Lecture 8: What additional tools are available from, and what are the applications of, the products

of molecular biotechnology. How are they important to human health, quality of life and society?

Directed Mutagenesis (Chapt 8: pp290-329)

Diversity and biodiversity
Protein Engineering (Chapt 8: pp290-329)
Therapeutic Agents (Chapt 10: pp379-399)

simple protein molecule

[proteins are the workhorse of the cell and body]
Goal is to mimic Nature, and use!

http://en.wikipedia.org/wiki/Epoetin

simple protein molecule

[proteins are the workhorse of the cell and body]
Goal is to mimic Nature, and use!

and improve
http://en.wikipedia.org/wiki/Epoetin

Mutagenesis

In vivo
In vitro

Mutagenesis

In vivo
In vitro

Two thoughts:
Mutations occur spontaneously
Single base changes in the genome
Indels
Duplications
Mutations occur in response to stimulus
Single base changes in the genome
Indels
Duplications
Mimic or expedite naturally occurring mutations
Attractive given recombinant DNA technology
Overproduction and purication
Make better version
What is the basis for changing parts of the genome? Cell? Individual?

Natural selection and growth: How?

[bacteria as a model]

Source of biodiversity
Mutations and Natural Selection/ Evolution
Biological perspective of mutations: resource limitation
Glucose- depleted
Fructose- not available
Maltose- not available
Lactose- available

or in the presence of selection event

Population overgrowth -> colicin

[Antibiotic challenge -> ampicillin]
George Church, @ISB symposium 10/10/08 natural and prevalent antibiotic resistance
Environmental challenge -> radiation, heavy metals, poisons
Natural selection vs adaptive evolution
Whole organism, population versus molecular level

Bacterial modeling 1

Random neutral mutations vs adaptive evolution

RLenski (MSU) long-term evolution experiment
1998: 12 strains E. coli
Feb 2011: 50,000 generations, sampled at every 500
SJGould: replay lifes tape (fossil record)
RWoods and JBarrick: slow and steady often win evolutionary race; hares might eventually lose
hidden potential mutations with no obvious advantages
Genetic interactions that reduce benet of certain regulatory mutations in losers-> outcompeted
Woods, Barrick, Cooper, et al. (Mar 11) Sci 331:1433

http://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benets-of-being-slow-and-steady/

Bacterial modeling 1

SJGould: replay lifes tape (fossil record)

Replay: 10 clones from each of four strains at 500 gen +900 gen
Vertical axis is measure of evolution, ie change from original
@1500 generation- many had two mutations (some dating to 500th gen)- winners
Both winners and losers were more competitive than 0th gen.
losers were better adapted than winners in competition after 350 gen
Winners accumulated mutations ~100 gen earlier than losers
Quicker and more dramatically
At 800 gen, made up for losers headstart and can outcompete
Not faster evolution, eg same number of mutations
Winners had mutation in spoT [affects DNA packaging] [ppGpp hydrolase]
Causes global genome changes, on/off
Losers had mutation in topA [controls packaging]
Negates spoT advantages and suppresses spoT mutations

http://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benets-of-being-slow-and-steady/

Bacterial modeling 1

SJGould: replay lifes tape (fossil record)

Replay: 10 clones from each of four strains at 500 gen; +900 gen
Vertical axis is measure of evolution, ie change from original
@1500 generation- many had two mutations (some dating to 500th gen)- winners
Both winners and losers were more competitive than 0th gen.
losers were better adapted than winners in competition after 350 gen
Winners accumulated mutations ~100 gen earlier than losers
Quicker and more dramatically
At 800 gen, made up for losers headstart and can outcompete
Not faster evolution, eg same number of mutations
Winners had mutation in spoT [affects DNA packaging] [ppGpp hydrolase]
Causes global genome changes, on/off
Losers had mutation in topA [controls packaging]
Negates spoT advantages and suppresses spoT mutations

http://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benets-of-being-slow-and-steady/

Bacterial modeling 2

Role of history in evolution

SJGould: innocuous historical events can have massive repercussions, often the difference
Between survival and extinction every genetic change is an accident of history
Evolution is fundamentally quirky and unpredictable (Wonderful Life)
Lifes tape replayed- evolution could go down very different paths
SCMorris: Any number of evolutionary routes, but destinations are limited
See convergence on same adaptations-> history has little effect on evolution
Lifes tape replayed- evolution gives the same results, more or less
Grow in low sugar so is limited after a period of time
Citrate present but cannot be used if oxygen is present
In 20 yrs of evolution, only 1 clone has done so

http://blogs.discovermagazine.com/notrocketscience/2008/06/02/history-restricts-and-guides-the-evolution-of-innovations/

