNA-directed RNA polymerase (RdRP). RdRP
is capable of RNA-directed RNA synthesis, using
RNA as a template for synthesizing complemen-
tary RNA molecules. RdRP is also capable of
accepting single-stranded DNA molecules as tem-
plates for RNA transcription. Vectors containing
RdRP operatively linked to regulatory elements
allow expression in prokaryotic and/or eukaryotic
host cells. RdRP and antagonists/inhibitors are
particuarly useful for transgenic plant cells and
plants.
Use with: universal (generic); plant cells;
plants
Patents: Related patent include U.S. 6,218,142
and 7,09,1397, Nucleic acid molecules encoding
polypeptides having the enzymatic activity of an
RNA-directed RNA polymerase (RdRP), as-
180
signed to Plant Bioscience Ltd.
Ltd.
283
Stratosome Biologics System;
Oilbody expression; Saffower
plant seed expression
SemBioSys Genetics Inc. - Licensor, primary
Description: Stratosome is a system for expres-
sion of proteins in safflower (Carthamus tinctorius
L.) plant seeds, attaching them to oily molecules
naturally occurring in safflower seeds, greatly
facilitating purification. Proteins are stable in
this form more or less indefinitely. Purification is
simplified by the separation of the oily structures
from homogenized seeds. Similar oil-protein
structures may be used for oral deliver of proteins.
SemBioSys is developing recombinant safflow-
er expressed insulin, which could could reduce
capital costs for an insulin facility by 70%, and
cost-of-goods by more than 40% compared with
current production methods. In 2006, the compa-
ny reported accumulation in safflower with 1.2%
of total seed protein, a level considered com-
mercially viable. For manufacture of Apo A1,
SemBioSys estimates the cost to grow a ton of
seed, about an acre of safflower which would then
produce about 2 kg of Apo A1, would be about
$800, compared to the current cost which is in the
range of about $400 per gram of Apo A1.
The Stratosome Biologics System is based
on the discovery that recombinant proteins can
be targeted to, or captured on, naturally occur-
ring oilbodies in seeds. A transgene encoding an
oleosin/proteinX fusion protein is transferred into
an oilseed plant; where proteinX is a protein
of interest (e.g. recombinant human insulin). As
the plant grows the oleosin/proteinX fusion is
naturally targeted to and captured on oilbodies in
the seed. Based on the principle that oil is lighter
than water, harvested seed is milled in an aque-
ous buffer and the oilbodies are purified from
seed-derived impurities through a series of simple
centrifugation and wash processes. An enzyme
or chemical that recognizes an engineered cleav-
age site between the oleosin/proteinX fusion is
added to the purified oilbodies cleaving proteinX
from the oilbody-associated oleosin. The oilbod-
ies are removed via centrifugation and proteinX,
now in the aqueous phase, is purified simply and
economically using conventional downstream
processing.
Stratosome Biologics System is the only trans-
genic system to offer comprehensive enabling
advantages in the low cost production of high vol-
ume recombinant proteins. High levels of expres-
sion coupled with an intrinsic, unique purification
process allows the system to offer both upstream
and downstream advantages in the production of
recombinant proteins over conventional (mi-
crobial, yeast and animal cell culture) and other
transgenic (plant and animal) approaches. includ-
ing lower production costs (up to 75% reduction
in unit costs); lower capital costs for production/
purification facilities (up to a 70% reduction); and
enhanced flexibility and scalability of production.
A large number of simple and complex pro-
teins, ranging from approximately one kilodalton
(nine amino acids) to over 100 kilodaltons, have
been produced at high levels using the Strato-
some Biologics System. From one ton of seed,
it is possible to recover more than one kilogram
of purified recombinant protein. The system can
be used with recombinant polypeptides, mul-
timeric-protein-complexes, heteromultimeric-
protein-complexes, multimeric-fusion-proteins,
heteromultimeric-fusion-proteins, immunoglobu-
lin-polypeptide-chains, immunoglobulins, redox-
fusion-polypeptides, and/or thioredoxin-related
proteins; in association with oil bodies.
Use with: safflower plants
ies
Benefits: Key advantages in oilbody/protein
manufacture, include:
Physiology - safflower is a highly productive
oilseed crop whose seed offers superior inventory
181
management advantages;
Regulatory - safflower is a low acreage crop
that is predominantly self-pollinating and can be
easily segregated from other safflower production.
SemBioSys is the only company to have pro-
duced transgenic safflower in field conditions;
having done so in Canada, the United States and
Mexico, and counter-seasonally in Chile for at
least four years. Current production results in 200
tons of seed harvested from 280 acres.
Patents: Related patents include 7,098,383,
Methods for the production of multimeric immu-
noglobulins, and related compositions, Szarka, et
al., Aug. 29, 2006, with its abstract stating, Im-
proved methods for the production of multimeric-
protein-complexes, such as redox proteins and
immunoglobulins, in association with oil bodies
are described. The redox protein is enzymatically
active when prepared in association with the oil
bodies. Also provided are related nucleic acids,
proteins, cells, plants, and compositions.
Other related patents include SemBiosSy US
Patents: 7,091,401 - Expression of epidermal
growth factor in plant seeds; 6,924,363 - Oil
bodies and associated proteins as affinity matri-
ces; 6,777,591 - Legume-like storage protein
promoter isolated from flax and methods of
expressing proteins in plant seeds using the pro-
moter; 6,761,914 - Immunogenic formulations
comprising oil bodies; 6,753,167 - Preparation
of heterologous proteins on oil bodies; 6,750,046
- Preparation of thioredoxin and thioredoxin
reductase proteins on oil bodies; 6,599,513
- Products for topical applications comprising
oil bodies; 6,509,453 - Oil bodies and associ-
ated proteins as affinity matrices; 6,288,304
- Expression of somatotropin in plant seeds;
5,948,682 - Preparation of heterologous proteins
on oil bodies; 5,856,452 - Oil bodies and as-
sociated proteins as affinity matrices; 5,792,922
- Oil body protein cis-elements as regulatory
signals; 5,650,554 - Oil body proteins as carri-
ers of high-value peptides in plants.
Licensing information: Contact SemBioSys,
which offers CMO services.
Products made using this tech.: SemBioSys

intends to scale up production of safflower-pro-
duced Apo A1 and perform the necessary preclini-
cal work in 2008 in order to initiate clinical trials
in 2009. The company has also produced authen-
tic human insulin in safflower, currently in early
trials.
284
Super-mas Plant Gene
Promoter; Gelvin promoter;
(Aocs)3AmasPmas - plants
Biotechnology Research and Development
Corp. (BRDC) - Licensor, primary
Purdue University, Purdue Research Founda-
tion - Assignee, patent
Description: The Super-mas Plant Gene Pro-
moter derived from Agrobacterium tumefaciens
(see related entry) is useful to drive high levels of
protein expression in plants, and is widely used
commercially. In particular, a hybrid promoter
combines a triple repeat of the octopine synthase
(ocs) activator sequence along with mannopine
synthase (mas) activator elements fused to the
mas promoter, forming (Aocs)3AmasPmas.
The promoter was invented by Dr. Stan Gelvin,
Purdue University, and is ofter referred to as the
Gelvin promoter.
In a head-to-head test, the new super-pro-
moter resulted in 156-fold more GUS activity
than did the -800 CaMV 35S promoter of pBI121.
CaMV 35S from cauliflower mosaic virus
(CaMV) is the most popular promoter in plant
molecular biology research.
5,955,646, Chimeric regulatory regions and
gene cassettes for expression of genes in plants,
Gelvin, et al., Sept. 21, 1999, assigned to Purdue
University [and exclusively licensed to Biotech-
nology Research and Development Corporation
(BRDC)]. Claim 1 states: A cassette for inducible
182
expression of a foreign gene comprising said for-
eign gene operably linked to a regulatory region
comprising a promoter derived from a mannopine
synthase gene of Agrobacterium tumefaciens,
an upstream activating sequence derived from
a mannopine synthase gene of Agrobacterium
tumefaciens, and at least one upstream activating
sequence derived from an octopine synthase gene
of Agrobacterium tumefaciens.
Availability:
Licensing information: Contact BRDC.
BRDC reports it has granted commercial li-
censes, or option to license, to nearly every major
agriculture biotechnology company in the world.
Licensees include Japanese, European, multina-
tional and American companies. BRDC has made
the promoter generally available to researches in
the field of plant genetic engineering and to date
over twenty-five research organizations have
taken research licenses. The promoter is already
being used in genetic engineered crops that are
being prepared for field trials.
Licensees reportedly include:
Agricultural Research Service (ARS), U.S.
Dept. of Agriculture (USDA) - Uses for manu-
facturing
Agrigenetics, Inc. - Uses, currently in R&D
Akkadix, Inc. - Uses for manufacturing
American Cyanamid Co. - Used for manufac-
turing
AstraZeneca - Uses for manufacturing
Biolex Therapeutics - Uses for manufacturing
CropTech Corp. - Uses for manufacturing
DNA Plant Technology Corp. - Uses for manu-
facturing
Exelixis Pharmaceuticals, Inc. - Uses for manu-
facturing
Icon Genetics (subsidiary of Bayer AG) - Uses
for manufacturing
Kirin Brewery Co. Ltd. - Uses for manufactur-
ing
Novartis Seeds AG - Uses for manufacturing
Pioneer Hi-Bred International, Inc. (now Du-
Pont) - Uses for manufacturing
Planet Biotechnology, Inc. - Uses for manufac-
turing
Plant Breeding International Cambridge Ltd.
- Uses for manufacturing
Purdue University, Purdue Research Founda-
tion - Uses for manufacturing
Seminis Vegetable Seeds, Inc. - Uses for manu-
facturing
Suntory Ltd. - Uses for manufacturing.
285
TransBacter Gene Transfer
System -plants; royalty-free
Cambia Biosystems, L.L.C. (CBL) - Licensor,
primary
Description: TransBacter is a collective name
given to non-pathogenic bacteria that have been
modified to be able to replace Agrobacterium (see
related entry) in plant cells transformation pro-
cesses. The bacteria for transforming plants usu-
ally contain binary vectors, such as a plasmid with
a vir region of a Ti plasmid and a plasmid with a
T region containing a DNA sequence of interest.
Using modified plasmids, at least three species
of bacteria, Sinorhizobium meliloti, Mesorhi-
zobium loti, and Rhizobium sp. NGR234 have
been shown able to transfer genes to plants under
certain circumstances.
Use with: plants
Benefits: This alternative to Agrobacterium-medi-
ated technology may provide two distinct advan-
tages:
1. It may lead to better use of natural bacte-
ria-plant interactions, for instance with benign
epiphytic bacteria, to achieve more efficient and
optimal plant transformation for species that have
proven challenging.
2. The cumbersome patent thicket that surrounds
Agrobacterium gene transfer technology has ren-
dered this tool largely unusable for most compa-
nies, institutions and individuals for commercial
purposes. The patents in the thicket explicitly
183
refer to and claim Agrobacterium. This technol-
ogy provides a comprehensive work-around to
these patents, as the species now capable of gene
transfer are very distinct from Agrobacterium.
Patents: Applications include US 20050289672
and US 20050289667, and PCT Publication WO
2006004914.
Licensing information: Following the open
source software model, vectors, strains and use
for applications in biotechnology are available
under a BiOS license, which allows both research
and commercial application in worldwide fields of
use royalty-free.
286
Ubiquitin linkage domain -
multiple proteins in transgenic
plants
University of Wisconsin - Assignee, patent
Description: Vectors containing a ubiquitin
linkage domain are useful for transformation
of plants, and enable coordinated expression of
multiple proteins in the same transformed plant or
plant cell. The ubiquitin linkage domain consists
of an entire sequence for ubiquitin monomer
preceded by the last 14 amino acids (C-terminus)
of an additional ubiquitin molecule, sandwiched
between two foreign genes of interest. In trans-
genic plants created with this construct, expres-
sion results in a fusion protein that is then cleaved
by endogenous ubiquitin-specific proteases. These
proteases, which naturally remove proteins from
the C-terminus of ubiquitin, release functional
proteins into the cytosol at approximately equal
molar levels.
Background: Since most current methods for
producing transgenic plants that synthesize
multiple proteins involve either simultaneous or
sequential introduction of individual genes, the
genetic independence of each locus hinders coor-
dinated protein expression.
Allows readily coordinated synthesis of
multiple proteins needed for creating novel com-
mercial plant varieties with complex biochemical
characteristics or multiple traits (e.g. pesticide and
herbicide resistance)
Because ubiquitin is highly conserved in
plants, this approach should work in a majority of
higher plant species
Inventors have demonstrated coordinated ex-
pression of two proteins, beta-glucuronidase and
luciferase, from a single construct in both tobacco
and Arabidopsis
6,455,759, Expression of multiple proteins in
transgenic plants, Vierstra, R.D., et al., with its
claim 1: A transgenic angiosperm plant compris-
ing a DNA construction inserted into the genome
of the plant, the DNA construction comprising 5
to 3: a promoter operable in plant cells; a cod-
ing region encoding a fusion protein, the fusion
protein comprising in a common reading frame,
in order: a coding region for a first protein of
interest; a coding region for a plant ubiquitin
monomer; and a coding region for second protein
of interest; a transcriptional terminator operable in
plants, the coding region encoding the fusion pro-
tein effective in the cells of the transgenic plant
to result in translation of the fusion protein in the
cells of the plant, the fusion protein being cleaved
at the ubiquitin monomer by enzymes present
in the cells of the plant separating the first and
second proteins of interest from each other in the
cells of the transgenic plant, neither the first nor
the second proteins of interest being ubiquitin.

184
287
Ubiquitin plant promoters
DuPont Corp. - Licensor, primary
Pioneer Hi-Bred International, Inc. - Assignee,
patent
Description: Ubiquitin promoters are widely used
in transgenic plants. The promoters have a TATA
to start sequence containing 64% or greater GC
content and an synthetic upstream element incor-
porating several OCS binding motifs and novel
flanking sequences. Upstream activating regions
(UARs) can further increase the constitutive tran-
scriptional activity when they are operably linked
to the promoter and/or the synthetic upstream
element. In particular, the nucleotide sequence
of the UAR of the maize Ubi-1 gene is useful in
expression cassettes and vectors containing these
promoter elements.
Patents: A patent rather broadly covering ubiq-
uitin promoters is U.S. 6,072,050, Synthetic
promoters, Bowen, B., et al., June 6, 2000, is
assigned to Pioneer Hi-Bred International, Inc.,
now DuPont
Licensing information: Contact DuPont.
288
Zara technology; Protein body-
inducing sequence (PBIS)
fusion proteins; Recombinant
protein body-like assembly
(RPBLA); StorPro organelles
(protein encapsulation);
Prolamin fusion proteins -
eukaryotes
ERA Biotech - Licensor, primary
Description: Zara is a suite of technologies use-
ful to improve the manufacturing productivity of
plant-made pharmaceuticals and other protein-
based products of high value. Zera assembles
Protein Bodies in plant cells to accumulate recom-
binant products, and eases their recovery. Desired
proteins are expressed as prolamin or prolamin
domain fusion proteins containing the peptide
or protein of interest and stably accumulate as
recombinant protein body-like assemblies (RPB-
LAs) . The fusion proteins containing the poly-
peptide of interest stably accumulate as recombi-
nant protein body-like assemblies (RPBLAs) in
the eukaryotic cells, which can be plant, animal,
fungal or algal cells.
The unique density of the StorPro organelles
facilitates easy recovery by a simple gradient
process: the starting biomass is mechanically
homogenized, then centrifuged, and the intact
product-containing organelles are recovered as
a characteristic fraction. Most of the non-prod-
uct biomass and cellular debris is eliminated by
this rapid and low-cost procedure which can be
followed by additional downstream wash steps,
enzymatic cleavage, and further affinity or chro-
matographic purification.
Benefits; Claims include:
Enablement of Difficult-to-express Products
Zera assembler peptides enable difficult pro-
teins such as toxic-to-cell molecules (enzymes,
other bioactive proteins), unstable or labile
products (peptides, antimicrobials) and membrane
proteins.
Works in most eukaryotic cells lines with hor-
mones, growth factors, peptides, enzymes, anti-
gens, membrane proteins.
Compatible with transient and stable transfection/
transformation.
Recovery by density delivers high pre-purification
concentrations which are expected to result in sig-
nificant operational cost-savings, size reduction
in downstream cGMP suites, and overall faster
process development.
Over a 10-fold increase possible in protein yield
on a per cell or biomass basis. In vivo encapsula-
tion improves the capacity of the cell factory to
185
assemble and accumulate complex products.
The unique structure of the StorPro organelle
matrix makes possible novel drug and vaccine
formats which take advantage of the synthetic
organelles physical properties.
20070243198, Production of biologically active
proteins , Heifetz, et al., Oct. 18, 2007. The
abstract states, A fusion protein that is expressed
in a recombinant protein body-like assembly
(RPBLA) in host eukaryotic cells and organisms
is disclosed. More particularly, a biologically
active polypeptide fused to a protein sequence
that mediates the induction of RPBLA formation
is expressed and accumulated in host cells after
transformation with an appropriate vector. The
eukaryotic host cell does not produce protein bod-
ies in the absence of the fusion protein. Methods
for preparing and using the RPBLAs and the fu-
sion protein are also disclosed, as are nucleic acid
molecules that encode the fusion proteins.
Licensing information: Contact ERA Biotech.
289
CleanGene plant
transformation
Life Technologies, Inc. - Retail seller
Description: CleanGene is a method for direct
transformation of plant cells using minimal vector
sequences. While maintaining all the desirable
benefits of bombardment, e.g., gene gun, technol-
ogy (genotype independence, ecological safety,
high co-transformation frequency), this technol-
ogy produces transgenic plants with very simple
integration patterns and low transgene copy
numbers, resulting in stable expression over many
generations and almost no transgene silencing.:
Benefits: Plant Bioscience claims, The Clean-
Gene technology represents a significant and
important improvement to conventional methods
of plant transformation using direct DNA transfer.
The use of minimal expression vectors, com-
prising linear DNA fragments containing only
promoter, transgene coding region and termina-
tor/polyadenylation sites used in the CleanGene
technology prevents illegitimate recombination
events. Through this approach the benefits of high
transformation efficiencies, simple integration
patterns and low copy numbers are combined with
high expression, reduction in gene silencing and
increased operational efficiency. From a regula-
tory perspective the minimal constructs integrated
into the plant genome reduce presence of unde-
sired DNA fragments that are frequently scruti-
nized by both government and non-government
bodies. The CleanGene technology therefore leads
to more efficient and safer transgenic plants.
Patents: Exemplary patents include, 6,846,970,
Christou , et al., January 25, 2005, Transfor-
mation method and transgenic plants produced
thereby, assigned to Plant Bioscience Ltd. The
abstract follows: This invention relates to meth-
ods for producing, at a high frequency, transgenic
plants that contain little if any vector sequences,
have simple integration patterns, contain few
copies of the transgene at each locus, express the
transgene at all stages of development and do not
exhibit transgene silencing. The method compris-
es introducing minimal transgene expression cas-
settes, which are substantially or totally devoid of
vector sequences, by direct DNA transfer, prefer-
ably by particle or microprojectile bombardment.
This invention also relates to transformed plant
cells, the transgenic plants regenerated therefrom,
and subparts of the transgenic plants produced by
the methods of this invention. The invention also
includes all progeny and subsequent progeny (i.e.,
all subsequent generations) derived from primary
transformants through selfing or crossing.
Also, see WO 01/05936. Other patent applica-
tions and divisional applications are pending.
Availability: Available from Life Technologies
Licensing information: Plant Bioscience Ltd.
regarding licensing.
Further info.: Loc, NT, et al (2002). Linear
transgene constructs lacking vector backbone
186
sequences generate transgenic rice plants which
accumulate higher levels of proteins conferring
insect resistance. Molecular Breeding, 9, 231-244.
Fu, X, et al (2000). Linear transgene constructs
lacking vector backbone sequences generate low-
copy-number transgenic plants with simple inte-
gration patterns. Transgenic Research, 9, 11-19.
290
Plastids (chloroplasts)
expression - plant cell culture
Rutgers University - Licensor, primary
Description: This technology provides DNA
constructs and methods for stably transforming
plastids of multicellular plants and expressing
recombinant proteins in transformed plastids.
Plastids are often present in a plant cell at a very
high copy number, with up to 50,000 copies per
cell present for the chloroplast genome. Thus,
through plastid transformation plant cells can
be engineered to maintain an introduced gene of
interest at a very high copy number.
Background: Plastids of higher plants, i.e.
chloroplasts, amyloplasts and chromoplasts, have
the same genetic content, and are believed to be
derived from a common precursor, known as a
proplastid. The plastid genome is circular and var-
ies in size among plant species from about 120 to
about 217 kilobase pairs (kb). The genome typi-
cally includes a large inverted repeat, which can
contain up to about 76 kilobase pairs, but which
is more typically in the range of about 20 to about
30 kilobase pairs.
Plant plastids are major biosynthetic centers. In
addition to photosynthesis in chloroplasts, plastids
are responsible for production of important com-
pounds such as amino acids, complex carbohy-
drates, fatty acids, and pigments. Plastids can also
express two or more genes from a single plastid
promoter region. A DNA sequence expressed in a
plastid may include a number of individual struc-
tural gene encoding regions under control of one
set of regulatory components. Thus, it is possible
to introduce and express multiple genes in a plant
cell, either from an engineered synthetic sequence
or from a pre-existing prokaryotic gene cluster.
This expression method makes possible large
scale and inexpensive production of certain
proteins and chemicals that are not practically
produced through standard plant nuclear transfor-
mation methods. In nuclear expression from in-
troduced genes, each encoding sequence must be
engineered under the control of a separate regula-
tory region, i.e., a monocistron. As a consequence,
gene expression levels vary widely among
introduced sequences, and generation of a number
of transgenic plant lines is required, with crosses
necessary, to introduce all of the cistrons into one
plant and to get proper coordinated expression in
the target biochemical pathway.
Benefits: An advantage of plant plastid trans-
formation over nuclear transformation is that the
plastids of most plants are maternally inherited,
and consequently heterologous plastid genes are
not pollen disseminated. This feature is particu-
larly attractive for transgenic plants having altered
agronomic traits, as introduced resistance or toler-
ance to natural or chemical conditions will not be
transmitted to wild-type relatives.
Patents: Exemplary patents assigned to Rut-
gers University include 5,877,402; 6,388,168;
6,987,215; and 7,176,355, all by Maliga, et al.
Licensing information: Contact Rutgers Univer-
sity.
187
Insects
291
High Five cell line (BTI-TN-
5B1-4, ATCC CRL 10859);
Trichopulsia ni cell line -
baculovirus host cells; insect
cell culture
- Licensor, primary
Description: The High Five cell line (BTI-TN-
5B1-4, ATCC CRL 10859) derived from Tricho-
plusia ni (cabbage looper) egg homogenate is
capable of high levels of recombinant protein
secretion when used with baculovirus vectors/ex-
pression systems. The cell line is susceptible to
various baculoviruses, including TnSNPV and
AcMNPV. The cell lines has been adapted to cul-
ture in serum-free media, have a 20 hour doubling
time, and grow in both monolayer and suspension
culture. As with many other insect cells lines use
with baculovirus expression systems (BEVS),
High Five provides N-linked glycosylation,
including the addition of terminal sialic acid resi-
dues to N-linked oligosaccharides.
High Five host cells have been shown to pro-
duce up to 25-times more human papillomavirus
(HPV) vaccine antigen for HPV vaccine manu-
facture than standard Sf9 (Spodoptera frugiperda)
baculovirus host cells.
Use with: Trichoplusia ni (cabbage looper) cells
Patents: Exemplary patents held by the Boyce
Thompson Institute for Plant Research include
U.S. 5,300,435, Trichoplusia ni Cell Line which
Supports Replication of Baculoviruses, concern-
ing a high production cell line of Trichoplusia
ni (cabbage looper), ATCC CRL 10859; and
6,403,375, Establishment of Trichoplusia ni
cell lines in serum-free medium for recombinant
protein and baculovirus production, concerning
cell lines from Trichoplusia ni eggs that may be
cultured in commercial serum-free medium.
Availability: Invitrogen markets High Five cells
Licensing information: Contact Boyce Thomson
Inst. BTI reports that High Five has been Widely
non-exclusively licensed with one exclusive field
of use for Human Papilloma Virus vaccine pro-
duction.
Products made using this tech.: Marketed or
nearing approval biopharmaceuticals manufac-
tured in High Five cells include:
a) HPV vaccine [Cervarix MEDI 501; human
papilloma virus (HPV) vaccine types 16 and 18
L1 virus-like particles, recombinant] largely de-
veloped by MedImmune, and licensed , manufac-
tured and marketed by GlaxoSmithKline Biologi-
cals S.A.
b) FavId [Id-KLH; tumor idiotype antigen, recom-
binant, personalized-keyhole limpet hemaggluttin
conjugate autologous priming of dendritic cells
plus granulocyte macrophage-colony stimulat-
ing factor (GM-CSF, rDNA); idiotype-pulsed
dendritic cell vaccination; B-cell non-Hodgkins
lymphoma autologous immunoglobulin idiotype-
KLH conjugate vaccine] - BEVS/High Five is
used to produce patient-specific tumor idiotype
antigen, which is conjugated to keyhole limpet
hemaggluttin and used with GM-CSF for cancer
immunotherapy.
Further info.: Granados, R.R., Guozun, L., Derk-
sen, A.C.G., and McKenna, K.A. 1994. A new
insect cell line from Trichoplusia ni (BTI-Tn-5B1-
4) susceptible to Trichoplusia ni
single enveloped nuclear polyhedrosis virus. J.
Invertebr. Pathol. 64: 260-266.
292
Insect cells glycosylation
University of Wyoming - Licensor, primary
University of Notre Dame - R&D
Chesapeake PERL, Inc. - R&D
Description: Baculovirus vectors incorporate
genes encoding mammalian N-glycan processing
enzymes into the insect cell lines commonly used
188
as hosts for baculovirus expression vectors. Ex-
pression plasmids and methods for stable transfor-
mation of lepidopteran insect cells were devel-
oped and used to produce transgenic insect cell
lines that constitutively express mammalian genes
encoding functions required for N-glycoprotein
sialylation, i.e., have humanized N-glycoprotein
processing pathways.
Mimic Sf9 Insect Cells (see related entry)
appear to have been developed using this technol-
ogy.
These cell lines have normal morphologies,
growth properties, and remain competent as hosts
for baculovirus expression vectors. The constitu-
tively expressed mammalian transgene products
participate in N-glycoprotein biosynthesis and
support the production of more authentic (mam-
malian- or human-like) products. These cell lines
offer a distinct advantage over unmodified hosts
for the baculovirus-mediated production of more
authentic recombinant N-glycoproteins.
Use with: insect cells
Patents: The University of Wyoming has three
relevant technologies available in its Baculovirus-
Insect Cell Expression System portfolio:
a) 99-001 - Modifying Insect Cell Glycosylation
Pathways with Baculovirus Expression Vectors
b) 04-042 - Production of Human Glycosylated
Proteins in Transgenic Insects
c) 04-043 - Post-Translational Modification of
Proteins in Transgenic Insects
Availability: Invitrogen Corp. has a license,
seemingly exclusive, from the Univ. of Wyoming
for non-commercial sales of glycosylating cell
lines.
Licensing information: The Univ. of Wyoming
(Dr. Jarvis, the primary inventor) is collaborat-
ing with Chesapeake PERL and the University
of Notre Dame in the long-term development of
human-like glycosylation capabilities for baculo-
virus expression systems.
293
Insect cells glycosylation
Human Genome Sciences, Inc. (HGSI) - Li-
censor, primary
Johns Hopkins University (JHU) - Assignee,
patent
Description: Host insect cells are modified to
contain and co-express glycosylation enzymes.
A cytidine monophosphate sialic acid (CMP-
SA)-synthase gene encodes a polypeptide that
catalyzes the formation of CMP-SA from SA
and cytidine triphosphate (CTP). The sialic acid
synthase (SAS0 gene encodes a polypeptide that
catalyzes the formation of sialic acid phosphate
from N-acetylmannosamine (ManNAc) and phos-
phoeylpyruvate (PEP).
Engineering intracellular sialylation pathways,
and 6,333,182, Human glycosylation enzymes,
both coassigned to Johns Hopkins University and
Human Genome Sciences, Inc.
U.S. application 20020142386, Engineering
intracellular sialylation pathways, assigned to
Human Genome Sciences, Inc., has an abstract
stating, Methods for manipulating carbohy-
drate processing pathways in cells of interest are
provided. Methods are directed at manipulating
multiple pathways involved with the sialylation
reaction by using recombinant DNA technology
and substrate feeding approaches to enable the
production of sialylated glycoproteins in cells of
interest. These carbohydrate engineering efforts
encompass the implementation of new carbohy-
drate bioassays, the examination of a selection
of insect cell lines and the use of bioinformatics
to identify gene sequences for critical process-
ing enzymes. The compositions comprise cells of
interest producing sialylated glycoproteins. The
methods and compositions are useful for heterolo-
gous expression of glycoproteins.
189
University and/or Human Genome Sciences, Inc.
294
Insect cells/Baculovirus
expression systems;
Baculovirus expression vector
systems (BEVS)
Description: Also, see the following more
specific entries. Baculovirus-based vectors and
baculovirus expression vectors (BEVS) are com-
monly used for transformation of insect cells. In
many insect species, there is a period of rapid
larval growth involving massive protein expres-
sion prior to pupation. This can be exploited for
recombinant protein expression. The baculovirus-
insect cell system has been reported to have used
to express over five hundred genes, with 95%
biologically active.
Insect cell lines can be an economical means
for obtaining high yields and large quantities of
recombinant proteins. Unlike many mammalian
cells used for this purpose, insect cells grow
readily in suspension, and do not require a special
atmosphere. Therefore, they can be grown in large
stirred tanks with minimal specialized equip-
ment. Yields of protein from baculovirus expres-
sion vectors in insect cell cultures can be 20-250
times higher than those from mammalian cells.
There are a number of protein-free media avail-
able from commercial supplies and the yields of
foreign gene products from cells grown in these
media generally have been comparable to yields
in serum-supplemented media.
Baculovirus is in the family Baculoviridae, a
diverse group of large dsDNA viruses that in-
fect insects, arachnids and crustaceans. They are
known to infect different species of insects and
are used as biopesticides. Baculoviruses pose no
risk to mammals. Baculoviruses are not pathogen-
ic to mammals and plants, and they have a very
restricted host range mostly limited to invertebrate
species. Pathogenic viruses do not transform in-
sect cells; therefore, minimal containment condi-
tions are required. Insect cells can be easily and
reproducibly scaled up for large-scale production
of recombinant proteins. For example, cell lines
derived from Spodoptera frugiperda can easily
be cultivated as suspension cultures, which is the
supposition for large-scale production in bioreac-
tors. The most used cell lines for BEVS applica-
tions are Spodoptera frugiperda (Sf9, Sf-21) and
Trichoplusia ni (see related entries).
The BEVS process involves the introduction
of a foreign gene into a nonessential region of the
circular baculovirus expression vector genome via
homologous recombination with a transfer vector
containing the cloned gene. The production of
foreign protein is then achieved by infection of
additional insect cell cultures with the resultant
recombinant virus vector. The translated proteins
obtained are glycosylated, acylated, and proteo-
lytically cleaved. Among the numerous baculo-
viruses, Autographa californica multiple NPV
(AcMNPV) is the most well studied and exten-
sively used. This system has been widely used for
the production of numerous recombinant proteins
in insect cells as it is associated with high levels
of protein expression; post-translational modifica-
tions; relevant cellular compartmentalization of
proteins; capacity of large cDNA inserts; simulta-
neous expression of multiple genes; and provides
quick and reliable results.
A main disadvantage of BEVS can stem from
the inherent insecticidal nature of baculoviruses.
Infected host cells may be viable only for several
days and then they die.
Protein Sciences, Inc. routinely uses BEVS/in-
sect cell technology to make products at a 500-L
scale. You may think thats pretty small but from
that we will be able to make one million doses of
an influenza vaccine in that one small fermenter,
Background: Protein poduction in BEVS largely
depends on the host being used. Historically,
the most widely used hosts were the established
insect cell lines IPLB-Sf21-AE (Sf21), origi-
nally derived from Spodoptera frugiperda ovaries
190
(Vaughn et al., 1977), and its clonal derivative,
Sf9 (Summers and Smith, 1987). In 1992, it
was reported that BTI-TN-5B1-4, an insect cell
line derived from Trichoplusia ni eggs, provides
higher levels of foreign protein production (Wick-
ham et al., 1992). Subsequent studies on a larger
sample of recombinant proteins have supported
this claim. As a result, BTI-TN-5B1-4 cells, more
commonly known as High Five cells (a trade-
mark of Invitrogen), have become widely-used
host for BEVs. Other newer baculovirus systems
can provide more extensive and human-like
N-glycosylation of glycoproteins. See various
related entries.
Benefits: The diversity of AcNPV-based and
other transfer vectors, combined with available
S. frugiperda Sf9 and Sf21 cell lines, have es-
tablished baculovirus expression as a commer-
cially-viable system for functional eukaryotic
gene expression and the large-scale production of
The baculovirus expression system offers
advantages over prokaryotic and other eukaryotic
systems:
High Level of Protein Expression. Yields of up
to 100 mg of protein per 10 9 cells.
Post-Translational Modifications. Including
disulfide bond formation, phosphorylation, glyco-
sylation, oligomerization and proper folding.
Relevant Cellular Compartmentalization of
Proteins. Secreted, membrane-bound, cytoplasmic
or nuclear.
Capacity of Large cDNA Inserts. Accommo-
dates genes up to 15 kb.
Simultaneous Expression of Multiple Genes.
With multiple promoter transfer vectors.
Patents: Many, if not all, of the original BEVS
and host cell lines developed by Texas A&M
University are or soon will be in the public do-
main (off-patent). See related entries. Texas A&M
University and its authorized licensing agent
MicroGeneSys, Inc., now Proteins Sciences, Inc.,
have been the primary source for many BEVS
technologies.
Availability: BEVS and host insect cells are
available for non-commercial use from multiple

vendors.
Licensing information: Contact Proteins Sci-
ences, Inc. regarding concerns about commercial
use and licensing of BEVS technologies.
295
PERLXpress; TRANSPILLAR
larvae; Trichoplusia ni larvae
expression - transformed
caterpillars
Chesapeake PERL, Inc. - Licensor, primary
- Assignee, patent
University of Maryland Biotechnology Institute
- Assignee, patent
Description: PERLXpress, involving recombi-
nant protein expression in insect larvae (not insect
cells) using baculovirus vectors, enables the pro-
duction of initial quantities of protein in as little
as 2-3 weeks followed by the rapid, linear scale-
up to gram and kilogram quantities. The system is
efficient and reproducibly results in high levels of
expression of target proteins.
Chesapeake PERL employs Baculovirus
Expression Vector System (BEVS; see related en-
tries) to produce recombinant proteins, but rather
than using cultured insect cells, uses the insects
themselves as living bioreactors. See also the
related Insect cells, per os (oral) baculovirus in-
fection entry, with this involving oral inoculation
of insect larvae with vectors. The system offers
speed, flexibility and scalability. Starting with a
recombinant baculovirus, onitial samples of pro-
tein-containing biomass for preliminary analysis
can be prepared in less than two weeks, and can
be scaled-up immediately to kilogram quantities.
Pre-occluded Virus (POV) vectors derived
from a polyhedrin-minus or granulin-minus (lack-
ing a functional polyhedrin or granulin gene)
baculovirus are fed to insect larvae per os (see
related entry) resulting in high infection rates.
The use of the POV form of polyhedrin-minus
191
baculoviruses is essential to this method of infect-
ing insect larvae per os using the POV form of
polyhedrin-minus baculovirus.
Once a baculovirus vector has been optimized
for the production of a particular protein in larvae,
a bulk inoculum is prepared. This is stable and
sufficient to inoculate several years worth of
larvae at full production, eliminating the need
for serial passage that can result in the accumula-
tion of defective virus as observed with cultured
cells. It also eliminates the need for the multiple
production intermediates. Production begins
with the companys patented oral inoculum. This
ensures optimized mass inoculation under natural
conditions that enable accurate calibration and
reproducible, optimized protein production at
large scale production levels. The majority of the
companys target proteins are expressed under
transcriptional control of either the baculoviral
polyhedrin or p10 promoters, which provide high
level transcription during very late stage infection.
Larvae for protein production are raised from
eggs hatched on a controlled diet. The manipula-
tion of this diet, as well as the dispensing of the
eggs, incubation and inoculation with baculovirus
are all automated for reproducible control of the
process. A small number of insect larvae are hand-
injected with recombinant baculovirus vector pre-
pared and amplified in culture. This preliminary
step is important in order to verify expression in
whole insects and to select optimal recombinants
for further development. It also is used to generate
the proprietary inoculum for mass oral infection
of production quantities of insect larvae. Larvae
that have reached the fifth instar of development
are infected orally by application of the viral
inoculum to the surface of the diet. Inoculated
larvae are incubated for 96 hours under care-
fully controlled conditions, harvested, frozen and
stored until needed for protein recovery.
Compared to the homogeneity of cultured cells,
the tissue heterogeneity of larvae can be a signifi-
cant advantage for difficult to produce proteins.
Insect larvae can out out-produce cultured cells
by significant margins for many proteins. The
company has an automated facility for the mass
rearing of insects under carefully optimized and
controlled conditions. With this facility, it can
produce protein-containing larvae at scales up to
tens of kilograms per week.
While yields vary significantly for different
types of proteins, 200 mg/larvae is an average
yield estimate. This indicates potential in-house
production capacity of ~200 grams of recombi-
nant protein per week.
Use with: Trichoplusia ni larvae
Benefits: Mammalian viruses and prions can-
not be propagated by insect larvae, eliminating a
concern with traditional culture production
The PERLXpress platform and recovery proce-
dures are capable of producing proteins with:
- typical expression levels that are 10-fold higher
per unit volume than insect cell culture systems,
with little or no protein-specific optimization of
the insect growth conditions
proper function, folding and structure
no detectable adventitious organisms
purity routinely >98%
protein functionality in vitro and in vivo
Patents: Patents/applications apparently exclu-
sively licensed by Chesapeake PERL include:
a) U.S. 6,090,379, Stable pre-occluded virus
particle for use in recombinant protein produc-
tion and pesticides, Wood, July 18, 2000, with its
abstract stating: A method of infecting insects is
disclosed; the method utilizes a form of a bacu-
lovirus which is highly efficient at establishing
infection and is normally destined to become
occluded within the polyhedrin or granulin--Pre-
occluded Virus (POV). Specifically, the POV as
derived from a polyhedrin-minus or granulin-mi-
nus (lacking a functional polyhedrin or granulin
gene) baculovirus is fed to insect larvae per os
resulting in high infection rates. The stabilization
and use of the POV form of polyhedrin-minus
baculoviruses for recombinant protein production
and as an insecticide is also disclosed.
b) U.S. 6,153,409, Process for continuous
optimized protein production in insect larvae,

192
Bentley, et al., Nov. 28, 2000, assigned to the
University of Maryland, with its abstract stating:
The present invention provides for a recombi-
nant insect larvae and a process of manufacturing
proteins utilizing insect larvae that allows for the
selection of individual larvae for harvest at the
point of their optimal expression of a protein of
interest. This invention also provides for a process
to manufacture proteins in larvae that does not re-
quire synchronization of the infection, growth and
harvest larvae to optimally manufacture a protein
of interest. The invention further provides for a
process of producing interleukin-2 in larvae.
Licensing information: Contact Chesapeake
PERL, Inc. concerning related CRO/CMO ser-
vices.
Products made using this tech.: More than
fifty different proteins have been expressed in
the PERLXpress system, representing a broad
range of protein classes including viral antigens,
esterases, virus-like particles and human growth
factors. Proteins produced in the PERLXpress
system have been shown to be functional when
studied in animals including mice, rats, guinea
pigs, sheep and monkeys. The company has
completed more than 50 projects, taking proteins
either to partial purification or to homogeneity.
Major completed projects include: a smallpox
subunit vaccine manufactured under a NIAID
contract, with four vaccinia proteins produced in
quantities ranging form several hundred milli-
grams to grams, all at purities greater than 99%; a
series of virus like particles with yields two orders
of magnitude greater than the client had been able
to produce on their own; butyrylcholinesterase
for use as a bioscavenger (neutralize cholines-
terase inhibitor pesticides and biological warfare
agents) and; growth hormone, beginning with a
DNA sequence, developing baculovirus vectors
that optimized the yield of a growth hormone,
with increases in yield ten-fold to more than 50
milligrams of recovered, 99.9% pure protein per
kilogram of larvae. The protein has been shown to
be processed properly, including cleavage, folding
and dimerization.
296
Trichoplusia ni (cabbage
looper) cell lines; BTI-TN-MG1;
ATCC CRL 10860; BTI-TN-5B1-
4; ATCC CRL 10859
- Licensor, primary
Description: Two new insect cell lines derived
from midgut (BTI-TN-MG1, ATCC CRL 10860)
and embryonic tissue (BTI-TN-5B1-4, ATCC
CRL 10859) of Trichoplusia ni (cabbage looper)
are useful with baculovirus expression systems
(BEVS). . These cell lines are susceptible, unlike
prior cell lines, to a much wider variety of baculo-
viruses, including TnSNPV and AcMNPV.
Use with: Trichoplusia ni cells
Patents: An exemplary patent is U.S. 5,298,418,
Cell line isolated from larval midgut tissue of
Trichoplusia ni, Granados, R.R., March 29,
1994, assigned to Boyce Thompson Institute for
Plant Research. The sole claims is 1. An isolated
cell line from the larval midgut tissue of Tricho-
plusia ni, having all the identifying characteristics
of BTI-TN-MG1, ATCC CRL 10860.
Availability: The cell line does not yet appear to
be available from major vendors.
Thompson Institute for Plant Research.
Products made using this tech.: This has been
non-exclusively licensed to a variety of compa-
nies. It has been exclusively to Medimmune Inc.
for the production of a human HPV vaccines.
MedImmune developed and GSK will market
(approval expected in 2009) Cervarix [MEDI 501;
human papilloma virus (HPV) vaccine types 16
and 18 L1 virus-like particles, recombinant]. HPV
L1 proteins are expressed from these insect cells
as virus-like particles (VLPs).
193
297
Trichoplusia ni (cabbage
looper) cell lines; H5CL-B and
H5CL-F; BTI-TN-5B1-4-derived
insect cell lines
- Licensor, primary
Description: H5CL-B and H5CL-F cell lines,
derived from BTI-TN-5B1-4 (High Five cell line;
see related entry) are improved cell lines use-
ful with baculovirus expression vector systems
(BEVs). These Trichoplusia ni (cabbage looper)
insect cell lines possess the properties of increased
production of baculovirus particles, increased
expression of foreign proteins using BEVs, and
increased resistance to cell culture stress.
Patents: An exemplary patent is U.S. 7,179,648,
Clonal cell lines derived from BTI-TN-5B1-4,
Granados, et al., Feb. 20, 2007, assigned to the
Boyce Thompson Institute for Plant Research.
Availability: H5CL-B and H5CL-F cell lines are
available from Invitrogen; and from ATCC with
accesssion numbers PTA-5635 and PTA-5636,
respectively.
298
Baculovirus expression vector
systems (BEVS)
Protein Sciences Corp. - Licensor, primary
Texas A&M University (TAMUS) - Licensor,
primary
Description: The original Baculovirus expression
vector system (BEVS), now or soon to be off-pat-
ent, developed by the Texas A&M University,
provides a proven plafform for BEVS and insect
cell expresion of recombinant (glyco)proteins.
The system is now a mainstream tool for the pro-
duction of proteins in cultured insect cells, insect
larvae and live insects. See also the generic Insect
cells/Baculovirus expression systems entry.
Benefits: Advantages include:
High level of production efficiency.
Large quantities of highly active protein.
Well-characterized virus/host interaction that
allows significant engineering of both the final
product and the metabolic pathways of the host to
maximize efficiency.
Glycosylation of proteins
Patents: Note, although the early, broad patents
covering BEVS and related insect cell culture
assigned to TAMUS are now expired, various
component and improvements remain patents (see
related entries).
Exemplary patents include U.S. 4,879,236 and
4,745,051 (both apparently now expired), both
entitled Method for producing a recombinant
baculovirus expression vector, covering aspects
of baculovirus transfer vector construction and
use in transient cell lines, by G.E. Smith and M.D.
Summers. U.S. 5,077,214, Use of Baculovirus
Early Promoters for Expression of Foreign Genes
in Stably Transformed Insect Cells, includes
promoters from Autographa californica nuclear
polyhedrosis virus and a stably-transformed insect
cell line, e.g., Sf9 of Spodoptera frugiperda or
High Five of Trichoplusia ni (see related entries),
and vectors capable of continuous expression
of a selected gene product, so the baculoviruses
do not kill the host cells (apparently expired in
2006). Other related patents include 5,023,328;
5,077,214; 5,155,037; 5,162,222; 5,169,784;
5,179,007; and 5,278,050.
In June 1999, MicroGeneSys, Inc., now Pro-
teins Sciences, Inc., and Texas A&M University
combined their intellectual property and know-
how regarding baculovirus expression systems.
Protein Sciences was exclusively authorized to
sublicense the combined BEVS technologies to
third parties.