Bacterial modeling 2

Role of history in evolution

SJGould: innocuous historical events can have massive repercussions, often the difference
Between survival and extinction every genetic change is an accident of history
Evolution is fundamentally quirky and unpredictable (Wonderful Life)
Lifes tape replayed- evolution could go down very different paths
SCMorris: Any number of evolutionary routes, but destinations are limited
See convergence on same adaptations-> history has little effect on evolution
Lifes tape replayed- evolution gives the same results, more or less
Grow in low sugar so is limited after a period of time
Citrate present but cannot be used if oxygen is present
In 20 yrs of evolution, only 1 clone has done so
Two explanations
1) Result of random mutation
2) Function of the particular history of the population

http://blogs.discovermagazine.com/notrocketscience/2008/06/02/history-restricts-and-guides-the-evolution-of-innovations/

prediction

Bacterial modeling 2

Two explanations
1) Result of random mutation
If so, could come up at any generation
2) Function of the particular history of the population
If so, appears in later generations, function of earlier mutation
Three replays, sampling from points in the past
Citrate users evolved much more often in later generations than earlier ones
Citrate use is not simple due to extremely rare mutation (1 in 10 trillion)
First mutants were not efcient at using citrate
Additional mutations needed
Divided population into sugar specialist and sugar+citrate generalist
Blount, Borland, Lenski (08). PNAS 105:7899

Experimental data

http://blogs.discovermagazine.com/notrocketscience/2008/06/02/history-restricts-and-guides-the-evolution-of-innovations/

Molecular evolution

MKimura, 1968: Neutral Theory of Molecular Evolution

Originally thought of as argument against Darwins theory of evolution by natural selection
Maintained, most agree, two theories are compatible
But provides a large role to genetic drift
Neutral: differences to not inuence tness of individual
1) Based on degenerate genetic code
Synonymous vs Non-synonymous mutations
GCC and GCA => Ala
2) Also based on majority of AAc changes being neutral

http://en.wikipedia.org/wiki/Neutral_theory_of_molecular_evolution

Evolution theory

1972 NEldredge and SJGould. Paleobiology 3:115: Punctuated Equilibrium

Degree of gradualism (commonly attributed to Darwin)
is virtually nonexistent in the fossil record stasis dominates the history of most fossil species
Misconceptions:
1) species originate more or less overnight, in a single step
rapidly is in the context of a geologist
2) species undergo no further evolutionary change once speciation is complete
is actually a form of gradualism
RDawkins: apparent gaps represented in fossil record document migratory events rather than
evolutionary events; evolution certainly occurred but probably gradually elsewhere
PE is a minor gloss, an interesting but minor wrinkle
Non-scientists: micro vs macro/evolution
EHDavidson: role of gene regulation (gene regulatory networks) in evolution; genome of sea urchin

http://en.wikipedia.org/wiki/Punctuated_equilibrium

Natural mutation: HBB, beta hemoglobin gene

Molecular, genetic and biochemical perspectives

1600 bp, three exons
mRNA 626 bp; 44 bp codes for AAc
Normal rbc ~120d; sickle cell ~10-20d
Left: hemoglobin, green and blue= alpha chains
Gold and aqua= beta chains
Gold spheres= phosphates; box= Glu6
Variant at 6= most common variant sickle cell HBS
Right: clumping of two hemoglobins with variant AAc
Several 100 HBB variants
Autosomal recessive

http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtml

Spontaneous chemical reactions

Naturally occurring mutations

Energy driven, eg UV light (tanning)
Chemical basis of mutations
Bases undergo random modications
If uncorrected, mutation is hard copied
SNP

Another perspective: people and mutations

Essay, NYTimes. Oct 9, 2007. BHLerner, MD

Longevity reects how the understanding of sickle cell disease has changed
Initially misdiagnosed in early 1960s- found its way into the public consciousness.
First complaints of joint aches- met with skepticism
At 16, diagnosed with both sickle cell anemia and beta-thalassemia
Combined condition may be less severe than pure sickle cell disease, contribute to longevity[?]
Course of disease: painful sickle cell crises, spleen removal, shoulder surgery, degeneration of hips
Fear of promoting drug addiction: under-prescribed pain meds, requiring blood tests rst

Site-directed mutagenesis

Theoretically, can modify protein at either the protein-level or the gene-level

@Protein-level: chemical, post-translational, modications are harsh, nonspecic and tedious
But, mirrors Post-translational and epigenomic
@Gene-level: Structure and predictive tools necessary
~easier
Directed mutagenesis -generate amino acid coding changes at DNA level

Evolution of genetic engineering

(playing god[s])

Experience
Bread, beer, agriculture and naturally occurring products and processes
[Modern] ways and means, biotechnology
Biology, genetics
[Ultra-modern] ways and means, biotechnology
Recombinant DNA technology, genomics, bioinformatics, funding
Improve upon Nature
out of context
More and inexpensive supply
[cadavers vs carcasses vs controlled products and processes]
Adaptation
[extremophile products and processes]
Genetic engineering
Directed mutagenesis
Protein engineering

Limited, now, by imagination and creativity

Recombinant DNA technology
Bacillus stearothermophilus, 90oC hot springs, -amylase
At high industrial-scale temperatures, converts starch to alcohol
Naturally occurring product or process not optimized for use
Small-scale to industrial scale
Yields, efciencies, stability
Directed evolution: Why?
Km and Vmax (substrate binding and catalytic efciency)
Thermal tolerance, pH stability (structure and function)
Activity in nonaqueous solvents (enhanced nonphysiological)
Remove or alter cofactor needs
Link enzymes/proteins/subunits together
Alter substrate-binding site and other binding/recognition sites
Alter resistance to proteases, extending half-life
Alter allosteric regulation
Alter trafcking and addressing, eg secretion/segregation/placement
Tracking or visualizing proteins
Purication issues
Directed evolution: How?
Mimic natural mutations
Alter amino acids as directed mutagenesis

Oligonucleotide-directed mutagenesis
[Example of imagination and creativity]

Biology and genetics

ssDNA M-13; E. coli plasmids; DNA replication and mutagenesis
Protein chemistry and biochemistry
Structure/function
Recombinant DNA technology [and synthetic chemistry]
Alter nucleotide sequence to alter amino acid sequence to alter structure/function
Results: 50-50 recovery of WT vs mutant

v2: Enrichment of mutated gene

Results: 50-50 recovery of WT vs mutant (in theory)
[[we dont know everything about Nature:]]
Only 1% of mutants recovered
Need to enrich

Another approach:
RF with cloned WT gene, grown in dut ung strain
[incorporates and leaves U]
Add oligo with mismatch nucleotide
- strand synthesized with U
Tf WT host
WT gene sequence is removed by repair mechanism

Mismatch repair: simple

Products and Processes

as biotechnological applications
DNA replication
DNA repair
dut/ung
Mismatch repair
Benecial to biotechnological applications
Deleterious to biotechnological applications

v3: Bypass multiple steps

Time-consuming multiple steps

Make RF form with WT gene
Go through ssDNA form
Recover mutated form as ds
Clone into plasmid to use

Another approach [Direct]:

Plasmid (ALTER) with cloned WT gene
Original ApS and TcR
Add three mismatched oligos:
Mutation + ApR and TcS
Add T4 DNA polymerase and T4 DNA ligase
Results: mutated gene with easy selection
[Repair mechanism of E. coli]
[[using a process as biotechnology]]

v4: Applying newer technology to simplify: PCR

Simpler and faster, and more versatile [more possibilities]

PCR to introduce mutation and enrich for mutated gene
Four types of mutations possible:
Point mutation
Deletion mutation
Insertion mutation
Large
Small

v5: Applying newer processes to maximize:

Error-prone PCR mutagenesis

Recall the basis for evolution (and natural selection)

Mechanism of molecular evolution
More versatile [more possibilities]
PCR to introduce multiple, random mutations
Taq pol inherently error-prone, lacks proofreading activity
From 1 to 20 errors in 10kb
[but 1-2 per 1,000 is limited number; usually one mutation per triplet codon]
Natural selection and subsequent articial selection
Replication biochemistry- enhancing errors
Mn++ for Mg++; nucleotide pools; others
Success: enzymes with improved solvent and temperature stability; specic activity

v6: Applying newer inexpensive technology to optimize:

Degenerate oligonucleotide primers

Simpler and faster, and more versatile [more possibilities] AND semi-controlled
DNA [Replication/Repair] Polymerization to introduce multiple, random mutations
At G position/vial, additional nucleotides provide random [mis]incorporation at G

v6: Applying newer inexpensive technology to optimize:

Degenerate oligonucleotide primers

Simpler and faster, and more versatile [more possibilities] AND semi-controlled
DNA [Replication/Repair] Polymerization to introduce multiple, random mutations
At G position/vial, additional nucleotides provide random incorporation at G
PCR primers used to synthesize novel gene
RE digests to cut out gene
Natural selection and subsequent articial selection

v7: Random insertion/deletion

Limitations of PCR error-prone mutagenesis

Alternative technique:
delete small number of nucleotides at random positions and
insert specic or random sequences into that position
Gene with RI-H3 ends [or any two unique]
Add non-P linker to one [[no P= no ligation, so gapped]]
Close circular DNA with T4 ligase and
repair degraded with T4Pol, extending nick to remove strand
Produces ss, with red insertion
Random nick ss with Cerium[IV]-EDTA complex
Ligate with RE-linker plus additional nucleotides
PCR amplify
Remove linkers with RE
Blunt end-lling and close/ligate into plasmid
Test for activity

v8: DNA shufing: creating hybrids or chimeras

Naturally occurring mutations and evolution, eg -interferon

Related genes with slightly different biological activity
Make hybrids containing parts or these genes
Goal is unique properties, eg, combine attributes of two or more originals
High enzymatic activity plus thermostability

v9a: DNA shufing: naturally occurring RE sites

Simplest solution:
Use RE fragments to generate hybrid
2 genes, ea with same 3 unique RE sites
14 hybrids

v9b: DNA shufing: random fragmenting

Alternative solution:
Use fragment several genes randomly with DNaseI
Select small fragments
PCR ll and amplify [will cross-amplify if hybridize]
Finish with terminal primers for ends
Can do this protocol with genes from different families, with limited homology
Can do variation protocol with genes from different families, with no homology

v9c: DNA shufing: nonhomologous random fragmenting

Can do variation of protocol with genes from different families, with no homology
Not PCR-based
Several DNA fragments combined and partially digested with DNaseI
Blunt lled with T4 DNA pol
Size fractionate
Add synthetic DNA fragment forming hairpin loop with specic RE
To prevent long concatenations
RE digest off hairpin ends
Put into vector