194
Availability: BEVS components and kits are
available for Invitrogen and other vendors (for
non-commercial use, where still covered by pat-
ents).
Licensing information: BEVS had been licensed
by TAMUS non-exclusively to more than 99 com-
panies in over 35 countries around the world for
development of recombinant therapeutics.
In June 1999, MicroGeneSys, Inc., now Pro-
teins Sciences, Inc., and Texas A&M University
combined their intellectual property and know-
how regarding baculovirus expression systems.
Protein Sciences was exclusively authorized to
sublicense the combined BEVS technologies to
third parties.
For biopharmaceutical manufacture (and to
better satisfy regulators), one still may want to
obtain cells from either of these sourceS vs. a
reagent vendor. And, it would probably be pru-
dent to check with these organizations regarding
their views on the need to take a license for your
particular application.
Products made using this tech.: MedImmune,
and later GlaxoSmithKine, its partner for com-
mercialization, manufacture and marketing,
licensed this technology (or other BEVS compo-
nents) from Texas A&M University, and uses this
technology for manufacture of Cervarix [MEDI
501; human papilloma virus (HPV) vaccine types
16 and 18 L1 virus-like particles, recombinant].
This HPV vaccine to be marketed by GSK is
awaiting approval at FDA.
Dendreon Inc. uses BEVS/insect cells for
manufacture of Provenge [Sipuleucel-T - prostatic
acid phosphatase (PAP)-granulocyte macrophage-
colony stimulating factor (GM-CSF) recombinant
fusion protein (PAP-GM-CSF; PA2024)-sensi-
tized autologous antigen-presenting cells (APCs);
PA2024-loaded APCs; APC8015], i.e,. manufac-
ture of prostatic acid phosphatase (PAP)-GM-CSF
fusion proteins as part of this prostate cancer
immunotherapysuing PAP-GM-CSF to activate
patients APCs, awaiting FDA approval.
Favrille, Inc. uses BEVS/insect cells for manu-
facture of FavId [Id-KLH; tumor idiotype antigen,
recombinant, personalized-keyhole limpet hemag-
gluttin conjugate autologous priming of dendritic
cells plus granulocyte macrophage-colony stimu-
lating factor (GM-CSF, rDNA); idiotype-pulsed
dendritic cell vaccination; B-cell non-Hodgkins
lymphoma autologous immunoglobulin idiotype-
KLH conjugate vaccine], i.e. for manufacture of
patient-specific tumor antigen-keyhole limpet
hemaggluttin conjugates as part of an patient-spe-
cific immunotherapy, awaiting approval at FDA.
299
InsectSelect Protein
Expression System - insect
cells; avoid baculoviruses
- Licensor, primary
University of British Columbia (UBC) - As-
signee, patent
Description: The InsectSelect Protein Expres-
sion System provides high-level expression of
recombinant proteins in insect cells without use of
baculovirus expression systems (BEVS). Insect-
Select is a non-lytic system that offers continuous
expression, analysis and purification of recom-
binant proteins. Using a stable, plasmid-based
vector, instead of baculovirus vectors, InsectSe-
lect simplifies protein expression by eliminating
the need to generate and amplify viral stocks and
reinfect host cells. Stable cell lines can be main-
tained with protein production (18 mg/liter) for 22
weeks in bioreactors.
Insect shuttle vectors and methods of using
these vectors are provided for stably transforming
disparate insect cell lines to express heterologous
proteins. The invention provides a transformed
insect cell selection system based on resistance to
the bleomycin/phleomycin family of antibiotics,
including the antibiotic Zeocin. Efficient promot-
ers derived from baculovirus immediate early
promoters direct expression of heterologous pro-
teins, including selectable markers, in transformed
insect cells. Transposon-based vectors provide
195
inducible transposition to optimize heterologous
protein expression and unobtrusive markers to
facilitate selection of desired transformants.
Patents: InsectSelect is a trademark of Invitrogen
Corp. The technology is the result of a research
collaboration between Drs. Tom A. Grigliatti,
Tom A. Pfeifer and Dwayne D. Hegedus in the
Department of Zoology, University of British
Columbia (UBC), and Dr. David A. Theilmann,
Pacific Agri-Food Research Centre, Agriculture
and Agri-Food Canada.
Relevant patents have been exclusively as-
signed to Research Corporation Technologies Inc.
(RCT). RCTs first licensee of the system was
InCell Technologies Inc., a startup company in
Vancouver.
An exemplary U.S. application is apparently
Insect expression vectors, 20020116723, Grigli-
atti, T.A., et al.
Availability: Available for non-commercial uses
from Invitrogen.
Licensing information: Contact Research Corpo-
ration Technologies Inc. (RCT) regarding com-
mercial use and licensing.
300
Mimic Sf9 Insect Cells -
mammalian-like glycosylation
Description: Mimic Sf9 Insect Cells are a deriva-
tive of the Sf-9 insect cell line (see related entry)
modified to stably express a variety of mamma-
lian glycosyltransferases. The cells can be used
to produce more mammalian-like proteins in both
baculovirus expression systems. Typically insect
cells are unable to process N-glycans to the extent
of mammalian cells. This can affect protein struc-
ture function antigenicity and enzymatic activity.
The addition of mammalian glycosyltransferases
to the Mimic Sf9 Insect Cells allows for produc-
tion of biantennary terminally sialyated N-glycans
from these insect cells. This is useful in produc-
ing correctly glycosylated and folded secreted
proteins that include both soluble glycoproteins
(enzymes, growth factors, hormones, etc.) and
membrane-bound glycoproteins.
Availabilty: Available from Invitrogen and other
vendors for non-commercial use.
Licensing information: Commercial use requires
a license from Protein Sciences Corp.
301
Polydnavirus vectors - insect
cells; baculovirus altnerative
Dept. of Agriculture (USDA) - Licensor, primary
Description: An insect cell lines transformed with
polydnavirus vectors, which is an insect virus
found in parasitic wasp species, are useful for
stable transformation and protein expresion. This
provides an alternative to the baculovirus expres-
sion system for foreign gene expression in insect
cells. Cell transformation is achieved by infecting
a susceptible insect cell culture with fluid contain-
ing polydnavirus DNA capable of integrating into
the host cell genome. The fluid is obtained from
female parasitoid wasps, such as Glyptapanteles
indiensis, and susceptible insect cells include both
Lepidopteran and Coleopteran cells.
Stable Insect Virus Cell Expression System,
Dougherty, E.M., et al., Nov., 7, 2000, assigned to
USDA. Claim 1 states, A method of transform-
ing an insect cell line, said method comprising: a)
isolating calyx fluid containing braconid polydna-
virus Glyptapanteles indiensis from a female para-
sitoid wasp capable of infecting insects: b) adding
said polydnavirus-containing fluid to a culture of
insect cells to infect the cells; and c) incubating
the infected cells for a time sufficient for polydna-
196
virus DNA to be incorporated into the genome of
the insect cells.
Licensing information: Contact USDA (docket
#0283.93).
302
Pre-occluded Virus (POV)
baculovirus vectors; Insect
cells, per os (oral) baculovirus
infection
- Licensor, primary
Description: Pre-occluded Virus (POV) derived
from a polyhedrin-minus or granulin-minus (lack-
ing a functional polyhedrin or granulin gene)
baculovirus are provided. The discovery of the
POV form of polyhedrin-minus baculoviruses is
essential to this method of infecting insect larvae
per os using the POV form of polyhedrin-minus
baculovirus to infect insect larvae (not cell lines;
see related PERLXPRESS entry). POVs are
highly efficient at establishing infection. POVs
derived from a polyhedrin-minus or granulin-mi-
nus (lacking a functional polyhedrin or granulin
gene) baculovirus is fed to insect larvae per os
(orally) resulting in high infection rates.
Methods of producing recombinant protein
involve selecting stabilized, pre-occluded bacu-
loviruses lacking a functional polyhedrin gene
wherein the baculovirus maintains and expresses
a foreign gene product but not a polyhedrin gene;
using the stabilized, pre-occluded baculoviruses
as an inoculum to infect insect larvae; and har-
vesting the infected insect larvae to collect the
recombinant protein. A method of infecting insect
larvae per os with a form of a baculovirus which
is normally destined to become occluded within
the polyhedrin involves: a. infecting an insect
host larva with baculoviruses lacking a functional
polyhedrin gene; b. waiting for the pre-occluded
virus particles derived from the baculovirus to
form in the insect host larva; c. harvesting the pre-
occluded virus particles from the insect host larva
prior to the liquefaction of the insect host larva;
d. stabilizing the infectivity of the pre-occluded
virus particles; and e. feeding the stabilized pre-
occluded virus particles per os to insect larvae.
Patents: Oral infection of larvae is covered by
patents including EP0638124, ORAL INFEC-
TION OF INSECT LARVAE WITH PRE-OC-
CLUDED BACULOVIRUS PARTICLES,
assigned to Boyce Thompson Institute for Plant
Research.
Licensing information: Exclusively licensed to
Chesapeake PERL
Contact Chesapeake PERL, Inc. and/or Boyce
303
Spodoptera frugiperda Sf-21
cell line - baculovirus host
insect cells
primary
Description: Spodoptera frugiperda Sf-21 cell
lines, including those adapted to serum-free me-
dia, are useful hosts with baculovirus expression
systems (BEVS), and are very commonly used.
The Sf21 cell line was cloned from pupal ovarian
tissue of the Fall Armyworm Spodoptera frugi-
perda by Vaughn et al, 1977). The Sf21 cell line
is highly susceptible to infection with Autographa
california nuclear polyhedrosis virus (AcNPV
baculovirus), and can be used with all baculovirus
expression vectors. Because Sf21 cells have a
large size they provide higher expression levels of
some proteins.
Patents: First reported in 1977, any patents held
by Texas A&M University (TAMUS) have now
expired.
Availability: Available for non-commercial
197
purposes (although being off-patent one could use
this commercially) from Invitrogen.
Licensing information: In June 1999, Micro-
GeneSys, Inc., now Proteins Sciences, Inc., and
Texas A&M University combined their intel-
lectual property and knowhow regarding baculo-
virus expression systems. Protein Sciences was
exclusively authorized to sublicense the combined
BEVS technologies to third parties. Those seeking
SF-21 cells for biopharmaceutical manufacture
may want to obtain these from either of these
sources vs. a reagent vendor.
304
Spodoptera frugiperda Sf-9 cell
line - baculovirus host insect
cells
primary
Description: The Spodoptera frugiperda (fall
army worm) Sf-9 cell line is a commonly-used
insect cell line used with baculovirus expression
systems (see related entries). Sf-9 cells are highly
susceptible to infection (and transformation) by
Autographa californica multinuclear polyhydrosis
virus (AcMNPV) and other baculoviruses. Cell
lines adapted to growth serum-free media are
available. Sf-9 was originally cloned from the
parent IPLB-Sf21 AE cell line, which originated
from pupal ovarian tissues of Spodoptera frugi-
perda. Besides being very effective in making
proteins, the SF-9 cell line is useful for producing
baculoviruses.
The other major insect source for baculovirus
culture, Trichoplusia ni, has some issues, largely
because the baculovirus is a biphasic infection
cycle, where the first part produces more virus-
like particles (VLPs). Trichoplusia organisms
have transposons that can inhibit the efficient
production of VLPs.
Patents: As with much of the original baculovirus
expression technology originally developed by
Texas A&M University, this is now in the public
domain (off-patent).
Availability: The cell line is available from many
vendors.
Licensing: In June 1999, MicroGeneSys, Inc.,
now Proteins Sciences, Inc., and Texas A&M Uni-
versity combined their intellectual property and
know-how regarding baculovirus expression sys-
tems. Protein Sciences was exclusively authorized
to sublicense the combined BEVS technologies to
third parties. For biopharmaceutical manufacture
(and to better satisfy regulators), one still may
want to obtain cells from either of these sources
vs. a reagent vendor.
305
NusA E. coli fusion proteins
- eukaryotes; protein
solubilization
Description: Fusion proteins from expression of
a sequence coding for an E. coli NusA carrier pro-
tein along with a desired gene for expression pro-
vide soluble fusion proteins in eukaryotics cells
and facilitate purification. Fusion proteins with
these carrier proteins have a predicted solubility
probability of at least 90%. This enables improved
purification of recombinant fusion proteins by
avoiding the initial expression of these fusion
proteins in E. coli as insoluble inclusion bodies.
This technology permit[s] the production of large
amounts of heterologous peptides or proteins in
a stable, soluble form in certain host cells which
normally express limited amounts of such soluble
peptides or proteins. The fusion proteins retain
the desirable characteristics of their carrier protein
(i.e., stability, solubility, and a high level of ex-
pression). The carrier protein portion of the fusion
protein may be readily enzymatically cleaved.
Patents: The NusA system is covered U.S.
5,989,868 assigned to the University of Okla-
homa. The abstract states, A fusion sequence
198
having a carrier protein which is preferably an E.
coli protein having a predicted solubility probabil-
ity of at least 90% fused to a target heterologous
peptide or protein, and a host cell (especially E.
coli) transformed with, or having integrated into
its genome, a DNA sequence comprising a DNA
encoding a carrier protein as defined herein fused
to the DNA sequence of a selected heterologous
peptide or protein. This fusion sequence is under
the control of an expression control sequence
capable of directing the expression of a fusion
protein in the cell. An objective of the present
invention is to improve the purification process
of recombinant fusion proteins by avoiding the
initial expression of these fusion proteins in E.
coli as insoluble inclusion bodies. The methods
and compositions of the present invention permit
the production of large amounts of heterologous
peptides or proteins in a stable, soluble form in
certain host cells which normally express limited
amounts of such soluble peptides or proteins. The
present invention produces fusion proteins which
retain the desirable characteristics of a carrier
protein (i.e., stability, solubility, and a high level
of expression).
mercial uses.
Oklahoma.
Other broad/universal and older
technologies
306
Enterokinase - fusion protein
cleavage
Wyeth - Licensor, primary
Description: Wyeth has patented recombinant
non-glycosylated enterokinase and variants and
their use for cleaving poly-histidine, C-LYTAG
and other fusion protein affinity tags (see related
entries) in E. coli and other expression systems.
First isolated in the late 1970s, Genetics Inst.,
now Wyeth, was the first to clone the enzyme.
Enterokinase (enteropeptidase) is useful as
a universal fusion protein cleaving enzyme.
Enterokinase is the physiological activator of
trypsinogen and cleaves with high specificity after
the sequence (Asp4)--Lys.
Used with: E. coli; universal
Patents: Exemplary patents include 6,746,859
and 5,665,566, Cloning of enterokinase and
method of use, assigned to the Genetics Inst., now
Wyeth. U.S. 5,665,566, 09/09/1997, Lavallle, et
al., has abstract, Provided are nucleic acid se-
quence sequences encoding enterokinase activity,
the expression products thereof, and methods for
using same.
Availability: Marketed by New England Biolabs
for non-commercial uses
Licensing information: Contact Wyeth regarding
307
Phage lambda promoters; PL
promoter; PR promoter - E. coli
Description: Lambda (bacterio)phage promoters,
PL and PR, are useful for protein expression in E.
coli.
Use with: E. coli
Availability: Available from various vendors and
culture collections.
Patents: Any relate patents have long expired.
Further info.:
Bernard et al, 1979, Construction of Plasmid
Cloning Vehicles that Promote Gene Expression
from the Bacteriophage Lambda PL Promoter
Gene, v5, 59-76.
Review : overexpression of protein under tran-
scriptional regulation of lambda pL promoter
system in Escherichia coli: consequences and
bioprocess improvement approaches, Journal of
Chemical and Natural Resources Engineering (1).
199
pp. 22-39, Dec 2007; online at http://eprints.utm.
my/4633/.
308
Phage T5 promoter
Stanford University - Assignee, patent
Universite Libre de Bruxelles - R&D
Description: The Phage T5 promoter useful in E.
coli vectors functions similar to the T7 promoter
(see related entry). By cleaving T5 phage and
selecting fragments specifically binding to RNA
polymerase, fragments containing promoters were
isolated.
Use with: E. coli
Patents: Exemplary patents include U.s.
4,495,280, Cloned high signal strength promot-
ers, Bujard, H.G., et al., Jan. 22, 1985, assigned
to Stanford University. Claim 1 states: A linear
DNA sequence having proximal to one end a
strong T5 phage promoter, proximal to the other
end a strong transcriptional terminator balanced
with said strong T5 promoter, and having interme-
diate said promoter and terminator at least one of
(1) a marker for selection adjacent to said termi-
nator or (2) a replication system foreign to T5,
wherein the direction of said promoter is away
from said terminator and said marker is expressed
at a frequency of less than about one-fourth the
frequency of a structural gene, when said struc-
tural gene is inserted between said promoter and
terminator, so as to be under the transcriptional
control of said promoter and to bridge said linear
DNA sequence to provide a circular DNA se-
quence.
main.
309
WI-38 cell line, Normal human
fetal lung fbroblasts
Description: This human fetal lung fibroblast-
derived cell line has long been used for culture of
viruses, including for non-recombinant vaccine
manufacture. This is not considered a continuous
cell line, with it becoming senescent after about
40-60 cell doublings; with cells doubling every
~24 hours, and can be used in continuous culture
for up to 1-4 months (generally not enough for
recombinant protein manufacture- transfection,
cloning, gene amplification, culture adaptation,
maintenace in production seed train, etc.)
The WI-38 cell line was developed in July
1962 from lung tissue taken from a therapeuti-
cally aborted fetus of about 3 months gestational
age. Cells released by trypsin digestion of the
lung tissue were used for the primary culture. The
cell morphology is fibroblast-like. The karyotype
is 46,XX; normal diploid female.
Availability: Most culture collections and other
cell line sources likely offer WI-38.
Licensing information: Developed in the 1960s,
any related patents (if there were any) have long
expired.
Products made using this tech.: Marketed bio-
pharmaceuticals, all non-recombinant, manufac-
tured using WI-38 include
1) Adenovirus vaccines - The prior U.S military
vaccine and the one replacing this.
2) Havrix (Hepatitis A Virus Vaccine, Inactivated)
from GlaxoSmithKline
3) Meruvax II [Rubella Virus Vaccine Live; Ger-
man measles vaccine] from Merck (and combina-
tion vaccines containing this)
4) Varivax II [Varicella Virus Vaccine Live (Oka/
Merck); chickenpox vaccine] from Merck (and
combination vaccines containing this); and
5) IMOVAX Rabies [Rabies Vaccine] from Sanofi
Pasteur.
200
310
Alkaline Phosphatase; Calf
Intestinal Alkaline Phosphatase
(CIAP) - prevent vector
recircularization
Description: Calf Intestinal Alkaline Phosphatase
(CIAP), usually E. coli-expressed but also from
shrimp, is useful to remove phosphate groups
from 5-ends of linear vectors to prevent recir-
cularization during cloning. Bacterial alkaline
phosphatase nonspecifically catalyzes the removal
of most phosphomonoester bonds, but it does not
degrade diphosphate or triphosphate linkages.
Shrimp Alkaline Phosphatase (SAP) nonspecifi-
cally catalyzes the dephosphorylation of most
phosphate monoesters, but it does not degrade
diphosphate or triphosphate linkages. It is irre-
versibly inactivated by heat denaturation at 65C
for 15 minutes.
dors.
Further info.: Reid, T.W. and Wilson, I.B. (1971)
in The Enzymes 3:373-416; Takanami, M. (1967)
J. Mol. Biol. 23:135-48; Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual,
pp 5.72, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
311
Benzonase; Serratia
marcescens endonuclease -
polynucleotides breakdown
Benzon Pharma A/S - Licensor, primary
Description: Although technically not an expres-
sion system, Benzonase endonuclease is useful to
degrade nucleotides remaining in culture media
or purified fractions. Benzonase is a unique,
genetically engineered endonuclease offering a
variety of advantages over existing methods of
nucleic acid removal.
A FDA DMF type II (no. BBMF 5403) has
been granted for Benzonase.
Patents: NycoMed Pharma A/S holds worldwide
patent rights. Merck Serono KGaA is the exclu-
sive patent licensee. Benzonase is covered by
several patents including U.S. 5,173,418 and EP
0229866 B1.
Availability: Benzonase is manufactured and ex-
clusively distributed by Merck Serono KGaA. It is
marketed for non-commercial uses by Invitrogen,
Novagen and other companies.
Licensing information: Contact Merck Serono
(processing@merck.de) regarding commecial
uses and licensing.
European Markets, Benzonase is used by MedIm-
mune for manufacture of Synagis (recombinant
humanized respiratory syncytial virus monoclonal
antibody. Other licensees include Amgen and
Celltech/UCB.
312
DNA Ligase (E. coli)
Description: E. coli DNA Ligase is useful to join
two fragments of DNA with cohesive ends. The
enzyme catalyzes the formation of phosphodiester
bonds between double-stranded DNA fragments
with 3-OH and 5-phosphate ends, in the pres-
201
ence of NAD as the cofactor. Unlike T4 DNA
Ligase, E. coli DNA Ligase can only ligate DNA
fragments with cohesive ends under standard
reaction conditions.
dors.
Further info.: Panasenko, S.M.et al. (1978) J.
Biol. Chem. 253:4590-2.
313
Site-directed mutagenesis
Description: As one of the basic and classic
methods of biological investigation and genetic
manipulation, there are many methods available
for site-directed mutagenesis, involving substi-
tutions, insertions and/or deletions of single or
sequences of nucleotides , whether gene sequence
targeted, e.g., using polymerase chain reaction
(PCR), or random, e.g., radiation exposure. Nu-
merous methods exist to generate different types
of mutations and to enhance for the selection of
the mutant. Examples of methods to enhance for
the selection of the mutant include positive antibi-
otic selection of the mutant strand, using a uracil-
containing DNA strand which can be selectively
degraded in vivo, and dNTP analog incorporation,
which can render one strand of heteroduplex DNA
impervious to digestion. Some approaches can be
combined, such as cassette mutagenesis and the
use of doped oligonucleotides to create a library
of random mutations in a small, defined region.
For a review of methods available, see http://
www.promega.com/pnotes/61/6222_12/6222_12_
core.pdf.
There also also many relatively new methods
available, including directed evolution and all
types of methods, e.g., phage display, useful for
generating mutations in genes and resulting pro-
teins and high-throughput screening and selecting
desired mutations.
In one classic/generic method for double-
stranded DNA using plasmids and T4 enzyme,
The DNA fragment of interest is recovered and
cloned into the plasmid vector. The dsDNA is
denatured to make the template strand available
for hybridization with the mutagenic and selection
oligonucleotides. The mutant strand is synthesized
by primer extension, using T4 DNA Polymerase,
from the hybridized oligonucleotides. T4 DNA
Ligase is used then to seal the resulting nicks at
the 5-ends of the primers. The heteroduplex is
propatated and selected by transforming a muta-
tion-deficient strain of E. coli, (i.e., mutS). Next,
the plasmid DNA is recovered, purified and used
to transform the final E. coli host strain. The re-
sulting heteroduplex is propagated by transforma-
tion in E. coli. T4 DNA Polymerase also exhibits
1,000-fold higher fidelity compared to other
polymerases that lack proofreading activity; there-
fore, it is useful in reducing undesirable secondary
mutations which can arise in strand synthesis. The
overall fidelity of T4 DNA Polymerase is estimat-
ed at one misincorporation per 10.sup.7 residues,
making it the enzyme of choice for site-directed
mutagenesis. An extension of this standard ap-
proach to site-directed mutagenesis includes those
that rely on DNA amplification, specifically the
polymerase chain reaction (PCR).
Patents: Many methods, now even including
basic PCR, date from the mid-1980s and 1970s
and are now in the public domain (any patents
expired). The original plasmid-based methods for
site-directed mutagenesis using oligonucleotides
for in vitro synthesis of mutant DNA date back to
the late 1970s.
Availability: Reagents and protocols are read-
ily available. Check the full documentation that
comes with products or check with the manufac-
turer/marketer to determine if the reagents/method
of interest is covered by patents or otherwise
requires licensing for commercial use.
202
314
T4 DNA Ligase
Description: T4 DNA Ligase, usually from E.
coli, is useful to join two fragments of DNA
(blunt-ended or with compatible overhangs), and
for adding linkers to blunt-ended DNA. T4 DNA
Ligase catalyzes the formation of phosphodiester
bonds between double-stranded DNA fragments
with 3-OH and 5- phosphate ends, in the pres-
ence of ATP.
dors.
now esaily be in the public domain, although
315
T4 RNA Ligase
Description: T4 RNA ligase is useful for lT4
RNA ligation of single-stranded RNA or DNA,
and 3-end labeling of RNA. T4 RNA ligase
catalyzes the formation of phosphodiester bonds
between single-stranded nucleic acids with 5-
phosphate and 3-hydroxyl termini. The enzyme
requires ATP for activity. The rate of activity is
highest for RNA-RNA ligation, lower for RNA-
DNA ligation and very low for DNA-DNA liga-
tion.
dors.
Romaniuk, P.J. and Uhlenbeck, O.C. (1983)
Methods Enzymol. 100:52-9.
England, T.E. et al. (1980) Methods Enzymol.
65:65-74.
203
More Specifc and
Component Technologies
316
Altogen transfection; RNAi
gene silencing
Altogen Biosystems - Licensor, primary
Description: Altogen Biosystems specializes in
and offers CRO services involving polymeric,
lipid- and nanotech-based (and also traditional)
methods for transfection of genes into mammalian
and bacterial cells, and use of RNA interference
(RNAi) for highly efficient gene silencing. The
company performs custom cell-line develop-
ment and designs kits for permanent and transient
transfection. Altogen Custom Services offer stable
cell line generation and RNA Interference (RNAi)
gene silencing services, including development
of efficient transfection reagents and optimization
of siRNA transfection conditions to enable highly
efficient gene silencing in a cell line or cultured
primary cells.
Use with: mammalian cells; bacterial cells
Patents: No patents assigned to Altogen were
retrieved from several simple searches.
Availability: Altogen Biosystems offers pre-op-
timized transfection kits for more than 60 cell
types. Each kit contains cell type specific Trans-
fection Reagent, Transfection Enhancer Reagent,
and recommended transfection protocol. Lipid-
based and polymer-based ALTOGEN In Vivo
Transfection Reagents enable delivery of func-
tional RNA and DNA molecules in vivo.
Licensing information: Contact Altogen regard-
ing CRO services, commercial uses and licensing.
Bacteria/Prokaryotes
317
Profuse vectors; Cisperone
chaperone fusion tags - E. coli;
Protheon Inc. - Licensor, primary
Description: Cisperone fusion tags with binding
affinity for RNA, which is used as a chaperone,
expressed by ProFuse vectors as fusion proteins
with the protein of interest offer proprietary
protein folding technology regarded as a corner-
stone in commercial production of biologically
active proteins in E. coli (and also S. cerevisiae).
Profuse expresses target protein having improved
solubility and folding efficiency, based on ex-
pression of a target protein as a fusion protein
and binding of the fusion protein with an RNA
molecule. The system avoid problems with E. coli
inclusion bodies. CleEZ (an enterokinase mutein;
see related entry) is used to cleave the fusion
protein. Cisperone in fusion proteins dramatically
increases solubility and the activity of the target
protein.
Protheon states, Our experience so far [has]
convinced us of its [ProFuse] distinctive advan-
tage over other commercially available vectors
employing GST, thioredoxin, or MBP as fusion
partners. Proteins which had been refractory
in E. coli are now produced in E. coli as active
forms. The success with our limited experience
is far-reaching: a variety of proteins of human or
viral origin is produced as soluble form, provid-
ing a robust platform for high-throughput protein
expression. Cisperone capitalizes on a novel
chaperone property of RNA molecule that dramat-
ically increases the solubility and the activity of
the target protein fused to RNA-binding module,
leading to development of the expression vector
ProFuse.
204
See also the related SecHancer vector entry
used primarily with S. cerevisiea.
Profuse also enables high level production in
S. cerevisiae, as has been demonstrated with hu-
man growth hormone (hGH), human Granulocyte
Colony Stimulating factor (hG-CSF), Hepatitis
B Surface Antigens (HBV PreS2), and site-spe-
cific protease enterokinase (CleEZ ). The vector
continues to expand its utility in production of cy-
tokines, chemokines, hormones, and viral antigens
for recombinant vaccines.
Complementing this technolgy, Protheon has
established a library of site-specific proteases
CleEZ, for efficient cleavage of proteins. This
allows testing of cisperone vectors to make the
choice of the most efficient production method
tailor-made to the protein of interest.
Use with: E. coli; Saccharomyces cerevisiae
Patents: Relevant Profuse patent applications
include US2006292667, US2004033564 and
KR20040016654, METHOD FOR PRODUC-
ING RECOMBINANT PROTEIN USING
RNA BINDING PROTEIN GENE AS FUSION
PARTNER, assigned to Protheon. Applica-
tions covering CleEZ (enterokinase) include
KR20030097036, RECOMBINANT EN-
TEROKINASE OF WHICH N-TERMINAL OR
C-TERMINAL OF LIGHT CHAIN IS MODI-
FIED, assigned to Protheon.
Licensing information: Contact Protheon, which
offers custom CloneXpress services for protein
expression.
318
Bacterial transcription
promoters
Description: Bacterial transcription enhancers
increase gene expression from promoters in most
or all bacterial systems. A means has been found
to increase the rate of transcription initiation from
a bacterial promoter by fusing a DNA sequence
containing an alpha subunit RNA polymerase
binding site to the promoter upstream of the sigma
subunit of RNA polymerase binding site; and to
screen for such promoters. The resulting DNA
sequence causes RNA polymerase to bind to the
promoter more efficiently, without the need for
transcription factors.
Use with: bacteria
Patents: A related patent is U.S. 6,605,431,
Promoter elements and methods of use,Gourse,
et al., August 12, 2003, provides methods and
kits for identification of compounds that alter
transcription, preferably decrease transcription,
of a polynucleotide, by the inventors Richard
L. Gourse, Shawn T. Estrem, Tamas Gaal, and
Wilma E. Ross, assigned to WARF.
This patent provides a method for detect-
ing whether a compound alters transcription of a
transcription unit. The method includes providing
in a reaction mixture a first polynucleotide that
includes a promoter operably linked to a tran-
scription unit, adding an amount of the compound
to be tested to the reaction mixture under condi-
tions effective to cause transcription, detecting an
amount of a transcription product, and comparing
the amount of the transcription product in the
presence of the compound to an amount of the
transcription product in the absence of the com-
pound under the same conditions. The promoter
includes an accessory promoter element and a
core promoter element, and the reaction mixture
includes at least one RNA polymerase. Option-
ally, the compound does not alter the amount of a
transcription product from a second polynucleo-
tide, where the second polynucleotide includes a
second promoter operably linked to the same tran-
scription unit that is operably linked to the first
promoter, where the second promoter includes the
same core promoter element of the first promoter
and not an accessory promoter element. Typically,
transcription of the transcription unit is decreased;
205
The polynucleotide can be present on a plasmid
vector.
319
BresaGen fusion protein
expression - E. coli.
BresaGen - Licensor, primary
Hospira - Parent co.
Description: BresGen reports using proprietary
bacterial (E. coli) fusion protein technology to
express and purify recombinant proteins on a
commercial scale and offers this to its CRO/CO
clients.
Likely associated with this, BresaGen has
reported scaled-up for GMP production for CBio
Ltd. of Chaperonin 10 at 100L fermentation scale
in less than 3 months; manufacture of thionyl
equine somatotropin (eST;EquiGen Injection; the
first approved equine growth hormone product) at
yields over 1 gram/L; and manufacture of E21R, a
human granulocyte-macrophage colony stimulat-
ing factor (GM-CSF) antagonist, with addition
of a short N-terminal leader sequence to increase
expression, expressed in inclusion bodies at >1.5
g/L.
Use with: E. coli
Patents: No relevant patents assigned to Brasa-
Gen were identified from simple searches.
Licensing information: Contact BresaGen, now
a subsidiary of Hospira, which is developing a
line of biosimilar/follow-on biologic products for
the U.S. and other major markets.
320
Cold-Induced expression -
Bacillus subtilis
PVA-MV AG - Licensor, primary
Description: A cold-induced expression system
using a promoter of the desaturase enzyme from
Bacillus subtilis for temperature regulated (cold-
induced) gene expression is particularly useful for
over expression of hydrophobic or thermo labile
proteins in B. subtilis. A prototype of a cold-active
expression system in Bacillus subtilis is offered.
The expression system is suited for the production
of proteins that tend to form protein aggregates,
are unable to go back to a native state, during the
overexpression process. This enables the pro-
duction of thermolabile proteins at temperatures
around 20 degrees C with substantial cost reduc-
tion.
Use with: Bacillus subtilis
Patents: No relevant patents assigned to PVA-
MV AG were retrieved from simple searches.
Licensing information: Available for licensing,
with or without exclusivity. Contact PVA-MV
AG.
321
desA promoter, iron-
regulated; DmdR repressors -
Actinomycetes
Institute of Biotechnology (INBIOTEC) - Li-
censor, primary
Description: DmdR (divalent metal dependent
repressor) that bind to the desA (desaturase) pro-
moter in presence of Fe2+ or other divalent ions
is useful for induction or repression of expression
in actinomycetes, e.g., Streptomyces and Rhodo-
coccus bacteria. This is a common mechanism of
iron-regulation in actinomycetes. Induction by
addition of the iron chelating agent 2,2-dipyridyl
206
results in strong expression. A 51 bp DNA frag-
ment of the desA promoter containing the 9 bp
inverted repeat is sufficient for binding of the
DmdR repressor.
Use with: actinomycetes, e.g., Streptomyces
and Rhodococcus
Patents: No patents or applications retrieved from
several simple searches.
Licensing information: Contact the Institute of
Biotechnology (INBIOTEC).
Further info.: Characterization of the iron-regu-
lated desA promoter of Streptomyces pilosus as
a system for controlled gene expression in acti-
nomycetes, Flores, F.J., et al., Microb Cell Fact.
2003; 2: 5 (Published online 2003 May 19. doi:
10.1186/1475-2859-2-5).
Flores, F.J., Iron-regulatory proteins DmdR1 and
DmdR2 of Streptomyces coelicolor
form two different DNA-protein complexes with
iron boxes, Biochem. J. (2004) 380, 497-50.3.
322
E. coli. vectors; Mnt-Arc
promoters; T1 and T2 rrnB
ribosomal terminators -
bacteria
ConjuGon, Inc. - Assignee, patent
Description: Tightly-regulated bacterial vec-
tors can express toxic proteins in a wide range
of Gram positive and Gram negative bacteria.
This particularly enables cloning and expression
of toxic proteins, RNA, and metabolites in vivo,
allowing expression of protein and RNAs that
were previously not able to be expressed. Vector
comprising one or more transcription termina-
tors (e.g.,rrnB ribosomal terminators T1 and T2),
a promoter (e.g., promoter/operator is a hybrid
mutant Mnt-Arc promoter operator), a cloning site
and a low copy number origin of replication (e.g.,
modified pSC101 origin of replication), with one
or more transcription terminators upstream of the
promoter.
Use with: bacteria
20050181395, Systems for tightly regulated gene
expression, 20050181395, Anthony, L., et al.,
Aug. 18, 2005.
Licensing information: Contact ConjuGon.
323
pAVEway expression - E. coli
and Pseudomonas
Avecia Biologics Ltd. - Licensor, primary
Description: pAVEway vectors with fully
repressible basic expression and the modulat-
able expression induction enables class-lead-
ing (>10g/L) microbial production and rapid
production cell line development. Using a novel
configuration of operators, promoters and repres-
sors, Avecia has created a range of vectors which
provide tightly controlled production of the target
proteins, while allowing very high expression lev-
els. These vectors are complemented by a range
of host strains.
pAVEway combines two unique features.
First, the basic expression levels can be fully
suppressed, so that no protein is produced before
induction. This allows high expression levels
even for proteins that would normally impair the
growth of the host cells. A second characteristic
is the ability to modulate the expression levels by
varying the concentration of inducer. The expres-
sion kinetics can be tightly regulated to match
the folding capabilities of the host cell, enabling
the soluble expression of proteins of high folding
complexity.
The system has been adopted to Pseudomonas
and E. coli. Avecia expects to expand the host
range to include at a minimum, yeasts and mam-
malian systems. High titres have been demon-
strated for soluble, insoluble or secreted proteins.
Generally, vector construction is completed in 2
weeks; shake flask experiments in week 3, and
fermentation can begin in week 4.
207
Use with: E. coli; Pseudomonas; yeasts; mam-
malilan cells
Fast track process creation and development
Reduced time to clinic, Reduced time to market
High yield protein production
Class leading fermentation productivity
Potential for reduced cost of goods
Can produce a wide range of biotherapeutic pro-
teins using a family of expression systems
Regulatory compliance - Well characterised sys-
tems with full development history
Patents: Avecia report various patent and applica-
tions cover aspects of this technology.
Availability: Avecia makes the pAVEway protein
production platform available as a service to its
CMO clients for cGMP manufacturing of their
protein. pAVEway technology was launched in
May 2007
Licensing information: Contact Avecia, which
offers this technology to its CMO clients, and
may make it available for out-licensing.
324
pTAT-HA plasmids - E. coli;
bacteria
Washington University - Licensor, primary
Description: The pTAT-HA plasmid allows for a
significant increase in transduction efficiency of
proteins and provides ease of use in E. coli and
other bacteria. This technology provides a new
inducible regulatory system, pTAT-HA, that pro-
vides for efficient transduction of proteins to their
target cells. The technology also provides a puri-
fication protocol yielding denatured proteins. The
pTAT-HA plasmid has the ability to transduce pro-
tein into multiple types of target cells in a concen-
tration-dependent manner to achieve maximum
protein concentrations in the target cells within 10
minutes. Transduction occurs in a concentration-
dependent manner, achieving maximum intracel-
lular concentrations in less than 10 min.
The LacZ protein is cloned at the XhoI site.
The pTAT-HA vector carries a T7 promoter (see
related entry), a 6xHis-tag fusion affinity purifica-
tion tag (see related entry) and Tat domain for the
expression and purification of the desired protein,
and a multiple cloning site.
Use with: E. coli; bacteria
Patents: Exemplary patents include
Availability: pTAT-HA plasmid may be available
from Invitrogen for non-commercial uses.
University (ref. no. CH0011)
Further info.: Methods Enzymol. 2000;322:508-
21.
325
Twin-arginine translocation
(Tat) system; Tat nanomachine
- protein folding, then
secretion; bacteria
John Innes Centre (JIC) - Licensor, primary
University of Pennsylvania - R&D, of tech.
Description: Use of Twin-arginine transloca-
tion (Tat) in vectors results in protein expression
with folding of the proteins first and then their
export by bacteria. This new paradigm could have
a huge potential for protein production in bacte-
rial species that are already used for this purpose
- including E. coli and Bacillus. The Twin-argi-
nine translocation (Tat) machinery represents a
recently discovered, widely conserved system
for bacterial protein secretion. This multi-subunit
nanomachine can transport fully folded proteins.
Tat nanomachine selects which proteins to secrete
by recognising a short signal sequence attached
to the end of the protein. The system can pro-
duce a greater variety of proteins than previously
thought, including proteins with lipid anchors and
large proteins.
Use with: bacteria
208
Background: Currently, the proteins used by
the biopharmaceutical industry are secreted by
bacterias general secretion pathway (GSP) which
first transports unfolded proteins. But this method
doesnt have a good capacity and allows limits the
proteins that can be producted.
By harnessing the Tat system, E. coli may be able
to manufacture a much extended range of pro-
teins.
Bacteria normally use TAT to secrete a range
of proteins that help them digest food and com-
pete with other microorganisms in the soil.
WO/2007/071996, TWIN-ARGININE TRANS-
LOCATION (TAT) STREPTOMYCES SIGNAL
SEQUENCES, PALMER, et al., June 28, 2007,
with abstract, Described herein are novel Tat
signal polypeptides and methods for using the Tat
signal polypeptides for producing heterologous
polypeptides. A novel reporter assay for testing
the biological activity of the secreted proteins is
also described.
Availability:
Licensing information: Contact the John Innes
Centre (JIC) regarding commercial use and licens-
ing.
The inventors (Dr. Tracy Palmer, et al.) have
joined the Tat Machine Project, an EU-funded
consortium of researchers from across Europe
studying the Tat system.
Further info.:
Widdick et al. (2006) The twin-arginine trans-
location pathway is a major route of protein
export in Streptomyces coelicolor. Proceedings
of the National Academy of Sciences 103 (47)
17927-17932 (http://intl.pnas.org/cgi/content/ab-
stract/103/47/17927).
Constitutive Expression of Escherichia coli tat
Genes Indicates an Important Role for the Twin-
Arginine Translocase during Aerobic and Anaero-
bic Growth, Journal of Bacteriology, March 2001,
p. 1801-1804, Vol. 183, No. 5.
326
VegI promoters - Bacillus
subtilis and E. coli
Hong Kong University of Science and Tech-
nology, - Licensor, primary
Description: Vegl endonuclease-protected
promoters allow very high expression of het-
erologous proteins to be achieved in bacteria,
particularly Bacillus subtilis and E. coli. An
efficient expression/secretion vector, pM2Veg,
was constructed for extracellular production of
heterologous proteins in Bacillus subtilis. The
cassette may be cloned into a modified B. subti-
lis/E. coli shuttle vector pRB373M2 to form an
expression/secretion vector named the vegl vector.
This novel expression cassette, particularly for
use in B. subtilis (and E. coli), allows very high
expression levels of heterologous proteins to be
achieved in B. subtilis. The putative vegI pro-
moter of the B. subtilis endonuclease restriction
site was identified, allowing the modification of
the palindromic restriction site to protect against
B. subtilis endonuclease activity and the use of a
modified vegI promoter.
A major problem encountered in B. subtilis is
that at the onset of the stationary (S) growth phase
it expresses large quantities of proteases which
are detrimental to the integrity of the heterologous
protein which is being expressed, and product ex-
pression during S-phase is unacceptably low. This
problem has been overcome in the past by the use
of protease-deficient and endonuclease-defective
strains (see related entries). However, although
able to express desired proteins at improved levels
during S-phase, these strains suffer from the prob-
lem of typically being slow-growing strains and
introducing additional expense and difficulties
into the manufacturing process.
The application of the cassette has successfully
used to promote efficient secretory production
of two widely different heterologous proteins, an
endoglucanase (Eng) of Cellulomonas fimi and
human epidermal growth factor (hEGF) in B. sub-
209
tilis. Moreover, the cassette was successfully used
to express and secrete proteins that were found to
be difficult to produce in B. subtilis, e.g. hEGF.
To construct pM2Veg, a synthetic cassette, the
Veg cassette carrying: (1) the strong vegetative
vegI promoter from B. subtilis, (2) the Escherich-
ia coli lac operator, (3) the B. subtilis consensus
ribosome-binding site, (4) the Staphylococcal
protein A leader sequence, (5) a cloning region for
insertion of foreign genes, (6) translational stop
codons in all three reading frames, and (7) the
gnt transcriptional terminator, was cloned into a
derivative of the stable pRB373 B. subtilis E. coli
shuttle plasmid, the pM2 vector.
Use with: Bacillus subtilis; E. coli
Benefits: Advantages include:
1. High stability
2. High levels of extracellular products provided
3. Efficient production of protocols and clones of
the two recombinant proteins (C. fimi Eng and
hEGF)
4. Avoid the use of protease-deficient strains
which are usually slow growing and difficult to
handle
5. Render the heterologous gene cassette to be
protected from the restriction of the host bacteria
6,146,848, Bacterial expression system, Wong,
W.K., et al., Nov. 14, 2000, assigned to Hong
Kong University of Science & Technology. The
first 2 of 26 claims state: 1. An endonuclease-
protected vegI promoter. 2. An endonuclease-
protected vegI promoter according to claim 1,
wherein it is a B. subtilis endonuclease-protected
vegI promoter.
Licensing information: Contact the Hong Kong
University of Science & Technology.
327
Xer-cise gene excision; Xer
recombinases - Bacillus
subtilis
Cobra Biomanufacturing plc - Licensor, pri-
mary
Description: Xer-cise, a simple, effective method
of unlabeled, stable gene insertion into bacterial
chromosomes, has been developed; and multiple
improvements have been made to improved Bacil-
lus subtilis for recombinant protein manufacture.
Xer recombinases, enzymes that naturally oc-
cur in most bacterial strains, are used to remove
(Xer-cise) target genes in E. coli, B. subtilis and
other bacteria. This enables genes to be switched
in and out of bacterial chromosomal DNA cleanly
and more efficiently than current methods, and
eliminates the need to express exogenous site-spe-
cific recombinases. Xer-cise methods for making
chromosomal modifications in Bacillus subtilis
along with expression plasmids and modifications
to B. subtilis 168 based on the Pgrac and Pxyl
promoters and the amyL secrection signal se-
quence and deletion of the sporulation sigma fac-
tor gene sigF and protease enzymes that degrade
secreted proteins improve recombinant protein
production in B. subtilis.
Xer-cise utilizes an insertion cassette consist-
ing of an antibiotic resistance gene flanked by dif
sites and regions homologous to the chromosomal
target locus. dif is the recognition sequence for the
native Xer site-specific recombinases responsible
for chromosome and plasmid dimer resolution
(XerC/XerD in Escherichia coli and RipX/CodV
in Bacillus subtilis). Following integration of the
insertion cassette into the chromosomal target
locus by homologous recombination, these recom-
binases act to resolve the two directly repeated dif
sites to a single site, thus excising the antibiotic
resistance gene. Previous approaches required the
inclusion of exogenous site-specific recombinases
or transposases in trans, but this is unnecessary,
since an effective recombination system is already
210
present in bacteria. The high recombination
frequency makes the inclusion of a counter-select-
able marker gene unnecessary.
B. subtilis protein expression has traditionally
relied on integration of single-copy chromosomal
expression cassettes, but elevated gene copy can
improve the yield and reduce fermentation time.
The Xer-cise genetic modifications avoid the
permanent insertion of antibiotic resistance genes.
Deletion of the sporulation-related sigF gene
creates strains that do not form spores, improving
their standing with regulatory agencies. Extracel-
lular protease mutations (the most available in any
B. subtilis strain at the time) are useful to evalu-
ate recombinant protein expression, as secreted
recombinant proteins are often degraded by these
proteases.
Model proteins studied applying these methods
included the B. anthracis protective antigen (rPA;
anthrax vaccine), the collagenase genes colG and
colH from Clostridium histolyticum and the HIV
CCR5 receptor blocker, RANTES.
Use with: E. coli, B. subtilis; bacteria
Benefits: Gene insertion and deletion is particu-
larly problematic in the more novel species of
bacteria of industrial interest such as Bacillus.
Xer-cise is of generic utility for this and is simple
to employ. Xer-cise allows genes to be inserted
or deleted efficiently without permanently inte-
grating antibiotic resistance genes. An additional
advantage of Xer-cise is the ubiquitous nature of
the Xer recombination system, which allows the
technology to be applied to a wide range of bac-
teria, including species for which only a limited
range of molecular biological techniques have
been developed.
U.S. 2007259430, Process for the Removal of
Selectable Marker Gene Sequence, Cranenburgh,
R.M., et al., Nov. 8, 2007, assigned to Cobra
Biomanufacturing plc. The abstract states, The
invention relates to a process for the removal of
selectable marker gene sequences, in particular
antibiotic gene sequences, from nucleic acid
molecules. The invention further relates to the ap-
plication of this process in the unlabelled integra-
tion and deletion of chromosomal genes and in
controlling gene expression.
Licensing information: Contact Cobra, which is
primarily a CRO/CMO offering manufacturing
services.
Further info.: An Efficient Method of Select-
able Marker Gene Excision by Xer Recombina-
tion for Gene Replacement in Bacterial Chro-
mosomes, Alexandra E. Bloor and Rocky M.
Cranenburgh, Applied and Environmental Micro-
biology, April 2006, p. 2520-2525, Vol. 72, No. 4.
328
Agrobacterium tumefaciens
RpoA co-expression
transcriptional activator -
E. coli
Rutgers University - Licensor, primary
University of Arkansas for Medical Sciences
- Licensor, primary
Description: Methods are provided for expres-
sion in E. coli of other bacterial proteins that
previously could not be expressed in E. coli. Al-
though E. coli presents many advantages as an ex-
pression host, it had not been possible to express
the genes of many bacteria because the E. coli
RNA polymerase does not recognize the promoter
sequences of the heterologous genes. This block
in expression can be overcome by co-expression
of the Agrobacterium tumefaciens rpoA gene
product (a sub-unit of the RNA polymerase) and
the transcriptional activator from the heterologous
organism.
Use with: E .coli
6,764,837, Agrobacterium tumefaciens rpoA
gene, Shouguang, J., et al., assigned to the
University of Arkansas for Medical Sciences
and Rutgers University. The abstract states, The
present invention is directed to a method for
211
expression of at least one heterologous gene in a
host cell comprising transforming a host cell with
at least one nucleic acid construct comprising a
complete ? subunit of an RNA polymerase or a
portion thereof of a hybrid nucleic acid containing
a portion of the ? subunit of an RNA polymerase
obtained from the same genus as the heterologous
gene, and transforming the host cell, with at least
one heterologous gene; and culturing the trans-
formed host cell. The present invention further
is directed to nucleic acid molecules used in the
present method, vectors containing these nucleic
acid molecules, and host cells containing the
nucleic acid molecules. The nucleic acid encod-
ing the alpha subunit of an Agrobacterium RNA
polymerase and the corresponding amino acid
sequence and portions thereof is disclosed.
Licensing information: This is reported as avail-
able for exclusive licensing. Contact the Uni-
versity of Arkansas for Medical Sciences and/or
Rutgers University.
329
Avidin affnity fusion protein
affnity tags; Biotin purifcation;
PinPoint Xa Protein Purifcation
System - E. coli
Biotechnology Research and Development
Corp. (BRDC) - Licensor, primary
Rohm and Haas Co. - Licensor, secondary
University of Illinois - R&D
Description: Vectors containing the gene for
biotin along with a desired protein are useful for
expression of biotin-protein fusion proteins in E.
coli useful for affinity purification using avidin
immobilized on chromatography media. The
biotin portions of the fusion protein bind to the
immobilized avidin, and are subsequently cleaved
using thrombin. Vectors have an encoded endo-
proteinase Factor Xa proteolytic site for separat-
ing the purification tag from the desired protein.
These vectors also carry a convenient multiple
cloning region for ease in construction of fusion
proteins. The system contains vectors in all pos-
sible sense reading frames. Related media, e.g.,
SoftLink Soft Release Avidin Resin from Pro-
mega, allows elution of the fusion protein under
non-denaturing conditions.
Use with: E. coli
Fusion proteins having a site for in vivo post-
translation modification and methods of mak-
ing and purifying them, Cronan, October 12,
1993, coassigned to Biotechnology Research and
Development Corp. and the University of Illinois.
Claim 1 states, . A transformed host cell into
which DNA has been introduced, or progeny of
said transformed host cell, the introduced DNA
comprising: (a) DNA coding for a fusion protein
comprising: (i) a first DNA sequence which codes
for a protein or polypeptide having an amino acid
sequence that allows for post-translation biotina-
tion of the fusion protein; and (ii) a second DNA
sequences joined end to end with the first DNA
sequence and in the same reading frame, the
second DNA sequence encoding a selected protein
or polypeptide; and (b) DNA coding for biotin
ligase; the DNA coding for the fusion protein and
the DNA coding for biotin ligase being operative-
ly linked to expression control sequence
Related chromatography media have been
patented by Rohm and Haas Co.. e.g., 5,276,062,
HPLC avidin monomer affinity resin, Haase,
Jan. 4, 1994, with its abstract stating, Novel,
improved ligand-containing media, a method of
preparation and use in the production of peptides,
proteins, and the like, by chromatographic separa-
tion, and more specifically media having perma-
nently attached via a covalent bond to an inert
solid substrate an avidin polypeptide ligand in the
dissociated renatured form which reversibly binds
to certain molecules such as proteins, peptide,
nucleotides, oligonucleotides, and the like and to
other molecules which bind to avidin via biotinyl-
ation or by way of their secondary/tertiary micro-
molecular structures.
212
Promega product literature mentions U.S.
6,072,039 (Hybrid polypeptide comparing a
biotinylated avidin binding polypeptide fused to
a polypeptide of interest, Haase, et al., June 6,
2000) but does not include any information about
what aspect of the product this covers. Claim 1
broadly claims: A composition of matter com-
prising at least one polypeptide of interest fused to
at least one biotinylated avidin binding polypep-
tide, wherein the polypeptide of interest and the
biotinylated avidin binding polypeptide each have
a N-terminus and a C-terminus and the fusion
takes place at the C-terminus of the biotinylated
avidin binding polypeptide.
Availability: Marketed as PinPointTM Xa Protein
Purification System by Promega for non-commer-
cial uses.
Licensing information: Contact BRDC and/or
Rohm & Haas before adopting this for commer-
cial uses.
330
Biogenerics manufacturing
technology packages - E. coli
ProSpec-Tany TechnoGene Ltd. - Licensor,
primary
Description: ProSpec-Tany TechnoGene, an
experienced CMO, offers technology packages for
specific biogeneric, biosmilars, follow-on biolog-
ics, etc., manufacture. The company has devel-
oped a simple, cost effective and easy to imple-
ment proprietary Technology for the production
of highly active bulk recombinant E.Coli derived
proteins. Packages include a comprehensive
course at the companys facility. The company
clams to have developed a simple, cost effective
and easy to implement its proprietary technology
for the production of highly active bulk recombi-
nant E.Coli derived proteins.
Technology packages include:
Interferon-Alpha 2a
Interferon-Alpha 2b
Interferon gamma 1b
Granulocyte Colony Stimulating Factor
Granulocyte Macrophage Colony Stimulating
Factor
Interleukin-2
Growth Hormone
Insulin Like Growth Factor
Epidermal Growth Factor
Fibroblast Growth Factor
Interleukin-11
Use with: E. coli
Use to make: biogeneric proteins
Patents: The exact contents of technology pack-
ages are unknown (company did not respond to
the authors inquiry), but likely include basic, now
off-patent, E. coli manufacturing technology.
Licensing information: Contact ProSpec-Tany
TechnoGene Ltd. The company claims 13+ years
experience manufacturing recombinant proteins
and offer in-house CMO (manufacturing) ser-
vices.
331
BL21(DE3) competent E. coli
cells
Life Technologies, Inc. - Licensor, primary
Description: BL21(DE3) is a highly competent
(transformable) E. coli cell line often used with
the T7 Expression System (see related entry).
Several E. coli strains prepared by this method
have transformation efficiencies of from 1 to 5 x
10.sup.8 transformants/ug plasmid DNA. Gener-
ally, frozen competent BL21(DE3) cells have
transformation efficiencies of about 1 x 10.sup.8
transformants/ug plasmid DNA. These competent
cells can be stored frozen at -70.degree. C. for
several months without significant loss of trans-
formation efficiency, and frozen cells cannot be
thawed and refrozen without significant loss of
transformation efficiency.
Use with: E. coli
Process of producing highly transformable cells
213
and cells produced thereby, Jessee, et al., Janu-
ary 1, 1991, assigned to Life Technologies, Inc.
Claim 1 of the 797 patent claims: A process for
producing transformable E. coli cells of improved
competence by a process comprising the follow-
ing steps in order: (a) growing E. coli in a growth-
conductive medium at a temperature of 18C to
32C; (b) rendering said E. coli cells competent;
and (c) freezing the cells. The abstract states,
This invention relates to a process for producing
cells of improved competency that are transform-
able. Competency refers to the ability of cells to
take up and incorporate exogenous DNA, and to
such competent cells. This invention particularly
claims Escherichia coli having competency for
plasmid DNA. The invention further relates to a
process to obtain these cells and to cells produced
by the process, which can be repeatedly frozen
and thawed without significant loss in transform-
ability.
In july 2007, a jury trial determined that
Invitrogens 4,981,797 patent is valid and that
Stratagene infringed this patent by making and
selling its competent E. coli cell products. Strata-
gene had previously modified its process for
manufacturing competent E. coli cell products. As
a result, Invitrogen agreed that Stratagene prod-
ucts sold in recent years and currently offered for
sale would not be affected by the jury verdict. The
jury awarded Invitrogen a 15% royalty rate on
Stratagene sales between the years 1997 and 2004
(for a total of $7.8 million in damages) and found
Stratagene to have willfully infringed the patent
only between the years 1997 and 2001.
Availability: Available from Invitrogen, Overex-
press and many other reagent suppliers
Licensing information: For commercial uses and
licensing, contact Invitrogen.
332
C-LYTAG affnity fusion
protein tag; Streptococcus
pneumoniae N-
acetylmuramoyl-L-alanine
amidase LytA - E. coli;
purifcation
Description: Vectors containing a C-LYTAG
sequence along with that for a desired protein
are useful for efficienct expression and purifi-
cation of C-LYTAG fusion proteins in E. coli
on C-LYTRAP chromatography media bearing
immobilized choline or analogs. The affinity of C-
LYTAG (C-LytA) for choline and other structural
analogs allows its use as an efficient affinity tag
for overexpression, immobilization and single-
step purification of proteins of interest (CLYTAG
fusion protein purification system). An enteroki-
nase recognition sequence enables cleavage of the
C-LYTAG moiety from the fusion protein using
enterokinase.
The C-LYTAG (3 moiety of the lytA protein of
Streptococcus pneumoniae) has high affinity for
choline and choline analogs (tertiary or quaternary
amines). C-LYTAG system enables the single-step
affinity purification of C-LYTAG fusion proteins
using C-LYTRAP resin, a simple and efficient
chromatographic support. Binding conditions are
gentle and do not involve covalent modifications,
with the fusion protein highly stable once bound
to the resin. C-LYTAG-fusion proteins are selec-
tively eluted using choline-containing buffers.
The C-LYTAG system yields high expression
levels when induced, but maintains low basal lev-
els prior to induction. As marketed as a reagent/kit
for non-commercial, the system integrates CAS-
CADE technology (see related entry), in which
the synergistic effect of using two transcriptional
regulators in a sequential cascade amplifies
expression levels nearly 20-fold compared to
expression from either promoter individually.
214
Use with: E. coli
Background: The choline-binding domain
(ChoBD) of the carboxy-terminal region of
the Streptococcus pneumoniae amidase LYTA
(C-LYTA) presents a strong affinity for tertiary
amines. As reported in Biotechnol Bioeng 74:
164-171, 2001. a method for single-step purifica-
tion of proteins expressed in the methylotrophic
yeast Pichia pastoris was based on the fusion of
C-LYTA to the protein of interest. C-LYTA was
efficiently expressed and secreted in this host,
with the system suitable for the purification of
recombinant proteins with high specificity for
industrial purposes.
Benefits: Advantages of C-LYTAG Purification
System
a) One-step purification from crude lysate to >
95% pure protein.
b) Tightly regulated expression.
c) Resin is simple, inexpensive and reusable.
d) Compatible with virtually all common buffers.
e) Purified fusion protein can rebind to the matrix.
f) Elution buffers do not interfere with protein
quantitation (Coomassie, UV-absorption, etc...).
g) The C-LYTAG moiety is easily refolded from
inclusion bodies into a functional conformation.
h) No covalent modification of the protein is
needed for efficient immobilization.
i) It has been successfully tested in many cases,
and in a wide range of protein sizes: from small
peptides (<10 aa) to large proteins (>1000 aa).
C-LYTAG has been employed successfully
with different chromatography supports. It is
regenerable -- in most cases, it is only necessary
to wash thoroughly with choline elution buffer
followed by equilibration buffer. To wash cell de-
bris, washes with 1M NaOH and 70% ethanol can
be performed. The eluent (choline) is a non-reac-
tive molecule so that, in principle, it is not neces-
sary to remove it from the purified sample, thus
avoiding additional purification steps (desalting,
dialysis, chromatography, etc.). The only known
incompatibilities are the presence of tertiary or
quaternary amines above 20 mM in the binding
and washing buffers, since they would induce the
premature elution of the protein. C-LYTRAP is
an affinity chromatography support with excellent
flow properties and high capacity for biomol-
ecules. NaCl washes above 0.5 M are effective
enough to eliminate all E. coli proteins bound to
C-LYTRAP. For this reason, it is not necessary
to use any gradient and a fixed universal choline
concentration can be used.
ES203271 (Spain), Process for immobilising
and/or purifying fusion proteins of bio-techno-
logical usefulness in a single step,
Availability: Kits and reagents are available from
Biomedal and Active Motif for non-commercial
uses.
Licensing information: Contact Biomedal re-
333
Campylobacter jejuni
glycosylation genes; OTase of
C. jejuni - glycosylation; E. coli
Imperial College - R&D
London School of Hygiene and Tropical Medi-
cine - R&D
Swiss Federal Institute of Technology (ETH),
Zurich - R&D
Description: Vectors providing Campylobacter
jejuni OTase genes glycosylation along with the
gene for a protein of interest are useful for E. coli
(and also eukaryotic) expression of glycosylated
proteins. Campylobacter jejuni is a gram-negative
bacterium that produces glycoproteins.
Use with: E. coli; eukaryotes
Background: Campylobacter jejuni uniquely
contains glycosylation machinery similar to the
type found in higher organisms.
U.S. 20040265954, System and method for the
production of recombinant proteins, Aebi, M.
and Wacker, M., with its first claim stating: A
215
system for the production of recombinant N-gly-
cosylated target proteins, the system comprising
a prokaryotic organism into which is introduced
a genetic information encoding for a metabolic
apparatus capable of carrying out the requested
N-glycosylation of the target protein, wherein said
prokaryotic organism also contains the genetic
information required for the expression of one or
more recombinant target proteins.
Licensing information: The research was done
by a team of scientists from the Swiss Federal In-
stitute of Technology, Zurich (ETH), the London
School of Hygiene and Tropical Medicine and Im-
perial College in London. Contact ETH (markus.
aebi@micro.biol.ethz.ch) and/or the Imperial
College in London (j.h.moore@ic.ac.uk).
Further info.: Wacker, M., Linton, D., Hitchen,
P. G., Nita-Lazar, M., Haslam, S. M., North, S. J.,
Panico, M., Morris, H. R., Dell, A., Wren, B. W.
and Aebi, M. (2002). N-linked glycosylation in
Campylobacter jejuni and its functional transfer
into E. coli. Science, 298: 1790-1793.
334
Chaperone expression
plasmids; DnaK, DnaJ and
GrpE chaperones - E. coli
Takara Mirus Bio, Inc. - Licensor, primary
HSP Research Institute, Inc. - Assignee, patent
Description: Vectors that coexpress multiple
chaperone proteins, e.g., DnaK, DnaJ and/or
GrpE, along with a target protein enable expres-
sion and proper folding of otherwise difficult-to-
express proteins in E. coli. Co-expression of a
target protein with one of these chaperone teams
increases the recovery of soluble proteins. These
endoplasmic reticulum stress transcription factors
are capable of regulating transcription-inducing
activity. Plasmids carry a pACYC-derived origin
of replication site, allowing co-expression with
other protein expression vectors such as pET (see
related entry) carrying a ColE1 ori.
As marketed by Takara, each plasmid carries
an origin of replication derived from pACYC
and a Cmr gene, which allows use with E. coli
expression systems utilizing ColE1-type plasmids
with an ampicillin resistance gene as a marker.
The chaperone genes are situated downstream of
an araB orPzt-1 (tet) promoter, thus, expression
of target proteins and chaperones can be induced
individually if the target gene is placed under the
control of other promoters (e.g. lac). These plas-
mids also contain the necessary regulator (araC
or tetr) for each promoter. This system works
well with the pCold expression system vectors
(see related entry). [This system cannot be used
in combination with chloramphenicol-resistant E.
coli host strains or expression plasmids that carry
a chloramphenicol-resistance gene. For example,
E. coli BL21(DE3), which is often used with pET
systems, is acceptable as a host strain, however E.
coli BL21(DE3) pLysS and BL21(DE3) pLysE,
which contain pLysS or pLysE plasmids that have
the pACYC replication origin and the Cmr gene,
cannot be used with this system].
Use with: E. coli
Patents: Originally developed by the HSP Re-
search Institute, Inc., with this organizations IP
acquired by Takara. Exemplary patents include
6,159,708, Chaperone expression plasmids,
Sogo, et al., Dec. 12, 2000, with its abstract stat-
ing: An artificial operon comprising polynucleo-
tides encoding each of chaperones DnaK, DnaJ
and GrpE; an expression plasmid carrying the
operon; a cotransformant prepared by introducing
the expression plasmid into E. coli together with
a foreign protein expression vector; and a method
for producing a foreign protein comprising using
the cotransformant.
Availability: Kits/reagents available from Takara
Licensing information: Contact Takara regarding
216
335
cis-Acting Peptide chaperones
- E. coli
Brookhaven National Laboratory - Licensor,
primary
Description: cis-Acting peptide chaperones
expressed as fusion proteins with the protein of
interest provide a method for increasing the yield
of soluble, properly folded recombinant proteins
from E. coli. Brief protease treatments of the
soluble fusion proteins cleave the peptide exten-
sions from the proteins of interest. Expressing
proteins fused to short peptide extensions causes
the protein to become either self-chaperoning in
vivo or more recoverable from inclusion bod-
ies in vitro. The peptide extensions eliminate the
need to alter to culture conditions to reduce the
extent or likelihood of inclusion body formation.
The peptide extensions enhance the recovery of
properly folded protein following chemical and
heat denaturation and renaturation of the protein
in vitro. Also, the peptide extensions are useful for
recovery of proteins in vitro from inclusion bodies
that continue to persist despite expression in fu-
sion with the extension. Proteins can be renatured
from inclusion bodies at concentrations exceeding
5mg/mL (5 g/L). Antibodies to the peptide exten-
sions can provide a simplified means of isolating
the expressed fusion proteins or monitoring the
proteolytic removal of the peptide.extension.
The chaperones are claimed to be effective in
any expression system in which inclusion body
formation is a problem.
Use with: E. coli and other prokaryotes; also
claims for yeast, insect and mammalian and
other eukaryotic cells
U.S. 20030134352, Facilitating protein fold-
ing and solubility by use of peptide extensions,
Freimuth, Paul I., et al., July 17, 2003, assigned to
Brookhaven National Laboratory.
Licensing information: Contact Brookhaven
National Laboratory.
336
Codon-Optimized, Expression-
Ready E. coli Clones
Bioclone Inc. - Licensor, primary
Description: Bioclone uses unique proprietary
technology to optimize codon, secondary struc-
ture, GC content etc. Due to the optimization,
expression levels can be dramatically increased,
from 2-100 times depending on different genes,
as compared with pre-optimization. These genes
have been cloned into vectors containing a T7
promoter (see related entry) ready for expression
in E. coli.
Use with: E. coli
Availability; Bioclone markets Hundreds of
codon-optimized cDNA clones...ready for high
expression in E.coli for non-commercial uses.
Licensing information: Contact Bioclone regard-
ing use for commercial applications.
337
Continuous culture - bacteria;
E. coli
Cornell University - Licensor, primary
Description: A system, including vectors, is
provided for the continuous culture and produc-
tion of recombinant proteins from Gram-negative
bacteria without plasmid loss. Production of the
desired protein product occurs while host cell
replication is inhibited, allowing immobiliza-
tion of the bacteia, proteins are excreted into the
culture medium, and there is no use of antibiotics
in culture media
217
5,942,421 and 5,747,281. U.S. 5,942,421, Sys-
tem useful for the production of proteins from
recombinant DNA in single celled organisms,
Shuler, et al., August 24, 1999. The abstract states,
A single celled organism, vector, and method
are provided for continuous production of ex-
creted proteins in the absence of substantial host
cell replication and without the necessity for the
addition of antibiotics to control cell replication.
The vector has (1) an inducible promoter capable
of activating a gene for a protein to be produced
under conditions that substantially inhibit host
cell replication, and (2) a hybrid gene containing a
signal sequence fused to a gene for the protein to
be produced. Large quantities of the transformed
single celled organisms containing the vector can
be grown in the absence of inducing conditions,
thereby reducing the problem of plasmid loss.
Further, the protein is produced under condi-
tions that substantially inhibit host cell replica-
tion, thereby allowing immobilization of the
single celled organism by entrapment or attach-
ment within or onto a solid support surface. The
single celled organism can be immobilized after,
before or simultaneously with induction. The
single celled organism advantageously may be an
encapsulated gram-negative bacterium which has
no outer membrane or an outer membrane that is
permeable to the proteins to be produced so that
the protein products are excreted into the media
thereby avoiding difficulty in recovering the
desired protein products due to the formation of
inclusion bodies or the presence of contaminants
produced by cell lyses.
Licensing information: Contact Cornell Univer-
sity (ref. no. D-523).
338
Disulfde isomerase
coexpression; DsbC and DsbG
- E. coli; disulfde bonds and
folding
Genentech, Inc. - Assignee, patent
University of Texas - Assignee, patent
Description: Co-expression of certain disulfide
isomerase enzymes, DsbC and DsbG, is useful for
bacterial (Escherichia or Salmonella) expression
of proteins with disulfide bonds. This enables E.
coli expression of eukaryotic polypeptides con-
taining disulfide bonds which are active, correctly
folded, and secreted from the bacterial cell to
provide economic and convenient means for the
recovery, isolation, and purification of the recom-
binant polypeptide of interest. In E. coli, DsbA
and DsbB provides a pathway for the formation,
secretion and folding of proteins with disulfide
bonds.
Use with: E. coli; Salmonella.
Use to make: proteins, disulfide bonds
Methods for producing heterologous disulfide
bond-containing polypeptides in bacterial cells,
Georgiou, et al., July 4, 2000, assigned to the
University of Texas and Genentech. The pat-
ent abstract states, Disclosed are methods and
compositions for producing heterologous disulfide
bond containing polypeptides in bacterial cells.
In preferred embodiments the methods involve
co-expression of a prokaryotic disulfide isomer-
ase, such as DsbC or DsbG and a gene encoding
a recombinant eukaryotic polypeptide. Exemplary
polypeptides disclosed include tissue plasminogen
activator.
Texas and/or Genentech.
218
339
Elastin-like polypeptide (ELP)
self-cleaving fusion protein
tags - chromatography-free
purifcation; E. coli
Princeton University - Licensor, primary
Description: Vectors containing elastin-like poly-
peptide (ELP) fusion tags along with a desired
protein provide controllable self-cleaving fusion
protein affinity tags useful for purification of
proteins expressed in E. coli. without use of chro-
matography. See also the other ELP self-cleaving
system. Self cleaving ELP tags consisting of re-
peating pentapeptides of VPGXG (X=any amino
acid) are fused to a controllable self-cleaving
intein. The ELP fusion tags effectively eliminate
the need for conventional affinity chromatogra-
phy or proteolytic tag removal in the purification
of arbitrary fusion proteins. This method allows
the recovery of native product proteins, without
additional amino acids on either terminus, and
has been used to purify ten structurally diverse
proteins with high purity, activity and reasonable
yield.
The system provides an engineered self-
cleaving intein to create a simple, economical
alternative to conventional methods of protein
purification. As shown in published work, the
target protein gene is fused to a gene encoding
a self-cleaving ELP-intein tag, and the resulting
fusion is overexpressed in E. coli. The soluble
ELP fusion precursor is then isolated from the
insoluble cell debris by centrifugation at a tem-
perature below the ELP T. The ELP-tagged fusion
is then heated in the presence of salt to a tem-
perature above its T, which causes the ELP tags
to self associate into an insoluble precipitate. The
precipitated ELP fusion can then be separated
from the soluble cellular components in a second
centrifugation step, and redissolved in a pH 6.0
buffer at low temperature. The intein self-cleav-
age reaction then releases the target protein from
the ELP tag, which can be subsequently removed
by an additional cycle of salt addition, heating
and centrifugation. Thus a highly purified native
protein is produced from a total cell lysate via a
simple mechanical means, without chromatogra-
phy.
The simple mechanical recovery of precipitat-
ed ELP fusion protein suggests a variety of means
for scale-up, including tangential flow microfiltra-
tion or continuous centrifugation. Alternatively
the method might be used in a robotic system to
purify protein libraries for screening. The simplic-
ity and self contained nature of this system prom-
ise a breakthrough in the production of purified
recombinant proteins in research and industrial
enzymes for commercial use.
Use with: E. coli
Patents: Note, this technology is very similar to
the other ELP fusion tag technology (from Duke
Univ.).
An exemplary U.S. application is
20060263855, Intein-mediated protein purifica-
tion using in vivo expression of an elastin-like
protein, Wood, D., with claim 1 stating: A
fusion protein comprising: (a) a product protein
domain, (b) a self-cleaving intein, and (c) at least
one aggregator protein domain capable of self-as-
sociation and precipitation that comprises one or
more elastin-like protein (ELP) domains; wherein
the intein is located between the product protein
domain and the aggregator protein domain.
Licensing information: Contact Princeton Uni-
versity (invention # 04-2188), which is currently
seeking industrial collaboration to commercialize
this technology.
Further info.: Banki,M., Feng,L., Wood, D.,
September 2005, Simple bioseparations using
self-cleaving elastin-like polypeptide tags, Nature
Methods, Vol. 2 No.9, 659-661.
Recombinant Protein Purification by Self-
Cleaving Aggregation Tag. Nature Protocols, 1,
2257-2262, 2006 [Wood, D., W.-Y. Wu, C. Mee,
F. Califano, and R. Banki].
Patel J, Zhu H, Menassa R, Gyenis L, Richman A,
Brandle J (2007) Elastin-like polypeptide fusions
219
enhance the accumulation of recombinant proteins
in tobacco leaves. Transgenic Res 16, 239.
340
ExpressProtect; p26, SicA, and
alpha-crystallin-type fusion
protein tags - E. coli
ECI Biotech - Licensor, primary
Worcester Polytechnic Institute - R&D, of tech.
Description: ExpressProtect involves a class
of proteins that stabilize and protect host pro-
teins from degradation, with these useful when
expressed as fusion protein tags in E. coli along
with a desired gene. p26, SicA, and other alpha-
crystallin-type fusion protein tags are provided.
These act as universal protein folders and broad
spectrum proteases inhibitors. The p26, SicA, or
alpha-crystallin type protein domain tags can be
removed by thrombin cleavage.
Use with: E. coli
Patents: Related U.S. patents include 6,861,403,
Method and device for improving protein stabil-
ity and solubility, Sanders, March 1, 2005, with
claim 1, A method for producing soluble and
active recombinant protein comprising: a) ex-
pressing an insoluble protein as a fusion protein
with an alpha-crystallin type protein or a frag-
ment thereof in bacteria; b) purifying said fusion
protein; and c) removing said alpha-crystallin type
protein or fragment thereof from said purified fu-
sion protein, thereby resulting in said soluble and
active recombinant protein.
Also, U.S. 7,074,757, Method and device
for improving protein stability and solubility,
Sanders, July 11, 2006, with claim 1, A method
for expressing proteins as a fusion chimera with
a domain of p26 or alpha crystallin type proteins
to improve the protein stability and solubility
when over expressed in bacteria such as E. coli
is provided. Genes of interest are cloned into the
multiple cloning site of the Vector System just
downstream of the p26 or alpha crystallin type
protein and a thrombin cleavage site. Protein ex-
pression is driven by a strong bacterial promoter
(TAC). The expression is induced by the addition
of 1 mM IPTG that overcomes the lac repression
(lac I.sub.q). The soluble recombinant protein is
purified using a fusion tag.
Both patents are assigned to Expressive Con-
structs, Inc. Much of the research was apparently
performed at the Worcester Polytechnic Institute.
Licensing information: Contact Expressive Con-
structs, Inc.
341
High copy number plasmids;
pBGP120 - E. coli
Chiron Corp., Novartis AG - Licensor, primary
Novartis AG - Parent co.
Description: DNA plasmids having high copy
numbers are modified such that readthrough
expression of heterologous DNA inserted in the
plasmid will not continue into the replication
primer strand. For example, plasmid (pBGP120)
exists in high copy number levels, approximately
10 to 20 plasmids per cell chromosome.
Use with: E. coli
Patents: U.S. 4,631,257, Stable high copy
number plasmids, assigned to Cetus Corp., now
Chiron Corp., now part of Novartis, issued Dec.
23, 1986. The abstract states DNA plasmids are
described which are selected mutants in which an
altered repressor gene leads to high copy number
replication. Elements in the plasmids are modi-
fied in such a way that readthrough expression of
heterologous DNA inserted in the plasmid will not
continue into the replication primer strand. Dele-
tions resulting from interference with replication
primer strand transcription are thereby avoided.
Licensing information: Patents now expired
- in public domain. Chiron Corp. (now part of
Novartis) has reported that it had received related
manufacturing-related patent licensing royalties
on sales of GlucaGen, recombinant glucagon from
220
Novo Nordisk A/S, and that royalties stopping in
late 2003 with the expiration of relevant patents.
342
High transformation effciency
(Hte) competency - E. coli cells
Stratagene Corp. - Licensor, primary
Description: E. coli with an hte (high transfor-
mation efficiency) gene are useful for their high
levels of protein expression. Gram negative bacte-
ria, e.g., E. coli, cells containing the Hte mutation
are highly competent for DNA transformation;
amd may be frozen so as to provide for prolonged
storage. Hte also enables expression of genes
which otherwise would be limited by the presence
of rare codons in mulitcellular insect, plant and
mammalian host cells.
Use with: E. coli; plant cells; insect cells; mam-
malian cells
Highly transformable bacterial cells and methods
for producing the same, Greener, et al., March
16, 2004, assigned to Stratagene, with claims con-
cerning E. coli competency and storage. Applica-
tions related to broader use, including expression
of genes limited by rare codon, and plants and an-
imals, include WO/2000/044926, HIGH LEVEL
EXPRESSION OF A HETEROLOGOUS PRO-
TEIN HAVING RARE CODONS, CARSTENS,
et al., assigned to Strategene.
mercial use from Strategene.
Licensing information: Contact Strategene re-
343
His-Patch ThioFusion System;
pThioHis vector - E. coli; fusion
proteins
Wyeth - Licensor, primary
Genetics Institute, Inc. - R&D
Description: The His-Patch ThioFusion System
provides thioredoxin-like fusion proteins with
increased solubility and simplified purification
of proteins expressed in E. coli, producing high
levels of stable and soluble fusion protein (avoid-
ing inclusion bodies), allowing recovery from
periplasmic extract or from the cell culture me-
dium. The fusion protein, located in the bacterial
cytoplasm, may be selectively released from the
cell by osmotic shock or freeze/thaw procedures.
The fusion tag can be readily be cleaved using
enterokinase to liberate the soluble, correctly
folded heterologous protein from the thioredoxin-
like portion.
The His-Patch ThioFusion System has the fol-
lowing features:
a) Recombinant proteins are fused to HP-thiore-
doxin for increased solubility and rapid purifica-
tion
b) Expression is driven by the trc promoter and
induced to high-levels with IPTG. Expression can
be carried out in commonly-used E. coli strains.
c) An enterokinase cleavage site downstream from
the HP-thioredoxin gene allows removal of the fu-
sion partner using enterokinase (see related entry).
d) The pThioHis vector is provided in three read-
ing frames for simplified subcloning in frame
with the HP-thioredoxin gene.
A protein may be fused to the amino terminus
of a thioredoxin-like sequence, the carboxyl ter-
minus of the thioredoxin-like sequence, or within
the thioredoxin-like sequence (e.g., within the ac-
tive-site loop of thioredoxin). The fusion sequence
may also contain a linker peptide between the
thioredoxin-like sequence and the selected peptide
or protein. This linker provides, where needed, a
selected cleavage site or a stretch of amino acids
221
capable of preventing steric hindrance between
the thioredoxin-like molecule and the selected
peptide or protein.
The His-Patch ThioFusion System improves
upon the original ThioFusion System by eliminat-
ing the need for specialized E. coli strains and
media, allowing easier subcloning of the gene of
interest, and simplifying purification of fusion
proteins. The vector used for expression, pThio-
His, contains a modified thioredoxin gene that
allows purification of fusion proteins on NTA
or other metal-chelating resins. Two amino acid
residues in the original thioredoxin gene were
changed to histidine residues--a Glutamic acid at
position 31 and a Glutamine at position 63. When
the resulting thioredoxin protein, His-Patch-(HP)-
thioredoxin is expressed, a histidine patch is
formed with the native His residue at position 7.
The histidine patch has a high affinity for divalent
cations (i.e. nickel) allowing simple, rapid purifi-
cation on NTA metal-chelating resins.
Used with: E. coli
Patents: Exemplary patents include 5,270,181
and 5,292,646, Peptide and protein fusions to
thioredoxin and thioredoxin-like molecules, as-
signed to the Genetics Inst., now Wyeth.
Availability: Marketed by New England Biolabs
for non-commercial uses
Licensing information: Contact Wyeth.
344
N(pro) fusion protein tag, self-
cleaving; Swine fever virus
N(pro) autoproteolysis; EDDIE
- E. coli
Austrian Center of Biopharmaceutical Technol-
ogy (ACBT) - R&D
Sandoz AG - Assignee, patent
Description: The gene for the autoproteolytic
function of N(pro) from classical swine fever
virus fused to a gene for a desired protein virus
enables prokaryotic expression, particularly E.
coli, of fusion proteins with subsequent self- or
auto-cleavage of the N(pro) tag. Proteins or pep-
tides expressed as N(pro) fusions are deposited as
inclusion bodies. Npro fusion proteins are isolated
from inclusion bodies, solubilized and self-cleave
during refolding. On in vitro refolding by switch-
ing from chaotropic to kosmotropic conditions,
the fusion partner is released from the C-terminal
end of the autoprotease by self-cleavage, leaving
the target protein with an authentic N-terminus.
The N(pro) expression system can be used as
a generic tool for the high-level production of
recombinant toxic peptides and proteins (up to
12 g/l) in E. coli without the need for chemical or
enzymatic removal of the fusion tag.
A tailor-made N(pro) mutant called EDDIE,
with increased in vitro and decreased in vivo
cleavage rates, has been used to express proin-
sulin, domain-D of staphylococcal protein A,
hepcidin, interferon-alpha1, keratin-associated
protein 10-4, green fluorescent protein, inhibitori-
al peptide of senescence-evasion-factor, monocyte
chemoattractant protein-1 and toxic gyrase inhibi-
tor, among others. In one example, yield of a toxic
protein was increased by 40-fold from 0.005 to
0.2 g/L; and for a peptide there as reduction in
COGs 20-30% at the 3000 L scale and 100% at
the 13,000 L scale, when compared to standard
fusion protein technology.
Use with: prokaryotes; E. coli
Effective and high yield expression technology
Controllable, autoproteolytic cleavage result-
ing in an authentic Nterminus
Proprietary technology - developed by Sandoz
in cooperation with the Austrian Center of Bio-
pharmaceutical Technology
Suitable for peptides and proteins
Production of proteins by autoproteolytic cleav-
age, Rumenapf, et al., May 27, 2008, assigned
to Biochemie GmgH (Sandoz AG), a subsidiary
of Novartis AG, with claim 1, A nucleic acid