Inserting non-standard (unusual amino acids) into mutant

Add non-standard amino acid with unique R group

Amino acids are modied post-translationally in eukaryotes
Different chemical and biochemical properties
example, OH-Pro, major component in collagen
Engineer E. coli host with novel tRNA
ex, tyr-tRNA synthetase from Methanococcus jannaschii
Adds novel amino acid at amber codon, UAG normally stop
O-methyl-L-Tyr
ex 2, aminobutyrate using Val-tRNA synthetase

Protein engineering

1000s of enzymes studies and biochemically characterized

~20 account for >90% used industrially
Why?
naturally-occurring enzyme is optimized for a niche
Not suited for highly specialized industrial application
ex, high temperature stability, organic solvents (many industrial processes)
Thermophilic organisms may not have particular desired catalytic function

Altering thermostability

Structure ~ Function
Not just thermostability
Often resistant to denaturation by organic solvents and nonphysiological conditions- ex, pH
Adding disulde bonds can increase stability
Problem: how do these perturb the native structure?

Altering thermostability: example, T4 lysozyme

Oligonucleotide-directed mutagenesis to increase structural stability at higher temperatures

Six variants with new di-S bonds
2, 4 or 6 AAc changes to Cys at the same time
Positions- spatially close to each other in the active enzyme structure
But not in active site
Generate 1, 2 or 3 di-S bonds
Activity not predictable, eg, 2 additional S-S in one case gives activity at higher temperature

Altering thermostability: example, xylanase

Use of computer modeling and crystallographic data

Paper making process

Wood pulp chemically treated [bleaching] to remove hemicellulose,
contributes to discoloration of paper product
Creates large amount of toxic efuent
Alternative is using enzyme: Bacillus circulans xylanase
But, follows hot alkali step
Computer modeling to determine placement of 1, 2 or 3 di-S bonds
All 8 mutants showed increase in thermostability, relative to WT
3 were as active as WT at 60oC
1 was twice as active at 60oC and retains 85% activity after 2 hr at 60oC
WT is unstable after 30 min

Adding di-S bonds to produce new application:

example, RNase

RNase or ribonuclease from bull semen can act as an anti-tumorigenic agent

in vivo and in vitro, dimeric form is internalized into tumor cells by
non-receptor mediated endocytosis
Selectively degrades rRNA in cytoplasm; Blocks protein synthesis --> Cell death
Anti-tumor activity due to dimer structure
Of all pancreatic-like RNase superfamily members, dimer structure only found in bull semen
But, clinical application generates human antibodies to bull semen RNase
Does not allow multiple applications or prolonged use
Engineer human pancreatic RNase to be a dimer [70% identical] to function as anti-tumor agent

Adding di-S bonds to produce new application:

example, RNase
[mammal cells are not all alike]

Engineer human pancreatic RNase to be a dimer [70% identical] to function as anti-tumor agent
Dimeric human RNase insoluble, segregated into inclusion body <-- bull cells different from human cells!
Renaturation yields slightly lower anti-tumor activity protein
But, does not interfere with normal human diploid broblast cells
Good candidate as human therapeutic agent

Protein engineering to increase stability:

temperature sensitive amino acids

Asn and Gln at high temperatures undergo deamidation to

Asp and Glu, different chemical and biochemical properties
structure ~ function changes
Example, yeast triosephosphate isomerase
Homodimer with 2 Asn per monomer, located at protein-protein interface
Changing either to Thr or Ile enhanced thermostability
Changing either to Asp decreased thermostability, as predicted
Changing both to Asp decreased stability at normal temperature and lowers activity
Another example, long-acting human insulin: Gly for Asp increased half-life
Recently approved for use as human therapeutic agent

Protein engineering to increase stability:

Are too many di-S bonds bad?

Expressed foreign protein maybe less active than expected

Human IFN- cloned and expressed in E. coli
10% antiviral activity of native glycosylated form
Yields good; Most of product as dimer or multimer --> inactive
Native has three Cys residues
Possible di-S bonds in E. coli forming multimers, but not in human
Change to Ser ->similar, but OH vs SH
Reasoned Cys-17 not involved in di-S bond in human, so changed it to Ser
Ser-17 form has specic activity similar to native and more stable during long-term storage

Protein engineering to increase enzymatic activity

Tyrosyl-tRNA synthetase (B. sterothermophilus)
Tyr + ATP -> Tyr-A +PPi
Tyr-A + tRNATyr + AMP

Given 3D structural information, including active site data

Increase activity by modifying substrate-binding specicity
Predictions of changing amino acids
Thr-51
Replace with Ala or Pro
OH of Thr-51 forms long H-bond with tyrosine adenylate
Removal of weak H-bond may improve afnity of enzyme for ATP

Theoretically, Pro distorts -helix

Should not work

Protein engineering: new enzymatic activity

Theory: mutagenesis plus select for new desired activity and against old activity
Example: endoprotease
Cleaves between adjacent Arg residues
Error-prone PCR; fuse to E. coli surface protein [blue], with negative charges
Two different substrates used to score selection
1) 3x Arg with 2 uorescent dyes, at ends of protein, with positive charges
2) 3x Arg with 1 dye, at one end of protein, with positive charges
Select for (1) and against (2) using uorescent cell sorter
1 clone found with >3Mx selectivity for new Ala-Arg over old Arg-Arg activity

Protein engineering: modifying metal cofactor requirements

Subtilisins, Ser proteases secreted into media

Widely used as bidegradable cleaning agent in laundry detergents (B. amyloliquefaciens)
Binds tightly to Ca++ which stabilizes enzyme
Industrial protocols include chelating agents
Strategy to remove Ca++ binding, then to increase stability of modied enzyme
Crystallographic data: residues 75-83 binds Ca++ --> delete but retains overall structure
Random mutagenesis: residues that interacted with 75-83 as targets --> 10 residues
Several rounds of stabilizing mutations but low levels of activity
Combine mutations for stabilized mutant without Ca++ requirement and high level of activity

Protein engineering: modifying metal cofactor requirements

Targets: N-term (2-5); omega loop (36-44); -helical region (63-85); -pleated region (202-220)
Mutate; grow and heat to 65oC/1 hr; assay for subtilisin activity
Use B. subtilis, product kills E. coli
After initial screening, 7 stabilizing mutations out of 10 positions targeted
Second round, combine mutations into one gene
Mutant is 10x more stable than native in absence of Ca++ and 50% more stable in presence of Ca++
-> complex properties involving large number of amino acids can be engineered

Decreasing protein sensitivity

Streptococcus streptokinase, 47 kDa protein that dissolves blood clots

Complexes with plasminogen to convert to plasmin, which degrades brin in clots
Plasmin also degrades streptokinase [feedback loop]
In practice, need to administer streptokinase as a 30-90 min infusion [heart attacks]
A long-lived streptokinase may be administered as a single injection

www-s.med.uiuc.edu; JMorrissey: Med Biochem 10/30/06

http://sandwalk.blogspot.com/2007/04/blood-clotting-basics.html

Protein engineering: decreasing protease sensitivity

Streptokinase (Streptococcus bacteria) is used as a blood clot-dissolving agent

Forms complex with plasminogen to convert plasminogen to plasmin
Plasmin degrades brin in clots
Plasmin degrades streptokinase [[example of feedback loop]]
Has to be administered as a 30 to 90-min infusion after heart attack
Desire single injection and rapid transportation to hospital
Plasmin cleaves at Lys-59 and also at Lys-386
Strategy: Lys (green) to Glu (red)
Similar side chain and not charged to retain 3D structure
Both single mutants plus double mutant have activities of native
Half-life in presence of plasmin increased, with double mutant ~21x more protease resistant

Protein engineering: modifying enzymatic activity,

eg, protein specicity through recognition

Catalytic activity includes recognition of specic substrate and subsequent action
>2,500 RE discovered
Many recognize same sequence -> 200 different recognition sites
Majority recognize 4-6bp sequences,
not useful for generating large fragments like 8+bp cutters
FokI endonuclease, relatively nonspecic (Flavobacterium okeanokoites)
Second type of DNA-binding proteins: Zn++ nger proteins, eg murine Zif268 with 3 ngers
Binds DNA with nger interacting with specic 3 nucleotides
Recombinant protein contains His tag to aid in protein purication
Under control of T7 promoter (expression cuts host DNA)
Results: two hybrid FokI REs that cut lambda DNA
#1 is specic at target site
#2 also cuts two other sites
Zn++ nger proteins recognize 3 bases but may also recognize 2 of 3
Promising [[Proof of concept]]

Protein engineering: modifying binding activity,

eg, protein specicity [non-enzymatic]

Antibodies
Binds antigens
Mostly same structure except hypervariable region
Unique and highly specic for antigenic determinant
Region called Fab fragment

http://en.wikipedia.org/wiki/Antibody

Protein engineering: modifying binding activity,

eg, protein specicity [non-enzymatic]

Antibodies
Mostly same structure except hypervariable region
Unique and highly specic for antigenic determinant
Region called Fab fragment
Binds in absence of rest of Ab
Two peptide chains: heavy and light
each with different hypervariable complementarity-determining regions (CDRs)
each with similar framework regions (FRs)
Modify CDRs to change binding/recognition specicity
As a dimer, complications for modication ->

Protein engineering: modifying binding activity,

eg, protein specicity [non-enzymatic]

As a dimer, complications for modication ->

6 CDRs total in two chains; modifying one or more amino acids changes specicity
Random mutagenesis PCR to mutate 3 CDRs of heavy-chain gene separately
Combine into single peptide
Example, Fab of monoclonal Ab specic for 11-deoxycortisol
(precursor to cortisol [stress hormone] and hypertensive)
New version recognized cortisol and not 11-deoxycortisol
Promising [[Proof of concept]]: screen library of mutagenized Fab genes for any antigenic determinant