222
molecule coding for a fusion protein comprising
a first polypeptide which is a pestivirus autopro-
tease N.sup.pro and a second polypeptide which
is covalently bound to the first polypeptide at the
C-terminus of the first polypeptide in a manner
such that the second polypeptide is capable of be-
ing cleaved from the fusion protein by the auto-
proteolytic activity of the first polypeptide, and
where the second polypeptide is a heterologous
with respect to the first polypeptide wherein said
C-terminus of said first polypeptide is a cysteine
at a position corresponding to position 168 of
SEQ ID NO:1.
Also, U.S. 6,936,455, Production of heter-
ologous proteins using an Npro autoprotease of a
pestivirus and inclusion bodies, August 30, 2005,
assigned to Sandoz AG.
Licensing information: Contact Novartis AG.
Npro fusion technology to produce proteins with
authentic N termini in E. coli, Nature Methods
- 4, 1037 - 1043 (2007) (onine doi:10.1038/
nmeth1116).
345
OverExpress C41(DE3) and
C43(DE3) - E. coli strains
AVIDIS SA - Licensor, primary
Medical Research Council (U.K.) (MRC) - As-
signee, patent
Description: These C41(DE3) and C43(DE3)
E. coli strains are derivatives of the E. coli B
strain, BL21(DE3; see related entry). C41(DE3)
and C43(DE3) were selected for their ability to
survive after inducing the expression of an oth-
erwises toxic recombinant protein. These strains
are effective in overexpressing toxic proteins from
all classes of organisms, including eubacteria,
archaea, yeasts, plants and mammals. There are
no intrinsic antibiotic resistances (or plasmids) in
either C41(DE3) or C43(DE3), and these gener-
ally grow in minimal media.
Like the parental BL21(DE3) strain, C41(DE3)
and C43(DE3) carry the lambda DE3 lysogen
which expresses T7 RNA polymerase (see related
entry) from the lacUV5 promoter after Isopropyl-
1-thio--D-galactopyranoside (IPTG) induction.
Use with: E. coli
Over-expression of proteins, Walker, et al.,
March 26, 2002, assigned to the Medical Re-
search Council, with this concerning methods for
selecting host cell mutants which are resistant to
expression system toxicity.
Licensing information: Contact AVIDIS S.A.
Three types of Commercial Use License are
available, for CMOs, for large companies, and
for small companies. All include an upfront and
annual fee (higher for large corporation than for
small ones) and a royalty scheme. There is also a
so-called intermediary product license, e.g. for the
production of target proteins.
It is possible that one may also need to sepa-
rately license T7 technology from Broohaven Nat.
Labs.
Products made using this tech.: Licensees have
been reported to include ProteinExpert, a French
CMO. The strains are widely used in research ap-
plications.
Miroux B, Walker JE. Over-production of proteins
in Escherichia coli: mutant hosts that allow syn-
thesis of some membrane proteins and globular
proteins at high levels. J Mol Biol. 1996 Jul 19;
260(3):289-298.
346
OxlT plasmids; Oxalate/
Formate Exchange Protein - E.
coli
primary
Description: Use of the OxlT transport protein in
combination with oxalylCoA decarboxylase and
formyl-CoA transferase in E. coli (and other or-
223
ganisms) expression cassettes provides an energy
generating system, extending culture longevity
and increasing product yield. Expression cas-
settes can be designed to express all or part of
the system in a target host. These energy gener-
ating systems can be used in industrial settings
that utilize fermentation, considerably extending
the lifetime of production runs, allowing greater
yields of biosynthetic products.
The protein, OxlT, isolated from the gram-
negative anaerobe, Oxalobacter formigenes, is
a transport protein responsible for the physical
movement of oxalate and formate across the
plasma membrane. This protein is part of the
three-protein oxalate/formate antiport exchange
system that allows for conversion of extracellular
oxalate to formate + CO2, providing a metabolic
energy system to cells.
Use with: E. coli
OxlT sequence and its use, Maloney, P.C., et
al., December 19, 2000, with its abstract stating
[truncated], A DNA sequence encoding a trans-
port protein responsible for the physical move-
ment of oxalate and formate across the plasma
membrane is provided. The protein, OxlT, can be
isolated from the gram negative anaerobe, Oxalo-
bacter formigenes. The protein is part of the three
protein oxalate/formate antiport exchange system
that allows for conversion of extracellular oxalate
to formate+CO2. The OxlT coding sequence can
be used to provide a metabolic energy system to
the cell. The system comprises the OxlT transport
protein in combination with oxalylCoA decar-
boxylase and formyl-CoA transferase. Expression
cassettes can be designed to express all or part
of the system in a target host. Such energy gen-
erating systems can be used in industrial settings
that utilize fermentation. The energy generating
systems of the invention extend culture longevity
and increase product yield...
University (JHU).
347
pBR322 - E. coli plasmid;
antibiotic selection
Genentech, Inc. - R&D
Centre de Investigacin en Biotecnologa
- R&D
Description: pBR322 is an ampicillin- and tet-
racycline-resistance conferring, general purpose
cloning vector. It is one of the most commonly
used E.coli cloning vectors. The convenience
of cloning pBR322 by inactivation of antibiotic
resistance genes, and various unique restriction
sites, offered a lot of flexibility, with pBR322
superior to its parental plasmid, pMB9. pBR322
was the first artificial plasmid created by Bolivar
and Rodriguez (how it got the PR designation).
pBR322 is a well-established multipurpose clon-
ing vector in laboratories worldwide. pBR322 was
constructed with a highly diminished ability to
propagate outside of controlled/culture conditions.
pBR322 was part of the first E. coli K2 system to
be certified as safe for manufacturing according
to the National Institutes of Health recombinant
DNA (RAC) guidelines.
A large number of derivatives have been creat-
ed for specific applications and research purposes,
including gene expression in its natural host, E.
coli, and few other bacteria. Portions of pBR322
are used in many, if not most, vectors, not only E.
coli vectors, but many inter-species shuttle vec-
tors. The early characterization of the molecule,
including its nucleotide sequence, replication and
maintenance mechanisms, and determination of
its coding regions, accounted for its success, not
only as a universal cloning vector, but also as a
provider of genes and an origin of replication for
other intraspecies vectors.
pBR322 is 4361 base pairs in length and
contains a replicon region (source plasmid pMB1)
and the tet gene (see related entry), encoding
the tetracycline resistance protein (source plas-
mid pSC101). It also contains the unique EcoRI
restriction site GAATTC and splices the DNA
224
between G and A from 5 to 3 position. The ori-
gin of replication in this plasmid is pMB1 (a close
relative of ColE1).
Use to make: proteins, nonglycosylated
Patents: pBR322 was constructed by Genentech
in 1975. Classic pBR322 vectors are now in the
public domain. No U.S. patents covering classic
pBR322 constructs were retrieved by this au-
thor. Genentech and others freely distributing the
plasmid and early pubication of its gene sequence
contributed to this being rapidly and widely
adopted, particularly by the research community.
The inventors article in Gene (see below) was
the most cited article from this periodical in 1990,
then with over 6,000 citations.
Availability: Widely available.
Licensing information: No need to license clas-
sic pBR322.
Further info.: Bolivar F, Rodriguez R L. Greene
PJ, Betlach M C, Heyneker H L, Boyer H W, Cro-
sa J H & Falkow S., Construction and character-
ization of new cloning vehicles. II. Amultipurpose
cloning system, Gene 2:95-113, 1977.
Balbas P. Soberon X. Merino E. Zuritu M, Lomeli
H, ValIe F. Flores N & Bolivar F. Plasmid vec-
tor pBR322 and its special-purpose derivatives-a
review. Gene 50:3.40. 1986.
348
pCold vectors; Cold Shock
Protein A (cspA) promoters - E.
coli, cold induction
University of Medicine & Dentistry of New
Jersey - R&D
Rutgers University - Parent org.
Description: pCold Expression Vectors utilize
the Cold Shock Protein A (cspA) promoter for
expression of high purity, high yield recombinant
proteins in E. coli. Overexpression of proteins in
Escherichia coli at low temperature improves their
solubility and stability. The cspA gene in pCold
vectors drives the high expression of cloned genes
upon induction by cold-shock. These vectors
selectively induce target protein synthesis at low
temperatures (15C) where the synthesis of other
proteins is suppressed and protease activity is
decreased, resulting in high yields of the target
protein (60% of intracellular protein). In addi-
tion to the cspA promoter, the vectors contain a
lac operator (for control of expression), ampicil-
lin resistance gene (ampr; for selection), ColE1
origin of replication, M13 IG fragment, and
multiple cloning sites (MCS). These vectors work
well with chaperones. pCold vectors are highly
complementary to the widely used pET vectors
(see related entry).
Use with: E. coli
Patents: pCold was apparently developed by Dr.
Masayori Inouye, Univ. of Med. and Dent. of
NJ and Takara Mirus Bio, Inc.. Related patents
include U.S. 6,479,260, assigned to Takara, and
5,981,280; 6,686,174; and 6,333,191 assigned to
the University of Medicine & Dentistry of New
Jersey and exclusively licensed to Takara.
Availability: Marketed by Takara for non-com-
mercial use.
Licensing information: Contact Takara regarding
Further info.: Cold-shock induced high-yield
protein production in Escherichia coli, Nature
Biotechnology 22, 877 - 882 (2004) (online: 13
June 2004; | doi:10.1038/nbt984).
349
pET Expression System; T7
promoter; pET Directional
TOPO Cloning; Champion pET
Expression Vectors; T7 RNA
polymerase (T7 RNAP) - E. coli
Brookhaven National Laboratory - Licensor,
primary
225
Invitrogen Corp. - Licensor, primary
Description: This classic, widely-used expression
system basically combines use of a competent
(tranformable) E. coli strain with use of a pET
vector with IPTG induction of T7 polymerase.
When the T7 RNA polymerase is present and the
lac operator is not repressed, transcription pro-
ceeds rapidly. Because T7 is a viral promoter, it
transcribes rapidly and profusely for as long as the
T7 RNA polymerase is present. The pET System
provides six possible vector-host combinations
that enable tuning of basal expression levels to
optimize target gene expression. These options are
necessary because no single strategy or condition
is suitable for every target protein.
The pET vector is a bacterial plasmid designed
to enable the quick production of a large quan-
tity of any desired protein when activated. This
plasmid classically contains several important
elements - a lacI gene which codes for the lac
repressor protein; a T7 promoter which is specific
to only T7 RNA polymerase (not bacterial RNA
polymerase) and also does not occur anywhere in
the prokaryotic genom; a lac operator which can
block transcription; a polylinker; an f1 origin of
replication (so that a single-stranded plasmid can
be produced when co-infected with M13 helper
phage); an ampicillin resistance gene; and a
ColE1 origin of replication. pLysS plasmids may
code for T7 lysozyme, a natural inhibitor of T7
RNA polymerase, to prevent leaky expression.
Control of the pET expression system is ac-
complished through the lac promoter and opera-
tor. Before a desired gene can be transcribed, T7
polymerase must be present. The gene on the host
cell chromosome usually has an inducible lac
gene promoter which is activated by isopropyl--
D-thiogalactopyranoside (IPTG), which displaces
the repressor from the lac operator. Since there are
usually lac operators on both the gene encoding
T7 polymerase and gene for desired protein, IPTG
activates both genes. When IPTG is added to the
cell, the T7 polymerase is expressed, and quickly
begins to transcribe the targe gene which is then
translated.
The lac genes express enzymes which are in-
volved in the breaking down of lactose, and there-
fore, the presence of lactose (or analog) triggers
the initiation of transcription of lac genes. IPTG
an analog of lactose, displaces the lac repressor.
Prokaryotic cells do not produce T7 RNA
polymerase RNA, and therefore the T7 RNA
polymerase must be added. T7 RNA polymerase
can be introduced to the cell through methods
other than incorporation a gene in the host chro-
mosome. It can be introduced through infection of
the original host cell with lambda CE6. Usually,
the host cell for this expression system is a bacte-
ria, E. coli, which has been genetically engineered
to incorporate the gene for T7 RNA polymerase,
the lac promoter and the lac operator in its
genome. When lactose or a molecule similar to
lactose is present inside the cell, transcription
of the T7 RNA polymerase is activated.. Classi-
cally, the host cell used is competent E. coli strain
BL(DE3).
For more stringent control of expression, hosts
carrying either pLysS or pLysE are available. The
pLys plasmids encode T7 lysozyme, with pLysE
hosts producing much more enzyme, providing
the more stringent control.
Use with: E. coli and other bacteria.
Background: The pET system was developed
in 1986 by W. F. Studier and B. A. Moffatt,
Brookhaven National Laboratory, who created an
RNA polymerase expression system which was
highly selective for bacteriophage T7 RNA poly-
merase. The initial system involved two different
methods of getting T7 RNA polymerase into cells
(E. coli). In one method, a lambda bacteriophage
is used to insert the gene which codes for T7 RNA
polymerase. In the other, the gene for T7 RNA
polymerase is inserted into the host chromosome..
This expression system has become known as the
pET Expression System, and is now widely used
because of its ability to mass-produce proteins,
and the specificity involved in the T7 promoter
which only binds T7 RNA polymerase. The
system allows for the easy manipulation of how
226
much of the desired protein is expressed and when
expression occurs.
T7 RNA polymerase, the product of T7 gene,
is a protein produced early in T7 infection. It is
a single-chain enzyme with a molecular weight
close to 100,000. The selectivity of the T7 RNA
polymerase is due to the interaction of the RNA
polymerase with a relatively large promoter se-
quence, a sequence large enough that it is unlikely
to be found by chance in any unrelated DNA. In
the case of T7, the highly conserved promoter
sequence appears to consist of ~23 continuous
base pairs, which includes the start site for the
RNA chain. If exact specification of even as few
as 15 of these base pairs were required for initia-
tion of chains, chance occurrence of a functional
promoter would be expected less than once in a
billion nucleotides of DNA. The stringent speci-
ficity of T7-like RNA polymerases for their own
promoter sequences is naturally used by these
phages to direct all transcription and replication to
their own DNAs during infection. After the phage
RNA polymerase is made, other phage gene prod-
ucts inactivate the host RNA polymerase, leaving
all transcription in the cell directed by the phage
enzyme.
Benefits: The pET System is claimed to be the
most powerful system yet developed for the
cloning and expression of recombinant proteins
in E. coli....pET System has been used to express
thousands of different proteins.
T7 protein expressions systems are noted
for their reliability and adaptability. Even gene
products that are difficult to express (e.g., toxic
products) can be successfully produced using
these systems. Claims include:
Highest expression levels, tightest control over
basal expression
Choice of vectors and host strains to control
basal and induced expression levels
Precise control of induced expression with
various hosts
May be used with various N-terminal and C-
terminal fusion tags for detection, purification and
localization
Expanded multiple cloning sites in all three

reading frames
f1 origin of replication for mutagenesis and
sequencing
E. coli-based system provides rapid results
Convenient restriction sites for subcloning
from other vectors
Choice of methods for purification
Patents: This technology will likely be going
off-patent soon, if not already. The exemplary
patent including claims for use of the T7 pro-
mote is 4,952,496, Aug. 6, 1990, Cloning and
expression of the gene for bacteriophage T7
RNA polymerase, Studier, et al., assigned to
Associated Universities, Inc., the patent agent for
Brookhaven National Labs. The exemplary claim
(no. 1) broadly claims: A process for obtaining
clones of a DNA sequence encoding an enzy-
matically active RNA polymerase of any T7-like
bacteriophage, comprising the steps of: obtain-
ing from the DNA of said T7-like bacteriophage
a fragment that contains the complete coding
sequence of a gene for said bacteriophage RNA
polymerase; eliminating from the fragment, or
inactivating, any promoters that may lie to the 5
side of the 5 terminus of said coding sequence
and which could direct the synthesis by any host
cell RNA polymerase of active mRNA from the
cloned gene; eliminating from the fragment, or
inactivating, any promoters that lie to the 5 side
of the 5 terminus, to the 3 side of the 3 terminus
and between said termini of said coding sequence
and which are recognizable by said phage RNA
polymerase, but retaining within the DNA frag-
ment a sequence encoding an enzymatically active
phage RNA polymerase; and cloning said DNA
fragment containing a sequence encoding enzy-
matically active phage RNA polymerase, but not
containing functional promoters recognizable by
said phage RNA polymerase, in a plasmid or other
vector that contains no promoters recognizable
by said phage RNA polymerase, and at a site in
the vector where functional mRNA for said phage
RNA polymerase would be only minimally tran-
scribed by any host cell RNA polymerase.

227
Other BNL patents include 5,693,489 and
5,869,320.
It appears that the the primary 4,952,496 has
or will soon expire, and this system will be in the
public domain (no license needed).
Availability: Vectors, cells and other components
are available for non-commercial use from many
reagent suppliers, including Invitrogen and Strat-
egene.
Licensing information: Associated Universities,
Inc.handles licensing for Brookhaven National
Laboratory. For commercial laboratories, a re-
search use license agreement must be entered into
prior to purchase of the products incorporating
T7. Even if you determine that this invention is
in the public domain, it would still be prudent to
contact BNL, if only to obtain related biological
materials from their source (better satisfy regula-
tory agencies).
Further information: Dunn JJ, and FW Studier,
Complete nucleotide sequence of bacteriophage
T7 DNA and the locations of T7 genetic elements,
J. Mol. Biol. 166, 477-535 (1983).
350
pMAL Protein Fusion and
Purifcation System; Maltose
binding (MBP) fusion affnity
tags; pMAL plasmids - antibody
fragments; E. coli
New England Biolabs - Licensor, primary;
Retail seller
Temple University - Assignee, patent
Description: Maltose binding (MBP) fusion af-
finity tags are useful for improved protein expres-
sion and purification, including affnity purication,
of antibody fragments (e.g., as MBP-scFv fusion
proteins), with the MBP-scFv fusion proteins
exported to the bacterial periplasm where anti-
body disulfide bonds may form. The cloned gene
is inserted into a pMAL vector down-stream from
the malE gene, which encodes maltose-binding
protein (MBP). This results in the expression of
an MBP-fusion protein. The technology uses the
strong Ptac promoter and the translation initiation
signals of MBP to
express large amounts of the fusion protein. The
fusion protein is then purified by a one-step affin-
ity purification for MBP, e.g., using matrix-im-
mobilized amylose, and the MBP portion of the
fusion protein enyzmatically cleaved off.
In one version, the system uses the pMAL vec-
tors which are designed so that insertion interrupts
a lacZ gene allowing a blue-to-white screen for
inserts on X-gal. The vectors include a sequence
coding for the recognition site of a specific
protease. This allows the protein of interest to be
cleaved from MBP after purification, without add-
ing any vector-derived residues to the protein. The
polylinker includes a restriction site superimposed
on the sequence coding for the site of the specific
protease. This is where the gene of interest is
inserted.
Expression from the pMAL vectors readily
yields up to 100 mg fusion protein from a liter
of culture. In most cases, the expressed protein
is soluble, as fusion to MBP has been proven to
enhance the solubility of proteins expressed in E.
coli. The pMAL Protein Fusion and Purification
System gives substantial yields of protein in more
than 75% of the cases tested.
Use with: E. coli
Background: MBP is a natural periplasmic E.
coli protein well expressed in the bacterial cyto-
plasm where it enhances solubility of proteins that
are fused to it. MBP has also been referred to as a
chaperone.
Benefits: An advantage of cytoplasmic expres-
sion of MBP fusions is that the expression level
is much higher when compared to that of MBP
fusions that are exported to the periplasm.
5,643,758, Production and purification of a pro-
tein fused to a binding protein, Guan, et al, July
1, 1997, coassigned to New England Biolabs, Inc.
and Temple University.
228
Availability: New England Biolabs markets re-
lated reagents for non-commercial uses.
Licensing information: A license to use the
pMAL vectors for commercial purposes is avail-
able from New England Biolabs.
Licensors include Avid Bioservices, Inc., a CMO.
Products made using this tech.: This technol-
ogy is used for manufacture of Hylenex [Enhanze
SC; Cumulase; Chemophase; rHuPH20; PH-20
hyaluronidase, recombinant human] by Halozyme
Therapeutics, Inc. with marketing by Baxter
and other companies for other indications. As
described in U.S. 5,721,348, which may only par-
tially reflect large-scale manufacture, a full-length
clone for PH-20 is subcloned into an E. coli
expression vector, pMAL-c. PH-20 is expressed
as a fusion protein, the N-terminal fusion partner
being the maltose binding (MBP) protein of E.
coli. The PH-20-MPB fusion protein is found in
the cytoplasm and does not form disulfides. Using
pMAL-c carrying strains of E. coli, expression
of PH-20 is high. To purify the human PH-20 fu-
sion protein, the MBP-PH-20 fusion protein was
bound to an amylose resin (to which MBP binds)
and eluted with maltose.
Guan, C., Li, P., Riggs, P.D. and Inouye, H.
(1987) Vectors that facilitate the expression and
purification of foreign peptides in Escherichia coli
by fusion to maltose-binding protein. Gene, 67,
21-30.
351
Polyhydroxybutyrate (PHB)
fusion tags, self-cleaving
coexpressed with affnity
medium - E. coli; universal
Princeton University - Licensor, primary
Description: Self-cleaving fusion protein affinity
purificatin tags are useful for the efficient puri-
fication of recombinant proteins, with bacterial
cells producing both the affinity resin and tagged
target fusion protein. The host cells simultane-
ously produce both a polyhydroxybutyrate (PHB)
tagged affinity fusion protein along an easily
recovered granular affinity carrier with affinity for
PHB (fusion proteins). The self-cleaving capabil-
ity of the affinity tag allows the easy recovery of a
native target protein at reasonable yield and very
low cost.
The tagged product proteins are expressed
in E. coli that also produce intracellular PHB
granules, where they bind to the granules via
the PHB-binding tag. The granules and attached
proteins can then be easily recovered following
cell lysis by simple mechanical means. Once puri-
fied, the product protein is self-cleaved from the
granules and released into solution in a substan-
tially purified form. By allowing the bacterial
cells to effectively produce both the affinity resin
and tagged target protein, the cost associated with
the purification of recombinant proteins could be
greatly reduced.
This simple technique is likely to be portable
to numerous other expression hosts and useful
for large-scale production of recombinant protein
products. This system has been successfully used
at laboratory scale to purify several active test
proteins at reasonable yield. It is expected that
this combination of improved economics and sim-
plicity will constitute a significant breakthrough
in both large-scale production of purified proteins
and enzymes and high-throughput proteomic stud-
ies of peptide libraries.
This technology combines two well established
technologies. The first is the production of poly-
hydroxybutyrate (PHB) granules in engineered
strains of E. coli. The second is a recently devel-
oped group of self-cleaving affinity tags based on
protein splicing elements known as inteins. By
combining these technologies with a PHB-binding
protein, a self-contained protein expression and
purification system has been developed.
Use with: E. coli; universal
20060141570, Intein-mediated protein purifica-
tion using in vivo expression of an aggregator
229
protein, Wood, D.W., et al., 06/29/2006.
Licensing information: Contact Princeton Uni-
versity. Cite ref. no. 04-2076. The university tech.
transfer office reports, We are currently pursing a
non-exclusive licensing strategy for these tech-
nologies. Our PI involved is very involved with
the development of these technologies and is open
to collaboration both from industry and academia.
Several collaborations are ongoing.
Novel and economical purification of recombi-
nant proteins: Intein-mediated protein purification
using in vivo polyhydroxybutyrate (PHB) matrix
association, Mahmoud Reza Banki, Tillman U.
Gerngross and David W. Wood, Protein Science
(2005), 14:1387-1395.
352
Red/ET recombination; ET
cloning/ET recombination; GET
recombination; Recombineer-
ing; f Red-mediated recombi-
nation; lambda-mediated
recombination - E. coli
Novozymes Delta A/S - Licensor, primary
Gene Bridges - Licensor, secondary
European Molecular Biology Labs. (EMBL)
- R&D
Description: The Red/ET recombination sys-
tem, used instead of conventional cloning, offers
a methods for DNA manipulation (homologous
recombination) in E. coli, allowing for cloning,
precise manipulation and modification of DNA
molecules independent of the presence of restric-
tion sites and the size of the DNA molecule to be
modified. Red/ET recombination allows specific
modifications such as insertions, deletions, fu-
sions or point mutations at any chosen position
regardless of inconvenient restriction sites. It
also does not produce unwanted mutations and is
quicker than previous techniques. Consequently,
Red/ET is more efficient than traditional recombi-
nation techniques. RecE/T and/or Red.alpha./.beta
bacterial recombinases can be used to clone, sub-
clone, propagate, and amplify a polynucleotide or
mixture of polynucleotides of interest using a vec-
tor comprising short regions of DNA homologous
to sequences flanking a designated target DNA
sequence of interest and an origin of replication.
Any recipient DNA in a host cell, from high copy
plasmid to the genome, is amenable to precise
alteration.
Gene Bridges claims the technology has be-
come the method of choice for engineering the E.
coli chromosome. Red/ET recombination was
developed in Dr. Stewarts lab. at EMBL
Red/ET Recombination enables the cloning of
extremely large DNA sequences. With traditional
methods, cloning is limited to sequences of up to
about 20 kilo bases (kb); with Red/ET sequences
of well over 120 kb can be cloned. Cloning with
Red/ET Recombination is precise. It is an in vivo
process involving the endogenous proofreading
and repair mechanisms of the bacterium cell, and
avoids PCR-derived in vitro mutagenesis. The
cloning of the target DNA is a one step procedure.
A length of homology of 30-60 bp is recom-
mended.
Gene Bridges states Red/ET recombination
makes it trivial to direct changes to a chosen
DNA sequence or to introduce point mutations at
any chosen site of a target DNA molecule regard-
less of its size. Methods can be used to clone,
subclone, propagate, and amplify a polynucleotide
or mixture of polynucleotides of interest using a
vector comprising short regions of DNA homolo-
gous to sequences flanking a designated target
DNA sequence of interest and an origin of replica-
tion.
Endogenous homologous recombination sys-
tem (termed RecA-dependent recombination) in
E. coli typically has many limitations. For exam-
ple, it is impossible to use linear DNA molecules
as they become rapidly degraded. Furthermore,
recombination of circular molecules requires
relatively long homology regions and the ratio
of intended to unwanted recombination products
230
is extremely low. None of these limitations are
encountered with Red/ET recombination. Red/ET
modifies DNA by utilizing the natural process of
homologous recombination. This process has been
used before for genetic engineering, but earlier
techniques suffered from an inability to use linear
DNA molecules as they degraded too quickly.
Use with: E. coli
Background: Red/ET recombination was de-
veloped in Dr. Stewarts lab. at EMBL. Red/ET
Recombination technology (e.g. Angrand et al
1999) has been published under several names:
ET cloning/ET recombination (e.g. Muyrers et
al. 2000); GET recombination (e.g. Nefedov et
al. 2000); Recombineering (e.g. Copeland et al.
2001); and lambda-Red-mediated recombination
(e.g. Murphy and Campellone 2003).
Advantages: Claimed advantages include:
Unknown sequences can be cloned, with only
the sequence information of the homology arms
needed.
Short sequence motifes like restrictions sites,
loxP, FRT, IRES can be introduced together with
the actual DNA modification step
Cloning from 1bp up to 250 kb can be used. Sub-
cloning size dependent on origin of replication,
with subcloning of 80 kb confirmed.
6,355,412, Methods and compositions for
directed cloning and subcloning using homolo-
gous recombination, Stewart, et al., March 12,
2002, assigned to EMBL.; and 6,509,156, DNA
cloning method relying on the E. coli recE/recT
recombination system, Stewart, et al., Jan. 21,
2003, assigned to EMBL. These and related pat-
ents have been licensed to Gene Bridges.
Licensing information: Gene Bridges offers
licenses and related services, including on a fee-
for-service basis for customers who do not want
to work with the technology in-house, and also
sells Red/ET kits for non-commercial uses.
Genencor and BASF both took licenses in
April 2007. Other licensees include Sanofi-Aven-
tis, Dupont, Novartis, Cubist, Merck & Co. and
Nucleis.
353
Skp and DsbC chaperone
fusions - E. coli; secretion
control
primary
Description: This invention provides fusion poly-
nucleotides (gene fusions) in which a polynucleo-
tide encoding a desired target protein is linked to
one or more chaperone protein domains, Skp and
DsbC, with or without the signal sequence. This
permits the expressed proteins to be transported to
the periplasm or to be retained in the cytoplasm,
respectively .
These vectors have been used to successfully
express significant amounts of such difficult to
express proteins as Hif1a, Folliculin (fol), a Fol-
liculin domain (FD), Wnt5a, Endostatin, YopD,
IL13 and IFN-Hybrid3.
Use with: E. coli
Background: Production of large quantities of
soluble and correctly folded proteins is essential
for a variety of applications ranging from func-
tional analysis and structure determination to
clinical trials. E. coli is a widely used expression
system that offers the advantages of ease of han-
dling, cost-effectiveness and the ability to produce
proteins in high yield. However, the enhanced
production obtainable with E. coli expression
systems is frequently accompanied by problems
of protein insolubility, production host non-viabil-
ity and aberrant protein folding. Many strategies
have been proposed to address these problems, in
particular the use of fusion vectors that mediate
the expression of a target gene linked to a peptide
signal sequence or to a ``chaperone or ``car-
rier protein that is capable of ``escorting the
fusion protein out of the cytoplasm and into the
periplasmic space. However, there remains a need
for methods that provide soluble proteins that are
correctly folded and in functional form without
unacceptably diminishing the yield of recovered
231
protein or requiring complex host strains.
Benefits: increased expression and secretion of
properly-configured proteins
Provisional Application No. 60/564,982 filed 26
Apr 2004. Inventors are Deb K. Chatterjee and
Dominic Esposito.
Licensing information: NIH states, These fu-
sion vectors are available for licensing and are
useful tools for the expression of commercially
viable amounts of functional proteins of therapeu-
tic and scientific interest, i.e., available for most
any type of licensing. Contact NIH (ref. no. E-
103-2004/0). The technology is available for fur-
ther development through collaborative research
with the inventors via a Cooperative Research and
Development Agreement (CRADA).
354
Tac promoters - E. coli
Description: In the early 1980s, two hybrid pro-
moters that are functional in Escherichia coli were
developed. These hybrid promoters, tacI and tacII,
were derived from sequences of the trp and the lac
UV5 promoters. The tacI and the tacII promoters,
respectively, direct transcription approximately 11
and 7 times more efficiently than the derepressed
parental lac UV5 promoter and approximately 3
and 2 times more efficiently than the trp promoter
in the absence of the trp repressor. Both hybrid
promoters can be repressed by the lac repres-
sor and both can be derepressed with isopropyl
beta-D-thiogalactoside (IPTG). Thus, these hybrid
promoters are useful for the controlled expression
of foreign genes at high levels in E. coli.
In contrast to the trp and the lac UV5 promot-
ers, the tacI promoter has not only a consensus
-35 sequence but also a consensus Pribnow box
sequence. This may explain the higher efficiency
of this hybrid promoter with respect to either one
of the parental promoters.
In the first hybrid promoter (tacI), the DNA
upstream of position -20 with respect to the tran-
scriptional start site was derived from the trp pro-
moter. The DNA downstream of position -20 was
derived from the lac UV5 promoter. In the second
hybrid promoter (tacII), the DNA upstream of
position -11 at the Hpa I site within the Pribnow
box was derived from the trp promoter. The DNA
downstream of position -11 is a 46-base-pair
synthetic DNA fragment that specifies part of the
hybrid Pribnow box and the entire lac operator. It
also specifies a Shine-Dalgarno sequence flanked
by two unique restriction sites (portable Shine-
Dalgarno sequence).
Use with: E. coli
Patents: First reported in 1983, any related pat-
ents are expired.
Availability: Marketed by various vendors.
Further info.: The tac promoter: a functional
hybrid derived from the trp and lac promoters,
deBoer et al. Proc. Natl. Acad. Sci. U.S.A. 80:21-
25 {1983}.
355
Tryptophan (trp) promoters -
E. coli
Searle, G.D. - Assignee, patent
Description: Tryptophan (trp) promoters, now in
the public domain, are commonly used in E. coli.
Use with: E. coli
Benefits: familiar; royalty-free
Plasmid vectors, production anduse thereof,
Carey, et al., Sept. 14, 1982, assigned to G.D.
Searle & Co., later acquired by Monsanto, later
merged into/became Pharmacia, later acquired
and merged into Pfizer.
Availability: Any related patents are now expired.
Further info.: Tacon et al. Mol. Gen. Genet.
177:427-38, 1980.
232
356
WACKER Secretion System;
E. coli K12-based secretion
system - antibody fragments
Wacker Biotech - Licensor, primary
Description: Wackers proprietary E. coli K12-
based expression system consists of a series of
high-expression plasmids and a host strain that
can transfer periplasmic correctly folded proteins
in a native conformation across the outer mem-
brane into the culture broth. Exhibiting highly
stable performance during fermentation, the strain
is used routinely in commercial-scale fermenters.
The E. coli strain is stable during fermentation,
reducing downstream processing operations.
Wackers E. coli secretion system is a commer-
cialized and well-established technology for pro-
tein production, with Wacker using it in-house for
its contract manufacturing. This secretion technol-
ogy is successfully used for technical applications
on a 4000 L scale with yields of up to 7 g/l protein
product in the culture broth. This technology can
also be applied to biologics manufacturing and a
yield of 5 g/l of a therapeutic protein has already
been achieved. The system is a platform technol-
ogy for production of antibody fragments achiev-
ing yields of > 2 g/L.
In 2005, Wacker reported expression high
yields of secreted and active antibody fragments
from MorphoSys AG, with yields exceeded 2
grams per liter in the supernatant. This result
was remarkable since the antibody fragments are
assembled from two different sub-units and have
intramolecular disulfide bridges.
Use with: E. coli
Use to make: proteins; antibody fragments
Licensing information: Available to Wackers
CMO clients. Presumably, also available for non-
Wacker has licensed BCE E. coli expression
technology from Xoma (see related entry) to pro-
duce antibodies and antibody fragments. XOMA
receives royalties from Wacker.
357
Flavivirus vectors; Kunjin
replicon vectors - prokaryotes;
Streptomyces; prokaryotes
Replikun Biotech Pty Ltd. - Licensor, primary
Description: Vector derived from the Kunjin vi-
rus are useful for recombinant protein expression
in prokaryotes, particularly Streptomyces. Kunjin
replicons can be delivered as plasmid DNA, as
naked RNA, and as naked RNA packaged into
virus like particles (VLPs). Regardless of the
delivery mode, once in the cytoplasm, Kunjin
replicon RNA initiates self-replication leading
to production of large amounts of replicon RNA
resulting in the production of high levels of heter-
ologous protein. The vectors are derived from the
Kunjin virus, a benign RNA flavivirus endemic to
Northern Australia.
Genes for Kunjin structural proteins have been
removed from the vector, and may be replaced by
a gene of interest. The Kunjin replicon also en-
codes seven non-structural Kunjin proteins (NS1-
NS5) that guide the production of therapeutic
protein and self-replication of the replicon in vivo.
Although the Kunjin replicon retains the ability to
self-replicate inside cells, the absence of structural
genes means that Kunjin replicon transfected cells
are unable to produce infectious viral particles or
escape and infect other cells.
Use with: prokaryotes; Streptomyces
Patents: Replikun Biotech claims that the tech-
nology is protected by four patent families, which
extend product and manufacturing protections as
far as 2026.
Flavivirus expression and delivery system,
Westaway, et al., May 17, 2005, with abstract,
The present invention provides a gene expression
system comprising: a) a self-replicating expres-
sion vector of flavivirus origin which includes the
flavivirus 5 untranslated region (UTR), at least
a portion of the 5 coding region for flavivirus
233
core protein, the nucleotide sequence coding for
the flavivirus non-structural proteins, and the
complete or most of the 3-terminal sequence of
the flavivrus 3UTR, required for self-replication
of flavivirus genomic material, which vector is
adapted to receive at least a nucleotide sequence
without disrupting its replication capabilities;
and b) at least a second vector that is capable of
expressing flavivirus structural protein(s) and any
other proteins required for packaging of the self-
replicating expression vector into flavivirus viral
particles which vector is engineered to prevent
recombination with the self-replicating vector
when in its presence.
Licensing information: Contact Replikun Bio-
tech Pty Ltd.
358
Streptomyces inducers
Centro Informtico Cientfico de Andaluca
- Licensor, primary
Description: The tipA gene is useful in vectors
for transformation of Streptomyces bacteria to
promote high level protein expression upon induc-
tion with (addition of) radamicine, a tiopeptidic
antibiotic-like molecule produced by a new strain
of Streptomyces. Radamicine (radamycin) does
not have antibiotic activity (is not toxic) against
Gram positive bacteria, unlike other tiopeptide
molecules. It presents a high induction activity of
the tipA promoter without showing any antibiotic
activity. Radamycin is the first inductor of this
promoter which does not present antibiotic activ-
ity, and this is its major advantage
Use with: Streptomyces
Benefits: This avoids the addition of active an-
tibiotics to the culture medium and the presence
of antibiotic resistance genes in the host strain,
which can induce undesirable physiological modi-
fications. Another unwanted side effect antibiotic
activity that is avoided is uncontrolled distribution
of resistance genes to tiopeptide compounds with
antibiotic activity.
Patents: Applications include ES 200102609, ap-
plied for 2002-01-11.
Licensing information: Contact Centro
Informtico Cientfico de Andaluca.
359
Streptomyces lividans strains;
xysA promoters
Spanish Center on Biotechnology (CSIC) - Li-
censor, primary
Description: Streptomyces lividans bacterial host
strains with diminished extracellular proteases
and new promoters are useful for recombinant
protein expression. Streptomyces with deficiency
in extracellular protease activity permit sustanted
extracellular stability of secreted proteins, provid-
ing improved performance on an industrial scale
in the production of proteins. Plasmid vectors
contain several variants of the promoter from the
xysA gene (xylanase Xys1) of Streptomyces hal-
stedii JM8, controlling the expression of the ORFs
of interest. The expression of genes under the
control of this promoter is constant and regulated
by the carbon source present in the media. Glu-
cose represses its expression while xylan, xylose
and other carbon sources act as inducers. This
response occurs in both minimal and rich media.
The system permits high expression of the ORFs
expressed under xysA promoter control. The ex-
pression is carbon source dependent. The promot-
ers are also useful with Thermus, Actinomyces
and any other species with similar codon usage.
Use with: Streptomyces; Actinomyces; Ther-
mus
WO/2004/016730, BACTERIA THAT CAN
OVERPRODUCE AND SECRETE PROTEINS,
MELLADO, R.P., et al., assigned to CONSEJO
SUPERIOR DE INVESTIGACIONES CIENT-
FICAS, has abstract, The invention relates to
bacteria that can overproduce and secrete proteins.
234
In one form of the invention, the extracellular pro-
tease activity of the Streptomyces lividans strain
is greatly reduced and, as a result, the stability of
the proteins in the extracellular medium increases
over time.
Licensing information: Contact the Spanish
Center on Biotechnology (CSIC).
Yeasts
360
AlcoFree Yeasts;
KOY.TM6* strains
Gothia Yeast Solutions AB - Licensor, primary
Description: AlcoFree Yeast strains of Saccha-
romyces cerevisiae do not produce ethanol under
standard fermentation conditions (aerobic cultiva-
tion at high glucose concentrations). The native
hexose transporters of yeast are replaced with a
chimeric transporter that has low transport capac-
ity (the AlcoFree genotype). This shift in respi-
ratory metabolism results in increased biomass
yields; increased heterologous protein yields;
faster fed-batch fermentation times; respiratory
quotient remains approximately one throughout
batch culture on glucose and; relief from glucose
repression.
The glycolytic flux sustained by this chime-
ric transporter is sufficient to maintain respira-
tory metabolism and a high growth rate, without
substantial spillover from pyruvate into ethanolic
fermentation. Chimeric Hxt transporters control
the rate of glycolysis to a high degree. Strains
produce functional chimeras between the hexose
transporters Hxt1 and Hxt7, each of which has
distinct glucose transport characteristics. The
strains display a range of decreasing glycolytic
rates resulting in a proportional decrease in etha-
nol production.
AlcoFree Yeast 01 is derived from the CEN.PK
strain family, which is widely used by experimen-
tal yeast biotechnologists. The basis of the Alco-
Free strains unique physiology is genetic modi-
fication of its sugar uptake system. This results in
an altered control of the glycolytic pathway such
that sugar substrates are metabolized solely via
respiration under aerobic conditions, irrespective
of the external sugar concentration. The higher ef-
ficiency of respiratory metabolism of these strains
leads to an increased biomass yield and to simpli-
fied cultivation procedures. Heterologous proteins
can be manufctured at higher yield and with less
byproducts.
Saccharomyces cerevisiae KOY.TM6*P
strain was the first reported AlcoFree strain of
S. cerevisiae (a second strain has recently been
transformed), which almost completely redirects
the flux of glucose from ethanol fermentation to
respiration even at high external glucose concen-
trations/
Use with: S. cerevisiea
Background: The yeast Saccharomyces cerevi-
siae predominantly ferments glucose to ethanol
at high external glucose concentrations, irrespec-
tive of the presence of oxygen. In contrast, at low
external glucose concentrations and in the pres-
ence of oxygen, as in a glucose-limited chemostat,
no ethanol is produced. The importance of the
external glucose concentration suggests a central
role for the affinity and maximal transport rates
of yeasts glucose transporters in the control of
ethanol production.
Saccharomyces cerevisiae is a Crabtree-posi-
tive yeast. Under aerobic conditions it displays
fermentative metabolism at high growth rates and
extracellular glucose concentrations. For biotech-
nological applications that require fermentation
products (e.g. brewing and baking) this is a desir-
able trait. However, for production of biomass or
biomass components, the Crabtree effect is unde-
sirable, as it results in lower yields and waste of
substrate. Crabtree metabolism is conventionally
avoided by complex and cumbersome cultivation
strategies.
235
Patents: Exemplary pending applications include
U.S. 20040058429, Recombinant saccharomyces
cerevisiae expressing chimeric glucose transport-
ers, assigned (or exclusively licensed) to Gothia
Yeast Solutions AB.
Licensing information: Contact Gothia Yeast
Solutions AB.
361
ALEU2 marker; AHSB4
promoter; Arxula adeninivorans
expression
mary
Institut fur Pflanzengenetik und Kulturpflan-
zenforschung - Assignee, patent
Description: Vectors, the ALEU2 gene and
AHSB4 promoter are useful in Arxula adenini-
vorans yeast-based expression systems. The
ALEU2 gene, encoding beta-isopropylmalate
dehydrogenase, is used as a selectable marker.
The constitutive AHSB4 promoter gene, encoding
histone H4, is strongly and constitutively ex-
pressed, maintaining this expression profile under
salt-stress conditions, and provides an attractive
component for constitutive heterologous gene ex-
pression under salt-free and salt-stress conditions.
This technology includes stable integration of
recombinant expression vectors and/or cassettes in
desired copy number. A variety of selection mark-
ers is available designed for low or high copy
integration. This feature together with different
promoters established for recombinant gene ex-
pression forms the basis for an optimized expres-
sion level. Expression cassettes can be designed
and applied using no any bacterial sequences.
Use with: Arxula adeninivorans
Patents; WO/2008/052797, EXPRESSION VEC-
TORS FOR MULTIPLE GENE INTEGRATION
AND OVEREXPRESSION OF HOMOLO-
GOUS AND HETEROLOGOUS PROTEINS IN
YEASTS OF THE GENUS ARXULA, KUNZE,
G., assigned to Institut fur Pflanzengenetik und
Kulturpflanzenforschung, has an abstract stating,
The present invention relates to expression vec-
tors which can be utilized in yeasts of the genus
Arxula, especially in the yeast Arxula adeniniv-
orans, for multiple, chromosomal gene integration
and, as a result, for higher production of heterolo-
gous proteins. The invention additionally relates
to methods for expressing heterologous proteins,
methods for producing host cells of the genus
Arxula, and kits, host cells and pharmaceutical
compositions.
Licensing information: Contact Artes Biotech.
regarding this and other Arxula technology, and
related CRO/CMO services. Artes has licensed
Arxula technolgies from Institut fur Pflanzenge-
netik und Kulturpflanzenforschung.
Products made using this tech.: Basic proof-of-
principle for recombinant protein production has
been achieved, including by expresion of a scFv
antibody fragment and Interleukin-6 (IL-6).
362
Aspergillus niger expression;
A. niger A4 promoters -
humanized antibodies
Genencor International - Assignee, patent
Danisco A/S - Parent co.
Description: Genencor has reported expression of
multiple humanized antibodies in the fungus As-
pergillus niger, and has received a U.S. patent for
A. niger promoters. As reported in Characteriza-
tion of Humanized Antibodies Secreted by Asper-
gillus niger, Appl Environ Microbiol. 2004 May;
70(5): 2567-2576, both light and heavy chains
were initially synthesized as fusion proteins with
native glucoamylase. After antibody assembly,
cleavage by A. niger KexB protease allowed the
release of free antibody. Purification by hydro-
phobic charge induction chromatography proved
effective at removing any antibody to which
glucoamylase remained attached. No significant
difference between the glycosylated antibody
236
produced by Aspergillus and that produced by
mammalian cell cultures was observed in tests for
affinity, avidity, pharmacokinetics, or antibody-
dependent cellular cytotoxicity function.
Use with: Aspergillus niger
ies
Aspergillus niger promoter for expressing genes
in host cells, Kim, S., et al., May 27, 2008, as-
signed to Genencor, concerning the Aspergillus
niger A4 long promoter (A4-L promoter), Asper-
gillus niger A4 promoter (SEQ ID NO: 2) and the
Aspergillus niger A4-5 promoter.
Licensing information: Contact Genencor.
363
Aureobasidin A vectors (pAUR)
- selectable marker in yeasts
Description: Aureobasidin A vectors (pAUR)
include six E. coli-yeast shuttle vectors, each
constructed for a particular application in either S.
cercevisiae, S. pombe or Aspergillis nidulans. The
vectors include a novel drug-resistance selective
marker that confers Aureobasidin A resistance in
transformed yeast species.
Aureobasidin A is a cyclic depsipeptide anti-
biotic with a molecular weight of approximately
1,100 Da. It is isolated from Aureobasidium pul-
lulans R106. Aureobasidin A is toxic to fungi and
yeast at low concentrations, including Saccharo-
myces cerevisiae, Schizosaccharomyces pombe
and Candida albica
Use with: yeasts
6,022,949, Fungal aureobasidin sensitivity gene
products, Okado, et al., Feb. 8, 2000, assigned
to Takara Shuzo Co., Ltd. The abstract states, A
gene associated with sensitivity to the antimycotic
agent, aureobasidin A, has been isolated. The
gene can be detected in a variety of cell types,
and variant forms of the gene have been identified
in mutant yeast strains. The proteins encoded by
these genes are useful in diagnosing and treating
mycoses. Also see U.S. 6,348,577, Regulation
of aureobasidin sensitivity in fungus, Okado, et
al., Feb. 19, 2002,
Availability: Reagents available for non-commer-
cial use from Takara Shuzo Co., Ltd.
Licensing information: Contact Takara Shuzo
Co., Ltd. regarding commercial use and licensing.
Further info.:
1. Takesako, K. et al. (1993) J. Antibiot. 46:1414-
20.
2. Nagiec, M.M. et al. (1995) J. Biol. Chem.
272:9809-17.
364
Calnexin chaperone -
Hansenula polymorpha; yeasts
mary
Rhein Biotech Ltd. - Assignee, patent
Description: The introduction of additional gene-
copies of yeast calnexin into a yeast cell line can
further increase yields from otherwise optimized
yeast cell lines. Two- to five fold boosts of pro-
ductivity have been reported with proteins already
secreted at g/L levels. With the integration of high
copy numbers of multiple expression cassettes in
one cell, Hansenula is an ideal host for this com-
binatory optimization.
approach.
Use with: yeasts; Hansenula
Background: Yeast calnexin represents the coun-
terpart to the mammalian calreticulin/calnexin
chaperon system that plays a major role in quality
control and folding assistance of newly synthe-
sised glycoproteins passing the ER. Publications
describe yeast calnexin to act on a much broader
237
subset of substrates being finally assembled in the
secretory pathway. Differentiating the gene doses
of Calnexin in yeasts may represent a core adjust-
ing screw to improve the capability of host cells
to produce foreign proteins.
EP1666602, Method for production of a het-
erologous protein using yeast type host cells,:
June 7, 2006, Gellisen, et al., assigned to ARTES
Biotechnology GmbH and Rhein Biotech GmbH,
now part of Dynavax. The abstract states, Prepa-
ration of a heterologous protein (I) comprises
preparing yeast-type host cells, which in addition
to their endogenous, homologous DNA sequences
encoding a first calnexin, also comprise additional
recombinant DNA sequence encoding (I), and ad-
ditional recombinant DNA sequence encoding at
least one second calnexin; inducing the host cells
to express (I) and to overexpress calnexin; induc-
ing the host cells to secrete (I); and isolating the
secreted (I). Preparation of a protein comprises :
(a) preparing yeast-type host cells, which in ad-
dition to their one or more endogenous, homolo-
gous DNA sequences encoding a first calnexin,
also comprise at least one additional recombinant
DNA sequence encoding the protein, and at least
one additional recombinant DNA sequence encod-
ing at least one second calnexin, and where the
protein is a heterologous secretable protein; (b)
inducing the host cells to express the heterologous
protein using the sequence encoding the protein
and to overexpress calnexin using the sequence
encoding the second calnexin; (c) inducing the
host cell to secrete the heterologous protein; and
(d) isolating the secreted heterologous protein.
Licensing information: Contact Artes Biotech.
Increase of calnexin gene dosage boosts the
secretion of heterologous proteins by Hansenula
polymorpha, Klabunde, J., et al., FEMS Yeast
Res. 2007 October; 7(7): 1168-1180 (Pub-
lished online 2007 July 6. doi: 10.1111/j.1567-
1364.2007.00271.x).
365
Chitin synthase (CHS1), Yeast
growth factor, chitin synthase
(CHS1) - yeast promoter
discovery
Millennium Pharmaceuticals, Inc. - Licensor,
primary
Description: Chitin synthase (CHS1) is an en-
zyme essential for cell wall synthesis and yeast
cell growth. A maltose responsive promoter
(MRP) with CHS1 provides vectors useful for
isolation of eukaryotic regulatory polynucleotides,
i.e., promoters. The vectors are useful to identiy
promoter regions by complementing the growth
of an auxotrophic host cell containing the vector,
which includes a promoter region operably linked
to a promoterless auxotrophic gene. The vector
is introduced into the host cell chromosome by
targeted integration.
Use with: eukaryotes; yeasts
Identification of eukaryotic growth-related genes
and promoter isolation vector and method of use,
Koltin, et al., Sept. 18, 2001, assigned to Millen-
nium Pharmaceuticals, Inc.
Licensing information: Contact Millennium
Pharmaceuticals, Inc.
366
Estradiol-dependent enhancer;
Gal4-ER-VP16 - yeasts
Description: An improved system using a Gal4-
ER-VP16 chimeric transactivator (promoter) with
estradiol-dependent regulation of gene expression
in yeasts allows a 250-fold induction of the regu-
lated gene, without increasing the basal activity of
the target promoter, and achieves a 12-fold higher
regulation efficiency than a previous configura-
238
tion. Enhanced regulation of recombinant genes is
driven by the GAL1 promoter. The basal expres-
sion level of this system is as low as that of native
GAL-driven genes in glucose-containing media.
Added regulatory elements allowing a simul-
taneous control of both the abundance and the
intrinsic activity of the Gal4-ER-VP16 chimeric
transactivator.
The tightly regulated yeast GAL1-10 promoter
is commonly used. However, induction of the
GAL system requires the presence of the rather
expensive inducer galactose and the absence of
glucose in the culture media. An alternative to
regulate transcription driven by GAL promoters,
free of general metabolic changes, is the incorpo-
ration of the hybrid Gal4-ER-VP16 protein. This
chimeric protein provides galactose-independent
activation of transcription from GAL promoters
in response to estradiol, even in the presence of
glucose.
yeast
Use with: yeasts
Patents: No published applications assigned to
Biomedal were retrieved from simple search-
es. A potentially related application is U.S.
20080034445, Ligand-regulable transactivation
systems, methods of use thereof, methods of de-
tecting estrogen receptor ligands, and methods of
differentiating estrogen receptor ligand agonists
and antagonists, Gambhir, S.S., affiliated with
Stanford University Medical Center, no assignee
reported on the published application, with claim
1, A ligand-regulable transactivation system
comprising: a reporter polynucleotide that in-
cludes a binding sequence, a promoter sequence,
and a reporter sequence, wherein the binding
sequence is connected with the promoter sequence
and the promoter sequence is connected with the
reporter sequence; and an activator fusion protein
that includes a DNA binding domain, an estrogen
receptor folding domain, and a transactivation
domain, wherein the DNA binding domain is con-
nected to the estrogen receptor folding domain,
and the estrogen receptor folding domain is con-
nected with the transactivation domain.
Further info.:
An improved system for estradiol-dependent
regulation of gene expression in yeast, Quintero,
M.J., et al., Microb Cell Fact. 2007; 6: 10. (Pub-
lished online 2007 March 20. doi: 10.1186/1475-
2859-6-10).
367
Formaldehyde dehydrogenase
(FLD1) promoter; Formaldehyde
selection - Pichia pastoris;
yeasts
- Licensor, primary
Description: A promoter for increased expresion
of formaldehyde dehydrogenase (FLD1) is useful
for increased protein expression in Pichia pastoris
(see related entry for the system from RCT). FLD
genes confer resistance to formaldehyde and are
therefore useful as a selectable marker in methy-
lotrophic yeasts. The FLD promoter sequences
are strongly and independently induced by either
methanol as sole carbon source (with ammonium
sulfate as nitrogen source) or methylamine as sole
nitrogen source (with glucose as carbon source).
Induction under either methanol, methylamine or
both provides levels of heterologous gene ex-
pression comparable to those obtained with the
commonly used alcohol oxidase I gene promoter
(P.sub.AOX1).
The FLD promoter of Pichia pastoris (P.sub.
FLD1) is an attractive alternative to P.sub.AOX1
(see AOX1 entry) for expression of foreign
genes in Pichia pastoris and other yeasts, allow-
ing regulation by carbon (methanol) or nitrogen
(methylamine) source within the same expression
strain. Yeast strains, expression cassettes, expres-
sion vectors, and host cells comprising an FLD
gene promoter and 3 termination sequence are
also provided. The primary role of FLD in me-
thylamine metabolism appears to be for protecting
239
cells from the toxic effects of formaldehyde and
not for generating carbon or energy.
Use with: Pichia pastoris; yeasts
6,730,499, A strong nitrogen source-regulated
promoter for controlled expression of foreign
genes in the yeast Pichia pastoris, Cregg, as-
signed to Research Corporation Technologies, Inc.
(RCT)..
Licensing information: Contact RCT.
368
GAPFL promoter;
Glyceraldehyde-3-phosphate
dehydrogenase-derived
promoter - yeasts
Swiss Federal Institute of Technology (ETH),
Zurich - R&D
Description: The GAPFL, apparently developed
by Ciba AG (now Novartis AG) is used with Sac-
charomyces cerevisiae for commercial manufac-
ture of Desirudin, a recombinant Hirudo medici-
nalis (leach)-derived protein for prophylaxis of
deep vein thrombosis.
Use with: yeasts; S. cerevisiae
Patents: No patents were retrieved from several
simple searches.
Licensing information: Contact Novartis AG
Products made using this tech.: This promoter
is used in the manufacture of Iprivask [Desirudin
- Revasc; desulfatohirudin, recombinant; hirudin
(Hirudo medicinalis isoform HV1), 63-desulfo-]
by Novartis AG for marketing by Sanofi Aventis
S.A.
As reported in Biopharmaceutical Products in
the U.S. and European Markets, Desirudin is
produced by S. cerevisiae strain 1454 transformed
with plasmid pDP34/GAPFL-YHIR. The inserted
coding sequence for desirudin was synthesized
chemically using the known amino acid sequence
of hirudin variant I (HVI) from the leech Hirudo
medicinalis along with the signal sequence of
yeast acid phosphatase (PH05). The expression
of desirudin by yeast is controlled by the GAGPL
promoter ligated to the coding sequence for the
PH05 signal sequence. The resulting 1.1. kb
expression cassette was cloned into the high copy
number yeast shuttle vector pDP34. The signal
sequence targets the expressed desirudin protein
to the S. cerevisiae endoplasmic reticulum, where
a peptidase enzyme cleaves the signal sequence,
and desirudin is secreted into the culture me-
dium.
Further info.: Mode of cultivation is critical for
the optimal expression of recombinant Hirudin
by Saccharomyces cerevisiae, Biotechnology
Letters, Volume 15, Number 7 / July, 1993, with
authors from Ciba AG and the Swiss Federal
Institute of Technology (ETH), Zurich.
369
Gene Design, algorithmic -
yeasts
GENEART AG - Licensor, primary
Description: Gene Design uses sophisticated,
empirically proven algorithms to increase expres-
sion and genetic stability in different expression
systems by codon adaptation and increasing RNA
stability. The technology has particularly been ap-
plied to yeast expression, including, Saccharomy-
ces cerevisiae, Hansenula polymorpha and Pichia
pastoris. Expression studies directly comparing
performance of natural and optimized human
genes revealed up to 100-fold increase. In con-
trast, natural genes are often hard to obtain and/or
difficult to express due to codon usage differences
between host and source; and cis-acting elements,
such as splice site,s may go unnoticed in screen-
ing systems, but may demolish expression down-
stream.
240
Gene Design provides genes that are ideally
adapted to the genetic rules of specific yeast
strains. This includes codon optimization; GC co-
nent adaptation; inhibition of internal splicing and
premature polyadenylation; prevention of creation
of stable RNA secondary structures; avoidance
of direct DNA repeats and thereby recombination
events; and finding the most efficient signal pep-
tide/gene combination. In many cases, a dramatic
increase in protein production has been reported,
which regularly exceeded wildtype driven gene
expression by a factor of 2 to 3 and in some cases
even by a factor of 10.
Use with: yeasts; Pichia; Saccharomyces cere-
visiae, Hansenula polymorpha; Pichia pastoris
Patents: WO 2004/059556, METHOD AND
DEVICE FOR OPTIMIZING A NUCLEOTIDE
SEQUENCE FOR THE PURPOSE OF EX-
PRESSION OF A PROTEIN, RAAB, D., et al.,
describes the method of optimising the nucleotide
sequence for expression of a protein.
Licensing information: Related services are
available from Geneart AG.
370
Hypermutable yeast
Morphotek Inc. - Licensor, primary
Eisai Co., Ltd. - Parent co.
Johns Hopkins University (JHU) - R&D
Description: Hypermutable yeast can be made
by altering the activity of endogenous mismatch
repair activity of host cells. Dominant negative
alleles of mismatch repair genes, when introduced
and expressed in yeast, increase the rate of spon-
taneous mutations by reducing the effectiveness
of endogenous mismatch repair-mediated DNA
repair activity, thereby rendering the yeast highly
susceptible to genetic alterations, i.e., hypermu-
table. Hypermutable yeast can then be utilized to
screen for mutations in a gene or a set of genes in
variant siblings that exhibit an output trait(s) not
found in the wild-type cells.
Use with: yeasts
6,656,736, Methods for generating hypermutable
yeast Nicolaides, et al., Dec. 2, 2003, coassigned
to the Johns Hopkins University and Morphotek,
Inc.
Licensing information: Contact Morphotek, Inc.
and/or Johns Hopkins University.
371
PH05 promoter; Phosphate
induction -yeasts
Novartis AG - Licensed/used for manuf. of:
Description: The PH05 yeast promoter used as
an expression control sequence can be repressed
or derepressed solely by increasing or decreasing
the concentration of inorganic phosphate in the
medium.
Use with: yeast; Saccharomyces cerevisiae
Licensing information: Reported over 20 years
ago by investigators with Ciba AG, now Novartis
AG. Now in the public domain.
Products made using this tech.: This promoter
is used in the manufacture of Iprivask [Desirudin
- Revasc; desulfatohirudin, recombinant; hirudin
(Hirudo medicinalis isoform HV1), 63-desulfo-]
by Novartis AG for marketing by Sanofi Aventis
S.A.
As reported in Biopharmaceutical Products in
the U.S. and European Markets, Desirudin is
produced by S. cerevisiae strain 1454 transformed
with plasmid pDP34/GAPFL-YHIR. The inserted
coding sequence for desirudin was synthesized
chemically using the known amino acid sequence
of hirudin variant I (HVI) from the leech Hirudo
medicinalis along with the signal sequence of
yeast acid phosphatase (PH05). The expression
of desirudin by yeast is controlled by the GAGPL
promoter ligated to the coding sequence for the
PH05 signal sequence. The resulting 1.1. kb
expression cassette was cloned into the high copy
241
number yeast shuttle vector pDP34. The signal
sequence targets the expressed desirudin protein
to the S. cerevisiae endoplasmic reticulum, where
a peptidase enzyme cleaves the signal sequence,
and desirudin is secreted into the culture me-
dium.
Further info.: The yeast PH05 promoter: Phos-
phate-control elements and sequences mediating
mRNA start-site selection, Proc. Natl. Acad. Sci.
USA, Vol. 84, pp. 1340-1344, March 1987.
372
Vesicular fusion factor 2
protein (Vff2p) enhancer -
yeast; bacteria; CHO cells
Actis Biologics Inc - Licensor, primary
Salk Institute for Biological Studies - R&D, of
tech.
Description: Vectors containing vesicular fusion
factor 2 protein (Vff2p) sequences along with
an expression cassette can increase the protein-
producing, particularly the secretion, capacity
of yeast, bacteria and CHO cells by eight times.
This protein (seemingly a chaperone) is useful for
increasing cell mass and secretion of expressed
proteins in yeast. Vff2p is useful in Saccharo-
myces cerevisiae, Schizosaccharomyces pombe,
Yarrowia lipolytica, Pichia pastoris, Hansenula
polymorpha, or Kluyveromyces lactis.
Use with: yeast; bacteria; CHO cells
Sequence and method for increasing protein
expression in cellular expression systems, Lat-
terich, et al., May 10, 2005, assigned to the Salk
Institute.
Licensing information: Contact Actis. Dr Martin
Latterich and Dr Kendall Powell developed VFF2
in 1999 at Salk Institute and the technology was
excusively licensed to Actis in 2005.
373
Vesicular fusion factor 2
protein (Vff2p) enhancer -
yeasts
Actis Biologics Inc - Licensor, primary
Salk Institute for Biological Studies - R&D
Description: Vectors containing vesicular fusion
factor 2 protein (Vff2p) sequences along with
an expression cassette can increase the protein-
producing, particularly the secretion, capacity
of yeast, bacteria and CHO cells by eight times.
This protein (seemingly a chaperone) is useful for
increasing cell mass and secretion of expressed
proteins in yeast. Vff2p is particularly useful in
Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Yarrowia lipolytica, Pichia pastoris, Han-
senula polymorpha, or Kluyveromyces lactis.
Use with: yeasts; bacteria; CHO cells
Sequence and method for increasing protein
expression in cellular expression systems, Lat-
terich, et al., May 10, 2005, assigned to the Salk
Institute.
Licensing information: Contact Actis Biolog-
ics. Dr Martin Latterich and Dr Kendall Powell
developed VFF2 in 1999 at Salk Institute and the
technology was excusively licensed to Actis in
2005.
374
Xplor Vector System, yeast
optimization
mary
Institute of Plant Genetics and Crop Plant Re-
search (IPK) - R&D, of tech
Description: The Xplor Vector System is claimed
to be the ideal tool to allow for an initial selec-
tion of the optimal host from available yeast
platforms. With quantitative questions addressed
242
at a later stage - applying strong homologous
and inducible promoter elements - expression
from the Xplor Vector System addresses relevant
qualitative aspects, such as processing, stability
and post-translational modification. The Xplor
Vector System is composed of a fixed backbone
for propagation in E. coli and individual modules
for integration into the yeasts rDNA; selection of
integrants (auxotrophic and dominant markers);
transient or permanent autonomous replication;
and recombinant gene expression (constitutive,
wide range promoter and terminator). From the
resulting vector portfolio, expression cassettes can
be excised for single or multi-copy integration.
The expression cassettes can furthermore be de-
signed in such a way that the generated integrants
are void of any bacterial sequences. These expres-
sion cassettes are stably integrated into the rDNA
of yeast genomes.
This approach for comparative expression
from a single vector source is designed for virtu-
ally any yeast platform and has been verified for
Hansenula polymorpha; Arxula adeninivorans
(budding and filamentous cells); Saccharomyces
cerevisiae; Pichia pastoris; Yarrowia lipolytica;
and Debaryomyces polymorphus and hansenii.
Further yeast expression systems to be included
are Kluyveromyces lactis, Candida boidinii,
Pichia stipitis and Schwanniomyces occidentalis.
Especially for the yeasts P. stipitis, S occidentalis
and S.cerevisiae, a general proof-of-principle for
an autonomous wide range vector has already
been successfully applied.
Use with: yeasts; Hansenula polymorpha;
Arxula adeninivorans; Saccharomyces cerevi-
siae; Pichia pastoris; Yarrowia lipolytica; and
Debaryomyces polymorphus and hansenii
Licensing information: Primarily offered as a
service to CRO/CMO clients, but may be avail-
able for out-licensing.
375
Antibody fragment expression
- Saccharomyces cerevisiae
Delta Biotechnology Ltd. - Licensor, primary
Novozymes Delta A/S - Parent co.
Description: Novozymes has developed propri-
etary Saccharomyces cerevisiae based technol-
ogy for the cost effective production of fully
functional antibody fragments. See als the Sac-
charomyces cerevisiae expression entry also from
Novozymes Delta. The company claims, Unsur-
passed expertise in the production of yeast based
recombinant proteins has enabled us to engineer
a scalable technology which is a superior alterna-
tive to other microbial systems; with this unique
and versatile expression technology delivering
cost effective, fully functional antibody fragments
with proven half-life extension. See the compa-
nies other technologies, including Saccharomyces
cerevisiae disintegration vectors.
Use to make: glycopoteins; antibody fragments
Benefits: The expression system delivers:
Animal-free manufacturing processes
Fragment yield in excess of 5g/L
Stably folded proteins with retained antigen bind-
ing specificity
Secreted fragments eliminating refolding hurdles
Proven scalability from 10L to 8000L
Extremely competitive cost of production
A safer process avoiding the use of hazardous
solvents
Licensing information: Contact Novozymes
Delta A/S, which offers this technology to its
CMO clients and, perhaps, makes it available for
out-licensing.
243
376
Saccharomyces cerevisiae,
cold induction
National Institute of Advanced Industrial Sci-
ence and Technology (AIST) - Licensor, primary
Description: AIST has briefly reported develop-
ing Saccharomyces cerevisiae that only express
desired proteins at low temperature. In the expres-
sion system, target proteins can be produced by
lowering culture media temperature. A produc-
tion yield of a protein in the expression system
was higher than that in the existing expression
systems in yeast at moderate temperature. Several
proteins that were expressed as insoluble forms in
Escherichia coli could be produced in functional
and soluble forms in this system. This expres-
sion system is expected to greatly contribute to
proteomics study and large-scale production of
pharmaceutical and industrial proteins.
WO/2004/003197, YEAST-ORIGIN PRO-
MOTER AND VECTOR AND EXPRESSION
SYSTEM USING THE SAME, Jan. 8, 2004,
SAHARA, T., et al., asigned to National Institute
of Advanced Industrial Science and Technology
(AIST), with abstract, A DNA fragment having
a cold-inducible promoter activity of a yeast and
showing an elevated activity in a low temperature
region is provided by identifying a DNA fragment
which occurs in the nontranslational region in
the 5-upstream side of a gene selected from the
group consisting of cold-inducible genes of Sac-
charomyces cerevisiae and has the function of a
cold-inducible promoter.
Licensing information: Contact AIST.
377
Secretion Enhancer Vector
System (SEVS); SecHancer
vector - Saccharomyces
cerevisiae
Protheon Inc. - Licensor, primary
Description: The Secretion Enhancer Vector Sys-
tem (SEVS), including the SecHancer vector with
a secretion enhancer peptide derived from the hu-
man interleukin 1, is useful for expression of pro-
teins in eukaryotic hosts, particuarly S. cerevisiae.
SecHancer enhances secretion in active conforma-
tion, with folding and precessing on exit from the
cell. SEVS overcomes the inherent low levels of
protein expression in S. cerevisiae, and has been
successfully applied for high level production in
S. cerevisiae, including human growth hormone
(hGH), human Granulocyte Colony Stimulating
factor (hG-CSF), Hepatitis B Surface Antigens,
and site-specific protease enterokinase. SecHan-
cer continues to expand its utility to production
of cytokines, chemokines, hormones, and viral
antigens for recombinant vaccines.
See also the related Profuse system primarly
used with E. coli.
Licensing information: Contact Protheon Inc.
Further info.:
N-Glycosylation of secretion enhancer peptide as
influencing factor for the secretion of target pro-
teins from Saccharomyces cerevisiae, Biochemi-
cal and Biophysical Research Communications,
Volume 337, Issue 2, 18 November 2005, Pages
557-562.
Recombinant enterokinase light chain with affin-
ity tag: Expression from Saccharomyces cerevi-
siae and its utilities in fusion protein technology,
Biotechnology and Bioengineering, Volume 75
Issue 6, Pages 718-724.
244
378
Mab Xpress Antibody
Production System; Pichia
pastoris - glycosylated
antibodies
Alder Biopharmaceuticals Inc. - Licensor,
primary
Keck Graduate Institute - Assignee, patent
Description: Building upon the established Pichia
pastoris yeast expression system (see related
entry), Alder has developed technology for Pichia
expression of full-length glycosylated antibodies.
The technology can also be used for the discov-
ery of novel antibodies. Commercial expression
strains can be developed in just 5 weeks. Mab
production ramp times are reported to be: 1 day
- seed to 1 L; 2 days - 1-10 L; 3-5 days - 10-100
L; and 5-14 daus - 100-1000 L.
Use to make: antibodies; glycoproteins
Benefits: Yeast is an inexpensive raw material.
Alder claims it can produce antibodies for $20 to
$40 a gram, roughly one-tenth of the $300 a gram
for the mammalian cell-based methods. Yeast
cells naturally divide much more quickly than
cells from mammals, so Alder believes it can run
commercial-sized tanks to manufcture antibodies
in three to five days, instead of three to five weeks
for mammalian expression systems. Besides being
cheaper and faster to make, Alders antibodies
may be better because one can select for those
that bind more tightly with target immunogens.
Claimed benefits the Mab Xpress Antibody
Production System include:
Generates full-length function modifying anti-
bodies at high expression levels much faster than
traditional mammalian systems
Employs a safe and reliable yeast system with
a proven record in man
Requires minimal media for improved purifi-
cation and reduced cost
Allows accelerated development cycles with-
out cell banking, viral testing, or extensive master
cell bank generation

Uses readily available microbial manufactur-
ing capacity for volume production
Reduces traditional antibody production royal-
ties
Scales rapidly up to 50,000 Liters
Employs technology that is easily transferable
to partner facilities
Patents: Dr. J Cregg, Keck Graduate Inst., is
a key inventor of this technology, and also the
primary inventor for Pichia technology avail-
able for RCT. Related applications include U.S.
20060270045, Methods of synthesizing hetero-
multimeric polypeptides in yeast using a haploid
mating strategy, Cregg, et al., coassigned to Keck
Graduate Institute and Alder Biopharmaceuticals,
Inc. The abstract states, Methods are provided
for the synthesis and secretion of recombinant
proteins preferably large mammalian proteins or
hetero-multimeric proteins at high levels and for
prolonged time in polyploid, preferably diploid
yeast. These methods use various mating compe-
tent yeast, including Pichia. In a preferred em-
bodiment, a first expression vector is transformed
into a first haploid cell; and a second expression
vector is transformed into a second haploid cell.
The transformed haploid cells, each individually
synthesizing a non-identical polypeptide, are iden-
tified and then genetically crossed or fused. The
resulting diploid strains are utilized to produce
and secrete fully assembled and biologically func-
tional hetero-multimeric protein.
Licensing information: Contact Alder regarding
commercial use and licensing. Various companies
have licensed the system, including Schering-
Plough Corp. and Genmab A/S. Alder is work-
ing on up to 10 antibody products for Scher-
ing-Plough. Other licensees evaluating or using
the system include Seattle Genetics. Alder has
reported contacting many companies seeking to
license its system, with many apparently waiting
for results fromt its early clinical trials before con-
sidering taking a license.
Products made using this tech.: An antibody,
ALD518, developed by Alder has shown promise
in Phase I clinical testing.