Protein engineering:
increasing enzyme stability and specicity

tPA, tissue plasminogen activator

Multidomain Ser protease
Dissolves blood clots [like streptokinase]
Cleared rapidly from body, so needs to be infused
Requires high initial concentration to be effective
Side effect of causing nonspecic internal bleeding
Desire: long half-life; increased specicity for brin; decreased nonspecic
Method: directed mutagenesis
Results:
Thr-103 to Asn -> 10x longer life in rabbit plasma;
Lys-His-Arg-Arg (296-299) to Ala-Ala-Ala-ala -> more specic for brin
Asn-117 to Gln -> brinolytic activity as native
All three into one mutant
[2010]: testing to validate as substitute for native tPA

Protein engineering:
increasing enzyme stability II
[Application and example of evolution and selection theory]

Fructosyl-amino acid oxidase

Glycation- nonenzymatic addition of glucosyl residues on surfaces of blood proteins
ex, hemoglobin, albumin
Increases in diabetics with high blood glucose levels
Does not follow food intake
May be a good way to monitor diabetes patients during therapy
ex, hemoglobin A1c (HbA1c) level measures Val glycation
Can use Fructosyl-amino acid oxidase (Corynebacterium sp)
Specic for D-fructosyl-L-Val on hemoglobin;
No activity for N-fructosyl-L-Lys on albumin
But Fructosyl-amino acid oxidase (Corynebacterium sp) enzyme is unstable
Clone into E. coli and repeated rounds of in vivo mutagenesis/screening for stability 47oC/10 min
simple directed evolution in four rounds

Protein engineering:
Changing enzyme specicity III

Enteropeptidase (or enterokinase), membrane-bound Ser protease

From duodenal mucosa; two polypeptide chains converting trypsinogen to trypsin
Bovine or porcine versions used to excise poly-His tags from recombinant proteins from E. coli
But also cleaves other sites too- lowers yield/modies recombinant proteins [[nonspecic]]
Difference between lab-scale vs industrial-scale (worry more about yields)
Alternatives: medaka- freshwater sh (Oryzias latipes)
Comparable enzymatic activity, same recognition site
10% activity against similar [nonspecic secondary] sites vs bovine/porcine versions
Study medaka enzyme to understand basis for strict specicity requirements
Look for amino acids conserved in four different mammalian enzymes not in medaka
5 sites
Mutate each in medaka to reect mammalian version, see 1 with less nonspecic activity
Generates enzyme with altered specicity and more biotechnological applications

Protein engineering:
Changing multiple properties simultaneously

Generally, directed mutagenesis addresses 1 property at a time

1 amino acid change may alter structure ~ function, other parameters
Requires compensating mutations
Labor-intensive and tedious; alternatives-
Molecular breeding: novel protein from set of existing similar proteins
Does not require prior knowledge of structure/function
Proof of Concept expt:
26 different subtilisin genes from different Bacillus strains
DNA shufe to produce library of chimears
Subset of 654 clones into B. subtilis
Assay secreted proteins, plus parentals
23oC activity; thermostability, solvent stability, pH dependence
[most useful in industrial applications]
Natural selection rarely selects for optimal activity at 23oC activity and 70oC
Results: 77 as well as or better at 23oC activity
Sequenced-> all were chimeric
1 had 8 crossovers with 15 amino acid substitutions

Laundry, detergent and mushrooms

First to combine two site-directed mutagenesis techniques with gene shufing and sorting procedures
Directed evolution
JCherry at Novo Nordisk Biotech/Davis, CA
. deliberate and random mutations can be screened for a commercial product..
-Maxygen Inc/Redwood City, CA
[Broad Institute: Coprinus cinereus 37.5 Mb genome sequenced]

http://www.wildaboutbritain.co.uk/gallery/g; http://www.education.umd.edu/EDMS/mislevy/Drawings/washing.jpe; http://www.fotosearch.com

Protein engineering:
Changing multiple properties simultaneously

Generally, directed mutagenesis addresses 1 property at a time
1 amino acid change may alter structure ~ function, other parameters
Requires compensating mutations
Labor-intensive and tedious; alternatives-
#1 DNA shufing to isolate hybrids-
need two or more similar genes
#2 Random mutagenesis or error-prone PCR-
essentially make two or more similar genes
Combine #1 with 1 of #2
Biotechnological application: Peroxidase
Ink cap mushroom Coprinus cinereus
Dye transfer inhibitor in laundry detergent
Oxidizes [decolorizes] leached dues and
prevents re-staining other clothes
Need high pH, temperature and peroxide levels

http://en.wikipedia.org/wiki/Coprinopsis_atramentaria
http://www.nature.com/nbt/press_release/nbt0499.html