245
379
Pichia expression, rhamnose
induction
MorphoSys - Assignee, patent
Description: A well-developed strong rhamnose-
inducible Pichia-based expression system using
rhaBAD promoters provides yield of protein at
levels up to 6 g/L in Pichia yeast.
EP1824980 (and WO/2006/061174 ), RHAM-
NOSE PROMOTER EXPRESSION SYSTEM.
Brass, J., et al., Sept. 29, 2007, coassigned to
Lonza AG and MorphoSys AG. The abstract
states, The present invention relates to new vec-
tors expressible in a host comprising the rhaBAD
promoter region of the L-rhamnose operon oper-
ably linked to a transcriptional unit comprising a)
a nucleic acid sequence which is heterologous to
said host b) a prokaryotic signal sequence oper-
ably linked to said nucleic acid sequence, whereas
said prokaryotic signal sequence is selected from
signal peptides of periplasmatic binding pro-
teins for sugars, amino acids, vitamins and ions
and, whereas the expression of said nucleic acid
sequence is controlled by said promoter region.
Also disclosed are: the use of said new vector for
the regulated heterologous expression of a nucleic
acid sequence in a prokaryotic host; an isolated
and purified nucleic acid sequence expressible
in a host comprising the promoter region of the
L-rhamnose operon; and a method for producing a
polypeptide in a host using said vector.
Licensing information: Lonza presumably offers
this to its CMO clients. For out-licensing inter-
ests, contact Lonza and/or MorphoSys.
380
Pichia GlycoSwitch System;
Glycoswitch plamids - yeasts;
glycosylation
- Licensor, primary
Flanders Interuniversity Institute for Biotech-
nology (VIB) - R&D
Ghent University - R&D
Description: The glycosylation of the Pichia
Expression System (Pichia pastoris; see related
entry) can be modified using Pichia GlycoSwitch
to produce uniform, small ASN-linked glycans
on any glycoprotein of interest. This also avoids
hyperglycosylation. Glycosylation enzyme genes
have been inserted into Pichia to create strains
that glycosylate proteins much the same as mam-
malian cells. The resulting glycans are pre-
dominantly GlycoSwitch M5 (Man5GlcNAc2),
GlycoSwitch M5GN (GlcNacMan5GlcNAc2) or
GlycoSwitch M8 (Man8GlcNAc2). These glycans
have the same structure as processing intermedi-
ates of the mammalian N-glycosylation pathway.
Use with: Pichia (yeast)
Use to make: glycoproteins
Patents: Exemplary application include U.S.
080009037, Protein glycosylation modification
in methylotrophic yeast, Contreras, R., et al.,
Jan. 10, 2008, assigned to Flanders Interuniversity
Institute For Biotechnology.
Licensing information: This is available to
Pichia Expression System licensees (see related
entry).
381
Pichia pastoris antibody
expression
Description: Fab, Fv, scFV, diabody and other re-
combinant antibody fragments (not fully-formed,
2-chain antibodies) may be expressed in large-
246
scale in transformed yeasts, particularly Pichia
pastoris (see the related entry concerning technol-
ogy available from RCT).
Use with: yeasts; Pichia pastoris
Use to make: antibody fragments
Patents: The technology description provided
by the U. of California, ref. 1999-101, does not
include inventor or other specific information al-
lowing identification of relevant patents.
California (ashwant.vaishnav@ucop.edu). Users
will still likely have to license Pichia technology
from RCT (see related entry).
382
Pichia pastoris AOX1
promoters
- Licensor, primary
Phillips Petroleum - R&D
Description: This concerns original AOX1 pro-
moters useful for recombinant protein expression
in Pichia, e.g., the Pichia pastoris expression sys-
tem available from RCT (see related entry). The
Alcohol Oxidase 1 promoter (AOX1) from Pichia
pastoris is recognized as among the most power-
ful and tightly regulated promoters known.
In the Pichia system, most foreign genes are
expressed under the transcriptional control of
the P. pastoris alcohol oxidase 1 gene promoter
(P.sub.AOX1). The promoter is tightly repressed
during growth of the yeast on most common car-
bon sources, such as glucose, glycerol, or ethanol,
but is highly induced during growth on metha-
nol. For production of foreign proteins, P.sub.
AOX1-controlled expression strains are initially
grown on a repressing carbon source to generate
biomass and then shifted to methanol as the sole
carbon and energy source to induce expression
of the foreign gene. One advantage of the P.sub.
AOX1 regulatory system is that P. pastoris strains
transformed with foreign genes whose expression
products are toxic to the cells can be maintained
by growing under repressing conditions.
nal antibodies
Background: The methylotrophic yeast Pichia
pastoris has two alcohol oxidase genes, AOX1
and AOX2. The AOX2 gene is transcribed at a
much lower level than the AOX1 gene. Apart
from this difference in expression levels, the two
genes are regulated similarly. Methanol-utiliz-
ing yeasts use methanol as a carbon and energy
source. The methanol utilization pathway begins
with the oxidation of methanol to formaldehyde.
That oxidation step is catalyzed by the enzyme
alcohol oxidase (AOX, EC 1.1.3.13). While AOX
is expressed at low levels in glucose-containing
media, AOX accounts for 30% of solubilized in-
tracellular proteins in methanol-containing media,
driven primarily by the AOX1 promoter. Pichia
pastoris, a methanol-utilizing yeast, naturally
carries both AOX genes (the AOX1 gene and the
AOX2 gene). The AOX1 promoter and AOX2
promoter differ markedly in activity. AOX activ-
ity in Pichia pastoris is due mostly to expression
at the AOX1 locus. Strains of Pichia pastoris in
which a heterologous gene is substituted at the
AOX1 locus grow slowly in methanol-containing
media, requiring a lengthy cultivation period, be-
cause the methanol utilization is based on AOX2
activity alone. Among the two AOX genes coding
for AOX, the AOX1 promoter, which is the regu-
latory region of the AOX1 gene, has high activity
and can be used for the expression of heterolo-
gous proteins in high yields in methanol-utilizing
yeasts. However, the natural AOX2 promoter
is weak in activity and hence unsuitable for the
expression of heterologous proteins.
Benefits: Strong promoter in Pichia
4,808,537, Methanol inducible genes obtained
from pichia and methods of use, Stroman, et
al., Feb. 28, 1989, assigned to Phillips Petroleum
Co., which has licensed this and other Pichia
technology to RCT. The patent abstract states,
A method for isolating and cloning methanol
247
inducible genes from Pichia is taught. The regula-
tory regions are useful for the methanol regulated
expression of heterologous genes.
Availability: Marketed by various vendors for
Licensing information: The U.S. and other
patents covering AOX1 use in Pichia have or will
soon expire, allowing commercial use of AOX1
without a license. However, before initiating com-
mercial use, it is best to touch base with RCT.
Further info.: Functional characterization of the
two alcohol oxidase genes from the yeast Pichia
pastoris, J M Cregg, K R Madden, K J Barringer,
G P Thill, and C A Stillman, Mol Cell Biol. 1989
March; 9(3): 1316-1323.
383
Pichia pastoris super
yeast; Pichia pastoris AOX1
promoters
VTU Technology GmbH - Licensor, primary
Description: Dr. R. Weis, VTU Technology
GmbH, recently reported an optimised expres-
sion system has been developed which turns the
Pichia pastoris yeast strain into a super yeast
and a proprietary library of synthetic variants of
one of the strongest promoters known, the Al-
cohol Oxidase 1 promoter (AOX1) from Pichia
pastoris. This enables individually tuning expres-
sion strength and promoter characteristics along
the time course of cultivation. This is particularly
convenient for controlling the occurrence of
chaperones for correct protein folding. This event
should precede the actual expression of a target
protein in order to ensure a defined ratio of helper
to acceptor protein, ultimately leading to in-
creased space-time-yield of target protein. AOX1
promotesr include those allowing the production
of protein using a reduced amount of methanol or
no methanol.
Use with: yeasts
Patents: Exemplary IP includes WO 2006089329
(EP1851312), MUTANT AOX1 PROMOT-
ERS, assigned to VTU.
Licensing information: Contact VTU.
384
Aminoglycoside
adenylyltransferase (aadA1)
promoter - bacteria;
eukaryotes
primary
Description: A gene cassette containing the
aadA1 (aminoglycoside adenylyltransferase) gene
that increases protein expression levels when
incorporated into a bacterial or eukaryotic host
genome. In bacterial systems, the inventors have
shown that this gene cassette induces enhance-
ment of protein production and accumulation.
This technology offers increased product yields,
and is not restricted by the nature of the vector,
induction system or nature of protein. In particu-
lar, this invention has yielded 3-fold upregulation
of anti-HIV peptide expression levels in a live
microbial microbicide involving bacteria express-
ing an HIV fusion inhibitor protein. .
The system has been validated in bacterial
cells. Development is underway for use in eukary-
otic expression systems.
Use with: bacteria; eukaryotes
Patents: U.S. Provisional Application No.
60/571,943 filed 18 May 2004 (HHS Reference
No. E-261-2003/0-US-01). PCT Application No.
PCT/US2005/17001 filed 17 May 2005, which
published as WO 2005/116222 on 08 Dec 2005
(HHS Reference No. E-261-2003/0-PCT-02). The
primary inventors are Shankar Adhya and Sudesh-
na Kar (National Cancer Inst.)
Licensing information: Contact NIH.
Further info.:
S Rao, S Hu, L McHugh, K Lueders, K Henry,
Q Zhao, RA Fekete, S Kar, S Adhya, DH Hamer.
Toward a live microbial microbicide for HIV:
248
commensal bacteria secreting an HIV fusion
inhibitor peptide. Proc Natl Acad Sci U S A. 2005
Aug 23;102(34):11993-8. [Epub 2005 Jul 22, doi
10.1073/pnas.0504881102].
385
ColE1 plasmids, E. coli -
prolonged viability
primary
Description: Expression vectors with modified
ColE1 origin of replication for control of plasmid
copy number provide an improved expression
system with prolonged bacterial viability during
fermentation. A host-vector system that uses the
RNA-based copy number control mechanism of
ColE1-type plasmids for regulating the expres-
sion of a marker gene allows for antibiotic-free
selection of plasmids. ColE1-based vectors
include a mutation in the loop regions of both the
RNAI and RNAII gene, which are homologous
to uncharged tRNAs. This provides a strategy to
deliberately manipulate the degree of homology
and ultimately tune the rate of plasmid replication.
Use with: E. coli
Expression vectors with modified ColE1 origin
of replication for control of plasmid copy num-
ber, Bayer, K., et al., assigned to Boehringer
Ingelheim International GmbH. The abstract
states An an expression vector having a ColE1
replication system, the homology of the RNAI
and RNAII of the ColE1 origin of replication to
uncharged tRNAs is modified mutations in the
coding region of the RNAI gene and correspond-
ing mutations in the RNAII gene. The mutation
results in one or more base exchanges in loop 1
and/or loop 2 and/or loop 3 of RNAI and RNAII.
In methods using this vector for producing recom-
binant proteins, plasmid copy number is stably
maintained. In methods for plasmid production,
high plasmid copy numbers can be obtained. The
company has also filed U.S. 20060063232, Host-
vector system for antibiotic-free CoIE1 plasmid
propagation, Grabherr, et al., published March
23, 2006.
Ingelheim.
Reported to have been licensed by Lonza.
Mammals
386
CMV/R Promoter - eukaryotes
primary
Description: Expression vectors with the CMV/R
promoter provide higher levels of protein expres-
sion than vectors currently in use. The elevated
levels of expression are achieved through use of
a specific promoter, known as CMV/R, in which
the Human T-Lymphotrophic Virus (HTLV-1)
Long Terminal Repeat (LTR) R-U5 region is
substituted for a portion of the intron downstream
of the CMV immediate early region 1 enhancer.
Sequences of 95% or better homology to CMV/R
can be used as well.
CMV/R vectors are currently being used in a
number of products in clinical trials, including
vaccines against West Nile Virus, Ebola virus,
and HIV, and achieving promising results. The
CMV/R vector can be used for any DNA vaccine
or for the production of recombinant proteins in
high yields.
Patents: Related patents/applications include U.S.
Patent No. 7,094,598 issued 22 Aug 2006 [HHS
Reference No. E-241-2001/1-US-01 (CMV/R)],
applications pending in EP, JP, CA, and AU; U.S.
Patent Application No. 10/491,121 filed 23 Aug
2004 [HHS Reference No. E-241-2001/0-US-07
(Ebola DNA vaccine)], applications pending in
EP, JP, CA, and AU; U.S. Patent Application No.
249
11/632,522 filed 16 Jan 2007 [HHS Reference
No. E-267-2004/1-US-08 (HIV DNA vaccine)]
Licensing information: Contact NIH (Susan
Ano, Ph.D.; 301/435-5515; anos@mail.nih.gov).
387
piggyBac transposon -
eukaryotes; insect cells
Dept. of Agriculture (USDA) - Licensor, pri-
mary
University of Notre Dame - R&D, of tech.
Description: The TTAA-specific transposon pig-
gyBac is a transposon from the cabbage looper
moth, Trichoplusia ni, particularly useful a a pro-
moter with insect cell/baculovirus and many other
expression systems. The piggyBac transposon
was first identified as one of several insertional
mutations in cell culture passaged baculoviruses.
The helper-dependent trans-mobilizing capabili-
ties of this transposon for gene vectoring was
first established in baculovirus expression system
(BEVS). Later applications of this transposon to a
diversity of organisms including protists, inver-
tebrates, and vertebrates has established it as a
highly versatile tool for genetic engineering. The
unique properties of piggyBac mobilization pro-
vide novel capabilities including saturation gene
tagging, promoter/enhancer trapping, relatively
large carrying capacity, and precisie excision from
insertion points within a genome
The piggyBac element is 2.4 kb in length and
terminates in 13 bp perfect inverted repeats, with
additional internal 19 bp inverted repeats located
asymmetrically with respect to the ends. The pig-
gyBac element has a potential RNA polymerase II
promoter sequence configuration, typical Kozak
translational start signal, and two apparently over-
lapping long open reading frames.
Use with: eukaryotes; insect cells
Background: Several different mobile host DNA
insertions have been identified within the FP locus
of the baculoviruses AcMNPV and GmMNPV.
The insertions most extensively studied are those
now designated as tagalong (formerly TFP3)
and piggyBac (formerly IFP2). These insertions
exhibit a unique preference for TTAA target sites,
whether inserting within the viral FP-locus or at
other regions of the viral genome. Both of these
elements are part of a larger family of TTAA-tar-
get site specific insertion elements that includes
the T. ni derived piggyBac and tagalong elements.
Patents: Invented by Dr. M.J. Fraser, affiliated
with the University of Notre Dame, and with
patents assigned to USDA. Exemplary patents
include 6,962,810 and 7,105,343, Methods and
compositions for transposition using minimal
segments of the eukaryotic transformation vector
Piggybac, Fraser, M.J., Nov. 2005 and Sept. 2006,
respectively. The abstract states: The present
invention provides efficient transfer of genes into
host cells or embryos to transform the cells or em-
bryos by transposition vectors using the minimal
amount of nucleotide sequences in the transposon
piggyBac required for gene transfer. The trans-
formed cells or embryos may also be developed
into transgenic organisms. Other related patents
include 7,129,083, Methods and compositions
for transposition using minimal segments of the
eukaryotic transformation vector Piggybac, Han-
dler, A.M., assigned to USDA.
Availability: available for res. using an MTA
form
Licensing information: Contact USDA and/or
Dr. Fraser, Malcolm.J.Fraser.1@nd.edu
Further info.: Grossman GL, Rafferty CS, Fraser
MJ, Benedict MQ. 2002. The piggyBac element
is capable of precise excision and transposition
in cells and embryos of the mosquito, Anopheles
gambiae. Insect Biochem Mol Biol. 32(4):487.
Li X, Heinrich J C, Scott M J. 2001. piggyBac-
mediated transposition in Drosophila melano-
gaster: an evaluation of the use of constitutive
promoters to control transposase gene expression.
Insect Mol Biol. 10(5):447-55.
Li X, Lobo N, Bauser CA, Fraser MJ. 2001. The
minimum internal and external sequence require-
250
ments for transposition of the eukaryotic transfor-
mation vector piggyBac. Mol Genet Genomics.
266(2):190-8.
388
Tax-inducible expression;
Bovine leukemia virus (BLV)
promoter - mammalian cells
University of Wisconsin - Assignee, patent
Description: An inducible mammalian promoter
expression system uses the bovine leukemia virus
(BLV) promoter. The system includes a vector
containing this retroviral promoter and a factor to
induce the retroviral promoter. The desired gene
product is expressed in proportion to induction
of the promoter. For example, the system may
include a Tax-inducible expression vector that
uses the bovine leukemia virus (BLV) promoter
and contains the foreign gene of interest. The
BLV promoter alone typically produces low levels
of gene expression. However, the transcriptional
activator Tax induces the BLV promoter. Thus,
when a second vector containing Tax is intro-
duced into a host cell, very high levels of gene
expression are achieved.
Exceptionally tight on/off regulation of expression
Extremely low background expression
Highly inducible
Fast response times
High absolute expression levels
Activates promoter, rather than represses it, to
control expression
7,297,536, Inducible protein expression system,
Harms, et al., Nov. 20, 2007, assigned to Wiscon-
sin Alumni Research Foundation.
389
BacMam; pBacMam vectors
- mammalian vectors,
baculovirus-based
Partners Healthcares Research Ventures &
Licensing (RVL) - Licensor, primary
General Hospital Corp. (The) - Assignee, pat-
ent
Massachusetts General Hospital - Parent co.
GlaxoSmithKline Inc. (GSK) - R&D
Description: Recombinant BacMam baculovirus
vectors modified to contain mammalian cell-ac-
tive expression cassettes deliver their DNA and
mediate gene expression in mammalian cells,
providing a new approach for protein expression
in mammalian cells. This system takes advantage
of the finding that baculovirus-budded virions are
able to enter a very large range of mammalian cell
types, typically at a very high efficiency. When
baculovirus carries a gene driven by an appropri-
ate mammalian promoter, foreign proteins can be
expressed at high levels in virtually every cell in
the culture. Thus, the baculovirus functions as a
gene entry vehicle for the transduction of a mam-
malian expression cassette (promoter and gene).
Because the baculovirus own genes are not
expressed in mammalian cells, there is no produc-
tion of infectious baculovirus virions in the mam-
malian culture. Mammalian cells transduced with
baculovirus vectors do not exhibit cytopathology,
and the expression of foreign genes can continue
for many days in culture.
BacMam viruses transduce a variety of estab-
lished and transformed cell lines and primary cells
without overt deleterious effects. Gene expression
in transduced populations is modulated by varia-
tion of viral multiplicity, or use of inhibitors of
histone deacetylase, e.g., trichostain and sodium
butyrate. The virus does not replicate in mam-
malian cells and is rapidly inactivated by serum
complement, giving it a favorable biosafety pro-
file compared to other recombinant viral vectors.
Gene delivery is accomplished with simple liquid
251
addition steps making the BacMam system com-
patible with automated high throughput screen-
ing platforms. Libraries of viruses can be easily
generated and stored for delivery to appropriate
cells in a variety of formats, providing a versatil-
ity in assay configuration not matched by usual
paradigms such as stable cell lines.
Benefits: Advantages of the BacMam system
were outlined including its lack of cytotoxicity, its
ability to transduce a host of cell lines, the pos-
sibility to perform transient or stable transfections
and its large capacity for gene copy insertion.
Patents: Originally developed by GlaxoSmith-
Kline Inc. (GSK) and the The General Hospital
Corp. (Massachusetts General Hospital). Exem-
plary patents include U.S. 5,731,182, Non-mam-
malian DNA virus to express an exogenous gene
in a mammalian cell, Boyce, F.M., assigned to
General Hospital Corp.
by EMD Biosciences.
Licensing information: Contact Partners Health-
cares Research Ventures & Licensing (RVL), the
licensing agent for Massachusetts General Hospi-
tal.
Products made using this tech.: GlaxoSmith-
Kline Inc. (GSK) has apparently used this system
for in-house recombinant (glyco)proteins manu-
facture.
Further info.: Baculovirus as versatile vectors
for protein expression in insect and mammalian
cells, Nature Biotechnology 23, 567 - 575 (2005)
- by GSK authors.
390
Calnexin, calreticulin, Erp57,
Hsp40, and Hsp70 chaperones
- mammalian cells
Bayer Corp. - Licensor, primary
Description: Bayer has patented a method for
increased production of secreted, recombinant
protein in mammalian host cells using multiple
coexpressed chaperones. Various novel calnexin,
calreticulin, Erp57, Hsp40, and Hsp70 chaperones
are provided.
7,244,616, Use of molecular chaperones for the
enhanced production of secreted, recombinant
proteins in mammalian cells, Chan, et al., July
17, 2007, assigned to Bayer
Licensing information: Contact Bayer.
391
CCT promoters - mammalian
cells
University of Iowa Research Foundation
(UIRF) - Licensor, primary
Description: The CCTp208 and CCTp240 mu-
rine-based promoters stimulate the expression of
genes in a variety of cell types. Their activity is
robust with several fold greater activity compared
to existing viral promoters (e.g., SV40 promoter).
The promoters are able to retain enhanced expres-
sion of desired genes in a stable fashion. Because
the promoters are not viral-based sequences, they
circumvent effects of inflammatory host respons-
es that turn off activity of commonly available
promoters. These promoters can be used to gener-
ate recombinant proteins, and to express reporter
genes in gene therapy and vaccine products. The
promoters naturally direct transcription of CTP:
phosphocholine cytidylyltransferase (CCT), a key
ubiquitous regulatory enzyme present in nucleated
(eukaryotic)
cells involved in phospholipid synthesis.
CCT protein expression promoter, Mallampalli,
R.K., 06/12/2007.
Iowa Research Foundation (UIRF).
252
392
ClonePixFL Selection -
antibodies, mammalian cells
Genetix - Licensor, primary
Description: This is an automated, high through-
put cell identification and selection system useful
for its proven ability to identify and isolate cell
lines producing MAbs. Generation of stable lines
or re-cloning a failing cell line is easily achieved
by sequential rounds of sub-cloning. The system
is compatible with a broad range of suspension
and adherent cells including hybridomas, myelo-
mas, CHO, HEK293 and mouse ES cells.
ClonePixFL automatically images over 1000
colonies per hour, ranks the clones in terms of
relative protein expression, then picks the selected
clones into 96-well destination plates. For mam-
malian cell colonies that secrete unlabelled, native
proteins or express fluorescently labelled proteins,
ClonePixFL automatically screens, identifies,
isolates and stratifies the highest producing colo-
nies within a population. Results can be viewed
and reported as cell confluence measurement,
cell number estimation and loci of growth. Using
the loci of growth function allows decisions to be
made on both clonality and viability.
Used with: hybridomas, myelomas, CHO,
HEK293 and mouse ES cells
Use to make: protein; Mabs in CHO and other cell
lines
Benefits: Genetix claims this is The most power-
ful technology currently available for rapidly
isolating rare-event high producing clones with
minimal hands-on time. ClonePixFLAllows
direct screening of large numbers of secreting
clones, resulting in the identification of higher
expressing cell lines; ncreases the precision and
documentation of clone picking; and can be used
with dual detectors to separate recombined clones
from non recombined clones following a site
specifc recombination reaction.
Licensing information: Contact Genetix.
393
Hsp60, Hsp70, Hsp90, Hsp100
chaperones - mammalian cells
censor, primary
Hoffmann-La Roche Inc. - Assignee, patent
Description: Hsp60, Hsp70, Hsp90 and Hsp100
chaperone small heat shock proteins and isomer-
ases coexpressed in expression vectors increase
and improve proper folding of desired proteins
expressed in mammalian expression systems,
and have also been reported useful in cell-free
expression systems. See also the GroEL,GroES
chaperones entry concerning prokaryotic versions
of HSP60.
Use with: eukaryotes; mammalian cells
Background: Heat shock protein 60 (HSP60)
is a mitochondrial chaperonin that is typically
responsible for the transportation and refolding of
proteins from the cytoplasm into the mitochon-
drial matrix. In addition to its role as a heat shock
protein, HSP60 functions as a chaperonin to assist
in folding linear amino acid chains into their
respective three-dimensional structure. HSP60 has
two main functions with respect to mitochondrial
protein transport. It catalyzes the folding of pro-
teins destined for the matrix and maintains protein
in an unfolded state for transport across the inner
membrane of the mitochondria.
Method for the expression of proteins in in vitro
translation systems with coexpression of folding
helper proteins, Buchner, August 16, 2005. The
abstract states The present invention concerns
a method for the expression of target proteins in
in vitro translation systems, characterized in that
folding helper proteins are co-expressed in this
system. The co-expressed folding helper proteins
are selected from one or several of the following
protein classes: Hsp70, Hsp60, Hsp90, Hsp100
protein family, the family of small heat shock
proteins and isomerases.
253
tein Expression Group (RPEG).
394
IRF-1 estrogen receptor
promoter; Estradiol induction
- mammalian cells
Gesellschaft fur Biotechnologische Forschung
mbH (GBF) - Licensor, primary
Description: A promoter-transactivator system
using an IRF-1 estrogen receptor-based promoter
with induction by binding of estradiol (added to
culture medium) achieves regulated high-level
gene expression in proliferation-controlled mam-
malian cells. By the simple addition of medium
supplements, expression levels can be achieved
above those from conventional promoters. Com-
posite promoters contain constitutive enhancer
elements that allows a basal expression level as
high as the levels achieved with a very efficient
viral promoter. In addition, the promoter encodes
sequences that are bound by a transactivator
whose activity can be regulated.
Patents: Exemplary patents include WO0065074,
PROMOTER-TRANSACTIVATOR SYSTEM
FOR INDUCIBLE HIGH-LEVEL MAMMALI-
AN GENE EXPRESSION WITH THE OPTION
OF CELL GROWTH CONTROL, Mueller, et
al., assigned to Gesellschaft fur Biotechnolo-
gische Forschung mbH (GBF).
Licensing information: Contact GBF.
395
Osteoclast-associated
receptor (OSCAR) promoter -
mammalian; CHO cells
University of Edinburgh - Licensor, primary
Description: The OSCAR promoter is claimed
to be able to produce high levels fo proteins and
Mab at level at levels that outperform commer-
cial systems, and also offer simpler and qucker
gene amplification. The OSCAR system produces
high levels of protein in cultured CHO cells in a
single step.
Use with: mammalian cells; CHO cells
Background: Osteoclasts are multinucleated
cells that resorb bone and are essential for bone
homeostasis. The osteoclast-associated receptor
(OSCAR) is a member of the leukocyte receptor
complex (LRC) protein family that plays critical
roles in the regulation of both innate and adaptive
immune responses. Different from the other LRC
members, OSCAR expression is detected specifi-
cally in preosteoclasts or mature osteoclasts. OS-
CAR may be an important bone-specific regulator
of osteoclast differentiation.
high yield of protein
rapid selection fo high-expressing clones
siimple selection and amplification in one step
no need for special media
Patents: The initial priority filing was made in
the UK in 2000 (Ref No. PCT/GB00/04959), and
is now in the national phase of prosecution in
Canada, USA and Europe
Licensing information: The University of Ed-
inburgh seeks partners for collaborative devel-
opment of OSCAR as a platform technology.
Samples are available under an MTA.
Further info.: Melton et al. A One-Step Gene
Amplification System for Use in Cultured Mam-
malian Cells and Transgenic Animals. Transgen-
ic Research 2001 10:133-142.
396
pAccAB vectors - antibodies;
mammalian cells
Accuro Biologics Ltd - Licensor, primary
Description: For its CRO/CMO clients requiring
expression of full antibody molecules, Accurobio
254
uses its pAccAB vector system for immuno-
globulin genes assembly from genomic DNA.
Antibody heavy and light chains are carried in the
same plasmid driven by separate high expression
promoters. Expression vectors may be compiled
to enable expression using alternative selection
schemes and provide a choice of constant region
isotypes.
nal antibodies
Patents: No relevant patents or applications re-
trieved from simple searches.
Licensing information: Contact Accuro Biolog-
ics Ltd.
In May 2007, Accuro Biologics concluded a 2-
year agreement with UniTargetingResearch AS to
provide the UniTargetingResearch Clone Consor-
tium (an EU-funded project) with mammalian cell
line development and other services for increasing
protein production.
In May 2008, Accuro Biologics Ltd. and Mil-
leGen SA, a biotech company focused on Di-
rected Molecular Evolution (DME), entered into
a strategic collaboration to combine MilleGens
DME platform MutaGen and Accuro Biologics
know-how and expert capabilities in antibody
engineering, T cell epitope identification and anti-
body expression.
397
RNA transport elements (CTE and RTE) -
mammalian cells
National Institutes of Health (NIH)
Description: Vectors combining a constitutive
RNA transport element (CTE) with a mutant form
of another RNA transport element (RTE) results
in a synergistic effect on stability of mRNA tran-
scripts, which in turn leads to increased expres-
sion levels. The functions of CTEs and RTEs are
conserved in mammalian cells, and this technol-
ogy can be used with diverse plasmids and viral
vectors for mammalian cells. This provides simple
and useful way of obtaining high levels of expres-
sion of otherwise poorly expressed genes .
Using HIV-1 gag as reporter mRNA, one
mutated RTE in combination with a CTE was
found to improve expression of unstable mRNA
by about 500-fold. Similarly this combination of
elements led to synergistically elevated levels of
HIV-1 Env expression.
0070031921, Potent combinations of mRNA
transport elements, Felber, B., et al., Feb. 8,
2007, assigned to National Institutes of Health
(NIH)
Licensing information: Contact NIH (HHS Ref-
erence No. E-223-2003/1-US-03).
398
RP Shift; Senescence
induction; PACE Expression
Vector - mammalian cells;
expression enhancement;
antibodies
Clonex Development, Inc. (CDI) - Licensor,
primary
Description: RP Shift technology is based on
conditionally producing a phenotypic change,
conditionally producing premature senescence,
in biopharmaceutical-producing cells in culture.
RP Shift increases cell stability, and allows higher
concentration of secreted products. Cells undergo-
ing RP Shift stop dividing, increase their volume,
increase numbers of mitochondria, expand their
endoplasmic reticulum, and increase their syn-
thesis and secretion of protein. These cells have
longer lifespan and are also substantially more re-
sistant to environmental stresses, such as lowered
pH, loss of serum factors, osmotic changes and
other impedance that triggers cell death in prolif-
erating populations.
CDI has routinely demonstrated 5-10-fold RP
Shift enhancement in the production of protein
255
from fibroblasts, Chinese hamster ovary, and
mouse myeloma cells. CDI has also shown greater
than 30-fold increase in the production of a mono-
clonal antibody from hybridoma cells in a static
culture system.
CDI has generated CHO, NS0 and Sp20 cell
lines with RP Shift into which any protein ex-
pressing construct may be cloned. CDI has also
prepared a PACE Expression Vector with promot-
er elements that are activated in cells to undergo
the RP Shift. Customers can simply clone their
recombinant gene into the PACE Expression Vec-
tor to increase its transcription.
CDI has produced plasmid systems containing
the RP Shift constructs that may be readily intro-
duced into any mammalian cell line. A timeline
of approximately ten weeks is necessary to isolate
clones ready for testing for RP Shift enhancement
of protein production. An additional 3-6 months
is required for complete analysis of the RP Shift
enhancement at analytical and commercial scales.
CDI routinely achieves 3-7 fold increases in
protein production from commercial cell lines. In-
creases of this magnitude likely translate to costs
savings of 6-10% of total sales.
Use with: mammalian cells; CHO, NS0 and
Sp20 cells
ies
Methods and compositions for increasing protein
yield from a cell culture, Bucciarelli, T., et al.,
10/21/2003, assigned to Clonex Development,
Inc. The abstract states, Disclosed herein are
compositions and methods for increasing pro-
tein production from a cell culture. By switching
the cells from a replicative to a productive state
(RP switch), protein biosynthesis is extended.
The productive state is a pseudo-senescent state.
This pseudo-senescent state can be induced by
transforming the cells with a vector expressing a
cell cycle inhibitor. Expression of the cell cycle
inhibitor within the cell, because it does not cause
cell death, allows for cells to be maintained in cul-
ture for longer periods. The invention allows for
controlled enhanced protein biosynthetic produc-
tivity of cell lines for commercial and research
purposes.
Licensing information: Contact CDI. CDI gener-
ates its revenue through licensing and royalties on
use of its RP Shift technology in the biomanufac-
turing process.
399
Semliki forest virus (SFV)
vectors - mammalian and
insect cells
Bio-Xtal S.A. - Licensor, primary
Description: Semliki forest firus (SFV) vec-
tors offer advantages for large-scale recombi-
nant (glyco)protein manufacture in mammalian
and insect cells. SFV vectors overcome folding
limitations and post-translational modification
in mammalian proteins; infect a broad range of
mammalian and insect cells; provide rapid infec-
tion/transformation and gene expresion, with fast
scale-up in suspension cell cultures and; can co-
express multiple proteins in the same cells with
high reliability and reproducibility.
An efficient expression system has been engi-
neered for SFV by splitting the genome on two
plasmid vectors. The expression vector carries the
nonstructural (replicase) genes and the foreign
gene of interest. The helper vector carries the SFV
structural genes. Rapid high-titer virus production
can be achieved by in vitro transcription of RNA
from both vectors followed by co-transfection
of BHK-21 cells. The broad host range of SFV
allows infection of various mammalian cell lines
and primary culture cells. The SFV technology
has been scaled up for expression of large quanti-
ties of recombinant proteins in suspension cul-
tures of mammalian cells.
SFV vectors have been used for efficient over-
expression of a variety of topologically different
proteins. Intracellular, transmembrane and se-
creted proteins have been expressed at high levels.
The authenticity (post-translational modifications,
256
well folded and native protein) of recombinants
products can be guaranteed because of the possi-
bility of using different type of cells.
Dr. Lundstrom, seemingly an inventor of this
technology, then cited as affilitated with Hoff-
mann-La Roche AG, has presented a talk, Rapid
Large-scale Production of Recombinant Mem-
brane Receptors using Semliki Forest Virus Vec-
tors, concerning use of SFV vectors for transient
gene expression of membrane receptors. High-
titer virus stocks can be rapidly generated (in <2
days) and applied for infection of numerous mam-
malian cell lines and primary cell cultures. SFV
vectors have turned out to be particularly good
for expression of G protein-coupled receptors and
ligand-gated ion channels (>150 pmol receptor/
mg protein; >5 million receptors/cell). Transfer
of the SFV technology to suspension cultures of
mammalian cells growing in serum-free medium
with yields of (1-5 mg receptor / liter culture) has
allowed purification of hundreds of milligrams of
receptor protein for structural studies.
Use with: mammalian cells; insect cells
Background: SFV is a positive-stranded RNA
virus of the genus Alphavirus of the family Toga-
viridae
Patents: No relevant patents or applications were
retrieved from simple searches (Bio-Xtal, Lund-
strom, etc.).:
Licensing information: Bio-Xtal S.A. offers
expression evaluation services, along with pro-
teomic services, crystallography services and re-
combinant protein production using SFV vectors.
Further info.:
Semliki Forest virus vectors for rapid and high-
level expression of integral membrane proteins,
Lundstrom K., Biochim Biophys Acta. 2003 Feb
17;1610(1):90-.
Chinese hamster ovary (CHO)
cells
400
CHEFl expression; CHO
elongation factor-la (EF-Ialpha)
promoter - CHO cells
Icos - Licensor, primary
Lilly, Eli & Co. - Parent co.
Description: The CHEFl expression system is
based on the CHO elongation factor-la (EF-Ial-
pha) promoter. CHEFl provides CHO cell expres-
sion levels over six to 25 times greater than those
obtained with other promoters, such as CMV,
without amplification. ICOS is using CHEFl
vectors to express cellular proteins that may be
rate-limiting for expression/ folding/secretion/
modification of the protein of interest.
Use with: CHO cells
Patents: No relevant patents or application were
retrieved by the author from simple searches.
Licensing information: ICOS was acquired by
Eli Lilly in 2007. Contact Lilly.1.
401
CHO cells - antibodies; serum-
free media
Polymun Scientific Immunobiologische Forsc-
hung GmbH - Licensor, primary
Description: Recombinant CHO-cells for manu-
facture of human monoclonal antibodies have
been adapted from serum supplemented medium
to a protein-free medium. Reproducable adapta-
tion protocols have been established which can be
applied to any other CHO cells.
Long term stability tests in laboratory scale
cultures, production tests in repeated fed-batch
(500L scale) as well as long term continuous
perfusion (more than 3 months) in fluidized bed
257
reactors have shown stability of antibody secre-
tion. In fed-batch culture up to 600g antibody/m3
was accumulated, while in a continuous perfused
system up to 100g/m3 were achieved.
Use with: CHO cells
Cost effective and safer production
Unlimited production scale
Elimination of animal products in media
Licensing information: Contact Polymun, which
offers CMO services for mammalian and micro-
bial cell-based manufacture.
Polymun developed a CHO cell line as well
as a fermentation and purification process for the
production of a biosimilar/follow-on biologic
version of recombinant human erythropoietin
(EPO) on an exclusive contract for AS Biotech
AG. Three consecutive industrial batches have
been manufactured under GMP conditions. The
EPO will be registered and commercialized by
AS Biotech AG in Europe and other international
markets.
For Baxter International Inc. Polymun manu-
factured the mouse monoclonal antibody HPC4
which is used for the immunoaffinity purification
of Ceprotin (recombinant activated Protein C) by
Eli Lilly & Co.
402
CHO cells dhfr RNA
interference; RNA silencing
vectors - CHO cells
National Tsing Hua University - Licensor,
primary
Description: Use of RNA interference (RNAi)
targeting to dihydrofolate reductase (DHFR)
genes is useful to improve recombinant protein
expression and stability of CHO cells. CHO cells
and dhfr/MTX gene amplification system are
routinely used to generate high-producing stable
CHO cell clones for recombinant protein expres-
sion (see DHFR and related CHO cell entries).
RNA silencing vectors targeting both the mouse
and Chinese hamster dhfr genes provide high-
producing stable cell clones from CHO-dhFr
cells and CHO-K1 (dhfr-competent) cells through
DHFR/MTX gene coamplification and selection.
Use of the dhfr-targeting RNA interference pro-
vided by a silencing vector also results in increas-
ing the stability of recombinant protein expression
in stable CHO cell clones grown in MTX-free me-
dium. This strategy may be applied to obtain high
producer cell clones for recombinant antibody
expression in both dhfr deficient and wild-type
CHO cells.
Co-amplification of an expression vector and
a RNA silencing vector targeted to dhfr gene to
obtain stable producer clones in dhfr-deficient and
wild-type CHO cells has been shown to induce an
efficient knockdown of both exogenous (mouse)
and endogenous (Chinese hamster) dhfr gene
expression in CHO/dhfr-| and CHO-K1 cells,
respectively. Combining the dhfr-targeted RNA
silencing vector and expression vectors yields sig-
nificant improvements for obtaining high-produc-
ing CHO cell clones with an enhanced stability.
in MTX-free medium.
Use with: CHO cells
ies
Benefits: The advantages of using silencing vec-
tors targeted to dhfr gene transcripts include pow-
erful inducer activity, high transfection efficiency,
low cytotoxicity,, and dhfr-directed target gene
co-amplification through stepwise MTX selection.
The dhfr-targeted silencing vectors not only in-
duce effective shRNA against the dhfr gene tran-
scripts, but also yield a permanent integration of
a silencing cassette, resulting in co-amplification
of the dhfr-directed target gene throughout out
stepwise MTX selection to increase the strength
of RNAi specific to the dhfr gene transcripts.
Patents: No patents or applications were retrieved
by the author. Applications may not have been
published yet.
Licensing information: Contact National Tsing
Hua University. The inventors are Drs. Willy W.L.
Hong and Suh-Chin Wu.
258
Further info.: A novel RNA silencing vector
to improve antigen expression and stability in
Chinese hamster ovary cells, Willy W.L. Hong,
Suh-Chin Wu, Vaccine 25 (2007, 4103-4111.
000000
403
CSL4S-342 CHO cells (CHO-K1
CSL4S-342)
CSL Ltd. - Licensor, primary
Charles River Tektagen, Inc. - Retail seller
Description: The CSL4S-342 CHO cell line (a
derivative of CHO K1 cells) originally developed
by CSL and prepared by Tektagen Inc. is used by
BioMarin for manufacture of Naglazyme [Galsul-
fase - N-acetylgalactosamine 4-sulfatase, recom-
binant; Aryplase; chondroitinase; rhASB], e.g.,
see U.S. 6,972,124.
Use with: CHO cells
Patents: No patents or application concerning
CSL4S-342 were retrived.
Licensing information: Contact CSL Ltd.
404
Osteoclast-associated
receptor (OSCAR) promoter -
mammalian; CHO cells
University of Edinburgh - Licensor, primary
Description: The OSCAR promoter is claimed
to be able to produce high levels of proteins and
Mabs at level at levels that outperform commer-
cial systems, and also offer simpler and qucker
gene amplification. The OSCAR system produces
high levels of protein in cultured CHO cells in a
single step.
Use with: mammalian cells; CHO cells
Background: Osteoclasts are multinucleated
cells that resorb bone and are essential for bone
homeostasis. The osteoclast-associated receptor
(OSCAR) is a member of the leukocyte receptor
complex (LRC) protein family that plays critical
roles in the regulation of both innate and adaptive
immune responses. Different from the other LRC
members, OSCAR expression is detected specifi-
cally in preosteoclasts or mature osteoclasts. OS-
CAR may be an important bone-specific regulator
of osteoclast differentiation.
high yield of protein
rapid selection for high-expressing clones
siimple selection and amplification in one step
no need for special media
Patents: The initial priority filing was made in
the UK in 2000 (Ref No. PCT/GB00/04959), and
is now in the national phase of prosecution in
Canada, USA and Europe
Licensing information: The University of Ed-
inburgh seeks partners for collaborative devel-
opment of OSCAR as a platform technology.
Samples are available under an MTA.
Further info.: Melton et al. A One-Step Gene
Amplification System for Use in Cultured Mam-
malian Cells and Transgenic Animals. Transgen-
ic Research 2001 10:133-142.
405
Pangen CHO expression
PanGen Biotech Inc. - Licensor, primary
Neurotech Pharmaceuticals - Parent co.
Description: Vector constructs and high yielding
CHO cell lines can be developed after only two
steps of methotrexate (MTX) selection, which
greatly shortens the screening time, with the am-
plified sequences stably maintained in the absence
of a selective agent. The use of MTX, a potential
carcinogen, can be avoided for large-scale produc-
tion of therapeutic proteins.
The expression system is believed to confer
position-independent expression of the integrated
sequence. Regardless of the integration site and
nature of surrounding sequences, the productiv-
259
ity seems to be primarily dependent on the copy
number of amplified gene alone. The presence
of an efficient transcription terminator in the
vector eliminates problems associated with pos-
sible read-through and improves the stability of
mRNA, which leads to increased protein yield.
Proteins for research purposes can be obtained
in sufficient amounts in usually 2-3 months.
Stable CHO cell lines for biopharmaceutical pro-
ductions can be obtained in 4-6 months.
In the conventional CHO cell technology,
multiple colonies have to be separately cultured in
a prolonged process to obtain the best one, since
it is often difficult to know which colony is the
best until all the colonies are fully grown. In most
cases using PanGens vector, significant amounts
of desired protein can be synthesized from the
pool of transfectants prior to colony isolation.
About 8 to10-fold increase in protein production
is observed during the early stage. A large pro-
portion of transfectants (70-80%) synthesize the
recombinant protein at a relatively uniform level
as the expression seems to occur in a position-in-
dependent and copy number-dependent manner.
Semi-automation of the whole cell culture process
is currently underway, which will reduce the labor
cost even further.
Use with: CHO cells
Patents: No as yet published patents/applications
assigned to Pangen were retrieved.
Availability: PanGen offers a complete spectrum
of services for cell lines and proteins including
cloning, expression, production, and purification.
Licensing information: Contact Pangen.
406
StableFast Biomanufacturing
System; pBFdfhr.2 Expression
Vector - CHO cells; antibodies
BioFactura, Inc. - Licensor, primary
Description: The StableFast expression system
includes a DHFR- (see related entries) transfec-
tion vector, pBFdfhr.2 Expression Vector, and an
novel approach to generating stable mammalian
cell lines, e.g., CHO cells. Methotrexate, a potent
competitive inhibitor of DHFR, may be used for
amplification. The system is particularly useful
for antibody expression.
In Jan. 2007, BioFactura received a $200,000
investment through the MdBio Project Accelera-
tor Program targeted at accelerating the develop-
ment of the StableFast Biomanufacturing System.
Use with: CHO cells; mammalian cells
nal antibodies
Benefits: Claimed advantages include
a) Proven Commercial System, e.g,. CHO DG44
cells (see related entry)
b) High Stability/High Yield
c) Increased Expression with Optimized pCMV
MIE Intron A Promoter Sequence
d) Favorable MCS for Cloning of Monoclonal
Antibody Sequences
e) Non-Drug Metabolic selection
d) High Fidelity - Faithful Glycosylation and
Post-Translational Modification of Human Pro-
teins and Antibodies
e) Commercially Scalable (with appropriate
licenses)
f) Small Business-Friendly Licensing/Royalty
Terms (no details)
Patents: No relevant yet published patent applica-
tions assigned to BioFactura were retrieved. .
by BioFactura.
Licensing information: Contact BioFactura
260
407
Tandem Chimeric Antibody
Expression (TCAE) vectors;
ANEX vectors; CHO cell
line TCAE 8 (ATCC 9119);
Kozak sequences, impaired -
antibodies
Biogen Idec, Inc. - Licensor, primary
Description: Tandem Chimeric Antibody Expres-
sion (TCAE) vectors are useful with CHO cells
for monoclonal antibody expression, including in
CHO cells. The vectors allow for inserting DNA
encoding non-human variable antibody regions
with light and heavy chain variable regions
incorporated into the vector. A most preferred
TCAE vector, used to generate immunologically
active chimeric anti-CD20 antibodies (apparently
for commercial manufacture of Rituximab) for
therapeutic treatment of lymphomas, is TCAE 8,
a derivative of a vector owned by Biogen Idec re-
ferred to as TCAE 5.2. In TCAE 5.2, the transla-
tion initiation start site of the dominant selectable
marker (neomycin phosphostransferase, NEO)
is a consensus Kozak sequence, while for TCAE
8, this region is a partially impaired consensus
Kozak sequence. Impaired consensus Kozak
sequences are useful with dominant selectable
markers of transcriptional cassettes as part of an
expression vector.
TCAE 8 comprises four transcriptional cas-
settes, and these are in tandem order, i.e., a human
immunoglobulin light chain absent a variable
region; a human immunoglobulin heavy chain
absent a variable region; DHFR; and NEO. Each
transcriptional cassette contains its own eukary-
otic promoter and polyadenylation region.
Use with: CHO cells
Patents: Details regarding the impact of the
initiation start site of the dominant selectable
marker of the TCAE vectors (also referred to
as ANEX vectors) for protein expression are
presented in U.S. 5,648,267, assigned to Idec
Pharmaceuticals, now Biogen Idec, Inc. Claim 1
states: 1. An expression vector which expresses
at least one protein of interest in a recombinant
host cell wherein said expression vector com-
prises: (i) a translationally impaired neomycin
phosphotransferase (NEO) dominant selectable
marker gene which has been translationally im-
paired by modification of the region of the NEO
gene which includes the NEO translation initia-
tion start codon such that said modified region of
the NEO gene which includes the NEO translation
initiation start codon has the following nucleotide
sequence: which translationally impaired NEO
gene is operably linked to a promoter and polyad-
enylation sequence; and (ii) at least one heterolo-
gous DNA which encodes for at least one protein
of interest, wherein said heterologous DNA is
operably linked to a promoter and polyadenyl-
ation sequence different from the promoter and
polydenylation sequence operably linked to the
NEO gene, and wherein said heterologous DNA
and said promoter and polyadenylation sequence
operably linked to said heterologous are inserted
into an intronic insertion region contained in the
NEO gene.
U.S. 5,736,137, Anderson, et al., Therapeutic
application of chimeric and radiolabeled antibod-
ies to human B lymphocyte restricted differentia-
tion antigen for treatment of B cell lymphoma,
, assigned to Idec Pharmaceuticals, now Biogen
Idec, Inc., although concentrating on claiming
CD20 Mabs (e.g. rituximab; see below), includes
a detailed description of TCAE 8 vector for hu-
manized antibody expression.
Licensing information: Contact Biogen Idec
U.S. application 20040063911, Protein re-
modeling methods and proteins/peptides produced
by the methods, assigned to Neose Technolo-
gies, Inc., describes use of TCAE 8 CHO cells
for expression of humanized CD20 monoclonal
antibodies, with Neose apparently having licensed
TCAE technology from Idec/Biogen Idec.
Products made using this tech.: Rituxan [Ritux-
261
imab - IDEC-C2B8; MabThera; CD20 monoclo-
nal antibody, recombinan] manufactured by Bio-
gen Idec and also Genentech with U.S. marketing
by Biogen Idec is is produced in batch culture us-
ing a plasmid-transformed Chinese hamster ovary
(CHO) cell line (apparently TCAE 8 deposited as
ATCC 9119) cultured in serum free medium.
408
UTR Clone Generation;
UTRtech; Cell Factories;
Gaussia luciferase
signal peptides - Mabs
supersecretion; CHO cells
UniTargetingResearch AS - Licensor, primary
University of Barcelona - R&D
University of Bergen - R&D
University of Newcastle-upon-Tyne - R&D
Description: Signal peptide 3UTRs and other
powerful protein targeting and trafficking se-
quences derived from Gaussia luciferase (IV)
provide increased intracellular protein and anti-
body expression in CHO and other mammalian
cells. Using this method of inserting appropriate
targeting elements, both the efficiency of protein
synthesis and its secretion can be significantly en-
hanced. A seamless cloning method is provided
that does away with linkers that could affect the
functionality of the signals and/or encoded pro-
tein. Targeting elements are easily incorporated
into various vectors and cell systems to create
high producer clones. The localization of mRNAs
to specific ER sub-domains facilitates protein
targeting within the endomembrane system.
This approach has demonstrated yield im-
provements 30% - 60% above those obtained in
highly optimized industrial cell culture systems.
Increased productivities of up to 50-fold have
been observed using model proteins secreted by
recombinant cells. UTRtech can bring improve-
ments incremental to results previously achieved
with other high expression approaches, such as
modulation of cell growth conditions or transcrip-
tional enhancement. Researchers at UTR have
demonstrated that different signal peptides vary
greatly in the efficiency with which they direct
secretion of a reporter protein, with one particular
class of signal peptides being surprisingly effi-
cient. (The efficiency also varies depending on the
type of protein used.)
Use with: CHO cells; mammalian cells
ies
Patents: Related patents include WO2005001099,
PROTEIN EXPRESSION SYSTEM, HES-
KETH, et al., assigned to UniTargetingResearch
AS and the inventors, with claims including the
signal peptides from Guassia princeps or Vargula
hilgendorfii luciferase.
Availability: UniTargetingResearch works with
companies to customize solutions for protein pro-
duction. UTR works with customers to determine
the best set of these key elements for the particu-
lar protein of interest and then genetically modi-
fies cells to optimize the protein production. Kits
are marketed for non-commercial use by Japan
EnviroChemicals (Osaka and Paracelsian (Ithaca,
NY) and Abraxis Kits (Warminster, PA).
Licensing information: Contact UniTargetin-
gResearch AS. The company commercializes
technology from an EU-funded project between
Profs. Ian Pryme (University of Bergen), John
Hesketh (University of Newcastle-upon-Tyne;
Tyne, U.K.), and Albert Tauler (University of
Barcelona).
This technology has or is being used by com-
panies including UCB-Celltech, Angel Biotech-
nology Plc, Selexis SA and Inovio Biomedical
Corp. (for gene therapy).
409
DNA microinjection -
transgenic animals, chickens
Roslin Institute - Licensor, primary
Description: DNA microinjection into the cyto-
262
plasm of the germinal disk is a commonly-used
method for generation of transgenic animals,
e.g., chickens. The nucleus of a somatic cell is
transferred into a germ cell. In chickens, the chick
zygotes are removed from the oviduct of laying
hens before the first cleavage division, transferred
to surrogate shells, manipulated, injected and
cultured through to hatching. Using these meth-
ods, the Roslin scientists achieved a world first
in creating birds that breed true, meaning the
added human genes are passed on from generation
to generation. This enables creation of indus-
trial-scale flocks and offers a potentially unlim-
ited cheap source of recombinant proteins. ISA
Browns, a common breed of egg-laying hen, have
each had human genes added to their DNA to
enable them to produce complex human proteins
that are secreted into the whites of the birds eggs,
from which the proteins can be easily extracted.
Other technologies, e.g., nuclear transplanta-
tion (see related entry) have largely replaced this
for large-scale commercial protein manufacture.
Use with: animals; chickens
nal antibodies
Licensing information: Like other transgenic
animal technologies developed by Roslin Insti-
tute, this had been licensed to Viragen, but has
been returned to Roslin. Contact Roslin concern-
ing commercial uses and licensing.
HEK-293 cells
410
293ST-3F6 cell line; HEK-293
adapted to SFM
sor, primary
Description: See the HEK293 cell line entry.
NRC- BRI has developed a HEK-293 cell line
adapted to suspension and serum-free media
which grows at high cell density. See also a
related NRC-BRI entry concerning an HEK-293
expression system.
Benefits: Protein production at higher volume,
purity and biological activity.
regarding licensing. This invention has apparently
been licensed by one or more companies.
411
CRE-inducible expression;
cyclic AMP response elements
(CREs) - HEK-293 cells
sor, primary
Description: The CRE-inducible expression
system including use of cyclic AMP inducible/
sensitive promoters, derived from vasointestinal
peptide (VIP) promoter, useful in HEK-293 cells,
provides expression levels over 50% higher than
using a CMV promoter. The expression system
has the advantage of having a low basal level and
high induced level.
The cAMP inducible promoter consist of mul-
tiple cyclic AMP response elements (CREs) up-
stream of a fragment of the vasointestinal peptide
(VIP) promoter and the sequence for the desired
gene. The CRE sequence used is derived from the
vasointestinal peptide (VIP) promoter (see J. Biol.
Chem., 1987, 262:8743 and Proc. Natl. Acad. Sci.
USA, 1988, 85:6662). The cyclic AMP inducible
promoter comprises between 1 and 9 CREs.
Use with: HEK-293 cells; other mammalian
cells
nal antibodies
Background: The second messenger cyclic ad-
enosine monophosphate (cAMP) can induce tran-
scription by activating transcription factors acting
through cAMP-responsive elements (CREs) found
263
in various gene promoters.
6,596,508, CRE-inducible expression system,
Durocher, July 22, 2003. Claim 1 states: A
recombinant human embryonic kidney 293 cell
(HEK293) transfected with a DNA construct for
the expression of a desired gene, wherein said
construct comprises a vasointestinal peptide pro-
moter (VIP) fragment consisting of residue 230
to 521 of SEQ ID NO:3 and at least 6 additional
cyclic AMP response elements (CREs), said pro-
moter being operably linked to a DNA sequence
comprising the coding region of the desired gene,
and wherein said expression of the desired gene is
inducible to a level of activity of at least 50% of
that obtained by a control CMV promoter.
Licensing information: Contact NRC- BRI.
412
HEK-293 expression
Ecole Polytechnique Fdrale de Lausanne
(EPFL) - Licensor, primary
ExcellGene S.A. - Licensor, secondary
Description: See the other HEK-293 entries. An
improved HEK293 cell line has been developed
for transient expression and optimized for rapid
growth and high level protein synthesis in suspen-
sion culture. ExcellGene claims to be able to go
From gene to product in days. The system has
been used at scales up to 100 L.
Licensing information: Developed and available
for licensing from Ecole Polytechnique Fdrale
de Lausanne (EPFL). Related commercial cell
line development and other CMO services are
available from ExcellGene S.A.
413
HEK-293 expression
Kemp Biotechnologies, Inc. (KBI)- Licensor,
primary
Description: Kemp Biotechnologies reports
having developed a method for the cost-effective
production of recombinant proteins using tran-
sient transfection methods in HEK-293 cells, and
offers related CMO services. Coupling a novel
transfection reagent with stirred-tank bioreactor
technology, KBI can produce 1 to 1000 milli-
grams of recombinant protein in as little as 4 to 6
weeks. Protein expression is monitored in a pilot
experiment and during the bioreactor process to
ensure optimal results.
After receiving a plasmid or glycerol stock
containing the expression plasmid of interest,
Kemp produces a sufficient quantity of low-en-
dotoxin plasmid to perform a 2, 10, or 100 liter
transient transfection using HEK-293, HEK-293E,
or HEK-293T cells. The transient expression is
performed in a stirred-tank bioreactor using a
protocol developed by KBI. The cell pellet and/or
culture supernatant are harvested and shipped to
the customer or held at KBI for purification.
ies
Licensing information: Primarily available as a
CRO/CMO service. Contact KBI.
414
pTT vectors for HEK-293E cells
sor, primary
Description: PTT vectors provides enhanced
production of recombinant proteins by transient
transfection of suspension-growing mammalian
cells, particularly HEK-293E cells (stably ex-
pressing theEBNA1 protein or a fragment; see
264
related entry). This is an efficient, more economi-
cal system for transient transfection and recom-
binant protein production, allowing higher yields
to be attained. pTT vector, an oriP-based vector
has an improved cytomegalovirus (CMV) expres-
sion cassette. Using this vector, 10- and 3-fold
increases in SEAP expression have been reported
in 293E cells (HEK-293E cells; see related entry),
compared with pcDNA3.1 and pCEP4 vectors,
respectively. Also, intracellular proteins have been
reported to be expressed at levels as high as 50
mg/l, representing up to 20% of total cell proteins.
Patents: pTT vectors are disclosed in, EN-
HANCED PRODUCTION OF RECOMBINANT
PROTEINS BY TRANSIENT TRANSFECTION
OF SUSPENSION-GROWING MAMMALIAN
CELLS, Durocher, Y., et al., assigned to the Na-
tional Research Council of Canada.
Licensing information: Contact the Biotechnol-
ogy Research Institute, National Research Council
of Canada (NRC- BRI).
NRC-BRI reported in 2005, Five new licenc-
es for the pTT Vectors technology have been
granted to large pharmaceutical firms, bringing
to 11 the total number of licences issued for this
technology.
Further info.: High-level and high-through-
put recombinant protein production by transient
transfection of suspension-growing human 293-
EBNA1 cells, Nucleic Acids Research, 2002,
Vol. 30, No. 2 e9
Plants
415
Magnifection; magnICON;
Transgene Operating System
(TOS) - antibodies; plants and
plant cells
Icon Genetics AG - Licensor, primary
Mapp Biopharmaceutical, Inc. -
Description: Magnifection (magnICON) enables
transient (no permanent gene alteration) expres-
sion of mammalian monoclonal antibodies in
plants and plant cells. This simplified system, es-
pecifically useful for antibodies), does not require
an integrase or recombination. A first generation
platform provides fast (1-2 weeks), high-yield (up
to 5 g per kilogram fresh leaf weight) production
of biopharmaceuticals based on pro-viral gene
amplification in a non-food host.
The process relies on synchronous coinfection
and coreplication of two viral vectors, e.g., each
expressing a separate antibody chain. Tobacco
mosaic virus (TMV) and PVX transcripts, derived
from their respective provectors containing pro-
moter, recombination, and processing elements,
coexist in the cells of Nicotiana benthamiana
(tobacco) and efficiently transcribe their respec-
tive nonviral genes, i.e., the heavy chain (HC) and
light chain (LC) of an antibody. The two vectors
are derived from two different plant viruses that
were found to be noncompeting. Unlike vec-
tors derived from the same virus, noncompeting
vectors effectively coexpress the heavy and light
chains in the same cell throughout the plant body,
resulting in yields of up to 0.5 g of assembled
Mabs per kg of fresh-leaf biomass.
Plant cells expresing full length, assembled
Mabs may be rapidly developed (i.e., 14 days
from gene delivery to harvested plant cells, with
grams of Mabs in 14-20 days). Even in this
early stage of development, the approach easily
can accommodate gram-level production [and]...
the shortened time frame to gram-level produc-
tion described in this article may accelerate this
process greatly. Moreover, the ability to increase
the volume of mAb-containing biomass does not
appear to involve any change in growing condi-
tions, infection procedures, or equipment. Finally,
concerns about transgenic containment and envi-
ronmental contamination are vastly minimized,
because optimum growth conditions appear to be
in controlled growth rooms and there are no trans-
genic seeds or viable transgenic plants resulting
265
from the process.
In one example (see citation below), human
growth hormone (hGH; somatropin) was pro-
duced with yield of up to 10% of total soluble
protein or 100 mg per gram of fresh weight leaf
biomass.
Use with: plant cells; plants; tobacco
nal antibodies
Background: In general, a serious limitation
affecting transfecton of genes into plant cells has
been the size of the foreign gene that could be
accommodated by Agrobacterium. magnICON
improved viral vectors described are delivered in
Agrobacterium, allowing expression of transcripts
>1,500 bases, thus permitting Mab expression.
Benefits: Magnifection combines advantages of
three biological systems: the vector efficiency and
efficient systemic DNA delivery of Agrobacte-
rium, the speed and expression level/yield of plant
RNA viruses, as well as the post-translational
capabilities and low production costs of a plant.
Icon provides an attractive solution that combines
in one platform low cost, versatility and scalabil-
ity of plant production systems with the benefits
that were earlier available only either through
microbial fermentation (yield and speed) or
through mammalian cell culture (posttranslational
modification).
Patents: Related international patents/applica-
tions include:
WO 06/079546,2006-08-03
Production of hetero-oligomeric proteins in plants
WO 05/054478,2005-06-16
Controlling Gene Expression in Plastids
WO 05/049839,2005-06-02
RNA-Virus-Derived Plant Expression System
WO 04/108934,2004-12-16
Safe Production of a Product of Interest in Hybrid
Seed
WO 04/101797,2004-11-25
Process of Producing a Plastid-Targeted Protein in
Plant Cells
WO04/067748,2004-08-12
Plant Transformation with in vivo Assembly of a
Trait
WO04/067749,2004-08-12
Plant Transformation with in vivo Assembly of a
Sequence of Interest
WO 04/046359,2004-06-03
Method of Controlling Processes in Plants or
Plant Cells
WO 04/046360,2004-06-03
Method of Controlling Cellular Processes in
Plants
WO 04/046361,2004-06-03
Method of Controlling a Cellular Process in a
Multi-Cellular Organism
WO 04/015115,2004-02-19
Plastid Transformation Using Modular Vectors
WO 03/102197,2003-03-21
Transgenic Plants with Controlled Distribution of
a Trait to Progeny
WO 03/020927,2003-03-13
Creation of Artificial Internal Ribosome Entry
Site (IRES) Elements
WO 03/020928,2003-03-13
Identification of Eukaryotic Internal Ribosome
Entry Site (IRES) Elements
WO 03/020938,2003-03-13
Method of Protein Production in Plants
WO 03/012035,2003-02-13
Commercial Use of Arabidopsis for Production of
Human and Animal Therapeutic and Diagnostic
Proteins
WO 03/004658,2003-01-16
Gene Expression in Plastids Based on Replicating
Vectors
WO 03/001900,2003-01-09
Process of Controlled Shuffling of Chromosome
Fragments for Plant Breeding
WO 02/101006 ,2002-12-19
Production of Proteins in Plants
WO 02/101060 ,2002-12-19
Processes and Vectors for Producing Transgenic
Plants
WO 02/096192,2002-12-05
Process of Producing Environmentally Safe
Transgenic Organisms
266
WO 02/097080 ,2002-12-05
Amplification Vectors Based on Trans-Splicing
WO 02/088369,2002-11-07
Processes and Vectors for Amplification or Ex-
pression of Nucleic Acid Sequences of Interest in
Plants
WO 02/083867 ,2002-10-24
IRES Enabled Gene Trapping in Plants
WO 02/079481,2002-10-10
Method of Encoding Information in Nucleic Acids
of a Genetically Engineered Organism
WO 02/077246,2002-10-03
Site-targeted Transformation Using Amplification
Vectors
WO 02/068927,2002-09-06
Using Viruses to Detect or Purify Proteins
WO 02/068664,2002-09-06
Recombinant Viral Switches for the Control of
Gene Expression in Plants
WO 02/057466,2002-07-25
Processes and Vectors for Plastid Transformation
of Higher Plants
WO 02/055651 ,2002-07-18
Processes and Vectors for Plastid Transformation
WO 02/46440,2002-06-13
Processes and Vector Systems for Producing
Transgenic Plants
WO 02/29068,2002-04-11
Vector Systems for Plants
WO 01/70939 ,2001-09-27
Methods for Transforming Plant Plastids and
Making Transplastomic Plants
Licensing information: Contact ICON/Bayer.
Further info.:
Rapid high-yield expression of full-size IgG
antibodies in plants coinfected with noncom-
peting viral vectors, Giritch A, Marillonnet S,
Engler C, van Eldik G, Botterman J, Klimyuk V,
Gleba Y., Proc Natl Acad Sci U S A. 2006 Oct
3;103(40):14645-6.
Magnifection--a new platform for expressing re-
combinant vaccines in plants, Gleba Y, Klimyuk
V, Marillonnet S., Vaccine. 2005 Mar 18;23(17-
18):2042-8.
High-yield production of authentic human
growth hormone using a plant virus-based expres-
sion system, Plant Biotechnology Journal 3 (6),
613-62.
Plant Biotechnology Journal 3 (6), 613-620.
416
MARs PLUS; Matrix attachment
regions PLUS - plants
Rega Institute, Katholieke Universiteit Leuven
- Assignee, patent
Description: MARs Plus is an Extreme high-
level protein expression platform for plants now
adapted for use in non-mutant plants. Matrix
attachment regions (MARs; see related entry)
are genetic tools increasing not only expression
levels of transgenes in plants but also the stabil-
ity of their expression. Previous work on MARs
has yielded mixed results. Plant transforming
standard expression vectors flanked with MARs
in which post transcriptional gene silencing has
been blocked leads to very dramatic increases in
expression levels. The MARs PLUS technology,
using RdRP mutants (see related entry) with ma-
trix attachment regions, increases protein levels
on average 35-fold with some cell lines reaching
a 100-fold increase. The MARs PLUS technol-
ogy can accompany Plant Bioscience Ltd.s RdRP
technology, which can be used to provide the
blocking of post-transcriptional gene silencing.
Benefits: Advantages of the MARs Plus system
include:
Extreme high level expression using standard
plant expression constructs and nuclear transfor-
mation
Amongst the very highest level plant expres-
sion systems reported to date
Applicable to all plants through use of selec-
tively silencing RDR6
Uniformity of high level expression in differ-
ent transgenic events