Protein engineering:
Changing multiple properties simultaneously

Biotechnological application: Peroxidase
Ink cap mushroom Coprinus cinereus
Dye transfer inhibitor in laundry detergent
Oxidizes [decolorizes] leached dues and prevents re-staining other clothes
Need high pH, temperature and peroxide levels
Methods:
Site-directed mutagenesis to replace solvent-exposed amino acids with
nonoxidizable side changes
Introduce stabilizing features, eg, di-S bridges
Error-prone PCR to identify other benecial mutations
Results: successful mutations combined into 1 hybrid-
114x thermal stable; 2.8x oxidative stable
But, not optimal under actual wash conditions
Additional DNA shufing: 174x thermal stable; 100x oxidative stable
Used as dye transfer inhibitor in laundry detergent
Also, model for other enzyme development
[SJKimBKSong (Mar10) Biocatalysts and Bioreactor Design]
[CherryPedersen (99) Nat Biotech
Directed evolution of a fungal peroxidase]

Therapeutic agents

Human health, quality of life and society
Prior to recombinant DNA technology, most human protein pharmaceuticals were available
in limited quantities [cadavers, carcasses]
Costly to produce, modes of action not well-characterized
HIV, Hep-B from blood-derived products (hemophiliacs)
Evolution of therapeutic agents
Natural products
Accidental discovery/use of mixtures to isolation/use to synthesis by Nature to

Organic Chemistry (Age of Industrialization) to proteins (and antibodies) to
Derived and modied natural products
recombinant DNA technology (Molecular cloning/Protein engineering) to
Back to natural products (Bioprospecting)
Enhanced understanding and identication/characterization/development for use
Diversity
Biodiversity
Culture diversity

Protein therapeutic agents

Protein therapeutic agents

Prior to recombinant DNA technology
Most human protein pharmaceuticals were available in limited quantities,
Extremely costly to produce and biology not well-characterized
Animal sources
Horse/cow sera- antibodies; inuenza vaccine
Pig insulin prior to 1982
Blood donors- blood, blood components (clotting agents/hemophilia)
Cadavers- human growth hormone from pituitary glands; blood clot factors
Recombinant DNA technology
Sufcient quantities for efcacy testing and human use
Several thousand different proteins clones (2009)
500 undergoing clinical testing
250 biotech drugs approved in US and/or EU
2006, estimated annual global market for recomb protein drugs at $60B
10 blockbuster drugs accounted for 50% of this
Rituxan (rituximab)- monoclonal Ab for non-Hodgkin lymphoma @$4B
Recombinant insulin @$2.5B
1985: Genentech- FDA approval to sell rst biotech industry product,
recombinant human growth hormone
Hepatitis B vaccine
Recombinant human insulin

Pharmaceuticals: proteins, cDNAs and therapeutics

General strategies for obtaining cDNA (clone)- early era

Purify protein; determine partial amino acid sequence; make oligo and search libraries
Purify protein; make antibody against protein; screen libraries
Considerations include quantity of natural state protein and location
ex, Insulin is major protein synthesized by islets of Langerhans of the pancreas;
70% of its mRNA
Prior to whole human genome sequencing, difcult to isolate low copy or uncharacterized genes
Human interferon proteins: , , - each with different biological activity
Genomics, bioinformatics, systems biology, technology

Pharmaceuticals: interferon cDNAs

Human interferon proteins: , , - each with different biological activity
1980s IFN thought to be 1 protein
Site of synthesis unknown; low concentration
E. coli eukaryotic protein expression vectors not readily available

Interferon cDNAs: genomics and bioinformatics,

and medicine

Human interferon proteins: , ,

and synthesized in cells that have been exposed to viruses or viral RNA
synthesized in response to cell growth-stimulating agents
coded by 13 different but similar genes; encoded by 2; by 1
Antiviral activities vary
1 and 2 are similar when challenged with a virus-challenged bovine cell
2 is 7x more effective than 1 when human cells are treated with virus
2 is 30x less effective than 1 when mouse cells are used in this assay

Interferon cDNAs: genetic engineering

Gene shufing to obtain hybrid protein with novel properties
1 and 2 as parentals, use REs to shufe or PCR-based
Hybrids expressed in E. coli
Test for extent of protection against viruses; antiproliferative activity against various human cancers
Some hybrids have passed clinical trials and approved for use as human therapeutic agents

Interferon cDNAs, optimization

Some hybrids have passed clinical trials and approved for use as human therapeutic agents
Longer-acting interferon- minimizes side effects, lower dosage, lower frequency of treatment
Method: PEG (polymers)
Method: fuse with a stable protein, eg, human albumin
Results: native is removed after 2 days; hybrid effective for two weeks
Hepatitis C (Phase III, 2006)

Pharmaceuticals: human growth hormone

HGH or somatotropin is a 191 amino acid pituitary hormone, MW 22,125 daltons
Stimulates production of insulin-like growth factor 1
Children: controls growth; Adults: controls metabolism
Children growth usage @$10,000-$30,000/yr
One of rst recomb proteins to be approved for human use
1985 Genentech Protropin; discontinued in late 90s
Produced in E. coli and identical to native
Can re-engineer to augment of constrain mode of action
ex, native HGH binds to GHR and prolactin R
Side effects
Site-directed mutagenesis to change properties, ie structure/function