267
Patents: Patent applications include WO
2005/054483, ENHANCED EXPRESSION,
CAMMUE, B.P., et al., with abstract, Disclosed
herein are methods and means of achieving en-
hanced expression of a target nucleotide sequence
in a transgenic organism, which methods com-
prise the steps of: (i) providing an organism in
which post-transcriptional gene silencing (PTGS)
is suppressed, (ii) associating said target nucleo-
tide sequence with one or more heterologous Ma-
trix Attachment Region (MARs), and (iii) causing
or permitting expression from the target nucleo-
tide sequence in the organism. Unexpectedly,
the MARs do not merely relieve gene silencing,
but can actually lead to expression levels higher
than can be achieved in wild-type organisms and
higher than expression levels in organisms in
which PTGS is suppressed but where the MARs
are not employed.
Ltd. (Dr Lars von Borcke; lars@pbltechnology.
com)
Further info.:
Butaye, K.M.J. et al. (2004) Stable high-level
transgene expression in Arabidopsis thaliana us-
ing gene silencing mutants and matrix attachment
regions. The Plant Journal 39: 440-449.
Sels, J., et al. (2007) Use of a PTGS-MAR ex-
pression system for efficient in planta production
of bioactive Arabidopsis thaliana plant defensins
Transgenic Research 16: 531-538.
De Bolle, M.F., et al (2007) The influence of ma-
trix attachment regions on transgene, Plant Mo-
lecular Biology, Volume 63, Number 4 / March,
2007, 533-543.
417
Concert Plant-Cell-Produced
system - tobacco plant cell
culture
Dow AgroSciences LLC - Uses, currently in
R&D
Description: Not technically an expression
system or genetic engineering technology, the
Concert Plant-Cell-Produced System involves
large-scale culture of genetically-modified plant
cells in large steel fermenters currently being used
for vaccine manufacture and development.
Dow Agrosceinces claims, Concert Plant-
Cell-Produced vaccines even eliminate the envi-
ronmental concerns associated with other plant-
made methods because the Concert System uses
only plant cells - not the whole plant - to produce
vaccines. The plant cells are grown in a culture
medium comprised primarily of sugar, salt and
water. No seeds are planted, no whole plants are
grown and no pollen is produced, eliminating con-
cerns about transfer of seed, pollen or plant parts
into the environment. Instead, Dow AgroSciences
plant-cell-produced vaccines are grown in stain-
less steel tanks in a bio-contained facility. Addi-
tionally, these cells are non-propogative, meaning
they cannot grow outside the culture medium.
Use with: plant cells
Licensing information: Contact Dow AgroSci-
ences.
Products made using this tech.: Dow manufac-
tures a USDA-approved recombinant Newcastle
disease vaccine for prevention of this disease in
poultry using Concert. On January 26, 2006, Dow
AgroSciences received the first regulatory ap-
proval for a plant-made vaccine.
268
418
Phyton plant cell fermentation
Phyton Biotech - Licensor, primary
Description: Phyton has developed technologies
for large-scale plant cell culture manufacture of
recombinant proteins and small molecules (sec-
ondary metabolites). One of the key attributes of
Phytons cGMP plant cell fermentation technol-
ogy is that it can be scaled up to any size needed.
Phyton owns and operates the worlds largest
cGMP plant cell culture facility with bioreactors
up to 75,000 L in size and has the only regulatory
approved (in all major markets, including the US,
Europe and Japan) plant cell culture process for
the production of a pharmaceutical product (pacli-
taxel, the active agent in Bristol-Myers Squibbs
TAXOL).
Use with: plant cells
Patents: Advantages of Phyton Biotechs plant
cell culture technology include:
Higher yields of correctly folded, post-transala-
tionally modified, and functional proteins.
Certified cell banks for long-term production.
Completely contained cGMP production.
Proven scalability to meet demand.
Licensing information: Contact Phyton, which
offers CMO services.
Phyton has exclusively licensed technology for
glycosylation of plant-expressed proteins from
Dow Chemical, and plans to further develop this
through a research collaboration with the Dutch
research institute Plant Research International
(PRI), part of Wageningen University & Research
Centre, one of the innovators of the technology
platform.
Phyton is developing a plant-based method for
manufacture of IPLEX [Mecasermin rinfibate -
SomatoKine; insulin-like growth factor I--insulin-
like growth factor-binding-3 protein complex, Re-
combinant; IGF-1/IGFBP3 complex], a currently
marketed product from manufactured by Insmed
(as Avecia as CMO) and marketed by Insmed.
419
ExpressTec expression;
ExpressPro; ExpressMab - rice
and barley; antibodies
Ventria Bioscience - Licensor, primary
Description: ExpressTec provides high-level
protein, including antibodies, expression using the
self-pollinating crops of rice and barley. Recom-
binant protein expression at up to 1% of the rice
grains weight has been attained. For many can-
didate proteins, the expression level is between
0.1 and 1.0% of grain weight (10g/kg with brown
rice). Such high expression levels, coupled with
the large-scale production of cereal grains, could
make the transgenic production of truly large
volumes of recombinant proteins and peptides a
reality.
Ventria has used the ExpressTec production
system as a basis for forming more specialized
systems which produce specific molecules. These
platforms include ExpressPro for production of
proteins, ExpressTide for production of peptides
and ExpressMab for production of monoclonal
antibodies ExpressTec has proven its versatility
using these platforms to express the following
range of molecules: large molecules (larger than
50 kD); small molecules (less than 10 kD); mul-
tiple sub-unit molecules (e.g. fibrinogen); struc-
tural/functional proteins or enzymes; and products
resulting from metabolic engineering.
Use with: rice; barley
ies
Benefits: The system offers several advantages:
high and stable expression levels of recombinant
proteins, tissue-specific expression in the grain
endosperm, rapid scalability to metric-ton quanti-
ties, prevention of gene flow with self-pollinat-
ing crops, low capital investment and production
costs, and efficient processing and recovery.
- Proven scalability for large volumes of recom-
binant proteins/peptides with minimal capital
269
investment compared with other systems.
- Free of infectious or toxic contaminants.
- Self-pollinating crops provide a safe and closed
production system.
- Efficient supply management - the protein/pep-
tide can be stored in its lowest cost form for
multiple years.
- Utilizes a proven production and processing
infrastructure.
Patents: Ventria owns product and enabling tech-
nology rights from its internal development effort
and by license, assignment, or exclusive option
agreements as follows: 5 issued United States pat-
ents relating to protein expression and products; 4
foreign patents relating to protein expression and
products; over 10 filings relating to ExpressTec;
and over 10 filings for products, their formula-
tions, and uses.
Licensing information: Contact Ventria Biosci-
ence.
Ventria currently manufacttures lactoferrin and
lysozyme using this system.
Further info.:
High-Level Protein Expression System Uses Self-
Pollinating Crops As Hosts , BioProcess Interna-
tional, January 2004, Volume: 2, Number: 1: pp
54-59.
Insects
420
AcMNPV p35 apoptosis
inhibition; Sf9P35AcV5-1 and
Sf9P35AcV5-3 - insect cells,
apoptosis resistance
- Licensor, primary
Description: Sf9-derived cells, Sf9P35AcV5-1
and Sf9P35AcV5-3, express genes that encode
suppressors of apoptosis (SA), particularly Ac-
MNPV p35, conferring increased resistance to
apoptosis or programmed cell death, and express
certain recombinant proteins at increased levels.
These cell lines also have increased resistance to
many types of stress. The baculovirus P35 protein
is a caspase inhibitor that prevents the induction
of apoptosis during infection of Sf21 cells by
Autographa californica multicapsid nucleopoly-
hedrovirus (AcMNPV). P35 also inhibits the
induction of apoptosis in a broad range of cells
and circumstances. Increased levels of protein
secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3
cells result from a prolonged infection cycle and
accumulation of the secreted glycoprotein. The
cell lines are resistant to both exposure to inducers
of apoptosis, such as actinomycin D, and nutrient
deprivation.
Because some of the SA proteins inhibit apop-
tosis in a wide spectrum of organisms, these genes
may be inserted into other plant or animal cell
lines for a variety of purposes involving resistance
to apoptosis or resistance to stress.
Use with: insect cells; universal (generic)
Benefits: Cell live longer and make more protein.
WO/2001/066696, STABLE CELL LINES
RESISTANT TO APOPTOSIS AND NUTRI-
ENT STRESS AND METHODS OF MAKING
SAME, assigned to Boyce Thompson Institute
for Plant Research.
Thompson Institute for Plant Research regarding
commercial use, including nonexclusive licens-
ing. Reported to be under MTA evaluation by two
companies.
Further information: Stable cell lines express-
ing baculovirus P35: resistance to apoptosis
and nutrient stress, and increased glycoprotein
secretion., In Vitro Cell Dev Biol Anim. 2001
May;37(5):293-302.
270
421
AF 99 insect cell line
Expression Systems LLC - Licensor, primary
Description: Expression Systems LLC, primar-
ily a CRO/CMO, offers the AF 99 insect cell line
capable of culture in serum-free media and more
robust growth than the High Five cell line. AF 99
was established with embryonic tissue never ex-
posed to serum. It grows faster, it produces more,
and it never saw any serum.
Benefits: no bovine spongiform encephalitis
(BSE) concerns
Licensing information: Contact Expression
Systems LLC.
422
BL-Sf-21AE-Cl 3 cell line -
Insect cell lines, baculovirus
hosts
Dept. of Agriculture (USDA)
Description: Spodoptera fugiperda insect cell
lines, including I IBL-Sf-21AE-Cl 3 (ATCC
PTA-2207), are useful with baculovirus expres-
sion systems (BEVS). The cells produce large
quantities of baculovirus and large quantities of
expressed functional protein. Compared to pres-
ently available technology, these cloned cell lines
economically produce high yields of baculovirus
and large quantities of recombinant proteins, and
can grow in serum free medium. The cell lines
tolerate the expression of a wide range of recom-
binant proteins including known toxins.
Clones are derived from single cells obtained
from the insect cell line, IPLB-Sf-21-AE, origi-
nating from Spodoptera fugiperda. The clones
were obtained by picking isolated colonies from
agarose plates. Isolation was repeated three times
for each clone.
Selected Insect Cell Line Clones Providing In-
creased Yield of Baculoviruses and Gene Expres-
sion Products from Recombinant Baculoviruses,
April 30, 2002, assigned to U.S. Dept. of Agricul-
ture (USDA).
Availability: Available from ATCC for non-com-
mercial uses (ATCC PTA-2207).
Licensing: Contact ARS, USDA, regarding com-
mercial uses and licensing..
423
Cre/loxP Recombination-
Mediated Cassette Exchange
(Cre/loxP RMCE) - Drosophila
(mosquitos)
DuPont Corp. - Licensor, primary
BestGene Inc. - Contract services
New York University - R&D
Description: This is a modification of the Cre-
Lox system (see related entry). Cre/loxP RMCE
enables site-specific transgenesis in Drosophila
(mosquitos). RMCE transfers transgenes into
confined genomic locations. Cre recombinase
and lox sites can be used to replace the traditional
P-element system of Cre-Lox, so that insertion of
donor sequence is no longer random. Transgenic
lines at known genomic location can be created
without any position effect.
The service provided by BestGene using this
technology is similar to the P-element transforma-
tion. Donor plasmid is injected to landing site
into one or several cells, with injected larvae
provided to the client; or plasmid may be injected
into the stable lines or balanced lines.
Use with: Drosophila
Availability: Contact BestGene regarding con-
tract services, or the inventor at NYU:
Prof. Stephen Small
Associate Professor of Biology
271
New York University
Department of Biology
1009 Silver Building
100 Washington Square East
New York, NY 10012
Tel: +1-212-998-8244
Fax: +1-212-995-4015.
424
Drosophila expression
Carnegie Institution of Washington - Licensor,
primary
Description: An early Drosophila expression sys-
tem, now apparently superceded by the Drosoph-
ila Expression System (DES; see related entry),
was originally patented by the Carnegie Institu-
tion of Washington in 1987, with this technology
now off-patent and in the public domain
Use with: Drosophila
Patents: The exemplary patent is 4,670,388,
Method of incorporating DNA into genome of
drosophila, assigned to the Carnegie Institution
of Washington.
425
Drosophila melanogaster S2
cells; Drosophila-SFM.D.Mel-2
Cells; Schneider S2 Drosophila
cells; S2 cells, SFM - insect
cell culture
Life Technologies, Inc. - Licensor, primary
Description: This cell line is commonly used
with the Drosophila Expression System (DES),
a mosquito cell culture expression system (see
related entry). These cells are adapted to serum-
free media for the transient or stable expression of
Use with: Drosophila; insect cells
Background: The cells were isolated from
late-stage Drosophila melanogaster embryos and
adapted to suspension growth in Drosophila-SFM
media.
Patents: See the Drosophila Expression System
(DES) entry
Availability: Invitrogen markets S2 for non-com-
mercial use.
Licensing information: See the Drosophila Ex-
pression System (DES) entry.
426
IE-1 (BmNPV 1.2 kb fragment)
promoters; Bombyx mori actin
promoters - insect cells
DSM Biologics - Licensor, primary
New England Biolabs - Licensor, primary
Description: The yeast Kluyveromyces lactis is
useful as host cells for high yield recombinant
protein expression. K. lactis technology offers
high yield protein expression; rapid high cell den-
sity growth; can clone and express genes toxic to
E. coli; competent K. lactis cells are available; no
expensive antibiotics or methanol required; and
easy-to-use protocols for those inexperienced with
yeast systems. Acetamidase (amdS) expressed
from the pKLAC1 vector permits transformed
cells to utilize acetamide as a sole nitrogen source
on defined medium, allowing for simple positive
selection of transformed cells. Acetamide selec-
tion also promotes formation of cells containing
multiple integrations of pKLAC1 which results in
higher yields of protein. The technology includes
the K. lactis GG799 strain, an industrial isolate
that has no auxotrophies, rapidly grows to high
cell density, and efficiently secretes heterologous
proteins; pKLAC1 Vector; and pKLAC1-malE
Control Plasmid.
Proteins may be produced intracellularly or be
secreted using the integrative expression vector
pKLAC1. To achieve protein secretion, a gene
of interest is cloned downstream of the K. lactis
alpha-mating factor secretion domain, which is
272
eventually processed in the Golgi resulting in
secretion of the desired protein.
Use with: Kluyveromyces lactis (yeast)
Benefits: The K. lactis expression system offers
several advantages over other yeast and bacterial
protein expression systems. First, K. lactis has
been used to produce proteins at industrial scale in
the food industry for over a decade due to its abil-
ity to rapidly achieve high culture densities and
abundantly produce recombinant proteins. Sec-
ond, yeast expression is driven by a variant of the
strong LAC4 promoter that has been modified to
lack background expression in E. coli. Therefore,
genes toxic to E. coli can be cloned into pKLAC1
in bacteria prior to their expression in yeast.
Third, highly competent K. lactis cells make the
technology easy-to-use for those not accustomed
to working with yeast. Their high transformation
efficiency makes the system suitable for methods
that require large numbers of transformants, for
example, expression cloning using cDNA librar-
ies. Finally, proteins expressed in K. lactis have
access to eukaryotic protein folding and glycosyl-
ation machinery that E. coli and prokaryotic cells
do not possess, making it an important alternative
to bacterial expression systems.
Method for construction and use of Kluyvero-
myces lactis promoter variants in K. lactis that
substantially lack E. coli transcriptional capabil-
ity, Taron, C., et al., June 24, 2008, assigned to
New England Biolabs, Inc. The abstract states,
Methods and compositions are provided relating
to production of recombinant protein in yeast. A
modified PLAC4 is described where one or more
mutations may be introduced into the Pribnow
box-like sequences in the promoter. The modi-
fied promoter when placed upstream of a target
gene in a vector causes a significant reduction of
target gene expression in transformed bacteria but
produces efficient expression of the target gene in
yeast.
Availability: Related reagents are marketed for
non-commercial uses by New England Biolabs,
Inc.
Licensing information: A license to use this
system for manufacture of clinical grade material
or commercial purposes is available from New
England Biolabs, Inc., or DSM Biologics.
Further info.:
Kluyveromyces lactis LAC4 Promoter Variants
That Lack Function in Bacteria but Retain Full
Function in K. lactis, Paul A. Colussi and Chris-
topher H. Taron, Appl Environ Microbiol. 2005
November; 71(11): 7092-7098 (doi: 10.1128/
AEM.71.11.7092-7098.2005).
427
Insect cell lines - baculovirus
hosts
Dept. of Agriculture (USDA) - Licensor, pri-
mary
Description: A portion of the genome of polydna-
virus of the parasitic wasp Glyptapanteles indien-
sis (G. indiensis) stably integrated into the ge-
nome of Lepidopteran and Coleopteran insect cell
lines provides transformed insect cell lines useful
with baculovirus expression systems (see related
entries). A portion of braconid G. indiensis polyd-
navirus genome (GiPDV) is capable of integration
into the insect host chromosome in vitro and is
stably maintained. These cell lines may be used
for stable transformation with foreign genes when
inserted with the polydnavirus model systems,
and or developing viral-based insect-transforming
vectors.
In addition to cells from its most important host
insect L. dispar (gypsy moth), also provided are
cells from three families (Lepidoptera, Coleop-
tera and Hymenoptera) which had been isolated
from five different tissue sources. These include
Spodoptera frugiperda, Trichoplusia ni, Plodia
interpunctella and Diabrotica undecimpunctata.
These insects do not normally encounter GiPDV
in nature, but their cells are apparently susceptible
to GiPDV infection in vitro, suggesting a very
broad potential host range. Used with: insect cells;
273
Lymantria dispar; Spodoptera frugiperda; Tricho-
plusia ni; Plodia interpunctella; and Diabrotica
undecimpunctata
Patents: Exemplary patents include Stable
insect virus-cell expression system, 6,143,565,
Dougherty, et al., Nov. 7, 2000, assigned to the
Agricultural Research Service (ARS), U.S. Dept.
of Agriculture (USDA).
Licensing information: Contact ARS, USDA.
428
Insect cells, per os (oral)
baculovirus infection
Chesapeake PERL, Inc. - Licensor, primary
- Assignee, patent
Description: Methods and and apparatus are pro-
vided for rearing insects (whole insects, not insect
cell culture), including per os (oral) infection
of insect larvae with pre-occluded cirus (POV),
e.g., baculovirus vectors without the polyhedrin
gene. This forms much of the basis for the unique
whole-insect expression technology offered by
Chesapeake PERL, Inc. (see related entry).
In the oral larvae infection method, a baculo-
virus vector is used which is highly efficient at
establishing infection and is normally destined to
become occluded within the polyhedrin or granu-
lin. This Pre-occluded Virus (POV) derived from
a polyhedrin-minus or granulin-minus (lacking a
functional polyhedrin or granulin gene) baculovi-
rus is fed to insect larvae per os resulting in high
infection rates. The discovery of the POV form of
polyhedrin-minus baculoviruses is essential to this
method of infecting insect larvae per os using the
POV form of polyhedrin-minus baculovirus.
The methods and apparatus consider both the
physical needs and the behavior of the insect.
Information concerning the physical and dietary
needs of the insect as well as behavioral charac-
teristics are utilized to maximize the number of
larvae reared per unit surface area of diet and per
unit of rearing area while minimizing the amount
of labor and materials required. An enclosed
rearing unit is provided which can be located
within an appropriate environment for rearing the
insects. Ther are three sections within the rearing
unit: 1) a diet space, 2) a larval space, and 3) a
frass space. The diet space includes an appropriate
diet medium for the insects. The larval space is lo-
cated below the diet space and includes a series of
vertical partitions perpendicular to and in contact
with or nearly in contact with the diet medium
such that the insect larvae are able to disperse
themselves over the partitions. The frass space is
located below the larval space such that any frass
collects within the frass space as it is produced
and does not interfere with the larval space or the
diet space. The rearing system can further include
an emergence pan including an outlet for allowing
emerging adults to enter an oviposition cage. The
emergence pan replaces the frass collection pan
when all of the larvae have pupated. The rearing
unit is turned upside down such that the emer-
gence pan is located above the larval space and
the diet
space.
Use with: insect larvae
Benefits: Insect larvae can be reared at much
higher densities on considerably less diet with
better control of moisture difficulties and less
manual labor.
Patents: This is covered by patents assigned to
Boyce Thompson Institute for Plant Research and
exclusively licensed to Chesapeake PERL, Inc.
U.S. 5,351,643, High density rearing system for
larvae, Hughes, Oct. 4, 1994, covers insect rearing
methods and apparatus.
Licensing information: Contact Chesapeake
PERL, Inc. and/or Boyce Thompson Institute for
Plant Research.
274
429
Lymantria diapar
nucleopolyhedrovirus and
L. dispar 652Y (Ld652Y) cell
lines- baculovirus host cells
primary
Description: Lymantria diapar nucleopolyhedro-
virus and L. dispar 652Y cell lines are useful for
production of recombinant proteins in baculovirus
expression systems (BEVS). These cell lines pro-
duce foreign proteins in relatively high amounts
and that are non-allergenic has been discovered.
This provides an ability to generate both therapeu-
tic proteins and proteins for research purposes .
The potentially allergenic glycosylation
modification of alpha 1,3 fucosylation that is
common for other high expressing insect cells,
such as Trichoplusia ni, does not occur with this
system. In addition, the system appears to offer
much higher yields than other common insect cell
expression systems such as Spodoptera frugi-
perda. The L. dispar cells lacks the ability to add
GlcNAc, Gal, and Sialic Acid, (important glyco-
sylation events in mammalian cells) as do other
insect cell protein expression systems. However,
mammalian glycosylation can be achieved using
genetic modifications of the original host cell line
(see related entries). See the related insect cells
glycosylation technology from Johns Hopkins
Univ.
Benefits: These cell lines offer the potential
to produce high yields of fully-mammalianized
glycoproteins in an insect cell expression system,
the best combination of properties in a protein
expression system.
Patents: Patent applications by Slavicek & Beten-
baugh, JHU, are reported to be pending. Appar-
ently, these are new such that they have not yet
been published (could not be retrieved).
University (JHU).
430
pIEx baculovirus vectors; hr5
enhancer; ie1 immediate early
promoter - insect cells; avoid
baculovirus pathogenicity
primary
Novagen - Retail seller
Description: The pIEx series of vectors, e.g.,
pIE1, are designed for protein expression by
transient transfection of Spodoptera-derived insect
cells. To drive target gene expression, the vectors
employ an optimal combination of two transcrip-
tion elements derived from AcNPV baculovirus
-- the hr5 enhancer and the ie1 immediate early
promoter. Recombinants are constructed and used
with the same procedures as the classical polyhe-
drin-based vectors, except that foreign genes are
expressed much earlier in infection (e.g., begin-
ning at 4 hours vs. 24 hours).
This promoter/enhancer combination recruits
endogenous insect cell transcription machinery,
avoiding the need for using baculovirus and the
cytopathic effects associated with infection. The
vectors are designed with the option of produc-
ing unfused proteins or fusions with a variety of
tags, e.g, to facilitate purification. The vectors
also carry a high-copy pUC replicon to facilitate
the preparation of high-quality plasmid DNA for
transfection. The vectors can also be used for the
generation of drug-resistant stable cell lines by
cotransfection with antibiotic-resistance gene-
bearing vectors,e.g., with pIE1-neo selection
plasmid. The pIEx vectors contain a wide array
of restriction sites including an Nco I site that
provides the option for production of unfused tar-
get proteins. The extensive multiple cloning site
(MCS) regions in these vectors facilitate tradi-
tional subcloning.
275
Benefits: These immediate early baculovirus
expression vectors allow expression of target
genes before virus-induced cytopathic effects
compromise the function of protein processing
pathways, particularly those involved in glyco-
sylation and secretion. Therefore, although lower
amounts of protein are produced, higher levels of
biologically active protein may be expressed from
ie1-based vectors than from polh-based vectors.
Other advantages include: continuous expression
with minimal degradation of recombinant protein
products; creation of virus that is highly infec-
tious in vivo and (because it is occlusion positive)
resistant to inactivation in nature and; expression
of the foreign gene product during the immediate
early phase of infection.
Patents: Patents assigned to Texas A&M Univer-
sity (TAMUS) include 5,077,214 and 5,162,222.
U.S. 5,077,214, Use of baculovirus early pro-
moters for expression of foreign genes in stably
transformed insect cells, Guarino, et al., Dec.
31, 1991, provides an alternative strategy for
baculovirus-mediated foreign gene expression: the
promoters from immediate-early or delayed-early
baculovirus viral genes were used to obtain the
continuous expression of foreign genes in sta-
bly-transformed insect cells. The IE1 or the 30K
promoters from Autographa californica nuclear
polyhedrosis virus can drive the continuous ex-
pression of a foreign gene in insect cells.
mercial uses by Novagen.
Licensing information: In June 1999, Micro-
GeneSys, Inc., now Proteins Sciences, Inc., and
Texas A&M University combined their intel-
lectual property and knowhow regarding baculo-
virus expression systems. Protein Sciences was
exclusively authorized to sublicense the combined
BEVS technologies to third parties.
431
Rhopalosiphum padi virus
Internal Ribosome Entry
Sequence (IRES); Picorna-like
virus IRES; Drosophila IRES -
insect cells; plant cells
National Health Research Institutes - Licensor,
primary
National Taiwan University - Licensor, primary
Plant BioScience Ltd. - Licensor, primary
Description: Several organizations have filed
patent applications or received patents similarly
claiming Rhopalosiphum padi virus and related
Picorna-like viruses (PnV) Internal Ribosome
Entry Sequence (IRES) vectors and their use in
insect (and also plant) cell culture expression
systems.
The efficient scale-up of recombinant pro-
tein production in insect cell bioreactors using
baculovirus expression vectors is hampered by
reductions in yield with increasing viral passage,
the so-called passage effect. This phenomenon is
characterized by the generation and subsequent
accumulation of defective interfering baculovi-
ruses (DIs), which interfere with the replication of
genomically intact virus.
Plant BioScience Ltd. has reported [Journal of
Biotechnology, Vol. 123, Issue 1, 3 May 2006,
Pages 13-21] a stabilized baculovirus vector
expressing a heterologous gene and GP64 from a
single bicistronic transcript. A novel baculovirus
expression vector was developed with a bicis-
tronic expression cassette that allows the simul-
taneous expression of the recombinant gene (first
cistron) and an essential baculovirus gene (GP64,
second cistron) from a single messenger RNA
(mRNA). The translation of GP64 is mediated by
an internal ribosome entry site (IRES) element
from Rhopalosiphum padi virus (RhPV), while
the native GP64 gene is deleted. In this way,
dominant selection pressure is placed on the entire
bicistronic mRNA and on the maintenance of the
foreign gene. The bicistronic expression vector
276
was superior to a control baculovirus vector in
that expression remained at much higher levels
upon continued virus passage.
The versatility of this stabilized vector has
been demonstrated by its ability to propagate in
a number of insect cell lines including Sf21, Sf9
and High Five cells (see related entries). These
baculovirus vectors were concluded to be espe-
cially valuable for large-scale recombinant protein
production in insect-cell bioreactors where the
number of viral passages is high. In a related
patent application, the company also claims much
the same technology is useful for plant cells (see
below).
Patent applications from the National Taiwan
University claim perina nuda Picorna-like virus
(PnV) with IRES activity and their use in insect
cells.
A U.S. patent from the National Health Re-
search Institutes (with different inventors from
those listed by the National Taiwan University)
claims recombinant protein expression in a wide
range of cell types using an IRES sequence from
the Drosophila labial (lab) gene, or a variant or
fragment thereof, or alternatively, a homolog of a
lab IRES, or a variant or fragment thereof. Meth-
ods of using the compositions are also described.
Use with: insect cells; plant cells
Background: Rhopalosiphum padi virus (RhPV)
is an insect virus which infects a narrow range
of aphid species of the Rhopalosiphum and
Schizaphis families. Based on sequence data, it
is considered to be a member of a group of insect
picorna-like viruses.
Patents: An exemplary patent application from
Plant Biosciences is WO/2002/012522, GENE
EXPRESSION FROM THE INTERNAL RIBO-
SOME ENTRY SITE (IRES) OF RHOPALO-
SIPHUM PADI VIRUS (RHPV), Roberts and
Belsham, with it abstract stating: Polynucleotide
sequence capable of directing translation initiation
in a plant expression system in a cap-independent
manner, wherein the polynucleotide sequence
directs translation initiation from an AUG codon.
In particular, the invention relates to a polynucle-
otide sequence which comprises the 5 UTR of
Rhopalosiphum padi virus (RhPV) which com-
prises an internal ribosome enty site (IRES).
An exemplary application from Chung Yuan
Christian University/National Taiwan Univer-
sity, 20070065924, Isolated polynucleotide
sequence with IRES activity, Wu, Tzong-Yuan,
et al., March 22, 2007, has an abstract stating:
Provided herein is an isolated polynucleotide
sequence with internal ribosome entry site (IRES)
activity, which directs translation initiation in an
insect expression system in a cap-independent
manner. In particular, the invention relates to an
isolated polynucleotide comprises the 5 UTR of
perina nuda Picorna-like virus (PnV) that pos-
sesses IRES activity. Methods of identifying a
polynucleotide with IRES activity and methods of
expressing at least two polypeptides in an insect
system are also disclosed herein.
Licensing information: Contact Plant Biosceinc-
es and the universities.
432
Tni PRO; Trichoplusia ni cell
line
Description: Tni PRO is a proprietary Tricho-
plusia ni host cell line useful with baculovirus
expression systems (BEVS). This cell line has a
peak cell density of 9-10 x106/ml. The popula-
tion doubling time is 21 hours. Expression levels
are competitive to other Tni cell types. Animal
products-free media can be used for manufacture
under GMP conditions. This cell line was derived
in ESF AF and has never been exposed to animal
or animal-derived media components. The exact
date of extraction/isolation and passage number is
known and documented.;
in bulk.
Use with: Trichoplusia ni cells
277
Licensing information: Contact Expression
Systems LLC, which offers related CRO/CMO
services.
433
BAC-TO-BAC Baculovirus
Expression System;
baculovirus shuttle vectors;
bacmids - insect vectors
produced in E. coli
Monsanto Corp. - Licensor, primary
Description: The BAC-TO-BAC Baculovirus
Expression System (as marketed by Invitrogen)
enables vector production in E. coli and is use-
ful for recombinant protein expression in insect
cells. This system is claimed to be unique in that
the generation of recombinant baculovirus relies
on site- specific transposition (between transfer
and expression vector) in E. coli, as opposed to
homologous recombination in insect host cells.
Transposition between a bacmid (a form of
baculovirus genome that replicates in E. coli),
e.g., bMON14272, and transfer vector in E. coli
DH10Bac produces a recombinant bacmid DNA.
The basis of this system is the pFASTBAC
transfer vector containing the AcNPV polyhedrin
promoter, with (pFASTBAC HT) or without a
polyhistidine encoding gene (see His tags entries),
and the bacmid-containing E. coli host, DH10Bac.
A dual-promoter transfer vector, pFASTBAC
Dual, is also available for coexpression of two
heterologous proteins, each under the control of
the polyhedrin and p10 promoters.
Method of producing recombinant eukaryotic
viruses in bacteria, Lee, et al., Sept. 20, 1994, as-
signed to Monsanto. The abstract states, A meth-
od for producing infectious recombinant baculovi-
ruses in bacteria is described. A novel baculovirus
shuttle vector (bacmid) was constructed that
contains a low-copy-number bacterial replicon,
a selectable drug resistance marker, and a pre-
ferred attachment site for a site-specific bacterial
transposon, inserted into a nonessential locus of
the baculovirus genome. This shuttle vector can
replicate in E. coli as a plasmid and is stably in-
herited and structurally stable after many genera-
tions of growth. Bacmid DNA isolated from E.
coli is infectious when introduced into susceptible
lepidopteran insect cells. DNA segments con-
taining a viral promoter driving expression of a
foreign gene in insect cells that are flanked by the
left and right ends of the site-specific transposon
can transpose to the attachment site in the bacmid
propagated in E. coli when transposition func-
tions are provided in trans by a helper plasmid.
The foreign gene is expressed when the resulting
composite bacmid is introduced into insect cells
Availability: Reagents and kits are marketed by
Invitrogen for non-commecial use.
Licensing information: Contact Monsanto re-
434
BacTen System; p10 promoter
vectors - insect cells
Institut National de la Recherche Agronomique
(INRA) - Licensor, primary
Description: The BacTen baculovirus expres-
sion system is useful for the generation of p10
promoter-based baculovirus expression vectors.
The inserted gene can be expressed in the ab-
sence of the highly produced viral proteins P10
and polyhedrin. The BacTen system has been
designed to permit efficient expression of foreign
proteins in insect cells. In BacTen baculovirus
vectors, the polyhedrin promoter has been de-
leted. Foreign genes are expressed from a single
strong late promoter construct in the absence
of the highly produced viral proteins P10 and
polyhedrin. With polyhedrin gene expression is
abolished, the foreign gene expression driven by
the p10 promoter is placed in a favorable context
278
for optimal production of the recombinant protein.
The p10 promoter has been shown to be activated
earlier in infection time courses than the polyhe-
drin promoter allowing for the earlier initiation
of expression of some recombinant proteins. In
BacTen-based recombinants, no P10 polypeptide
is produced. This delays the lysis of infected cells
and allows for a greater cumulative expression of
Benefits: Higher yield
Patents: This is apparently covered by patents
assigned to Institut National de la Recherche
Scientifique Agronomique, including 5,939,285,
Method for regulating the expression of a gene in
a baculovirus using a retinoic acid receptor
Availability: Marketed by Qbiogene for non-
commercial uses.
Licensing information: Contact INRA regarding
commercial uses and licensing.
435
Baculovirus vectors and
promoters - glycosylation
University of Georgia - Licensor, primary
Description: Nuclear polyhedrosis virus-based
baculovirus vectors and promoters offer improved
expression levels, and allow proper post-transla-
tional modifications (glycosyloation) of the gene
product in insect cells. The novel promoters can
be stably incorporated at any region in the AcMN-
PV genome without concern for recombination
with other regions of the AcMNPV genome. The
modified promoters may also be designed to be
stronger than the polyhedrin promoter. Expression
is under the regulatory control of a modified late
or very later baculovirus promoter, with a modi-
fied baculovirus promoter comprising a poly-
adenylation signal opposite in orientation to the
direction of transcription directed by the modified
promoter, and the polyadenylation signal placed
3 to the transcription start site of the modi-
fied promoter. The modified promoter has been
incorporated into several different transplacement
plasmids
Baculovirus expression vectors, Miller, Sept.
14, 1993, assigned to the University of Georgia.
Georgia.
436
BestBac vectors; Autographa
californica nuclear
polyhedrosis virus (AcNPV)
vectors - insect cells
Description: BestBac DNA linearized Autogra-
pha californica Nuclear Polyhedrosis virus (Ac-
NPV) baculoviral DNA expression vectosr (for in-
sect cells) are improved versions of the BacPAK6
baculovirus vectors. BestBac DNA provides a
means for producing recombinant baculoviruses
with efficiencies close to 100%. The principle of
this technique lies in the production of baculovi-
rus DNA which contains a deletion in an essential
gene. Only co-transfection of insect cells with the
viral DNA and a complementing transfer vector
can reconstitute a viable virus. BestBac DNA is
derived from the E2 clone of AcMNPV which has
been alternately passaged in-vitro and in-vivo to
ensure no loss of viral integrity. The polyhedrin
locus of BestBac has been modified to remove
only the polyhedrin promoter and coding se-
quence. All polyhedrin gene locus-based baculo-
virus transfer vectors can be used to complement
the BestBac deletion.
Availability: Marketed by Expression Systems
LLC for non-commercial uses.
Licensing information: Conttact Expression
Systems LLC.
279
437
Drosophila sialyltransferases
- insect cells; glycosylation
primary
Description: Unique Drosophila sialyltransferase
enzymes are capable of efficient sialylation of
terminal N-acetyl-lactosediamine protein residues
(LacdiNac) either in vivo, by vector-mediated ex-
pression, in insect cells (e.g., use with baculovirus
expression systems/BEVs) or in vitro, by addition
of the enzyme. In vitro glycosylation of recom-
binant proteins suffers from a limited availability
of enzymes that display desired substrate speci-
ficities. These enzymes may be implemented in
large-scale enzymatic reactions both in vitro and
in vivo for the effi ient and cost-effective produc-
tion of proteins with specific sialylation patterns.
The use of transformed insect Sf9 cultured cells is
an example of such an approach.
Patents: No relevant U.S. patent applications or
patents from the inventors, Dr. V. Panin and Dr.
K. Koles, were retrieved. The university reports
a U.S. application has been filed. Patent applica-
tions may not have been published yet.
Licensing information: Contact Texas A&M
University regarding commercial use and licens-
ing.
438
Sapphire baculovirus
expression - disulfde bond
formation
Orbigen, Inc. - Licensor, primary
Description: Sapphire is a proprietary expression
technology offering up to 6-fold yield enhance-
ment. Sapphire was specifically designed for
efficient recombination with polyhedrin promoter-
based baculovirus transfer vectors. The Sapphire
DNA contains a lethal deletion of ORF1629,
which can only result in viable viral particles if
rescued by homologous recombination with a
polyhedrin promoter. This design leads to virtu-
ally 100% recombination efficiency and signifi-
cantly simplifies identification of recombinant
virus. In addition, the disulfide isomerase gene
was inserted at the p10 locus of the virus to ensure
proper protein disulfide bond formation. The Sap-
phire Baculovirus DNA vector is compatible with
most of the commercial polyhedrin-based transfer
vectors.
Background: Expression of the highly abundant
polyhedrin gene is nonessential in tissue culture
and its strong promoter can be used for the tran-
scription of foreign genes. The polyhedrin pro-
moter is maximally expressed very late stage in
infection when the lytic virus kills the host cells,
resulting in high levels of expression even for
certain toxic proteins.
- Enhanced protein folding. The disulfide isom-
erases gene was inserted at the p10 locus of the
virus to ensure proper protein disulfide bond
formation.
- High recombination efficiency. The Sapphire
baculoviral DNA contains a lethal deletion of
ORF1629 which can only result in viable viral
particles if rescued by homologous recombination
with a polyhedrin promoter-based transfer vector.
This design significantly reduces time and effort
in plaque assays.
- High level expression. The p10 promoter is
partially deactivated and the lytic p10 gene is de-
leted so that transcription levels are higher due to
reduced interference and healthier insect cells.
Patents: No patents or applications assigned to
Orbigen were retrieved in simple searches.
Licensing information: Contac Orbigen.
280
439
Vankyrin enhanced baculovirus
expression vector system (VE-
BEVS); Vankyrin-enhanced cell
lines (VE-CL)
ParaTechs Corp. - Licensor, primary
University of Kentucky - R&D
Description: The vankyrin enhanced baculovirus
expression vector system (VE-BEVS) and related
Sf9-derived insect cell lines (VE-CL) provide
marked improvements in protein expression levels
from baculovirus vectors while reducing the cost
of labor and materials. Cells infected with BEVS
expressing a vankyrin protein from the Campole-
tis sonorensis ichnovirus show increased longev-
ity and enhanced levels of recombinant protein
expression that can significantly enhance heterol-
ogous protein production. Vankyrin-enhanced cell
lines (VE-CL) are genetically engineered Sf9 cells
that contain a stably integrated vankyrin gene
and stably express vankyrin protein. Neomycin is
used for selection of stable lines. The cells offer
prolonged longevity after infection with a baculo-
virus expression vector system (BEVS).
The VE-cell lines allow for enhanced protein
expression from the BEVS (up to 15 fold) by
providing the vankyrin protein in trans. Further
enhancement (up to more than 20-fold) can be
obtained by using modified cells in combination
with the VE-BEVS transfer vector. When recom-
bined with traditional polyhedrin locus based
baculovirus vector systems, the vankyrin protein
is expressed in cys with the recombinant protein
cloned into the transfer vector.
Reduced cost and labor
Compatible with all conventional BEVS
Essentially no additional work or adaptation
required
Vectors and methods for enhanced cell longev-
ity and protein expression, 20060134743, Webb;
B.A. et al., assigned to the University of Kentucky
and exclusively licensed to ParaTechs Corp.
Availability: ParaTechs currently offers three
VE- cell lines (VE-CL 01, VE-CL-02 and VE-CL-
03) for non-commercial use. These cell lines show
a significant enhancement in protein expression
from BEVS over regular Sf9 cell lines.
Licensing information: Contact ParaTechs Corp.
Further info.: Fath-Goodin, A., Kroemer, J.A.,
Martin, S.B., Reeves, K., and Webb, B.A. (2006).
Polydnavirus genes that enhance the Baculovirus
Expression Vector System. Advances in Virus
Research, vol. 68, pp. 75-90.
Kroemer, J.A. and Webb, B.A. (2006). Diver-
gences in protein activity and cellular localiza-
tion within the Campolitis sonorensis ichnovirus
vankyrin family. Journal of Virology, 80 (24):
12219-12228.
Company/Organization Index
281
4-Antibody AG
WRO-1096.3
Schwarzwaldallee 215
CH-4002 Basel
phone: +41 (0)61 697 13 51
fax: +41 (0)61 697 13 50
255-HumanprimarypreBlymphocytes;
HupreBcells-humanmonoclonal
antibodies-Licensor,primary
Abbott Laboratories, Inc.
Abbott Park Road, Abbott Park, IL
60064; other facilities at North Chicago,
IL and Rocky Mount, NC
201-Expressionenhancers;Copy
numberincrease-eukaryoticcells-
Licensor,secondary
205-Tetracycline(Tc;Tet)Expression
Systems-eukaryotes-Assignee,patent
Abgenix, Inc.
recently acquired by Amgen
252-Cellfusion,NS0cells-antibodies
-Licensor,primary
Accuro Biologics Ltd
Daniel Ozanne PhD, MBA
Business Alliance Manager
Accuro Biologics Ltd
Crombie Lodge
Aberdeen Science & Technology Park
Balgownie Drive
Aberdeen AB22 8GU
UK
Tel: +44 (0)1224 707337
Fax: +44 (0)1224 820049
Email: daniel.ozanne@accurobio.com
396-pAccABvectors-antibodies;
mammaliancells-Licensor,primary
Actis Biologics Inc
5461 Goldenrod Drive
Livermore, CA 94551-4102
372-Vesicularfusionfactor2protein
(Vff2p)enhancer-yeast;bacteria;CHO
cells-Licensor,primary
Advanced BioNutrition Corp. (ABN)
7155 Columbia Gateway Drive
Columbia, MD 21046
Phone: 410-730-8600
Fax: 410-730-9311
David J. Kyle
DKyle@abn-corp.com
213-Shrimpexpression;Penaeus
stylirostrisexpression;Taura
SyndromeVirus(TSV)IRESvectors
-glycoproteins;antibodies-Licensor,
primary
Advanced Cell Technology, Inc.
11100 Santa Monica Blvd., #850
Los Angeles, CA 90025
Tel: (310) 481-5124
Fax: (310) 481-0833
info@advancedcell.com
212-Milkproteinpromoter-proteins
inmilkoftransgenicanimals-
Licensor,primary
276-NucleartransferCulturedinner
cellmasscells(CICM)-transgenic
animals;cloningfromsomaticcells
-Assignee,patent
Agricultural Research Service
(ARS), U.S. Dept. of Agriculture
(USDA)
June Blalock, Coordinator, Technology
Licensing Program
June.Blalock@ars.usda.gov
Phone: (301) 504-5257
Fax: (301) 504-5060
Brian Nakanishi, Technology Licensing
Specialist
Brian.Nakanishi@ars.usda.gov
Phone: (301) 504-4879
Fax: (301) 504-5060
301-Polydnavirusvectors-insect
cells;baculovirusaltnerative-
Licensor,primary
387-piggyBactransposon-
eukaryotes;insectcells-Licensor,
primary
427-Insectcelllines-baculovirus
hosts-Licensor,primary
Alder Biopharmaceuticals Inc.
11804 North Creek Parkway South
Bothell, WA 98011
Mark Litton, Ph.D.
Chief Business Officer
425.205.2920
litton@alderbio.com
378-MabXpressAntibodyProduction
System;Pichiapastoris-glycosylated
Altogen Biosystems
848 Rainbow Blvd #823
Las Vegas, NV 89107 USA
316-Altogentransfection;RNAigene
silencing-Licensor,primary
Ambrx, Inc.
10975 North Torrey Pines Road
La Jolla, CA 92037
Tel: (858) 875-2400
busdev@ambrx.com
132-Reconstitutingchemically
orthogonaldirectedengineering
(ReCODE)-Unnaturalaminoacids;
UAAs;E.coli;yeasts;mammalian
AmProtein, Inc.
355 North Lanatna Street #220
Camarillo, CA93010
Main: (805) 807 3362
Fax: (805) 987 8377
http://www.amprotein.com/
Email: bd_us@amprotein.com
215-AmProteinvectors-stongest
mammalianvectorset;universal-
Licensor,primary
AMRAD Corporation Ltd.
17-27 Cotham Road
Kew, Victoria 3101
Australia
118-Glutathione-S-transferase(GST)
fusionproteinaffinitytags;pGEX
vectors-R&D,oftech.
Apex Bioscience, Inc.
Durham, NC
175-ApoLifeYeastExpression;S.
cerevisiaeTwinCassettePlasmids-
antibodiesinyeast-R&D,oftech.
282
ApoLife
100 River Place
Suite #6800
Detroit, MI 48207
Anita Dean, MBA
Manager-Maketing & Business
Development
Phone: 313-446-2554
Email: adean@apolife.com
Email: anitadean28@yahoo.com
175-ApoLifeYeastExpression;S.
cerevisiaeTwinCassettePlasmids-
antibodiesinyeast-Licensor,primary
Apotex Holding Ltd.
Toronto, Canada
171-CANGENUS;Streptomyces
(lividansandgriseus)expression-
Parentco.
Artes Biotechnology GmbH
Dr. Melanie Drttboom
Business Development
Email: m.droettboom@artes-
biotechnology.com
182-Hansenulapolymorpha(yeast)
expression-E.colialternative-
Licensor,primary
361-ALEU2marker;AHSB4
promoter;Arxulaadeninivorans
expression-Licensor,primary
364-Calnexinchaperone-Hansenula
polymorpha;yeasts-Licensor,primary
374-XplorVectorSystem,yeast
optimization-Licensor,primary
ASA Spezialenzyme Ltd.
Am Exer 19c
38302 Wolfenbttel
Telefon: 05331 8825-30
Telefax: 05331 8825-32
E-Mail: service@asa-enzyme.de
Dr. Arno Cordes, Head or R&D
cordeswf@asa-enzyme.de
195-Chlamydomonasreinhardtii
expression-algae,single-cell-
Assignee,patent
Auburn University
270-ChloroplastTransformation
Technology(CTT)-plantcells-R&D,
oftech.
Austrian Center of
Biopharmaceutical Technology
(ACBT)
Muthgasse 18
A-1190 Vienna
Dr. Friedemann Hesse
Tel.: +43 1 36006 6806
Fax.: +43 1 3697615
friedemann.hesse@boku.ac.at
344-N(pro)fusionproteintag,self-
cleaving;SwinefevervirusN(pro)
autoproteolysis;EDDIE-E.coli-
R&D,oftech.
Avecia Biologics Ltd.
Tracy Kinjerski
Commercial Development Manager
301.845.8566 (office)
301.222.7744 (cell)
tracy.kinjerski@avecia.com
323-pAVEwayexpression-E.coli
andPseudomonas-Licensor,primary
AVIDIS SA
Michel Wurm, M.D.
Executive Director, Corporate
Development
AVIDIS SA
Biopole
63360 Saint-Beauzire, France
phone: +33 4 73 64 42 71
fax: + 33 4 73 64 43 93
mobile: + 33 6 09 97 81 04
e-mail: mwurm@avidis.fr
skype: michelwurm
345-OverExpressC41(DE3)and
C43(DE3)-E.colistrains-Licensor,
primary
Avigenics, Inc.
spun off from University of Georgia
Animal Science Complex
425 River Road
Athens, Georgia 30602 U.S.A.
Phone: 706-227-1170 Fax: 706-227-
2180
E-Mail: information@avigenics.com
Yesland,Kyle, attorney
248-Transgenicpoultry;avian
transgenesisandnucleartransfer
-proteinsinchickeneggs-Licensor,
primary
249-WindowingTechnology-
transgenicsavians/chickens-Licensor,
primary
BASF AG
Systems-eukaryotes-Parentco.
284-Super-masPlantGenePromoter;
Gelvinpromoter;(Aocs)3AmasPmas
-plants-Parentco.
Bayer Corp.
800 Dwight Way, P.O. Box 1986,
Berkeley, CA 94701
193-PlastidTransformation;
Translation-basedvectors(TBV)-
plants-Parentco.
266-HKB-11(HKB11)expression;
HybridofkidneyandBcells-HEK-
293alternative-Licensor,primary
277-NuclearTransformationSuite,
plants-Parentco.
-plants-Parentco.
390-Calnexin,calreticulin,Erp57,
Hsp40,andHsp70chaperones-
415-Magnifection;magnICON;
TransgeneOperatingSystem(TOS)
-antibodies;plantsandplantcells
-Parentco.
Benda Pharmaceutical, Inc.
China (PRC); parent of Shenzhen
SiBiono Gene Technologies Co.
264-HEK293celllines-Parentco.
Benzon Pharma A/S
now part of Nycomed A/S (see
Nycomed)
Hvidovre, Denmark,
158-Plasmidsstabilization-Former
inv.
311-Benzonase;Serratiamarcescens
endonuclease-polynucleotides
breakdown-Licensor,primary
BestGene Inc.
2918 Rustic Bridge,
Chino Hills, CA 91709
Phone: +1-909-628-0778
Fax: +1-909-628-7759
info@thebestgene.com
423-Cre/loxPRecombination-
MediatedCassetteExchange(Cre/loxP
RMCE)-Drosophila(mosquitos)-
Contractservices
283
Bio-Technical Resources (BTR)
Manitowoc, WI
Julie Maurina-Brunker
julie@biotechresources.com or 920-
684-5518
182-Hansenulapolymorpha(yeast)
expression-E.colialternative-
Licensor,primary
Bio-Xtal S.A.
2, Rue Thomas EDISON
67450 MUNDOLSHEIM - France
Mr. Etienne LHERMITE, Business
relations
Tel. +41 21 654 71 20
elhermite@bioxtal.com
399-Semlikiforestvirus(SFV)
vectors-mammalianandinsectcells
-Licensor,primary
Bioclone Inc.
7965 Silverton Ave. Suite 1309
San Diego, CA 92126
Tel: 1-800-860-5112 or (858)-547-
8349
Fax: (858)-547-8314
Email: info@bioclon.com
336-Codon-Optimized,Expression-
ReadyE.coliClones-Licensor,
primary
BioFactura, Inc.
9700 Great Seneca Hwy
Rockville, MD 20850
http://www.biofactura.com/
301-315-8002
dsampey@biofactura.com;lbranco@
biofactura.com
253-Cholesterol/3-ketosteroid
reductase(p3-KSR)expression-
cholesterolselection/inductionofNS0
406-StableFastBiomanufacturing
System;pBFdfhr.2ExpressionVector
-CHOcells;antibodies-Licensor,
primary
Biogen Idec, Inc.
Coney, Leslie (CLP)
Associate Director, Business
Development
Biogen Idec Inc.
14 Cambridge Center
Cambridge, MA 02142
Tel: (617) 679-2266
Fax: (617) 679-2804
Email: leslie.coney@biogenidec.com
Web: www.biogenidec.com
407-TandemChimericAntibody
Expression(TCAE)vectors;ANEX
vectors;CHOcelllineTCAE8(ATCC
9119);Kozaksequences,impaired-
Biolex Therapeutics
Biolex Therapeutics
158 Credle Street
Pittsboro, North Carolina 27312
919.542.9901 (Phone)
919.542.9910 (Fax)
Business Development: bizdev@
biolex.com
275-LEXSystem;Lemna(duckweed)
expression-algae,wholeplants-
Licensor,primary
BioMarin Pharmaceutical Inc.
105 Digital Drive
Novato, CA 94949
Tel: 415.506.6700
Fax: 415.382.7889
Business Development: bd@bmrn.com
http://www.biomarinpharm.com/
152-Flavobacteriumheparinum
expression-glycoproteins-Licensor,
primary
Biomedal, S.L.
Carlos Chinchilla
Marketing & Technology transfer
Manager
Avda. Americo Vespucio, 5-E
Planta 1 Modulo 12
Parque Cientifico y Tecnologico
Cartuja 93
41092 Sevilla (Spain)
Tel: +34 954 08 12 76
Fax: +34 954 08 12 79
E-mail: carlos.chinchilla@biomedal.
com
166-CASCADEexpression;pALEX1
plasmids-E.coli-Licensor,primary
167-Choline-bindingfusionaffinity
tags-bacteria-Licensor,primary
189-EASYEAST;Saccharomyces
cerevisiaestrains-easyproteinrelease
-Licensor,primary
332-C-LYTAGaffinityfusionprotein
tag;StreptococcuspneumoniaeN-
acetylmuramoyl-L-alanineamidase
LytA-E.coli;purification-Licensor,
primary
366-Estradiol-dependentenhancer;
Gal4-ER-VP16-yeasts-Licensor,
primary
Bioneer Corp.
49 - 3 Munpyeong - dong
Daedeok-gu, Daejeon 306-220
South Korea
Phone : 82 - 42 - 930 - 8777
Fax : 82 - 42 - 930 - 8688
sales@bioneer.com
also:
Bioneer Inc,
1000 Atlantic Avenue, Suite 102
Alameda, CA 94501-1147
Phone : 510 - 865 - 0330
Fax : 510 - 865 - 0350
order.usa@bioneer.com
156-P170Expression
System;Lactococcuslactisexpression
-Licensor,primary
Biotechnology Research and
Development Corp. (BRDC)
Peoria, Illinois
Grant Brewen
tel: 1-309-688-1188
fax: 1-309-688-1292
-plants-Licensor,primary
329-Avidinaffinityfusionprotein
affinitytags;Biotinpurification;
PinPointXaProteinPurification
System-E.coli-Licensor,primary
Biotechnology Research Institute,
National Research Council of
Canada (NRC- BRI)
Intellectual Property directorate
Daniel Desmarteaux, M. Sc., MBA
Business Development Officer, BRI
Tel: (514) 496-5300
Fax: (514) 496-5007
e-mail: daniel.desmarteaux@cnrc-nrc.
gc.ca
Photo Yves Durocher Dr. Yves
Durocher
Group Leader, Animal Cell Technology
Tel.: (514) 496-6192
Fax: (514) 496-7251
E-mail: yves.durocher@cnrc-nrc.gc.ca
170-Methylobacteriumextorquens
(bacterium)expression-Licensor,
primary
220-Cumategene-switch;Q-mate
InducibleExpression-mammalian
262-HEK293expression-Licensor,
primary
265-HEK293SFEcellline-Licensor,
primary
284
410-293ST-3F6cellline;HEK-293
adaptedtoSFM-Licensor,primary
411-CRE-inducibleexpression;cyclic
AMPresponseelements(CREs)-
HEK-293cells-Licensor,primary
414-pTTvectorsforHEK-293Ecells
-Licensor,primary
Blackstone Group
parent of Catalent Pharma Solutions
225-GPExGeneProductExpression
Technology-mammalian;CHO-
Parentco.
Boehringer Ingelheim Pharma KG
Boehringer Ingelheim Pharma GmbH
& Co. KG
Birkendorfer Strae 65
88397 Biberach/Riss, Germany
www.boehringer-ingelheim.com/
biopharm
biopharma@bc.boehringer-ingelheim.
com
163-Staphylococcuscarnosus
expresion-Licensor,primary
216-Anti-apoptosisexpressionsystem;
BCL-xLorBCL-2expressingcell
lines-CHO,NS0,BHK,SP2/0-Ag14
-Licensor,primary
233-BoehringerIngelheimHigh
ExpressionSystem(BIHEX);CHO-
DG44-Licensor,primary
385-ColE1plasmids,E.coli-
prolongedviability-Licensor,primary
Boyce Thompson Institute for Plant
Research
affiliated with Cornell University
Robert Granados, PhD
Phone: 607-254-1256
rg28@cornell.edu
126-Mucinpromoters;IIM14and
IIM22-vectorenhancers-Licensor,
primary
291-HighFivecellline(BTI-TN-5B1-
4,ATCCCRL10859);Trichopulsiani
cellline-baculovirushostcells;insect
cellculture-Licensor,primary
295-PERLXpress;TRANSPILLAR
larvae;Trichoplusianilarvae
expression-transformedcaterpillars
-Assignee,patent
296-Trichoplusiani(cabbagelooper)
celllines;BTI-TN-MG1;ATCCCRL
10860;BTI-TN-5B1-4;ATCCCRL
10859-Licensor,primary
297-Trichoplusiani(cabbagelooper)
celllines;H5CL-BandH5CL-F;BTI-
TN-5B1-4-derivedinsectcelllines
-Licensor,primary
302-Pre-occludedVirus(POV)
baculovirusvectors;Insectcells,peros
(oral)baculovirusinfection-Licensor,
primary
420-AcMNPVp35apoptosis
inhibition;Sf9P35AcV5-1and
Sf9P35AcV5-3-insectcells,apoptosis
resistance-Licensor,primary
428-Insectcells,peros(oral)
baculovirusinfection-Assignee,patent
BresaGen
8 Dalgleish Street
Thebarton, SA 5031
AUSTRALIA
http://www.bresagen.com.au
protEcol@bresagen.com.au
319-BresaGenfusionprotein
expression-E.coli.-Licensor,primary
Bristol-Myers Squibb Co. (BMS)
Debra Desmond
Bristol-Myers Squibb Company
Route 206 & Province Line Road
EST&L, Mail Stop B24-05
Princeton, NJ 08543-4000
Tel: 302-456-9431
Fax: 609-252-7337
Email: debra.desmond@bms.com
110-Cre-loxmediatedinvitro
recombination;Cyclization
Recombination/locusofX-overP1;
site-specificrecombination(SSR)
-universal;geneticrecombination-
Licensor,primary
Brookhaven National Laboratory
Christine L. Brakel
Licensing Associate
Office of Intellectual Property and
Industrial Partnerships
Building 475D
Brookhaven National Laboratory,
Upton, NY 11973
Tel: 631-344-7134
brakel@bnl.gov
335-cis-ActingPeptidechaperones
-E.coli-Licensor,primary
349-pETExpressionSystem;T7
promoter;pETDirectionalTOPO
Cloning;ChampionpETExpression
Vectors;T7RNApolymerase(T7
RNAP)-E.coli-Licensor,primary
Cambia Biosystems, L.L.C. (CBL)
Building 401B
Clunies Ross Street
Black Mountain, ACT 2601
Australia
Telephone & Fax
Tel. + 61 2 6246 4500
Fax. + 61 2 6246 4533
cambia@cambia.org
119-GUSreportersystem(beta-
glucuronidase);GUScontrolvector
-prokaryotes;plantcells;mammalian
cells,non-vertebrate-Licensor,
primary
285-TransBacterGeneTransfer
System-plants;royalty-free-Licensor,
primary
Cangene Corp.
Director, Strategic Development
& Intellectual Property
Cangene Corporation
155 Innovation Drive
Winnipeg, MB Canada
R3T 5Y3
Tel: (204) 275-4311
Fax: (204) 269-7003
e-mail:
strategicdevelopment@cangene.com
171-CANGENUS;Streptomyces
(lividansandgriseus)expression-
Licensor,primary
Cardinal Health, Inc.
see Catalent Pharma Solutions
Formerinv.
Carnegie Institution of Washington
Washington, DC
424-Drosophilaexpression-Licensor,
primary
Catalent Pharma Solutions
14 Schoolhouse Road
Somerset, NJ 08873
PH: 732 537 6200
Email: gpexinfo@catalent.com
Licensor,primary
285
CBD Technologies, Inc.
CBD Technologies, Inc.
P.O. Box 484
Bergenfield, New Jersey 07621
T: 201-816-9204
F: 201-816-9204
107-Cellulosebindingdomain(CBD)
fusionproteinaffinitytags;pET-CBD
-universal-Licensor,primary
Celanese AG
parent of Nutrinova
211-Tetrahymenathermophila
(protozoan)expression-Parentco.
Cell Culture Technologies LLC
Via al Chioso 12
6929 Gravesano/Lugano
Switzerland
Phone: +41 91 604 53 22
Fax: +41 91 604 53 26
http://www.cellculture.com/
260-Sp2/0-Ag14cells-protein-free
media;antibodies-Licensor,primary
261-HEK293cellline,protein-and
peptide-free(HektorG)media;human
embryonickidney(HEK293)cellline
--Licensor,primary
Cell Genesys, Inc.
now Abgenix, Inc.
223-Gene-Activated(GA)expression,
invivo-mammaliancells-R&D
Cellectis SA
102 route de Noisy
F-93235 Romainville Cedex
France
Isabelle Pelletier-Bressac
Vice President, Business Development
Mail: pelletier-bressac@cellectis.com
Tel.: +33 1 41 83 99 00
103-Invivolinearization;InVoLin;
MeganucleaseRecombinationSystem
(MRS)-improvedtransformation-
Licensor,primary
125-MeganucleaseRecombination
System(MRS)/I-SceI;homologous
recombination-universalgenetic
recombination-Licensor,primary
202-HomologousRecombination;
Knock-out/knock-inanimalsandcells
-Licensor,primary
Celliance Corp., Serologicals Corp.,
now Millipore
a wholly-owned subsidiary of
Serologicals Corp., now Millipore:
Christian Simonsen, Ph.D.
Vice-President Research and
Development
e-mail: csimonsen@celliancecorp.com
Tel.: 408-779-4533
vectors-Licensor,secondary
230-Ubiquitouschromatinopening
element(UCOE)expression-
Celltech Biologics plc
Slough, U.K.; now broken up;
Celltechs manufacturing establishment
in Slough, U.K., and some patents
now owned by Lonza Biologics;
other facilities and technologies were
retained by Celltech Group plc
127-NewCabilly;Cabilly-Boss;
Monoclonalantibody2-chains
expression-universal-Assignee,
patent
224-Glutaminesynthetase(GS)
System-NS0,CHO,mammaliancells
-R&D
Centre de Investigacin en
Biotecnologa
UAEM, Cuernavaca
Morelos
Mxico
347-pBR322-E.coliplasmid;
antibioticselection-R&D,oftech.
Centro Informtico Cientfico de
Andaluca
Apdo.de correos 1211
41012 Sevilla
Spain
buzon@cica.es
197-Streptomyceslividans-Licensor,
primary
358-Streptomycesinducers-Licensor,
primary
CEVEC Pharmaceuticals GmbH
Gottfried-Hagen-Str. 62
D-51105 Kln
Germany
waschuetza@cevec-pharmaceuticals.
com
250-CEVECAmniocyteProduction
(CAP)expression;Humanamniocytes,
immortalized-Licensor,primary
258-PER.C6expression;
Extremedensity(XD)Technology
-glycoproteins;antibodies-Patent
disputer
Chasin, Columbia University
Chasin, Lawrence
Professor
(212) 854-4645
lac2@columbia.edu
235-CHODG44cells,DHFR-;CHO-
DG44;DUK-XB11;CHOK1DUX
B11(DHFR-)cells;Dihydrofolate
reductaseselection/amplification,CHO
Chemicon International, Inc.,
Millipore Corp.
Roy J. Millender
Vice President Legal and Corporate
Affairs
Dept. of Technology Transfer
28820 Single Oak Drive
Temecula, CA 92590
28820 Single Oak Drive
Temecula, CA 92590 USA
(951) 676-8080 ext.274
(951) 699-4371 (fax)
E-mail: rmillender@chemicon.com
vectors-Licensor,primary
Chesapeake PERL, Inc.
8510 A Corridor Rd.
Savage, MD 20763
Tel: 301-317-9300 x 101
Fax: 301-317-9342
Bob Balcerzak, President
(301) 317-9300 x103
bbalcerzak@c-perl.com
292-Insectcellsglycosylation-R&D,
oftech.
larvae;Trichoplusianilarvaeexpression
-transformedcaterpillars-Licensor,
primary
428-Insectcells,peros(oral)
baculovirusinfection-Licensor,primary
286
Chiron Corp., Novartis AG
a subsidiary of Novartis AG; 4560
Horton Street, Emeryville, CA 94608;
Chiron acquired Cetus Corp. (located
across the street) in 1992
106-Aminoglycoside
phosphotransferasemarker;Neomycin
phosphotransferaseI(nptI)marker;
Geneticin(G-418)selection-universal
-Assignee,patent
341-Highcopynumberplasmids;
pBGP120-E.coli-Licensor,primary
Chlorogen, Inc
893 North Warson Road
St. Louis, MO 63141
info@chlorogen.com
T: 314 812 8151
F: 314 812 8080
Technology(CTT)-plantcells-
Licensor,secondary
Chromagenics BV
The Netherlands; acquired by Crucell
229-STabilizingAnti-Repression;
STARelements-expression
enhancement;mamaliancells-Former
inv.
Chromos Molecular Systems
220 - 980 W 1st Street
Mail Box # 8
North Vancouver, BC
Canada V7P 3N4
Telephone: 604.985.7100
Fax: 604 980 2501
Email: sg@chromos.com
232-ACEExpressionSystem-MAb-
expressingCHOcellslines-Licensor,
primary
Cilian AG
Johann-Krane-Weg 42
48149 Mnster
Germany
Ph: +49 251 6 20 31-0
http://www.cilian.com
207-CiliatePerformanceExpression
System;CIPEXsystem;Tetrahymena
(protozoa)expression-Licensor,
primary
Cistronics Cell Technology GmbH
Phone: +41 79 336 95 00
Fax: +41 86 079 336 95 00
P.O.B. 145, CH-8093 Zurich,
Switzerland
http://www.cistronics-cell-technology.
com/
198-Antibioticinduciblepromoters
-eukaryotes-Licensor,primary
City of Hope National Medical
Center
Duarte, CA
165-E.coliexpression/vectors-R&D
Clonex Development, Inc. (CDI)
3587 Anderson Street
Suite 103
Madison, WI 53704
Phone: 608-310-9575
Fax: 608-310-9579
Thomas Primiano, Ph.D., President and
CEO
ceo@cdibios.com
398-RPShift;Senescenceinduction;
PACEExpressionVector-mammalian
cells;expressionenhancement;
Cobra Biomanufacturing plc
Stephenson Building
The Science Park
Keele ST5 5SP UK
Oxford, UK
rocky.cranenburgh@cobrabio.com.
andrew.arrage@cobrabio.com
jason.rahal@cobrabio.com
327-Xer-cisegeneexcision;Xer
recombinases-Bacillussubtilis-
Licensor,primary
CODA Genomics, Inc.
Alan Carter
Sr. Vice President- Business
Development
CODA Genomics, Inc.
acarter@codagenomics.com
www.codagenomics.com
Phone: (949) 305-5505
140-TranslationEngineering
expression;CODA;Computationally
OptimizedDNAAssembly;HotRod
Genes;ControlledRibosomalPausing
Columbia University
GONZALO MERINO, Ph.D.
Associate General Counsel
E-mail: gm@gc.columbia.edu
Tel: (212) 854-0284
Fax: (212) 854-3156
109-Cotransformation,Columbia
University-eukaryotes,selection-
Licensor,primary
ConjuGon, Inc.
University Research Park
505 South Rosa Road, Suite 29
Madison, WI 53719
Phone: (608)441-2890
Fax: (608) 441-2891
Email: info@conjugon.com
Website: http://www.conjugon.com
322-E.coli.vectors;Mnt-Arc
promoters;T1andT2rrnBribosomal
terminators-bacteria-Assignee,
patent
Cornell University
Brenner, John A.
Senior Licensing Associate
Cornell Research Foundation, Inc.
20 Thornwood Drive
Suite 105
Ithaca, NY 14850
Tel: (607) 257-1081
Fax: (607) 257-1015
Email: jb77@cornell.edu
Web: www.crf.cornell.edu
337-Continuousculture-bacteria;E.
coli-Licensor,primary
Cornell University
Cornell Research Foundation, Inc.
Weill Medical College
418 East 71st St., Suite 61
New York, NY 10021
Phone: 212-746-1267.
134-SmallUbiquitin-likeModifier
(SUMO)fusionproteintags;Split
SUMOproSystem;Ubl-specific
protease-universal-Licensor,
secondary
287
Crucell Holland B.V.
Sven Lee
Director, Business Development
Crucell Biologics
Phone: +303 841 3923
E-Mail: Sven.Lee@crucell.com
229-STabilizingAnti-Repression;
STARelements-expression
enhancement;mamaliancells-
Licensor,primary
258-PER.C6expression;Extreme
density(XD)Technology-
glycoproteins;antibodies-Licensor,
primary
CSL Ltd.
Parkville, Victoria, Australia; formerly
Commonwealth Serum Labs., Ltd.
403-CSL4S-342CHOcells(CHO-K1
CSL4S-342)-Licensor,primary
Damon Biotech, Inc.
Needham Heights, MA
numberincrease-eukaryoticcells
-R&D
Danisco A/S
parent of Genencor International
185-Neurosporaexpression;cotA
promoter-fungi-Parentco.
362-Aspergillusnigerexpression;
A.nigerA4promoters-humanized
antibodies-Parentco.
Dartmouth College
Kan, Alla
Director, Technology Transfer
Dartmouth College
11 Rope Ferry Road
Hanover, NH 03755-1404
Tel: (603) 646-3027
Fax: (603) 646-3670
Email: technology.transfer@dartmouth.
edu
Web: www.dartmouth.edu
141-Whitecollarcomplex(WCC)
promoter;UVlightinduction-
universal-Licensor,primary
161-Ralstoniaeutrophaexpression;
Alcaligeneseutrophusexpression-
Licensor,primary
180-GlycoFitechnology;Next
GenerationBiotherapeutics-Pichia
pastoris;yeasts;glycosylation;
antibodies-R&D,oftech.
Delta Biotechnology Ltd.
Castle Court
59 Castle Boulevard
Nottingham
Nottinghamshire
NG7 1FD
United Kingdom
www.deltabiotechnology.co.uk
Dr Dermot Pearson
172-Saccharomycescerevisiae
375-Antibodyfragmentexpression
-Saccharomycescerevisiae-Licensor,
primary
Diosynth Biotechnology
a subsidiary of Schering-Plough. Corp.
Jenifer Wheat, Sr. Director
Commercial Development
Diosynth Biotechnology
101 J Morris Commons Lane
Morrisville, North Carolina 27560
1-866-762-6259 (U.S. toll free)
1-919-337-4477 (Outside the U.S.)
Fax: 1-919-337-0899
226-MARtech;MatrixAttachment
Regions;ScaffoldAttachmentRegions;
SARs-mammaliancells-Licensor,
primary
Dow AgroSciences LLC
9330 Zionsville Road
Indianapolis, IN 46268
Telephone: +1 317 337 3000
417-ConcertPlant-Cell-Produced
system-tobaccoplantcellculture-
Licensor,primary
Dowpharma, Dow Chemical Co.
5501 Oberlin Dr.
San Diego, CA 92121
Website: www.dowpharma.com
E-mail: dowpharma@dow.com
Phone: 866-332-5805
157-PfenexExpression;Pseudomonas
fluorescensbiovarI(MB101)-E.coli
alternative-Licensor,primary
Licensor,primary
DSM Biologics
Marcel Lubben
Vice President Marketing, Sales &
New Business Dev.
DSM Pharmaceutical Products
Phone: +31 46 47 73343
Fax: +31 46 47 73682
E-mail: marcel-m.lubben@dsm.com
183-Kluyveromyceslactisexpression;
pKLAC1;Acetamidase/Acetamide
selection;K.lactisGG799-yeast-
Licensor,primary
glycoproteins;antibodies-Licensor,
secondary
Duke University
Durham, NC
111-deltaPhase;Elastin-like
polypeptide(ELP)fusionproteintags
-chromatography-freepurification;
universal-R&D,oftech.
112-DirectedNucleaseEditor(DNE);
MeganucleaseDesign-universal-
R&D,oftech.
DuPont Corp.
Dr Robert Giaquinta
E. I. du Pont de Nemours and Co.
P.O. Box 80402
Wilmington, DE 19880-0402
Tel: 1-302-695-8096
E-mail: Robert.t.giaquinta@usa.
dupont.com,
or drew.e.van-dyk@usa.dupont.com
110-Cre-loxmediatedinvitro
recombination;Cyclization
Recombination/locusofX-overP1;
site-specificrecombination(SSR)
-universal;geneticrecombination-
Licensor,primary
287-Ubiquitinplantpromoters-
Licensor,primary
Licensor,primary
Dyadic International, Inc.
Rich Burlingame
1-561-743-8333 x19
technology@dyadic-group.com
177-Chrysosporiumlucknowense
expression;C1Express
HyperproducingProteinExpression
System-fungi-Licensor,primary
288
Dynavax Technologies Corp.
2929 Seventh Street, Suite 100
Berkeley, CA 94710
Phone: +1.510.848.5100
Fax: +1.510.848.1327
contact@dynavax.com
http://www.dynavax.com/
176-Arxulaadeninivoransexpression
-alternativeyeast-Parentco.
181-Hansenulaexpression(yeast)
-Parentco.
polymorpha;yeasts-Parentco.
ECI Biotech
85 Prescott Street
Worcester MA 01605
(Tel) 508 752-2209
(Fax) 508 752-4983
Business Development: busdev@
ecibiotech.com
Product/Technology Info.: nerd@
ecibiotech.com
340-ExpressProtect;p26,SicA,and
alpha-crystallin-typefusionprotein
tags-E.coli-Licensor,primary
Ecole Polytechnique Fdrale de
Lausanne (EPFL)
Kai Johnsson
Institute of Chemical Sciences and
Engineering
cole Polytechnique Fdrale de
Lausanne (EPFL)
1015 Lausanne, Switzerland
Fax: (+41) 21-693-9365
tel. +41 21 693 1111
SARs-mammaliancells-R&D,of
tech.
412-HEK-293expression-Licensor,
primary
Eisai Co., Ltd.
parent of Morphotek, Inc.
242-HumanMORPHODOMA;
Morphogenics;Hypermutation;
PMS2genescreening,modification
-humanmonoclonalantibodiesfrom
hybridomas-Parentco.
370-Hypermutableyeast-Parentco.
Entelechon GmbH
Entelechon GmbH Contact
Entelechon GmbH
St. Veit-Weg 2
93051 Regensburg
Germany
Tel. +49 941.9468.360
Fax +49 941.9468.366
protein@entelechon.com
www.entelechon.com
Assignee,patent
Licensor,primary
ERA Biotech
Parc Cientfic de Barcelona,
Baldiri i Reixac, 15-21
08028 Barcelona, Spain
tel +34 934 034 773
fax +34 934 034 772
Fran ois Arcand, farcand@erabiotech.
com
Fran ois Devesa, fdevesa@erabiotech.
com
288-Zaratechnology;Proteinbody-
inducingsequence(PBIS)fusion
proteins;Recombinantproteinbody-
likeassembly(RPBLA);StorPro
organelles(proteinencapsulation);
Prolaminfusionproteins-eukaryotes
-Licensor,primary
European Molecular Biology Labs.
(EMBL)
352-Red/ETrecombination;ET
cloning/ETrecombination;GET
recombination;Recombineering;f
Red-mediatedrecombination;lambda-
mediatedrecombination-E.coli
-R&D,oftech.
ExcellGene S.A.
Route de lile-au-bois 1A
CH-1870 Monthey, Valais
Switzerland
Email: contact@excellgene.com
telephone: +41 (0) 24-471-9660
fax : +41 (0) 24-471-9661
secondary
Expression Systems LLC
26 Harter Ave. Suite 2
Woodland, CA, 95776
sales@expressionsystems.com
phone: 530-662-2946
FAX: 530-662-5359
421-AF99insectcellline-Licensor,
primary
432-TniPRO;Trichoplusianicell
line-Licensor,primary
436-BestBacvectors;Autographa
californicanuclearpolyhedrosis
virus(AcNPV)vectors-insectcells
-Licensor,primary
Flanders Interuniversity Institute for
Biotechnology (VIB)
Belgium
380-PichiaGlycoSwitchSystem;
Glycoswitchplamids-yeasts;
glycosylation-R&D,oftech.
Fundamental Applied Biology, Inc.
(FAB)
Patricia Sinatra
VP, Business Development and
Commercial Strategy
Fundamental Applied Biology, Inc.
1455 Adams Drive, Suite 1015
Menlo Park, CA 94025
Phone: (650) 329-9374
Fax: (650) 329-9002
psinatra@cellfree.com
101-Cell-FreeProteinSynthesis
(Cell-Free)-Licensor,primary
Gala Biotech
merged into Cardinal Health in 20005
Assignee,patent
Gene Bridges
spun-off from the European Molecular
Biology Laboratory (EMBL)
licenses@genebridges.com
http://www.genebridges.com/
mediatedrecombination-E.coli-
Licensor,secondary
Gene Therapy Systems, Inc. (GTS)
parent of Genlantis
128-phCMVvectors;GenePORTER
-universal-Parentco.
289
GENEART AG
BioPark
Josef-Engert-Str. 11
D-93053 Regensburg
Germany
info@geneart.com
phone: +49 941 942 76-0
fax: +49 941 942 76-711
369-GeneDesign,algorithmic-
yeasts-Licensor,primary
Genencor International
a division of Danisco A/S
185-Neurosporaexpression;cotA
promoter-fungi-Licensor,primary
362-Aspergillusnigerexpression;
A.nigerA4promoters-humanized
antibodies-Assignee,patent
Genentech, Inc.
1 DNA Way, South San Francisco,
CA 94080-4990; majority owned by
Hoffmann-La Roche AG
127-NewCabilly;Cabilly-Boss;
Monoclonalantibody2-chains
expression-universal-Licensor,
primary
160-Quasi-syntheticvectors;
Syntheticgenesequencesinvectors
-bacteria-Licensor,primary
165-E.coliexpression/vectors-
Licensor,primary
187-Yeastexpressionsystems-
Assignee,patent
221-Dihydrofolatereductase
(DHFR)System-selectablemarker/
amplification;CHOandNS0cells-
Licensor,primary
221-Dihydrofolatereductase
(DHFR)System-selectablemarker/
amplification;CHOandNS0cells-
R&D,oftech.
338-Disulfideisomerase
coexpression;DsbCandDsbG-E.
coli;disulfidebondsandfolding-
Assignee,patent
347-pBR322-E.coliplasmid;
antibioticselection-R&D,oftech.
General Hospital Corp. (The)
55 Fruit St., Boston, MA; subsidiary or
Massachusetts General Hospital
389-BacMam;pBacMamvectors-
mammalianvectors,baculovirus-based
-Assignee,patent
Genetics Institute, Inc.
became a subsidiary of American
Home Products Corp., now Wyeth, in
1997
343-His-PatchThioFusionSystem;
pThioHisvector-E.coli;fusion
proteins-R&D,oftech.
Genetix Ltd.
Queensway
New Milton
Hampshire
BH25 5NN
sales@genetix.com
392-ClonePixFLSelection
-antibodies,mammaliancells-
Licensor,primary
Genlantis
sub. of Gene Therapy Systems, Inc.
(GTS)
10190 Telesis Court
San Diego, CA 92121 USA
Business Development:
licensing@genlantis.com
128-phCMVvectors;GenePORTER
Genzyme Corp.
One Kendall Square, Cambridge, MA
02139
inmilkoftransgenicanimals-Parent
co.
231-Wheyacidicprotein(WAP)milk
promoters-mammals-Assignee,
patent
Genzyme Transgenics Corp. (GTC)
Framingham, MA
inmilkoftransgenicanimals-
Assignee,patent
Geron Corp.
See also stART Licencing
-proteinsinchickeneggs-Assignee,
patent
Gesellschaft fur Biotechnologische
Forschung mbH (GBF)
Dr. Hansjrg Hauser
Mascheroder Weg 1
D-38124 Braunschweig
Phone: ++49-531-6181-232
Fax: ++49-531-6181-262
E-mail: hha@gbf.de
394-IRF-1estrogenreceptor
promoter;Estradiolinduction-
Ghent University
glycosylation-R&D,oftech.
GlaxoSmithKline Inc. (GSK)
5 Moore Drive Research Triangle
Park, NC 27709; U.S. subsidiary of
GlaxoSmithKline plc
196-DrosophilaExpressionSystem
(DES)-insectcellculture-Assignee,
patent
-R&D,oftech.
GlycoFi, Inc.
a wholly owned subsidiary of Merck
and Co. Inc. in may 2006
www.glycofi.com.
James Posada, Ph.D., MBA
603-727-5181
jposada@glycofi.com
GLYCOTOPE GmbH
Dr. Hans Baumeister
Director Business Development
Tel. +49 (0)30 941084-207, Fax +49
(0)30 941084-188
E-mail: hans.baumeister@glycotope.
com
http://www.glycotope.com/en/
254-GlycoExpresshumancelllines;
NM-F9cellline-glycolysis-Licensor,
primary
Gothia Yeast Solutions AB
Terrassgatan 7
411 33 Gothenburg
Sweden
http://www.gothiayeast.com/
Email: drarlek@yahoo.com
Phone: +46 (0)31 708 7710
FAX: +46 31 708 7711
360-AlcoFreeYeasts;Saccharomyces
cerevisiaeKOY.TM6*strains-
Licensor,primary
290
greenovation Biotech GmbH
Kyrill Schwarz-Herion
greenovation Biotech GmbH
Ndl. Freiburg
Btzinger Str. 29b
D-79111 Freiburg
Phone: +49 (0)761 47 099-111
Fax: +49 (0)761 47 099-190
E-Mail: kschwarz-herion@
greenovation.com
www.greenovation.com
273-Glyco-EngineeredMoss;
Physcomitrellapatensexpression;
mossexpressionpromotingregions
(MEPRs)-glycosylation;antibodies
-Licensor,primary
GTP Technology S.A.
Immeuble Biostep
Rue Pierre et Marie Curie - BP 48184
31681 Lab ge Cedex - France
Tel : +33 (0)5 61 28 70 20
Fax : +33 (0)5 61 28 70 21
contact@gtptech.com
153-LactococcuslactishtrA-
expression-proteasedepletion-
Licensor,primary
Health Research, Inc.
Cox, Dawn E.
Director, Technology Transfer and
Contract Programs
Health Research, Inc.
One University Place
Rensselaer, NY 12144-3456
Tel: (518) 431-1213
Fax: (518) 431-1234
Email: dec09@health.state.ny.us
Web: www.hrinet.org
108-ControllableSelf-CleavingIntein
Derivative;IMPACT-CN;pH-sensitive
self-cleavingfusionproteinaffinity
purificationtag-universal-Licensor,
primary
117-Fusionproteins,self-cleaving,
pH-sensitive-universal-Licensor,
primary
Hi-Genomics, LLC
1000 Jackson Street
Toledo, Ohio 43624
419-321-1307
psilverman@slk-law.com
271-Coupledregeneration/
transformation,plants-Licensor,
primary
Hoffmann-La Roche AG
Grams, Frank
License Manager
F. Hoffmann-La Roche Ltd.
Abt. PLR Bau 71533
Grenzacherstrasse 124
4070 Basel / Switzerland
SWITZERLAND
Tel: 41 61 688 72 37
Fax: 41 61 688 80 58
Email: Frank.Grams@Roche.com
169-GroEL,GroESchaperones;
Chaperonins-properfolding;
universal;E.coli-Parentco.
214-Transgenicrabbits-humanized
polyclonalantibodies-Parentco.
Hoffmann-La Roche Inc.
340 Kingsland Street, Bldg. 1, 2nd
floor, Nutley, NJ 07110
121-Histags;Poly-Histidinefusion
affinitytagtechnology;6xHistidine-tag
-univeral;purificationtags-Parent
co.
393-Hsp60,Hsp70,Hsp90,Hsp100
chaperones-mammaliancells-
Assignee,patent
Hong Kong University of Science
and Technology,
Clear Water Bay, Kowloon, Hong
Kong
+852 2358 7917 2358 1493
Email: ttcac@ust.hk
326-VegIpromoters-Bacillussubtilis
andE.coli-Licensor,primary
Hospira
parent of BressaGen
319-BresaGenfusionprotein
expression-E.coli.-Parentco.
HSP Research Institute, Inc.
company no longer exists. Patents/
technologies acquired by Takara.
334-Chaperoneexpressionplasmids;
DnaK,DnaJandGrpEchaperones-E.
coli-Assignee,patent
(HGSI)
Davis, James H.
Executive VP & General Counsel
14200 Shady Grove Rd
Rockville, MD 20850
Tel: (301) 251-6039
Fax: (301) 517-8831
Email: Jim_Davis@hgsi.com
293-Insectcellsglycosylation-
Licensor,primary
Hungarian Academy of Sciences
expressingCHOcellslines-Assignee,
patent
IBA Gene Technologies
IBA Headquarters
IBA GmbH
Rudolf-Wissell-Str. 28
D-37079 Gttingen
Germany
Phone: +49 (0) 551 50672-0
Fax: +49 (0) 551 50672-181
E-mail: info@iba-go.com
http://www.iba-go.com
135-Strep-tagaffinityfusionprotein
tag;Strep-Tactinpurification-
146-Tetracycline-inducedexpression
repression-prokaryotes-Licensor,
primary
Icon Genetics AG (subsidiary of
Bayer AG)
Biozentrum Halle
Weinbergweg 22, D-06120
Halle (Saale), Germany
gleba@icongenetics.de
193-PlastidTransformation;
Translation-basedvectors(TBV)-
plants-Licensor,primary
277-NuclearTransformationSuite,
plants-Licensor,primary
415-Magnifection;magnICON;
TransgeneOperatingSystem(TOS)
-antibodies;plantsandplantcells
-Licensor,primary
Icos
a subsidiary of Eli Lilly
400-CHEFlexpression;CHO
elongationfactor-la(EF-Ialpha)
promoter-CHOcells-Licensor,
primary
Imperial College
London, UK
333-Campylobacterjejuni
glycosylationgenes;OTaseofC.jejuni
-glycosylation;E.coli-R&D
291
InB:Biotechnologies, Inc.
Delaware Technology Park
9 Innovation Way, Suite 100
Newark, DE 19711
phone: 302-355-0650
fax: 302-356-1173
website: http://www.inb-
biotechnologies.com
Jennifer Kmiec
VP Business Development &
Marketing
E-mail jkmiec@inb-biotechnologies.
com
Tel 302-355-0650
274-iBioLaunchexpression;Launch
vectors-proteinsandMabsinplants
-Licensor,primary
Innovata plc (ML and Quadrant)
Innovata plc combines the businesses
formerly known as ML Laboratories
PLC,
Quadrant Technologies Limited and
Innovata Biomed Limited.
1 Mere Way
Ruddington
Nottingham
NG11 6JS
United Kingdom
Tel: +44 (0)115 974 7474
Fax: +44 (0)115 974 8494
Email: info@innovataplc.com
mammaliancells-Assignee,patent
Inproteo, Inc.
351 West 10th Street, Suite 316
Indianapolis, IN 46202
Peter T. Kissinger, President and CEO
pete@inproteo.com
122-His-tags;Poly-Histidinefusion
affinitytagtechnology-univeral;
purificationtags-Licensor,primary
Institut Fur Bioanalytik
Gemeinnutzige Gesellschaft MBH
Gottingen, DE
135-Strep-tagaffinityfusionprotein
tag;Strep-Tactinpurification-
universal-Assignee,patent
Institut fur Pflanzengenetik und
Kulturpflanzenforschung
also referred to as Leibniz Institute
of Plant Genetics and Crop Plant
Research (IPK):
Corrensstr. 3
06466 Gatersleben
Germany.
kunzeg@ipk-gatersleben.de
-alternativeyeast-R&D,oftech.
361-ALEU2marker;AHSB4
promoter;Arxulaadeninivorans
expression-Assignee,patent
Institut National de la Recherche
Agronomique (INRA)
Philippe Lenee
Director
INRA-Transfert
10 rue Vivienne
75002 PARIS - France
Tel.: +33 (0)1.55.35.26.30
lenee@paris.inra.fr
website : http://www.inra-transfert.
fr/uk_index.htm
Assignee,patent
434-BacTenSystem;p10promoter
vectors-insectcells-Licensor,
primary
Institut Pasteur
Paris, France
202-HomologousRecombination;
Knock-out/knock-inanimalsandcells
-Assignee,patent
Institute for Chemical and Bio-
Engineering, ETH
Zurich, Switzerland
198-Antibioticinduciblepromoters
-eukaryotes-Licensor,secondary
Institute for Molecular Biology and
Biotechnology/FORTH
Heraklion, Greece
142-Minostransposoncell
transformation-insectlarvae;also
eukaryotes-R&D,oftech.
Institute of Biotechnology
(INBIOTEC)
Instituto de Biotecnologa de Len
Avda. Real, n 1 - Parque Cientfico
de Len
24006- LEN - ESPAA
TEL: +34 987 210 308; FAX: +34 987
210 388
inbiotec@inbiotec.com
321-desApromoter,iron-regulated;
DmdRrepressors-Actinomycetes-
Licensor,primary
Institute of Plant Genetics and Crop
Plant Research (IPK)
http://www.ipk-gatersleben.de
374-XplorVectorSystem,yeast
optimization-R&D,oftech.
Invitrogen Corp.
Carlsbad,CA
Licensing & Collaborations
Traci Libby
Corporate Development
Phone: 760.603.6497
Email: traci.libby@invitrogen.com
protease-universal-Patentdisputer
RNAP)-E.coli-Licensor,primary
Invitrogen, Life Technologies, Inc.
Brawer, Rolando, Ph.D
Director Corporate Development
Invitrogen Corp.
1600 Faraday Avenue
Carlsbad, CA 92008
Tel: (760) 476-6246
Fax: (760) 602-6576
Email: rolando.brawer@invitrogen.
com
Web: www.invitrogen.com
(DES)-insectcellculture-Licensor,
primary
331-BL21(DE3)competentE.coli
425-DrosophilamelanogasterS2
cells;Drosophila-SFM.D.Mel-2
Cells;SchneiderS2Drosophilacells;
S2cells,SFM-insectcellculture-
Licensor,primary
Japan Tobacco Inc.
252-Cellfusion,NS0cells-
292
Jena Bioscience GmbH
Jena, Germany
info@jenabioscience.com
www.jenabioscience.com
208-LEXSY;Leishmaniatarentolae
expressionsystem-protozoa-
Licensor,primary
John Innes Centre (JIC)
Norwich Research Park
Colney
Norwich, NR4 7UH
UK
Telephone: +44 (0)1603 450000
FAX: +44 (0)1603 450045
jic.communications@bbsrc.ac.uk
325-Twin-argininetranslocation(Tat)
system;Tatnanomachine-protein
folding,thensecretion;bacteria-
Licensor,primary
Johns Hopkins University (JHU)
Tina Tudor
The Johns Hopkins University
100 North Charles Street, 5th Floor
Baltimore, Maryland 21201
Email: ttudor2@jhmi.edu
Phone:410-516-8300
Fax: 410-516-4411
102-Largeribosomalsubunit
proteins,E.coli-cell-freesystems-
Licensor,primary
hybridomas-R&D,oftech.
Assignee,patent
346-OxlTplasmids;Oxalate/Formate
ExchangeProtein-E.coli-Licensor,
primary
370-Hypermutableyeast-R&D,of
tech.
429-Lymantriadiapar
nucleopolyhedrovirusandL.dispar
652Y(Ld652Y)celllines-baculovirus
hostcells-Licensor,primary
Keck Graduate Institute
Keck Graduate Institute
www.kgi.edu
Diana Bartlett
Director of Corporate Partnerships
PH: 909/607-9864
FAX: 909/607-8598
Diana_Bartlett@kgi.ed
378-MabXpressAntibodyProduction
System;Pichiapastoris-glycosylated
Kemp Biotechnologies, Inc. (KBI)
7307 Governors Way
Frederick, MD 21704
Dr. Christopher W Kemp
301.620.7100
primary
Kentucky BioProcessing, LLC
(KBP)
3700 Airpark Drive
Owensboro, KY 42301
Phone: 270-689-2570
Fax: 270-689-2571
E-mail: info@kbpllc.com
Licensor,secondary
272-GENEWAREexpression;
Tobaccomosaicvirus(TMV)vectors
-tobacco;plants-Licensor,primary
Kings College
London, U.K.
mammaliancells-R&D,oftech.
Large Scale Biology Corp. (LBSC)
Daniel Tus, Ph.D.
Vacaville, CA 95688
Tel: 707.469.2316
Email: daniel.tuse@lsbc.com
272-GENEWAREexpression;
Tobaccomosaicvirus(TMV)vectors
-tobacco;plants-Assignee,patent
LifeSensors Inc.
LifeSensors Inc.
271 Great Valley Parkway
Suite 100
Malvern, PA 19355
610.644.8845 phone
610.644.8616 fax
info@lifesensors.com
protease-universal-Assignee,patent
Lilly, Eli & Co.
Lilly Corporate Center, Indianapolis,
IN 46285
122-His-tags;Poly-Histidinefusion
affinitytagtechnology-univeral;
purificationtags-Assignee,patent
400-CHEFlexpression;CHO
elongationfactor-la(EF-Ialpha)
promoter-CHOcells-Parentco.
London School of Hygiene and
Tropical Medicine
London, UK
Lonza Biologics plc
228 Bath Road, Slough, Berkshire, SL1
4DY, United Kingdom; with facilities
in Slough SL1 4DY, Berkshire, U.K,
and Portsmouth, NH; subsidiary of
(majority owned by) Alusuise-Lonza
Group
224-Glutaminesynthetase(GS)
System-NS0,CHO,mammaliancells
-Licensor,primary
239-GS-CHOProteinFreeSystem;
CHOK1SVcelllines-protein-free
media-Licensor,primary
379-Pichiaexpression,rhamnose
induction-Licensor,primary
Macquarie University
Access Macquarie Limited
Level 1, Dow Corning Building
3 Innovation Road
Macquarie University
NSW 2109
Australia
Phone:+61 2 9850 7111
mqinfo@mq.edu.au
186-Ophiostomaexpression-
ascomycetesfungi-Licensor,primary
MacroGenics, Inc
1500 East Gude Drive
Rockville, MD 20850
P: 301.251.5172
F: 301.251.5321
KoenigS@macrogenics.com
113-Dualexpressionvectors;
DualAffinityReTargeting(DART)
-antibodies;bacteriaandmammalian
293
Massachusetts General Hospital
Cambridge, MA
-Parentco.
Massachusetts Institute of
Technology (MIT)
Cambridge, MA
Assignee,patent
Max-Planck-Gesellschaft Zur
Forderung Der Wissenschaften E.V.
Hofgartenstr. 8
80539 Mnchen
Germany
146-Tetracycline-inducedexpression
repression-prokaryotes-Assignee,
patent
McMaster University
1280 Main Street West,
Hamilton, Ontario
L8S 4L8
Canada
264-HEK293celllines-R&D,of
tech.
Medicago, Inc.
1020, route de lglise, bureau 600
Qubec (Qubec), G1V 3V9
Canada
Ph: 418 658.9393
info@medicago.com
281-Proficiaexpression-transient
expression,plants-Licensor,primary
Medical Research Council, U.K.
London, UK
123-Hybridomas(Khlerand
Milstein)-monoclonalantibodies-
R&D,oftech.
Medical Research Council (U.K.)
(MRC)
London, U.K.
345-OverExpressC41(DE3)and
C43(DE3)-E.colistrains-Assignee,
patent
Merck & Co., Inc.
P.O. Box 4, Sumneytown Pike, West
Point, PA 19486; other facilities in
Liverpool, UK and Rensselaer, NY;
main HQ in Whitehouse, NJ
antibodies-Parentco.
Merck Serono International S.A.
Geneva, Switzerland
invivo-mammaliancells-Patent
dispute
Merck Serono KGaA
processing@merck.de
breakdown-Assignee,patent
Microbix Biosystems, Inc.
115 Skyway Ave
Toronto, Ontario, Canada M9W 4Z4
1-800-794-6694
1-416-234-1624
Fax: 416-234-1626
www.microbix.com
customer.service@microbix.com
264-HEK293celllines-Licensor,
primary
Millennium Pharmaceuticals, Inc.
40 Landsdowne Street
Cambridge, MA 02139
Phone: 617-444-1540
Fax: 617-374-7786
Email: BusDev@mpi.com
365-Chitinsynthase(CHS1),Yeast
growthfactor,chitinsynthase(CHS1)
-yeastpromoterdiscovery-Licensor,
primary
Millipore Corp.
290 Concord Road
Billerica, MA 01821
Phone : 800-645-5476
Fax : 800-645-5439
vectors-Parentco.
Minos Biosystems
Roger Craig
roger@minosbiosystems.com
142-Minostransposoncell
transformation-insectlarvae;also
eukaryotes-Licensor,primary
ML Laboratories
now Innovata and Cobra Bio
mammaliancells-R&D
MoBiTec GmbH
Lotzestrasse 22a
37083 Goettingen
Germany
phone: +49 551 707 220 (Central
Office)
phone: +49 551 707 2270 (Technical
Service)
fax: +49 551 707 2222
e-mail: info@mobitec.com
internet: http://www.mobitec.com
147-Bacillusmegateriumexpression
-E.colialternative-Licensor,primary
Monsanto Corp.
Technology Licensing Manager
Monsanto Company
800 North Lindbergh Blvd
St. Louis, MO 63017
Tel: (314) 694-5898
Fax: (314) 694-1671
Email: connie.m.armentrout@
monsanto.com; stefan.a.bledig@
monsanto.com
433-BAC-TO-BACBaculovirus
ExpressionSystem;baculovirusshuttle
vectors;bacmids-insectvectors
producedinE.coli-Licensor,primary
MorphoSys AG
379-Pichiaexpression,rhamnose
induction-Assignee,patent
Morphotek Inc.
210 Welsh Pool Road
Exton, PA 19341
610-423-6100
Kim Ilgenfritz
Executive Director Business
Development
610-423-6147
hybridomas-Licensor,primary
370-Hypermutableyeast-Licensor,
primary
294
National Health Research Institutes
Taiwan (ROC)
431-Rhopalosiphumpadivirus
InternalRibosomeEntrySequence
(IRES);Picorna-likevirusIRES;
DrosophilaIRES-insectcells;plant
National Institute of Advanced
Industrial Science and Technology
(AIST)
Tsukuba Central No.2 1-1-1 Umezono
Tsukuba City, Ibaraki Prefecture 305-
8568
Japan
Academic, Business, and
Governmental Cooperation Division
TEL: +81-29-861-9232
FAX: +81-29-862-6159
E-mail: aist-innovations@m.aist.go.jp
376-Saccharomycescerevisiae,cold
induction-Licensor,primary
National Institutes of Health (NIH)
Office of Technology Transfer
National Institutes of Health
6011 Executive Boulevard
Suite 325, Mail Stop 7660
Rockville, MD 20852-3804
Phone Number: 301-496-7057
E-mail: nihott@mail.nih.gov
100-Cell-freeexpression,ATP
regenerationsystem-Licensor,
primary
124-Lambdarecombinationprotein;
Homologousrecombination-Licensor,
primary
218-Celladhesionoptimization;
cdkl3,siat7e,andlama4genes-
antibody-expressingcells-Licensor,
primary
231-Wheyacidicprotein(WAP)
milkpromoters-mammals-Licensor,
primary
353-SkpandDsbCchaperonefusions
-E.coli;secretioncontrol-Licensor,
primary
384-Aminoglycoside
adenylyltransferase(aadA1)promoter
-bacteria;eukaryotes-Licensor,
primary
386-CMV/RPromoter-eukaryotes
-Licensor,primary
397-RNAtransportelements(CTE
andRTE)-mammaliancells-
Licensor,primary
National Taiwan University
Taiwan (ROC)
National Tsing Hua University
Wei-Chung Wang
Associate Dean
National Tsing Hua University
101, Sec. 2, Kuang Fu Road,
Hsinchu 30013, Taiwan, R.O.C.
E-mail: wcwang@pme.nthu.edu.tw
402-CHOcellsdhfrRNA
interference;RNAsilencingvectors
-CHOcells-Licensor,primary
Nature Technology Corp.
4701 Innovation Dr.
Lincoln, NE 698521
www.natx.com
Phone: (402) 472-6530
toll free:1-(888)WIzBANG
259-GenomeMassTransfer(GMT);
Retrotransposonvectors-transgenic
avian/chickencellculture-Licensor,
primary
Neugenesis Corp
849 Mitten Road
Suite 102
Burlingame, CA 94010
Tel: 650-689-2220
Email: info@neugenesis.com
184-NeuBIOSexpression;Neurospora
crassaexpression;NeuKARYON
-filamentousfungi;glycosylation;
Cabilly-Bossworkaround-Licensor,
primary
Neurotech Pharmaceuticals
parent of PanGen Biotech Inc.
405-PangenCHOexpression-Parent
co.
New England Biolabs
New England Biolabs, Inc.
240 County Road
Ipswich, MA 01938
Telephone (978) 927-5054
Toll Free (USA Tech)1-800-632-7799
Fax (978) 921-1350
e-mail: info@neb.com
www.neb.com
183-Kluyveromyceslactisexpression;
pKLAC1;Acetamidase/Acetamide
selection;K.lactisGG799-yeast-
Licensor,primary
350-pMALProteinFusionand
PurificationSystem;Maltosebinding
(MBP)fusionaffinitytags;pMAL
plasmids-antibodyfragments;E.coli
-Licensor,secondary
New York University
New York, NY
R&D,oftech.
NextGen Sciences Inc.
Building 56
Alconbury North Airfield
Alconbury, Huntingdon
CAMBS, PE28 4DA
t: +44 (0) 1480 410 850
f: +44 (0) 1480 410 858
US Office
4401 Varsity Drive, Suite E
Ann Arbor
Michigan 48108
t: +1-888-219-8471 (toll free)
f: +1-888-566-4383 (toll free)
http://www.nextgensciences.com
e: info@nextgensciences.com
115-expressionfactory;
baculoworkstation-automatedparallel
NIZO food research B.V.
P.O. Box 20, 6710 BA, Ede
The Netherlands
T: +31 (0) 318 659 511
F: +31 (0) 318 650 400
Igor Mierau
E-mail: igor.mierau@nizo.nl,
155-NIsin-Controlledgene
Expression(NICE);Lactococcuslactis
expression;nisApromoter-antibiotic
selection-Licensor,primary
164-SubtilinRegulatedGene
Expression;SUREcompetency-B.
subtilis-Licensor,primary
Novartis AG
Csendes, Ivan
Head, Licensing Drug Delivery & Out-
Licensing
Novartis Pharma AG
Business Development & Licensing
4002 Basel
SWITZERLAND
Tel: 41 61 324 2160
Fax: 41 61 324 2322
295
Email: ivan.csendes@pharma.novartis.
com
106-Aminoglycoside
phosphotransferasemarker;Neomycin
phosphotransferaseI(nptI)marker;
Geneticin(G-418)selection-universal
-Licensor,primary
236-CHOSSFcelllines-adherent
CHOcells;protein-freemedia-
Licensor,primary
341-Highcopynumberplasmids;
pBGP120-E.coli-Parentco.
Licensor,primary
368-GAPFLpromoter;
dehydrogenase-derivedpromoter-
yeasts-Licensor,primary
Novo Nordisk A/S
100 College Road West, Princeton, NJ
08540
137-TAGZyme;dipeptidyl
aminopeptidaseI(DPPI;DAPase
Enzyme;cathepsinC)-cleavageofHis
tags;universal-Licensor,secondary
Novozymes Delta A/S
Bagsvrd, DK-2880
Denmark
Mr. Hans Ole Klingenberg
Manager, Business Development
haok@novozymes.com
172-Saccharomycescerevisiae
expression-Parentco.
mediatedrecombination-E.coli-
Licensor,primary
375-Antibodyfragmentexpression-
Saccharomycescerevisiae-Parentco.
Nutrinova Nutrition Specialties &
Food Ingredients GmbH
subsidiary of Celanese:
Frankfurter Strae 111
61476 Kronberg im Taunus
Germany
Contact: Tatjana Spter
Phone: +49 (0)69 305 84771
Email: tatjana.spaeter@nutrinova.com
211-Tetrahymenathermophila
(protozoan)expression-Licensor,
primary
NycoMed Pharma A/S
Brett T. Watson, Ph.D.
Vice-President Project Coordination/
Evaluation
Nycomed
Dybendal All 10
DK-2630 Tstrup, Denmark
Tel: +45 4677 1701
Mobile: +45 2423 4632
Email: brett.watson@nycomed.com
net: www.nycomed.com
158-Plasmidsstabilization-Licensor,
primary
breakdown-Parentco.
Orbigen, Inc.
6827 Nancy Ridge Drive
San Diego, CA 92121 USA
1-858-362-2028
custom@orbigen.com
438-Sapphirebaculovirusexpression
-disulfidebondformation-Licensor,
primary
Origen Therapeutics
1450 ROLLINS RD
BURLINGAME, CA 940102307
rkay@origentherapeutics.com or
bheyer@origentherapeutics.com
246-Chickenrimordialgermcells
(PGCs)-Humanproteins/monoclonal
antibodiesinchickeneggs-Licensor,
primary
Oxford BioMedica plc
Oxford, UK
247-OVASystem;AvianTransgenic
Biomanufacturing-chickeneggs
expresssion-R&D,oftech.
PanGen Biotech Inc.
acquired by Neurotech Pharmaceuticals
(Korea) in Aug. 2007
pangen@chocell.com
Tel : 82-31-733-9165 Korea
Fax : 82-31-733-9167
www.chocell.com
405-PangenCHOexpression-
Licensor,primary
ParaTechs Corp.
A-205 ASTeCC Building
University of Kentucky
Lexington, KY 40546-0286
Phone: (859) 218-6541
Email: info@paratechs.com or sbass@
paratechs.com
FAX: (859) 257-2489
439-Vankyrinenhancedbaculovirus
expressionvectorsystem(VE-BEVS);
Vankyrin-enhancedcelllines(VE-CL)
-Licensor,primary
Partners Healthcares Research
Ventures & Licensing (RVL)
handles licensing for General Hospital
Corp. and Massachusetts General
Hospital
101 Huntington Avenue, 4th Floor
Boston, MA 02199
Tel: (617) 954-9500
e-mail: csrl@partners.org
website: www.techtransfer.
massgeneral.org
-Licensor,primary
Pennsylvania State University
University Park (formerly State
College), PA
Matthew D. Smith, 814-863-1122 /
mds126@psu.edu
190-Yeastcelllinesandvectors;
Saccharomycescerevisiaecelllines
-properfoldingandglycosylation-
R&D,oftech.
PERCIVA PER.C6 Development
Center
Percivia PERC.6 Development Center
One Hampshire Street
5th Floor
Cambridge, Massachusetts 02139
U.S.A.
Phone: 617-301-8800
E-Mail: info@percivia.com
glycoproteins;antibodies-R&D,of
tech.
Pfizer, Inc.
New York, NY; the worlds largest
pharmaceutical company, including in
terms of R&D and sales
200-Bovinegrowthhormone(bGH)
polyadenylationsequence-eukaryotes;
expressionenhancement-Parentco.
296
PharmedArtis GmbH
spun-off from Fraunhofer:
Forckenbeckstrasse 6
52074 Aachen
Germany
Prof. Dr. Gerd Gellissen, CSO
Phone +49-1 71-28 25 038
ggellissen@gmx.de
163-Staphylococcuscarnosus
expresion-Licensor,secondary
-alternativeyeast-Licensor,primary
178-CoMedsystem;UniversalYeast
vectors;pCoMedvectors;Arxula
adeninivorans-derivedTEF1promoter
-Licensor,primary
PhaseBio
4020 Aerial Center Parkway, Suite 101
Morrisville, NC 27560
Gabriel R. Cipau, PhD
Chairman and CEO
(919) 271-0926
contact@keyhealthcarepartners.com
111-deltaPhase;Elastin-like
polypeptide(ELP)fusionproteintags
-chromatography-freepurification;
Phillips Petroleum
Bartlesville, OK
191-Pichiapastorisexpression-
R&D,oftech.
382-PichiapastorisAOX1promoters
-R&D,oftech.
Phyton Biotech
German subsidiary, Phyton Biotech
GmbH, operates the worlds largest
commercial cGMP manufacturing
facility for plant cell fermentation
279 Princeton-Hightstown Road
East Windsor, NJ 08520-1401
+1 609-426-2520
info@phytonbiotech.com
418-Phytonplantcellfermentation
-Licensor,primary
Pioneer Hi-Bred International, Inc.
see DuPont Corp.
287-Ubiquitinplantpromoters-
Assignee,patent
Plant Bioscience Ltd.
Colney Lane
Norwich
Norfolk
NR4 7UH
UK
www.bltechnology.com
Karin Schofield
Email: karin@plantbioscience.com
Tracey Bettinson
Tel: +44 (0)1603 456500
tlb@pbltechnology.com
173-Streptomycesstationaryphase
expression;SPEsystem;Streptomyces
vectors;SecretedProteaseProduction
(SPP)System-Licensor,primary
282-RNA-dependentRNApolymerase
(RdRP)-universal-Licensor,primary
289-CleanGeneplanttransformation
-Licensor,primary
416-MARsPLUS;Matrixattachment
regionsPLUS-plants-Licensor,
primary
Plant BioScience Ltd.
Colney Lane, Norwich
Norfolk NR4 7UH
UK
tlb@pbltechnology.com
143-CoconutExpresscell-free
translation-plants-Licensor,primary
Polymun Scientific
Immunobiologische Forschung
GmbH
Prof. Dr. Hermann Katinger, CEO
Nudorfer Lnde 11
A-1190 Vienna
Austria
Phone: +43-1-36006-6203
Fax: +43-1-3697615
Email: office@polymun.com
401-CHOcells-antibodies;serum-
freemedia-Licensor,primary
Potomac Affinity Proteins
Rockville, MD
Bonnie Bryan
Email: Potomac_affinity@msn.com
Tel: 301-610-9687
145-Subtilisin(psub)fusionprotein
tagexpression;ProfinityeXact
expression-E.coli;bacteria;yeasts;
CHOcells-Assignee,patent
Precision BioSciences, Inc.
104 T.W. Alexander Dr., Bldg 7
Durham, NC 27713
Email; partner@precisionbiosciences.
com
112-DirectedNucleaseEditor(DNE);
MeganucleaseDesign-universal-
Licensor,primary
Princeton University
Laurie Tzodikov
Technology Licensing Associate
Princeton University
Office of Research and Intellectual
Property
4 New South Building
P.O. Box 36
Princeton, NJ 08544
Tel: 609-258-7256
Fax: 609-258-1159
E-mail: tzodikov@princeton.edu
339-Elastin-likepolypeptide(ELP)
self-cleavingfusionproteintags-
chromatography-freepurification;E.
351-Polyhydroxybutyrate(PHB)
fusiontags,self-cleavingcoexpressed
withaffinitymedium-E.coli;
ProSpec-Tany TechnoGene Ltd.
Weizmann Science Park
P.O Box 398
Rehovot 76103, Israel
Telephone (in US) +972-8-9318056
Fax +972-8-9460534
info@prospec.co.il
330-Biogenericsmanufacturing
technologypackages-E.coli-
Licensor,primary
Protalix BioTherapeutics, Inc
2 Snunit St., Science Park,
POB 455, Karmiel 20100
Israel.
Tel: +972-4-9028100
Email: info@protalix.com
280-ProCellExPlantExpression
-plantcells;glycosylation-Licensor,
primary
297
Protein Biopharma, Inc. (PDL)
34801 Campus Drive, Fremont,
California 9455; formerly Protein
Design Labs, Inc. (changed name in
Jan. 2006)
257-NS0-PFCFcells;NS0cells,
protein-andcholesterol-free-
antibodies-R&D,oftech.
Protein Sciences Corp.
Protein Sciences Corporation
1000 Research Parkway
Meriden, CT 06450-7159
Kroon, Hendrik A. III (O)
Chief Medical Officer
Tel: (908) 464-3264
Fax: (907) 464-3265
Email: henk-andre.kroon@rtp.ppdi.
com
Web: www.proteinsciences.com
298-Baculovirusexpressionvector
systems(BEVS)-Licensor,primary
300-MimicSf9InsectCells
-mammalian-likeglycosylation-
Licensor,primary
303-SpodopterafrugiperdaSf-21cell
line-baculovirushostinsectcells-
Licensor,primary
430-pIExbaculovirusvectors;
hr5enhancer;ie1immediateearly
promoter-insectcells;avoid
baculoviruspathogenicity-Licensor,
primary
Protemation, Inc.
11661 NE Sunset Loop
Bainbridge Island, WA 98110
rclark88@aol.com
104-Vectors,site-specificrecombinase
assembly-eukaryotes-Licensor,
primary
Protheon Inc.
B120E Yonsei Engineering Research
Complex
134 Shinchon-Dong, Seodaemun-Ku,
Seoul 120-749
KOREA
blseong@yonsei.ac.kr or mk2040@
protheon.com
317-Profusevectors;Cisperone
chaperonefusiontags-E.coli;
Saccharomycescerevisiae-Licensor,
primary
377-SecretionEnhancerVector
System(SEVS);SecHancervector-
Saccharomycescerevisiae-Licensor,
primary
Public Intellectual Property
Resource for Agriculture (PIPRA)
University of California
One Shields Avenue
Dept. Plant Sciences
Plant Reproductive Biology Building-
Mail Stop 5
Davis, CA 95616 USA
View a map
Tel: + (530) 754 2162
clchiham@ucdavis.edu
279-pPIPRAvectors-plants;public
domain-Licensor,primary
Purdue University, Purdue Research
Foundation
Purdue Technology Center, Suite C2-
100
(765) 494-8645 Phone
(765) 496-1146 Fax
-plants-Assignee,patent
PVA-MV AG
Joakim Ketonen
PVA-MV AG
Gerhart-Hauptmann-Strae 23
18055 Rostock
Fon + 49 381.497474-0
Fax + 49 381.497474-9
http://www.pva-mv.de/
j.ketonen@pva-mv.de
320-Cold-Inducedexpression-
Bacillussubtilis-Licensor,primary
QIAGEN GmbH
QIAGEN Strasse 1
40724 Hilden, Germany
Tel: +49 (0)2103-29-16221
Fax: +49 (0)2103-29-26221
Dr. Helmut Hilbert
E-mail: bd@qiagen.com
tags;universal-Licensor,primary
Rega Institute, Katholieke
Universiteit Leuven
Katholieke Universiteit Leuven
Prof. Jozef Ann
Minderbroedersstraat 10
3000 Leuven
Belgium
Phone: (32-16) 337371
Fax: (32-16) 337340
E-mail: jozef.anne@rega.kuleuven.be
416-MARsPLUS;Matrixattachment
regionsPLUS-plants-Assignee,
patent
Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown, NY 10591
914-345-7400
192-VelociMab;EESYRexpression
system;FASTRcelllines-CHO
expressionoptimization;antibodies
-Licensor,primary
Regensburg University
Regensburg, Germany
expression-algae,single-cell-R&D,
oftech.
Rensselaer Polytechnic Institute
Troy, NY
108-ControllableSelf-CleavingIntein
Derivative;IMPACT-CN;pH-sensitive
self-cleavingfusionproteinaffinity
purificationtag-universal-Assignee,
patent
RepliGen Corp.
41 Seyon Street
Building #1, Suite 100
Waltham, MA 02453
Phone: (781) 250-0111
Toll-free: (800) 622-2259
Fax: (781) 250-0115
E-mail: mpayer@repligen.com
Licensor,primary
Replikun Biotech Pty Ltd.
Level 1, 80 Jephson Street
Toowong Queensland 4066
AUSTRALIA
Phone: +61 7 3327 9717
Fax: +61 7 3720 9574
Dr Lavinia Proctor, Chief Operating
Officer
lavinia.proctor@replikun.com.au
357-Flavivirusvectors;Kunjin
repliconvectors-prokaryotes;
Streptomyces;prokaryotes-Licensor,
primary
298
Research Corporation Technologies
Inc. (RCT)
5210 East Williams Circle, Suite 240
Tucson, Arizona 85711-4410
Telephone: (520) 748-4400
Fax: (520) 748-0025
E-mail: dbramhill@rctech.com
150-Caulobactercrescentus
expression;PureProCaulobacter
ExpressionSystem-Caulobacter
bacteriahosts-Licensor,primary
191-Pichiapastorisexpression-
Licensor,primary
expressionenhancement-Licensor,
primary
299-InsectSelectProteinExpression
System-insectcells;avoid
baculoviruses-Licensor,primary
367-Formaldehydedehydrogenase
(FLD1)promoter;Formaldehyde
selection-Pichiapastoris;yeasts-
Licensor,primary
glycosylation-Licensor,primary
382-PichiapastorisAOX1promoters
-Licensor,primary
Rhein Biotech Ltd.
now a subsidiary of Dynavax
Technologies Corp.
Rhein Biotech GmbH
Dynavax Europe
Eichsfelder Strasse 11
D-40595 Dsseldorf
Phone: +49 (0)211 7 58 45 0
Fax: +49 (0)211 7 58 45 130
E-Mail: info(at)rheinbiotech.de
-alternativeyeast-Assignee,patent
181-Hansenulaexpression(yeast)
-Licensor,primary
polymorpha;yeasts-Assignee,patent
RheoGene, Inc.
wholly-owned affiliate of the
University of Pittsburgh Medical
Center (UPMC)
2650 Eisenhower Ave.
Norristown, PA
http://www.rheogene.com
Dr. Camille Jolly-Tornetta, Director of
Intellectual Property
610-650-8734 ext. 104
199-AttSiterecombinases-precise
geneinsertion;eukaryotes-Licensor,
primary
227-RheoSwitchMammalian
InducibleExpressionSystem;
RheoSwitchLigandRSL1promoter
;Ecdysonereceptorinduction
-mammaliancells;adjustable
RIKEN
Center for Intellectual Property
Strategies
2-1, Hirosawa, Wako
Saitama 351-0198
Japan
Ph: +81-48-462-5475
E-mail cips-kikaku@riken.jp
243-Cell-freesystem-glycoproteins;
hybridoma-Licensor,primary
Roche Protein Expression Group
(RPEG)
Indianapolis, IN
800.428.5433 x7525
indianapolis.rpeg@roche.com
http://www.roche-applied-science.
com/rpeg/
121-Histags;Poly-Histidinefusion
affinitytagtechnology;6xHistidine-tag
-univeral;purificationtags-Licensor,
primary
169-GroEL,GroESchaperones;
Chaperonins-properfolding;
universal;E.coli-Licensor,primary
393-Hsp60,Hsp70,Hsp90,Hsp100
chaperones-mammaliancells-
Licensor,primary
Rohm and Haas Co.
acquired by Dow Chemical in summer
2008
100 Independence Mall West
Philadelphia, Pa 19106
Phone: 877-288-5881
System-E.coli-Licensor,secondary
Roslin Institute
Roslin Biocentre
Roslin, Midlothian
EH25 9PS
Scotland, UK
Commercial Activities
Malcolm Bateman
Tel: +44 (0)131 527 4534
Fax: +44 (0)131 527 4499
expresssion-Licensor,primary
-proteinsinchickeneggs-Patent
disputer
409-DNAmicroinjection-transgenic
animals,chickens-Licensor,primary
Rutgers University
Castagno, James
Manager, Technology/Licensing
Rutgers University
Cook College, Martin Hall
88 Lipman Dr
New Brunswick, NJ 08901
Tel: (732) 932-1000 ext 593
Fax: (732) 932-4176
Email: jcastagno@ocltt.rutgers.edu
290-Plastids(chloroplasts)expression
-plantcellculture-Licensor,primary
328-AgrobacteriumtumefaciensRpoA
co-expressiontranscriptionalactivator
348-pColdvectors;ColdShock
ProteinA(cspA)promoters-E.coli,
coldinduction-Parentco.
Sabatier University
Toulouse, France
Assignee,patent
Salk Institute for Biological Studies
10010 North Torrey Pines Road
La Jolla, CA 92037
Attn.: Department of Intellectual
Property and Technology Transfer
Phone: 858-453-4100 ext 1275
Fax: 858-450-0509
Email: amueller@salk.edu.
222-Flp-Inexpressionsystem;
FLP-MediatedGeneModificationin
MammalianCells;FLPrecombinase
-mammaliancells-Licensor,primary
(Vff2p)enhancer-yeast;bacteria;
CHOcells-R&D,oftech.
(Vff2p)enhancer-yeasts-Licensor,
primary
299
Sandoz Inc./AG
Sandoz is now the name for the
(bio)generics subsidiary of Novartis.
Assignee,patent
Sangamo BioSciences, Inc.
VP, Corporate Development
501 Canal Blvd
Suite A100
Richmond, CA 94804
Tel: (510) 970-6000
Fax: (510) 236-8951
Email: pbluford@sangamo.com
Web: www.sangamo.com
recombination-Patentdisputer
206-ZincfingerDNA-bindingproteins
(ZFPs);ZFPTranscriptionFactors
-genemodification;mammaliancells;
plantcells-Licensor,primary
238-CHOZnCHODG44celllines
-antibodies-Licensor,primary
Scarab Genomics LLC
Tony A. Pharo
Director of Business Operations
Scarab Genomics
1202 Ann St.
Madison, WI53713
Tel: 888 513 7075
168-CleanGenomeE.coli;Stripped-
downE.coli-Licensor,primary
Schering-Plough Corp.
2000 Galloping Hill Road, Kenilworth,
NJ 07033
SARs-mammaliancells-Parentco.
Scripps Research Institute, The
10550 North Torrey Pines Road, TPC-9
La Jolla, CA 92037
Phone: (858) 784-8496
Fax: (858) 784-9910
Mathew B. Mitchell, Technology
Development Officer
858-784-9386
mathew@scripps.edu
cells-Assignee,patent
chloroplastexpression;promoter-
algae,single-cell-Licensor,primary
Searle, G.D.
Skokie, IL; later acquired by
Monsanto, later merged into/became
Pharmacia, later acquired and merged
into Pfizer
355-Tryptophan(trp)promoters-E.
coli-Assignee,patent
Selexis
Plan-les-Ouates 18
chemin des Aulx CH-1228
Switzerland
Phone: 0041228848342
Email: info@selexis.com
subtilis-Licensor,secondary
SARs-mammaliancells-Licensor,
primary
228-SelexisGeneticElements(SGEs)
-mammaliancelllines-Licensor,
primary
SemBioSys Genetics Inc.
Mr. Andrew Baum
President and Chief Executive Officer
SemBioSys Genetics Inc.
110, 2985 - 23rd Avenue N.E.
Calgary, AB T1Y 7L3
Telephone: 403.250.5424
Fax: 403.250.3886
Email: bauma@sembiosys.com
283-StratosomeBiologicsSystem;
Oilbodyexpression;Safflowerplant
seedexpression-Licensor,primary
Shire Pharmaceuticals Group plc
recombination-Parentco.
invivo-mammaliancells-Parentco.
Sigma-Aldrich (SIAL) (SAFC)
13804 W. 107th Street
Lenexa, Kansas 66215
Tel: +1 913-469-5580
Free Tel: 1 800-255-6032
Fax: +1 913-469-5584
E-Mail: info-na@sial.com
expressingCHOcellslines-Licensor,
secondary
SmithKline Beecham Corp. (SKB)
see GlaxoSmithKline Inc.
(DES)-insectcellculture-R&D,of
tech.
Spanish Center on Biotechnology
(CSIC)
Technology Licensing and
Commercialization
Head of Unit
Javier Etxabe Oria
Ph.+34 91 585 53 75
j.etxabe@orgc.csic.es
359-Streptomyceslividansstrains;
xysApromoters-Licensor,primary
Stanford University
Palo Alto, CA
101-Cell-FreeProteinSynthesis
(Cell-Free)-Assignee,patent
131-RecombinantDNA;Protein
Expression;Cohen-Boyer-Licensor,
primary
308-PhageT5promoter-Assignee,
patent
stART Licensing, Inc.
Exeter Life Sciences, Inc., owns a 50.1
percent stake and Geron Corp. a 49.9
percent stake.
2390 E Camelback Rd # 440
Phoenix, Arizona USA 85016-3448
Toll-Free: 877.667.0007 or
602.667.0007
Ben Carlson, 202-466-9633; ben@
turnerstrategies.com
-Licensor,primary
300
Stratagene Corp.
acquired by Agilent Technologies, Inc.
in June 2007
11011 N. Torrey Pines Road
La Jolla, CA 92037
(858) 373-6300
800.894.1304 (US/Canada)
342-Hightransformationefficiency
(Hte)competency-E.colicells-
Licensor,primary
RNAP)-E.coli-Patentdisputer
Swiss Federal Institute of Technology
(ETH), Zurich
ETH transfer
Tel: +41 (0)44 632 23 82
E-Mail: transfer@sl.ethz.ch
236-CHOSSFcelllines-adherent
CHOcells;protein-freemedia-R&D,
oftech.
368-GAPFLpromoter;
dehydrogenase-derivedpromoter-
yeasts-R&D,oftech.
Symphogen A/S
Elektrovej Building 375
DK-2800 Lyngby
Denmark
Jette Asboe Lassen
+45 4526 5057
jla@symphogen.com
240-Sympressexpression;Human
polyclonalantibodies(rpAB)-CHO
Takara Mirus Bio, Inc.
510 Charmany Drive
Madison, WI 53719
Toll-Free: 888-251-6618
Phone: 608-441-2844
Fax: 608-441-2845
Web: www.takarabiousa.com
techserv@takarabiousa.com
334-Chaperoneexpressionplasmids;
DnaK,DnaJandGrpEchaperones-E.
coldinduction-Licensor,primary
363-AureobasidinAvectors(pAUR)-
selectablemarkerinyeasts-Licensor,
primary
Technical University Carolo-
Wilhelmina at Brunswick
TU Braunschweig
38092 Braunschweig
Germany
147-Bacillusmegateriumexpression
-E.colialternative-R&D,oftech.
Temple University
Philadelphia, PA
350-pMALProteinFusionand
PurificationSystem;Maltosebinding
(MBP)fusionaffinitytags;pMAL
plasmids-antibodyfragments;E.coli
-Assignee,patent
TET Systems Holding GmbH & Co
KG.
Im Neuenheimer Feld 582
69120 Heidelberg
Tel. +49 6221 5 88 04 00
Fax +49 6221 5 88 04 04
info@tet-systems.com
license@tet-systems.com
Systems-eukaryotes-Licensor,
primary
Tetragenetics, Inc.
Cornell Business and Technology Park
95 Brown Road
Box 1010/Suite 220
Ithaca, NY 14850
John Reilly
jreilly@tetragenetics.com
Phone: 607-257-1199
210-TetraExpress;Tetrahymena
(protozoan)expression-Licensor,
primary
Texas A&M University (TAMUS)
College Station, TX
Janie C. Hurley, MBA
Licensing Manager -Agriculture and
Life Sciences
jhurley@tamu.edu
298-Baculovirusexpressionvector
systems(BEVS)-Licensor,primary
Licensor,primary
Licensor,primary
430-pIExbaculovirusvectors;
hr5enhancer;ie1immediateearly
promoter-insectcells;avoid
baculoviruspathogenicity-Licensor,
primary
437-Drosophilasialyltransferases
-insectcells;glycosylation-Licensor,
primary
Therapeutic Human Polyclonals Inc.
acquired by Roche for $56.5 million in
April 2007; originally funded by RCT
and SangStat Medical Corp.:
2105 Landings Drive
Mountain View, CA 94043
(650)-625-8840
214-Transgenicrabbits-humanized
polyclonalantibodies-Assignee,
patent
Transkaryotic Therapies, Inc. (TKT)
Cambridge, MA 02139; acquired in
April 2005 by Shire Pharmaceuticals
Group plc for ~$1.6 billion
invivo-mammaliancells-Assignee,
patent
invivo-mammaliancells-Licensor,
primary
TranXenoGen, Inc.
PO BOX 21
NORTHBORO, MA. 01532
Ph: 508 842 5036
business.development@tranxenogen.
com
245-Chickeneggexpression;
Transgenicanimals-Licensor,primary
Tsukuba Industrial Liaison and
Cooperative Research Center (ILC)
1-1-1 Tennodai, Tsukuba
Ibaraki 305-8577
Japan
TEL +81-29-853-2905/2912 FAX +81-
29-853-6565
E-mail : ilc ilc.tsukuba.ac.jp
http://www.ilc.tsukuba.ac.jp/
174-Streptomyceteshyper-
inducibleexpression;PnitA-NitR
system;Caprolactaminduction-
Streptomycetes-Licensor,primary
301
UniTargetingResearch AS
Avinoam Kadouri
Strategic Advisor
UniTargetingResearch
HIB, Thormhlensgate 55,
N-5008 Bergen,
Norway
Tel: +41 793014274
www.unitargeting.com
408-UTRCloneGeneration;
UTRtech;CellFactories;Gaussia
luciferasesignalpeptides-Mabs
supersecretion;CHOcells-Licensor,
primary
Universite Libre de Bruxelles
Brussels, Belgium
308-PhageT5promoter-R&D,of
tech.
Universithy of Freiburg
Freiburg, Germany
273-Glyco-EngineeredMoss;
Physcomitrellapatensexpression;
mossexpressionpromotingregions
(MEPRs)-glycosylation;antibodies
-R&D,oftech.
University of Arkansas for Medical
Sciences; UAMS Biomedical
Biotechnology Center (BBC)
4301 West Markham Street, Slot 718
Little Rock, Arkansas 72205
Phone: (501) 686-6696
Fax: (501) 686-8501
Timothy J. OBrien, Ph.D.
TJObrien@uams.edu
Charles Cook
CookCharlesA@uams.edu
328-AgrobacteriumtumefaciensRpoA
co-expressiontranscriptionalactivator
University of Barcelona
Barcelona,Spain
supersecretion;CHOcells-R&D,of
tech.
University of Bergen
Bergen, Norway
tech.
University of Birmingham
AL-RUBEAI, Mohamed
University of Birmingham
Dept. of Chemical Engineering
Birmingham B15 2TT
UNITED KINGDOM
Tel: +44-121-4143888
Fax: +44-121-4145324
E-mail: m.al-rubeai@bham.ac.uk
251-bcl-2(p21)overexpressingNS0
celllines-NS06A1(100)3cellline-
antagonizeapoptosis;Mabs-Licensor,
primary
University of British Columbia
(UBC)
Emily Vereker, Ph.D.
Technology Transfer Officer
UBC-University Liaison Office
103-6190 Agronomy Road
Vancouver, BC, Canada
V6T 1Z3 Canada V6T 1Z3
T:(604) 827-4507
F:(604) 822-8589
150-Caulobactercrescentus
expression;PureProCaulobacter
ExpressionSystem-Caulobacter
bacteriahosts-Assignee,patent
299-InsectSelectProteinExpression
System-insectcells;avoid
baculoviruses-Assignee,patent
University of Calgary
see University Technologies
International
426-IE-1(BmNPV1.2kbfragment)
promoters;Bombyxmoriactin
promoters-insectcells-R&D,oftech.
Office of Technology Transfer
Office of the President
1111 Franklin Street, 5th Floor
Oakland, California 94607-5200
Main Office Telephone: (510) 587-
6000
General Facsimile: (510) 587-6090
Website: http://www.ucop.edu/ott/
Cotton, Patricia, Director - (510) 587-
6023; patricia.cotton@ucop.edu
Weintraub, Silka, Sr Licensing Analyst
- (510) 587-6036; silka.weintraub@
ucop.edu
-universal-Assignee,patent
131-RecombinantDNA;Protein
Expression;Cohen-Boyer-Assignee,
patent
cells-Assignee,patent
136-Supercorepromoters;SCP1;
Corepromoters-thestrongest
[promoters]evermade-Licensor,
primary
140-TranslationEngineering
expression;CODA;Computationally
OptimizedDNAAssembly;HotRod
Genes;ControlledRibosomalPausing
-universal-R&D,oftech.
Assignee,patent
204-Light-switchablepromoters-
Licensor,primary
246-Chickenrimordialgermcells
(PGCs)-Humanproteins/monoclonal
antibodiesinchickeneggs-R&D,of
tech.
381-Pichiapastorisantibody
University of Central Florida
Dr. M.J. Soileau
Vice President for Research &
Commercialization
Millican Hall 243
University of Central Florida
4000 Central Florida Boulevard
Orlando, FL 32816
Phone: 407.823.5538
Fax: 407.822.1156
Email: mj@mail.ucf.edu
269-Chloroplastexpression-plants
-Licensor,primary
Technology(CTT)-plantcells-R&D,
oftech.
University of Colorado
Mary.Tapolsky@colorado.edu
303-735-2298
234-CHOcellline(Puck);CHO-K1
cellline-Licensor,primary
302
University of Edinburgh
Dr. Wendy Nicholson
Edinburgh Research and Innovation
University of Edinburgh
1-7 Roxburgh Streen
Edinburgh, EH8 9TA
Scotland, UK
wendy.nicholson@ed.ac.uk
404-Osteoclast-associatedreceptor
(OSCAR)promoter-mammalian;
CHOcells-Licensor,primary
University of Georgia
Gennaro J. Gama, Ph.D.
Technology Manager
University of Georgia Research
Foundation, Inc.
624 Boyd GSRC
Athens, GA 30602-7411
Ph: +706-583-8088
Fx: +706-542-3837
e-mail: GJG@uga.edu
-Licensor,primary
210-TetraExpress;Tetrahymena
(protozoan)expression-R&D,oftech.
-proteinsinchickeneggs-Assignee,
patent
435-Baculovirusvectorsand
promoters-glycosylation
University of Groningen
Groningen, Germany
148-Bacillussubtilis,super-
oxidizingstrains;Thiol-disulfide
oxidoreductases(TDORs);Thioredoxin
A(TrxA)depletion-disulfidebridge
optimization-Licensor,primary
subtilis-R&D,oftech.
University of Hawaii
184-NeuBIOSexpression;Neurospora
crassaexpression;NeuKARYON
-filamentousfungi;glycosylation;
Cabilly-Bossworkaround-Assignee,
patent
University of Illinois
University of Illinois
Research and Technology Management
Office
Fourth Floor Swanlund Building
601 East John Street
Champaign, IL 61820
Phone: (217) 333-7862
Fax: (217) 244-3716
System-E.coli-R&D,oftech.
University of Iowa Research
Foundation (UIRF)
214 Technology Innovation Center
Iowa City, Iowa 52242
319-335-4546
brenda-akins@uiowa.edu
219-CMV(human)promoter;
Cytomegaloviruspromoter;Complete
ControlInducibleMammalian
ExpressionSystem-mammaliancells
-Licensor,primary
391-CCTpromoters-mammalian
University of Kentucky
439-Vankyrinenhancedbaculovirus
expressionvectorsystem(VE-BEVS);
Vankyrin-enhancedcelllines(VE-CL)
-R&D,oftech.
University of Maryland
Biotechnology Institute
Jonathan Gottlieb, PhD, MBA
Director, Technology Transfer and
Commercialization
Office of Research, Innovation &
Commercialization
University of Maryland Biotechnology
Institute
9600 Gudelsky Drive, Suite 2105L
Rockville MD 20850
Phone: (240) 314-6506
Mobile: (443) 468-9875
Email: gottlieb@umbi.umd.edu
http://www.umbi.org
209-Perkinsusmarinusexpression
-protozoaexpresslargeproteins-
Licensor,primary
larvae;Trichoplusianilarvae
expression-transformedcaterpillars
-Assignee,patent
University of Massachusetts (UMass)
Commercial Ventures and Intellectual
Property
70 Butterfield Terrace
3rd floor
Amherst, MA 01003
Phone 413-545-3606
Fax 413-545-3632
cvip@research.umass.edu
-Assignee,patent
University of Medicine & Dentistry
of New
Jersey
Newark, NJ
coldinduction-R&D,oftech.
University of Milano-Bicocca
Industrial Liason Office
University of Milano - Bicocca
Piazza dellAteneo Nuovo
1 - 20126, Milano
Italy
ilo@unimib.it
http://www.unimib.it/go/Home/
English-version
188-Zygosaccharomycesbailii
expression;Zbleu2strain-yeast-
Licensor,primary
University of Naples
Naples Italy
http://www.dma.unina.it/
159-Pseudoalteromonashaloplanktis
TAC125(PhTAC125)-coldexpression
-Licensor,primary
University of Newcastle-upon-Tyne
Tyne, U.K
tech.
303
University of Notre Dame
Licensing Department
436 Grace Hall
Notre Dame, IN 46556
Phone: 574-631-9327
Email: ndlicens@nd.edu
292-Insectcellsglycosylation-R&D,
oftech.
387-piggyBactransposon-
eukaryotes;insectcells-R&D,oftech.
University of Oregon
Gerhart, Donald J., Ph.D.
Director, Technology Transfer
1238 University of Oregon
Eugene, OR 97403-1238
Tel: (541) 346-3176
Fax: (541) 346-5215
Email: dgerhart@techtran.uoregon.edu
138-TEVProtease;TobaccoEtch
Virus(TEV)NIaprotease-cleavage
offusionproteintags;universal-
Licensor,primary
University of Pennsylvania
Philadelphia, PA
325-Twin-argininetranslocation(Tat)
system;Tatnanomachine-protein
folding,thensecretion;bacteria-
R&D,oftech.
University of Pittsburgh Medical
Center (UPMC)
Pittsburgh, PA
199-AttSiterecombinases-precise
geneinsertion;eukaryotes-Assignee,
patent
University of Texas
Austin, TX
138-TEVProtease;TobaccoEtch
Virus(TEV)NIaprotease-cleavage
offusionproteintags;universal-
Licensor,secondary
338-Disulfideisomerase
coexpression;DsbCandDsbG-E.
coli;disulfidebondsandfolding-
Assignee,patent
University of Texas Southwestern
Medical Center
Hall, Stanley D.
Director of Venture Development
University of Texas
Southwestern Medical Center
5323 Harry Hines Blvd
B6.200
Dallas, TX 75390-8596
Tel: (214) 648-9101
Fax: (214) 648-6488
Email: stan.hall@utsouthwestern.edu
Web: www.utsouthwestern.edu
114-Expressionenhancers,
oligonucleotides-universal-Licensor,
primary
University of Toledo
Toledo, OH
271-Coupledregeneration/
transformation,plants-R&D,oftech.
University of Waikato-New Zealand
186-Ophiostomaexpression-
ascomycetesfungi-R&D,oftech.
University of Washington
Seattle, WA
Assignee,patent
University of Wisconsin
See WARF
149-Bacterialartificialchromosome
(BAC)expression;pBACvectors-
bacteria-R&D,oftech.
151-ClostridiumExpressionSystem;
NTNHpromoterfromClostridium
botulinum-R&D,oftech.
162-Rhodospirillumrubrum
(bacterial)expression-membrane
proteins-R&D,oftech.
downE.coli-R&D,oftech.
203-InternalRibosomeEntry
Sequences(IRES)RNAtranslation
enhancers;pCITEvectors;Cardiovirus
2AIRES;pIRES-eukaryotes-R&D,
oftech.
217-Autocatalyticcleavagesites;
Mengovirusvectors;Scissioncassettes
-mammaliancells-R&D,oftech.
286-Ubiquitinlinkagedomain-
multipleproteinsintransgenicplants
318-Bacterialtranscriptionpromoters
-R&D,oftech.
388-Tax-inducibleexpression;Bovine
leukemiavirus(BLV)promoter-
mammaliancells-Assignee,patent
University of Wyoming
WyomingInvents@uwyo.edu
Licensor,primary
University Technologies
International
Suite 130, 3553 - 31 Street NW
Calgary, AB
T2L 2K7
Phone: 403.270.7027
Fax: 403.270.2384
Email: info@uti.ca
426-IE-1(BmNPV1.2kbfragment)
promoters;Bombyxmoriactin
promoters-insectcells-Licensor,
primary
UNIZYME Laboratories
Jos Arnau, PhD
Business Development Manager
Unizyme Laboratories A/S
Dr. Neergaardsvej 17
DK-2970 Hrsholm
DENMARK
Email: ja@unizyme.dk
Web: http://www.unizyme.com
tags;universal-Assignee,patent
Upjohn Co.
now long merged into Pfizer
expressionenhancement-Assignee,
patent
Ventria Bioscience
Ning Huang
V.P., R&D
Ventria Bioscience
4110 North Freeway
Sacramento, CA 95834
Ph: 1-916-921-6148
Email: nhuang@ventriabio.com
419-ExpressTecexpression;
ExpressPro;ExpressMab-riceand
barley;antibodies-Licensor,primary
Viragen, Inc.
Rothberg, Melvin
Executive Vice President
Viragen, Inc.
865 SW 78th Avenue
Suite 100
Plantation, FL 33324
Tel: (954) 233-8746
Fax: (954) 233-8743
Email: mrothberg@viragen.com
expresssion-Formerinv.
304
Vivalis S.A.
Pierre Miniou, PhD
Business Development Manager
Tel. +33 (0) 228 07 37 16
Fax +33 (0) 228 07 37 11
Email: pierreminiou@vivalis.com
244-Ex-CellEBxexpression;EBx
cells;Chickenembryonicstemcells;
ChickenEBxcells;DuckEBxcells
-Licensor,primary
VTU Technology GmbH
Dr. Michael Koncar
Founder and Managing Director for
R&D
Parkring 18
8074, Grambach
Austria
Tel.: +43 (316) 4009-200
Fax: +43 (316) 4009-210
michael.koncar@vtu.com
383-Pichiapastorissuperyeast;
PichiapastorisAOX1promoters-
Licensor,primary
Wacker Biotech
a spin off of the state-owned Hans
Knll Institute in Jena, Germany
Wacker-Chemie GmbH
Hanns-Seidel-Platz 4
81737 Mnchen, Germany
Tel. +49 89 6279-01
Fax +49 89 6279-1770
info@wacker.com
356-WACKERSecretionSystem;E.
coliK12-basedsecretionsystem-
antibodyfragments-Licensor,primary
Washington Research Foundation
(WRF)
licensing agent for the University of
Washington and other organizations
Beth G. Etscheid, Ph.D.
Director of Licensing
Washington Research Foundation
2815 Eastlake Avenue E, Suite 300
Seattle, WA 98102
Tel: 206.336.5532
Fax: 206.336.5615
betschei@wrfseattle.org
Licensor,primary
Washington State University
Research Foundation (WSURF)
1610 NE Eastgate Boulevard
Pullman WA 99163
Telephone: 509.335.5526
Fax: 509.335.7237
278-Plantexpression-glycosylation
-Assignee,patent
Washington University
St. Louis, Missouri
Carl Morrell
morrellc@otm.wustl.edu
P: (314) 747-0922
F: (314) 362-5872
267-Agrobacteriumtumefaciens;Ti
plasmids-transgenicplants-Licensor,
primary
324-pTAT-HAplasmids-E.coli;
bacteria-Licensor,primary
Wisconsin Alumni Research
Foundation (WARF)
licensing@warf.org
-Licensor,primary
149-Bacterialartificialchromosome
(BAC)expression;pBACvectors-
bacteria-Licensor,primary
151-ClostridiumExpressionSystem;
NTNHpromoterfromClostridium
botulinum-Licensor,primary
162-Rhodospirillumrubrum
(bacterial)expression-membrane
proteins-Licensor,primary
downE.coli-Assignee,patent
203-InternalRibosomeEntry
Sequences(IRES)RNAtranslation
enhancers;pCITEvectors;Cardiovirus
2AIRES;pIRES-eukaryotes-
Licensor,primary
217-Autocatalyticcleavagesites;
Mengovirusvectors;Scissioncassettes
-mammaliancells-Licensor,primary
286-Ubiquitinlinkagedomain-
multipleproteinsintransgenicplants
318-Bacterialtranscriptionpromoters
-Licensor,primary
388-Tax-inducibleexpression;Bovine
leukemiavirus(BLV)promoter-
Worcester Polytechnic Institute
Worcester, MA
340-ExpressProtect;p26,SicA,and
alpha-crystallin-typefusionprotein
tags-E.coli-R&D,oftech.
Wyeth
401 North Middleton Rd., Pearl River
NY 10965; HQ in Madison, NJ;
formerly American Home Products
Corp. (AHP); Genetics Institute was
merged into Wyeth in 1997
306-Enterokinase-fusionprotein
cleavage-Licensor,primary
343-His-PatchThioFusionSystem;
pThioHisvector-E.coli;fusion
proteins-Licensor,primary
Xcellerex
170 Locke Drive
Marlborough, MA 01752
Tel: 508-480-9235
Fax: 508-480-9238
www.xcellerex.co
Geoffrey Hodge, VP, Tech. Dev.
ghodge@xcellerex.com
237-CHOSupercell;Targeted
transfection;CHODG44cellline-
Licensor,primary
Xoma Corp.
Tom Smart
XOMA (US) LLC
2910 Seventh Street, Berkeley,
California 94710 USA
510-204-7536 TEL
510-644-0537 FAX
smart@xoma.com or BizDevInfo@
xoma.com
144-Bacterialcellexpression
technology(BCE);araBpromoter
-antibodyfragments;E.coli;
prokaryotes-Licensor,primary
Yeast Protein Sciences, Inc.
Burlingame, CA
Robert Leach, VP, COO
190-Yeastcelllinesandvectors;
Saccharomycescerevisiaecelllines
-properfoldingandglycosylation-
Licensor,primary
Yissum Research Development Co.
licensing agent for the Hebrew
University of Jerusalem and other
Israeli R&D centers
-universal-Assignee,patent
Subject Index
305
293ST-3F6 cell line 410
3-ketosteroid reductase (p3 -KSR) expression 253
6xHistidine-tag 121
aadA1 384
ACE Expression System 232
Acetamidase/Acetamide selection 183
AcMNPV p35apoptosis inhibition 420
Actinomycetes Pabr promoters 198
Adenosine triphosphate (ATP) regeneration system 100
AF 99 insect cell line 421
Agrobacterium tumefaciens RpoA co-expression transcriptional
activator 328
Agrobacterium-mediated transformation, plants 267
Alcaligenes eutrophus expression 161
AlcoFree Yeasts 360
alcohol oxidase I (AOX1) methanol regulated promoter
382
ALEU2 marker 361
Alkaline Phosphatase 310
alpha-crystallin-type fusion protein tags 340
Altermonas haloplanktis 159
Altogen transfection and RNAi gene silencing 316
Aminoglycoside adenylyltransferase (aadA1) enhancer 384
AmProtein vectors, mammalian 215
Anti-Apoptosis Expression System 216
Antibiotic resistance marker genes, plants 268
antibodies 113
antibodies 123
antibodies 127
antibodies 144
antibodies 175
antibodies 180
antibodies 192
antibodies 213
antibodies 214
antibodies 218
antibodies 238
antibodies 240
antibodies 242
antibodies 246
antibodies 252
antibodies 255
antibodies 257
antibodies 258
antibodies 260
antibodies 273
antibodies 350
antibodies 356
antibodies 362
antibodies 375
antibodies 378
antibodies 381
antibodies 392
antibodies 396
antibodies 398
antibodies 401
antibodies 406
antibodies 407
antibodies 415
antibodies 419
Antibody fragment expression 375
(Aocs)3AmasPmas 284
AOX1 methanol regulated promoter 382
ApoLife Yeast Expression 175
apsbD promoter 194
araB promoter 144
Arxula adeninivorans expression 176
Arxula adeninivorans marker 361
Arxula adeninivorans-derived TEF1 promoter 178
Aspergillus niger A4 promoters 362
Aspergillus niger expression 362
ATCC 55366 170
ATCC 55962 342
ATCC 67525 203
ATCC 9119 407
ATCC CCL 10 241
ATCC CRL 10859 291
ATCC CRL 10859 296
ATCC CRL 10860 296
ATCC CRL 1580 260
ATCC PTA-5635 and PTA-5636 297
ATP regeneration system 100
AttSite recombinases 199
Aureobasidin A (pAUR) selectable vectors 363
Autocatalytic cleavage vectors 217
Autographa californica Multiple Polyhedrosis virus
(AcMNPV) with p 1p promoter 434
Autographa californica nuclear polyhedrosis virus (AcNPV)
vectors 436
avian transgenesis and nuclear transfer 248
Avian Transgenic Biomanufacturing 247
Avidin affinity fusion protein affinity tags 329
Subject Index
Index refers to Monograph numbers
306
Baby hamster kidney (BHK) 21 cells 241
BAC-TO-BAC Baculovirus Expression System. 433
Bacillus megaterium expression 147
Bacillus subtilis expression 326
Bacillus subtilis, super-oxidizing strains 148
BacMam vectors 389
bacmids 433
BacTen System (baculovirus expression) 434
Bacterial artificial chromosome (BAC) expression vectors
149
Bacterial cell expression technology (BCE) 144
Baculovirus expression vector system (BEVS) 294
Baculovirus expression vector system (BEVS) 298
Baculovirus host cells 291
baculovirus shuttle vectors 433
Baculovirus vectors and promoters 435
Baculovirus vectors, circular DNA 104
Basic E. coli Expression/Vectors 165
BCE 144
bcl-2 overexpressing NS0 cell lines 251
BCL-xL or BCL-2 expressing cell lines 216
Benzonase 311
BestBac vectors 436
beta-glucuronidase reporter 119
BEVS 294
BEVS 298
bGH polyadenylation sequence 200
BHK 21 cells 241
BI HEX expression 233
Biogenerics Manufacturing Technologies Packages 330
Biotin fusion protein purification 329
BL21(DE3) competent E. coli cells 331
BL21 (DE3) Star 349
BL21-CodonPlus 349
BmNPV 1.2 kb fragment promoters 426
bMON14272 bacmid 433
Boehringer Ingelheim High Expression System 233
Bombyx mori actin promoters 426
bovine growth hormone (bGH) polyadenylation sequence
200
Bovine leukemia virus (BLV) promoter 388
BresaGen fusion protein expression, E. coli. 319
BTI-TN-5B1-4 291
BTI-TN-5B1-4 296
BTI-TN-5B1-4-derived insect cell lines 297
BTI-TN-MG1 296
C-LYTAG fusion protein tag 332
C1 Express 177
Cabilly-Boss 127
Calf Intestinal Alkaline Phosphatase (CIAP) 310
Calnexin, calreticulin, Erp57, Hsp40, and Hsp70 chaperones
390
Calnexin chaperone, Hansenula and Arxula yeasts 364
calreticulin chaperones 390
Campylobacter jejuni genes for E. col glycosylation 333
CaMV 35S promoter 284
CANGENUS 171
CAP expression 250
Caprolactam induction 174
Cardiovirus 2A IRES 203
CASCADE expression 166
cathepsin C 137
Caulobacter Expression and Display System, bacteria 150
CBD fusion protein tags 107
CCT promoters, mammalian cells 391
cdkl3 genes 218
Cell adhesion optimization 218
Cell fusion, NS0 cells 252
Cell-free expression, ATP regeneration system 100
Cell-Free Protein Synthesis 101
Cell-free system for glycoproteins 243
Cellulose binding domain (CBD) fusion protein affinity tags
107
CEVEC Amniocyte Production (CAP) expression 250
Chaperone expression plasmids 334
chaperonins 169
CHEFl expression 400
Chicken EBx cells 244
Chicken eggs exprsession 247
Chicken eggs, transgenic 245
Chicken embryonic derived stem cells 244
Chicken Primordial germ cells 246
Chinese hamster ovary (CHO) cell line TCAE 8 407
Chlamydomonas reinhardtii chloroplast expression 194
Chlamydomonas reinhardtii expression 195
Chloroplast expression, plants 269
Chloroplast Transformation Technology (CTT) 270
chloroplasts expression 290
CHO cell line (Puck) 234
CHO cells dhfr RNA interference 402
CHO DG44 cell line 237
CHO DUXB11 cells, DHFR- 235
CHO EF-Ia promoters 400
CHO elongation factor-la (EF-Ialpha) promoter 400
CHO expression, antibodies in SFM 401
CHO K1 DUX B11 (DHFR-) cells 235
Subject Index
307
CHO Mab expression optimization 192
CHO SSF cell lines 236
CHO SSF3 cells 236
CHO SSF3 X9 cells 236
CHO Supercell 237
CHO-DG44 cells 235
CHO-DG44 expression 233
CHO-K1 CSL4S-342 403
CHOK1SV cell lines 239
Cholesterol selection of NS0 cells 253
Cholesterol/3-ketosteroid reductase (p3 -KSR) expression
253
Choline-binding fusion affinity tags 167
CHOZn CHO DG44 cell lines/vectors 238
Chrysosporium lucknowense expression 177
Ciliate Performance Expression System 207
CIPEX system 207
cis-Acting Peptide Chaperones 335
Cisperone chaperone fusion tags, E. coli 317
Clean Genome E. coli 168
CleanGene plant transformation 289
ClonePixFL Selection 392
Clostridium Expression System 151
CMV (human) Promoter 219
CMV/R Promoter 386
Coconut Express cell-free translation 143
CODA 140
Codon-Optimized, Expression-Ready E. coli Clones 336
Cohen-Boyer 131
Cold Shock Protein A (cspA) promoters 348
Cold-Induced expression system 320
ColE1 plasmids, E. coli 385
CoMed expression 178
Complete Control Inducible Mammalian Expression System
219
Computationally Optimized DNA Assembly 140
Concert Plant-Cell-Produced, tobacco plant cells 417
Continuous culture, bacteria vectors 337
Controllable Self-Cleaving Intein Derivative (affinity fusion
tag), pH-sensitive 108
Controlled Ribosomal Pausing 140
Copy number increasers 201
Core promoters 136
cotA promoter 185
Cotransformation 109
Coupled regeneration/ transformation, plants 271
Cre recombinase 110
CRE-inducible expression 411
Cre-lox mediated in vitro recombination 110
Cre/loxP Recombination-Mediated Cassette Exchange 423
Cre/loxP RMCE 423
Cricetulus griseus (CHO) cells 234
CSL4S-342 CHO cells 403
cspA 348
cyclic AMP response elements (CREs) 411
Cyclization Recombination/locus of X-over P1 110
cytomegalovirus promoter 219
D. Mel 2 Drosophila cell line 425
DAPase Enzyme 137
DE3 331
DE3 349
DE3 E. coli 331
deltaPhase 111
DES 196
desA promoter, iron-regulated 321
DG44 cells, CHO 235
DHFR System 221
Di-codons 140
Dihydrofolate reductase (DHFR) System 221
Dihydrofolate reductase selection marker, CHO cells 235
dipeptidyl aminopeptidase I 137
Directed Nuclease Editor (DNE) 112
disintegration vectors 172
Disulfide isomerases 338
DmdR repressors 321
DNA Ligase (E. coli) 312
DNA microinjection, transgenic chickens 409
DnaJ chaperones 334
DnaK chaperones 334
DPPI 137
Drosophila Expression System (DES) 196
Drosophila melanogaster S2 cells. 425
Drosophila S2 cells and vectors 196
Drosophila-SFM.D.Mel-2 Cells 425
DsbC and DsbG 338
DsbC chaperone fusions, E. coli 353
Dual expression vectors 113
Duck EBx cells 244
duckweed 275
DUK-XB11, CHO cells 235
E. coli BL21(DE3) 331
E. coli expression 328
E. coli. expression vectors 322
E. coli glycosylation 333
E. coli plasmid vector pBR322 347
EASYEAST 189
EBx avian cells 244
ECACC 85110503 256
308
ECACC 96022940 258
Ecdysone receptor inducible expression 227
EDDIE 344
EESYR expression system 192
Elastin-like polypeptide (ELP) fusion affinity tags 111
Elastin-like polypeptide (ELP) self-cleaving fusion protein
tags 339
ELP self-cleaving fusion protein tags 339
EMCV IRES 203
Enterokinase fusion protein cleavage 306
Erp57 chaperones 390
Estradiol induction 394
Estradiol-dependent enhancer 366
Estrogen receptor promoter 394
Eukaryotic cell transformation 142
Ex-Cell EBx platform 244
Expression enhancer sequences 201
Expression enhancers, oligonucleotides 114
expressionfactory 115
ExpressPro 419
ExpressProtect 340
ExpressTec expression, plants 419
FASTR cell lines 192
Flavivirus expression 357
Flavobacterium heparinum expression 152
FLD1 promoter 367
FLP recombinase 222
Flp-In expression system 222
FLP-Mediated Gene Modification in Mammalian Cells 222
Formaldehyde dehydrogenase (FLD1 promoter 367
Fungal expression systems 179
Fusion protein expression systems 116
Fusion proteins, self-cleaving, pH-sensitive 117
G-418 marker 106
GAL1 promoter 366
GAPFL promoter 368
Gaussia luciferase signal peptides 408
Gelvin promoter 284
Gene Design, algoritmic 369
Gene Product Expression Technology 225
Gene-Activated (GA) expression, in vivo 223
GenePORTER 128
Geneticin (G-418) selection 106
GENEWARE expression, tobacco 272
GET recombination 352
glucuronidase reporter 119
Glutamine synthetase (GS) System 224
Glutathione-S-transferase (GST) fusion proteins 118
Glyceraldehyde-3-phosphate dehydrogenase-derived yeast
promoter 368
Glyco-Engineered Moss 273
GlycoExpress human host cell lines, glyco-optimised 254
Glycoswitch plamids 380
Glycosylation, mammalian 105
GPEx 225
GroEL,GroES chaperones 169
GrpE chaperones 334
GS System 224
GS-CHO Protein Free System 239
GST fusion proteins 118
GUS reporter system 119
H5CL-B and H5CL-F 297
Hansenula Expression Platform 181
Hansenula polymorpha expression 182
heat shock Promoters (HSPs) 120
HEK 293 cell line 261
HEK 293 cell line, protein- and peptide-free (Hektor G) media
261
HEK 293 expression 262
HEK-293 adapted to SFM 410
HEK-293 alternative, SFM 266
HEK-293 cell lines 412
HEK-293 transient transfection 413
HEK293 Cell Lines 264
HEK293E cells, SFM 265
High copy number plasmids 341
High Five cell line 291
His-Patch ThioFusion System 343
Histidine fusion affinity tag technology 121
HKB-11 (HKB11) expression 266
Homologous recombination 124
homologous recombination 125
Homologous Recombination 202
Hot Rod Genes 140
hr5 enhancer and the ie1
immediate early promoter 430
HSP chaperones 120
Hsp40 chaperones 390
Hsp60, Hsp70, Hsp90, Hsp100 chaperones 393
Hsp70 chaperones 390
Hte competent E. coli 342
Human amniocytes, immortalized 250
Human antibody producing (HupreB) cells 255
Human embryonic kidney (HEK) 293 cells 264
Human MORPHODOMA 242
Human polyclonal antibodies (rpAB) 240
Human Proteins/Monoclonal Antibodies In Chicken Eggs
246
Subject Index
309
HupreB cells 255
Hybrid of kidney and B cells 266
Hybridomas for non-Mab protein expression, cell-free 243
Hybridomas (Khler and Milstein) 123
Hypermutable yeast 370
Hypermutation 242
iBioLaunch expression 274
IBL-Sf-21AE-Cl 3 cell line 422
IE-1 (BmNPV 1.2 kb fragment) promoters 426
ie1 immediate early promoter 430
IIM14 and IIM22 126
In vivo linearization 103
Insect cell, apoptosis resistant 420
Insect cell lines, baculovirus hosts, SFM 422
insect cells 196
Insect cells glycosylation 293
Insect cells, humanization of glycosylation 292
Insect cells, per os (oral) baculovirus infection 428
Insect cells/Baculovirus expression systems 294
Insect larvae expression 142
InsectSelect Protein Expression 299
Inteins, self-cleaving, pH-sensitive 117
Internal Ribosome Entry Sequences (IRES) translation
enhancer 203
InVoLin 103
IRES translation enhancer 203
IRF-1 Estrogen receptor promoter 394
Isopropyl-beta-galactosidase (IPTG) induction 154
K. lactis GG799 183
Kluyveromyces lactis expression 183
Knock-out/knock-in animals and cells 202
Kozak sequences, impaired 407
Kunjin replicon vectors 357
L21(DE3) derived competent E. coli strains 345
Lactococcus lactis expression 156
Lactococcus lactis htrA- expression 153
Lactose (lac) bacterial promoter 154
Lactose (lac) promoter 165
lama4 genes 218
Lambda recombination protein 124
lambda-mediated recombination 352
lambda-mediated recombination 352
Large ribosomal subunit proteins. E. coli 102
Launch vectors 274
Leishmania tarentolae protozoa expression 208
Lemna expression 275
LEX System 275
LEXSY 208
Light-switchable promoters 204
LNT01, Clostridium botulinum strain 151
Lymantria diapar cell lines 429
Lymantria dispar 652Y cell lines 429
Mab Xpress Antibody Production System 378
magnICON 415
Magnifection 415
maltose binding (MBP) fusion protein tags 350
Mammalian vectors 397
Mammalian vectors, baculovirus-based 389
MARs PLUS 416
MARtech 226
Matrix Attachment Regions 226
Matrix attachment regions PLUS 416
MB101 157
Meganuclease Design 112
Meganuclease Recombination System 103
Meganuclease Recombination System 125
Mengo virus vectors 217
metallothionein (MT) promoter 196
Methylobacterium extorquens expression 170
Milk protein promoter 212
Minos; transposon - insect larvae 142
Mnt-Arc promoters 322
Monoclonal antibody expression 127
MORPHODOMA 242
Morphogenics 242
mosquito cells 196
moss expression promoting regions (MEPRs). 273
MRS 103
MRS/I-Scel 125
Mucin enhancers 126
Multiple proteins in transgenic plants 286
N-(2-ethyl-3-methoxybenzoyl)-N-(3,5-dimethylbenzoyl)-
N-tert-butylhydrazine 227
N(pro) fusion protein tag, self-cleaving 344
neomycin phosphotransferase I 106
Neomycin phosphotransferase I (nptI) 106
Neomycin phosphotransferase marker 106
NeuBIOS fungal expression 184
NeuKARYON 184
Neurospora crassa expression 184
Neurospora fungal expression 185
New Cabilly 127
Next Generation Biotherapeutics 180
NICE 155
nisA promoter 155
NIsin-Controlled gene Expression, Lactococcus lactis 155
nitrilotriacetic acid (NTA) chromatography resins 121
Nitrogen source and light promoters, plants 281
310
NM-F9 cell line 254
Normal Human Fetal Lung Fibroblasts 309
NS0 cells 252
NS0 murine myeloma cell lines 256
NS0 protein-free, cholesterol-free cells 257
NS0-PFCF cells 257
NS06A1(100)3 cell line 251
NTNH promoter from Clostridium botulinum 151
Nuclear transfer, animals 212
Nuclear transfer, animals 276
Nuclear Transformation Suite, plants 277
NusA E. coli fusion proteins 305
Oilbody expression 283
Ophiostoma expression, ascomycetes 186
OSCAR System 404
Osteoclast-associated receptor (OSCAR) promoter 404
OTase of C. jejuni 333
OVA System 247
OverExpress C41(DE3) and C43(DE3) 345
Oxalate/Formate Exchange Protein 346
OxlT plasmids 346
p10 promoter vectors, baculoviruses 434
P170 Expression System 156
p21 overexpressing NS0 cell lines 251
p26 fusion protein tags 340
p3 -KSR vectors 253
pAccAB vectors 396
PACE Expression Vector 398
Pangen CHO expression 405
pAUR 363
pAVEway expression, E. coli and Pseudomonas 323
pBAC vectors 149
pBacMam vectors 389
pBFdfhr.2 Expression Vector 406
pBGP120 341
pBR322 347
pCITE 203
pCold Expression Vectors 348
pCoMed vectors 178
Penaeus stylirostris expression 213
PER.C6 expression 258
Perkinsus marinus expression 209
PERLXpress 295
pET 349
pET Directional TOPO Cloning 349
pET-CBD 107
pFASTBAC transfer vector 433
Pfenex Expression 157
pGEX vectors 118
pH-sensitive affinity purification tag 108
PH05 signal sequence 371
phage lambda promoters (PL and PR) 307
phage T5 promoter 308
phCMV vectors 128
Phosphate induction 371
PhTAC125 159
Physcomitrella patens expression 273
Phyton plant cell fermentation 418
Pichia expression, rhamnose-inducible 379
Pichia GlycoSwitch System 380
Pichia pastoris aglycosylated antibodies 378
Pichia pastoris AOX1 promoters 382
Pichia pastoris AOX1 promoters 383
Pichia pastoris expression 191
Pichia pastoris, fully human glycosylation 180
Pichia pastoris Mabs expression 381
Picorna-like virus IRES 431
pIEx baculovirus vectors 430
piggyBac transposon promoters 387
PinPoint Xa Protein Purification System 329
pIRES 203
pJIR1457 and pJIR1456 151
pKLAC1 183
PL promoter 307
Plant expression (glycosylation) 278
Plant vectors 279
Plastid Transformation 193
Plastids (chloroplasts) expression 290
pMAL Protein Fusion and Purification System 350
PMS2 gene screening, modification 242
pNEBR-R1 227
PnitA-NitR 174
Poly(A) Polymerase 129
Poly-Histidine fusion affinity tag technology 122
Poly-Histidine fusion affinity tag technology (6xHistidine-
tag) 121
Polydnavirus vector insect cell expression 301
Polyhydroxybutyrate (PHB) fusion tags, self-cleaving 351
pPIPRA vectors 279
PR promoter 307
Pre-occluded Virus (POV) baculovirus vectors 302
Primordial germ cells (PGCs) 246
Pro-vector assembly system 163
ProCellEx Plant Expression 280
Proficia protein expression 281
ProfinityTM eXact expression 145
Profuse vectors 317
Prolamin fusion proteins 288
Subject Index
311
Promoters 130
Protein body-inducing sequence (PBIS) fusion proteins 288
Protein Expression 131
Pseudoalteromonas haloplanktis TAC125 159
Pseudomonas fluorescens biovar I 157
pTAT-HA plasmids 324
pTT vectors for HEK-293E cells 414
pTT5 vector 262
PurePro Caulobacter Expression System 150
Q-mate Inducible Expression 220
Quasi-synthetic vectors 160
Ralstonia eutropha expression 161
ReCODE technology 132
Recombinant DNA 131
Recombinant protein body-like assembly (RPBLA) 288
recombinases, site-specific 104
recombinases, site-specific 199
Recombineering 352
Reconstituting chemically orthogonal directed engineering
(ReCODE) technology 132
Red/ET recombination 352
Retrotransposon vectors 259
RheoSwitch Ligand RSL1 promoter 227
RheoSwitch Mammalian Inducible Expression System 227
Rhodococcus rhodochrous expression 174
Rhodospirillum rubrum expression 162
Rhopalosiphum padi virus Internal Ribosome Entry Sequence
(IRES) 431
ribosomal subunit proteins. E. coli 102
RNA silencing vectors 402
RNA transport element (CTE) and RNA transport element
(RTE) 397
RNAi gene silencing 316
RP Shift expression enhancement 398
rpAB 240
S. cerevisiae Twin Cassette Plasmids 175
S2 cells, SFM 425
Saccharomyces cerevisiae cell lines 190
Saccharomyces cerevisiae, cold induction 376
Saccharomyces cerevisiae expression 172
Saccharomyces cerevisiae KOY.TM6* strains 360
Saccharomyces cerevisiae strains 189
Safflower plant seed expression 283
Sapphire baculovirus expression 438
SARs 226
Scaffold Attachment Regions 226
Schneider S2 Drosophila cells 425
Schneider S2 insect cells 425
SCP1 136
SecHancer vector 317
SecHancer vector 377
Secreted Protease Production (SPP) System 173
Secretion Enhancer Vector System (SEVS) 377
Selectable markers 133
Selexis Genetic Elements (SGEs) 228
Semliki forest firus (SFV) vectors 399
senescence-enhanced protein production 398
Serratia marcescens endonuclease 311
SEVS 377
Sf-21 cell line 303
Sf-9 cell line 304
Sf9-derived insect cell lines (VE-CL) 439
Sf9P35AcV5-1 and Sf9P35AcV5-3 420
SFV vectors 399
SGE 228
Shrimp expression 213
siat7e genes 218
SicA fusion protein tags 340
Site-directed mutagenesis 313
Skp and DsbC chaperone fusions, E. coli 353
Small Ubiquitin-like Modifier (SUMO) fusion protein tags
134
Sp2/0-Ag14 cells 260
SPE system 173
Spodoptera frugiperda Sf-21 cell line 303
Spodoptera frugiperda Sf-9 cell line 304
SPP System 173
Stabilized plasmids 158
STabilizing Anti-Repression 229
Stable Insect Virus Cell Expression System 427
StableFast Biomanufacturing System 406
STAR elements 229
StorPro organelles (protein encapsulation) 288
Stratosome Oilbody expression 283
Strep-tag Expression 135
Streptococcus pneumoniae N-acetylmuramoyl-L-alanine
amidase LytA 332
Streptomyces lividans 66 171
Streptomyces (lividans and griseus) expression 171
Streptomyces lividans vectors 173
Streptomyces lividans Y62 expression 197
Streptomyces Stationary Phase Expression System 173
Streptomyces strains and promoters 359
Stripped-down E. coli 168
Subtilin Regulated Gene Expression 164
Subtilisin (psub) fusion protein tag expression 145
SUMO fusion protein tags 134
Super core promoters 136
312
Super-mas Plant Gene Promoter 284
Superpromoter 206
SURE 164
Swine fever virus Npro autoproteolysis 344
Sympress expression 240
Synthetic gene sequences used in vectors 160
T1 and T2 rrnB ribosomal terminators 322
T4 DNA Ligase 314
T4 RNA Ligase 315
T5 promoter 308
T7 RNA polymerase (T7 RNAP) expression 349
tac promoter 354
tagalong promoters 387
TAGZyme 137
Tandem Chimeric Antibody Expression (TCAE) 407
Targeted transfection 237
Tat nanomachine 325
Taura Syndrome Virus (TSV) IRES vectors 213
Tax-inducible expression 388
TCAE 8 CHO cells and vectors 407
Tetracycline (Tc; Tet) Expression Systems 205
Tetracycline-induced expression repression, prokaryotes 146
TetraExpress 210
Tetrahymena expression 207
Tetrahymena expression 210
Tetrahymena thermophila (protozoan) expression 211
TEV Protease 138
TF chaperone 348
Thiol-disulfide oxidoreductases (TDORs) 148
Thioredoxin A (TrxA) depletion 148
Ti plasmids, Agrobacterium 267
tipA promoters 358
Tobacco Etch Virus (TEV) NIa protease 138
Tobacco mosaic virus (TMV) vectors 272
TransBacter Gene Transfer System 285
Transcription enhancers, bacteria 318
Transfection 139
Transgene Operating System (TOS) 415
Transgenic animals 245
Transgenic chickens 246
transgenic poultry eggs 248
Transgenic rabbits for humanized polyclonal antibodies 214
Transient expression, plants 281
Translation Engineering expression 140
Translation-based vectors (TBV) 193
TRANSPILLAR larvae 295
Trichoplusia ni (cabbage looper) cell lines 296
Trichoplusia ni (cabbage looper) cell lines 297
Trichoplusia ni larvae expression 295
Trichoplusia ni, Tni PRO, cell line 432
Trichopulsia ni insect cell line 291
Trigger Factor (TF) chaperone 348
Tryptophan promoter 165
Tryptophan (trp) promoters 355
Twin Cassette Plasmid 175
Twin-arginine translocation (Tat) system 325
UAAs 132
Ubiquitin linkage domain 286
Ubiquitin plant promoters 287
Ubiquitous chromatin opening element (UCOE) expression
230
Ubl-specific protease 134
UCOE expression 230
Universal Yeast vectors 178
Unnatural amino acids, yeast expression 132
UTR Clone Generation 408
UTRtech 408
UV light induction 141
Vankyrin enhanced baculovirus expression vector system
(VE-BEVS) 439
Vankyrin-enhanced cell lines (VE-CL), insect cells 439
VE-CL 01, VE-CL-02 and VE-CL-03 cell lines 439
vegI promoters 326
VelociMab 192
Vesicular fusion factor 2 protein (Vff2p) enhancer, yeasts
372
Vesicular fusion factor 2 (Vff2p) enhancer 373
VFF2 enhancer, yeasts 372
Vff2p enhancer 373
WACKER Secretion System 356
WAP milk promoter, mammals 231
WCC UV light promoter 141
Whey acidic protein (WAP) milk promoters 231
White collar complex (WCC) promoter 141
WI-38 cell line 309
Windowing Technology, avian transgenics 249
Xer recombinases 327
Xer-cise 327
Xplor Vector System, yeast optimization 374
xysA promoters 359
Yeast cell lines 190
Yeast (fungi) expression systems 187
Yeasts, fully human glycosylation 180
Zara technology 288
Zbleu2 strain (yeast) 188
Zinc finger DNA-binding proteins (ZFPs) 206
Zygosaccharomyces bailii expression 188
Subject Index
313
Animals, misc. - 212
Baby hamster kidney (BHK) cells - 241
Bacteria, misc. - 147
Cell-free systems - 100
Chicken eggs - 245
Chicken eggs - 246
Chicken eggs - 247
Chicken eggs - 248
Chicken eggs - 249
Chicken eggs - 250
Chickens - 244
Chickens - 409
Chinese hamster ovary (CHO) cells - 192
E. coli - 166
E. coli - 167
E. coli - 168
E. coli - 169
E. coli - 170
E. coli - 307
E. coli - 308
E. coli - 328
E. coli - 329
E. coli - 330
E. coli - 331
E. coli - 332
E. coli - 333
E. coli - 334
E. coli - 335
E. coli - 336
E. coli - 337
E. coli - 338
E. coli - 339
E. coli - 340
E. coli - 341
E. coli - 342
Primary Host/Oganism Index
E. coli - 343
E. coli - 344
E. coli - 345
E. coli - 346
E. coli - 347
E. coli - 348
E. coli - 349
E. coli - 350
E. coli - 351
E. coli - 352
E. coli - 353
E. coli - 354
E. coli - 355
E. coli - 356
E. coli - 385
E. coli - 165
Eukaryotes, misc. - 103
HEK-293 cells - 261
HEK-293 cells - 262
HEK-293 cells - 263
HEK-293 cells - 264
HEK-293 cells - 265
HEK-293 cells - 266
HEK-293 cells - 410
HEK-293 cells - 411
HEK-293 cells - 412
HEK-293 cells - 413
HEK-293 cells - 414
Human cells, misc. - 251
Index refers to Monograph numbers
314
Insect cells/organisms - 298
Insects cells/organisms - 142
Mammalian cells - 105
Microbial - 317
Murine hybridomas - 243
Myelomas, murine - 242
Plants - 193
Plants - 194
Plants - 195
Plants - 267
Plants - 268
Plants - 269
Plants - 270
Plants - 271
Plants - 272
Plants - 273
Plants - 274
Plants - 275
Plants - 276
Plants - 277
Plants - 278
Plants - 279
Plants - 280
Plants - 281
Plants - 282
Plants - 283
Plants - 284
Plants - 285
Plants - 286
Plants - 287
Plants - 288
Plants - 289
Plants - 290
Plants - 415
Plants - 416
Plants - 417
Plants - 418
Plants - 419
Prokaryotes, misc. - 144
Protozoa - 207
Protozoa - 208
Protozoa - 209
Protozoa - 210
Protozoa - 211
Saccharomyces - 189
Saccharomyces - 190
Saccharomyces - 375
Saccharomyces - 376
Saccharomyces - 377
Streptomyces - 171
Streptomyces - 172
Streptomyces - 173
Streptomyces - 174
Streptomyces - 197
Streptomyces - 357
Streptomyces - 358
Streptomyces - 359
Universal (generic) - 106
universal (generic) - 109
universal (generic) - 132
Yeasts - 175
Yeasts - 176
Yeasts - 177
Yeasts - 178
Yeasts - 179
Yeasts - 180
Yeasts - 181
Yeasts - 182
Yeasts - 183
Yeasts - 184
Yeasts - 185
Yeasts - 186
Yeasts - 187
Yeasts - 188
Yeasts - 360
Yeasts - 361
Yeasts - 362
Yeasts - 363
Yeasts - 364
Yeasts - 365
Yeasts - 366
Yeasts - 367
Yeasts - 368
Yeasts - 369
Yeasts - 370
Yeasts - 371
Yeasts - 372
Yeasts - 373
Yeasts - 374
Yeasts: Pichia - 191
Primary Host/Organism Index
315

Biopharma Expression Systems 080926

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FIRST EDITION

Biopharmaceutical Expression Systems

lymphoma vaccines. Blood 2007, 109, 3393-

this technology are available from PhaseBio for

Jahn, D., Malten, M., Deckwer, W.-D. (2006).

Advanced bioinformatics and functional ge-

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an exclusive, worldwide basis.

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Possible development of customized homog-

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prevention of white-spot disease in freshwater

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available for non-commercial use from multiple

B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es

B|opharmaceut|ca| Express|on Systems and Genet|c Eng|neer|ng Techno|og|es

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