Human growth hormone, optimization as

longer half-life

Relatively short half-life in plasma

Requires subcutaneous injection once a day
Inconvenient and expensive
Method: fuse extracellular domain of HGHR, promotes dimerization
In rats, promotes growth for 10 days [vs 1 day]
Dimerization stabilizes and allows it to circulate 300x native
Monomer is active and is slowly released from dimer

Human growth hormone, optimization

longer half-life

Relatively short half-life in plasma

requires subcutaneous injection once a day
inconvenient and expensive
Alternative method: fuse C-term with N-term of human serum albumin, Albutropin
produced in yeast
genetically modied to require minimal posttranslational modications
serum half-life 19 days
Effective in rats and monkeys, 5 days after injection
2009, Phase I clinical trials completed

Pharmaceuticals: tumor necrosis factor alpha

TNF- is a potent antitumor agent

Not widely used due to severe toxicity
Desire: deliver directly to site of action, less side effects
Method: add Cys-Asn-Gly-Arg-Cys-Gly [targets a tumor cell surface protein]
Mice: cytotoxic activities identical,
eg does not disrupt protein folding, trimerization, binding to receptor

Tumor necrosis factor alpha: potential drug

TNF- with Cys-Asn-Gly-Arg_Cys-Gly [targets a tumor cell surface protein]

Mice: cytotoxic activities identical,
eg does not disrupt protein folding, trimerization, binding to receptor
Modied version 12-15x more effective at inhibiting tumor growth
Higher percentage of mice with lymphoma survived after treatment with modied version
Mice that survived 30 days also survived a second and third challenge with lymphoma cells
2009, efcacy not yet demonstrated in humans

Genetic disease: genomics and medicine

Cystic brosis, hereditary disease

Progressive disability, early death [average life span 37 yrs]
Difculty breathing and lung infections, among symptoms
CF localized to cystic brosis transmembrane conductance regulator
Regulates components of sweat, digestive juices and mucous
One copy is enough for WT phenotype
1 in 25 European descent carry a mutation, Ashkenazi Jews

http://en.wikipedia.org/wiki/Cystic_brosis

Pharmaceuticals: Enzymes- DNaseI cloned

Cystic brosis patients

Highly susceptible to bacterial infections of lungs
Antibiotic treatments lead to resistance
Accumulation of mucous from bacteria-secreted alginate, DNA from bacteria and leukocytes
Genentech cloned DNaseI and expressed in CHO cells
Delivered as aerosol mist to lungs
1994 approved by FDA
2000 sales of $100M

Pharmaceuticals: Enzymes- DNaseI engineered

Actin binds very tightly to DNaseI

Inhibits enzymatic activity and limits therapeutic potential
Crystallographic data predicts residues to modify
Ala-144 to Arg or Tyr-65 to Arg
Decreased actin binding up to 10,000x
10-50x DNaseI activity
2009, clinical efcacy to be demonstrated

Pharmaceuticals: Enzymes- alginase cloned

Alginate is a polysaccharide produced by seaweed, and soil and marine bacteria

Cross-links to form gel with heavy viscosity
Pseudomonas aeruginosa excretes alginate into CF patient lungs
Provides a biolm that blocks antibiotics and provides anchorage
Alginate lysis gene from Flavobacterium species; cloned into E. coli

Cloning alginate lyase

Selection/scoring methods
Not simply overexpress and observe activity
Flavobacterium sp.
Clone bank in E. coli
Screen by plating onto medium plus alginate
Alginate lyaste requires cofactor Ca++
+/- Ca++
Ca++ + alginate = cross-linked opaque
Hydrolyzed alginate does not cross-link
Analysis and characterization of clones and alginate lyase

Alginate lyase[s]

ORF 69,000 Da
Precursor of three alginate lyases
-> 3,000 Da + 63,000 Da
63,000 Da lyses both bacterial and seaweed alginates
63,000 Da -> 23,000 Da seaweed effective + 40,000 Da bacterial effective
Clone bacterial activity portion

Alginate lyase[s]: Optimization of activity

Increase expression of 40,000 Da protein

PCR amplify and insertion behind strong promoter
B. subtilis plasmid, fused to a B. subtilis a-amylase leader peptide, directs secretion,
and a strong penicillinase gene promoter
Expressed and assayed for halo phenotype
Liquies alginates produced by P. aeruginosa isolated from lungs of CF patients
2003, additional trials to determine if effective therapeutic agent

State of the art, 2008-ish:

Cystic brosis treatment follow-up

What is dornase alfa???

State of the art, 2008-ish:

Cystic brosis treatment follow-up

Treatment of genetic and metabolic disease: Phenylketonuria (PKU)

Autosomal recessive genetic disorder in phenylalanine hydroxylase (PAH)

Phe accumulation, decreases other large, neutral AAc in brain, needed for
protein and neurotransmitter synthesis
Brain development; progressive mental retardation and seizures
Incidence ~1/15,000; But, varies: 1/4,500 Ireland and 1/100,000 Finland
12q22-q24.1
Macaque genome: PAH gene sequence identical to a human PKU mutation

wikipedia

Phenylketonuria treatment[s]

Traditional treatment: diagnosis at birth or prenatal test

Controlled semi-synthetic diet with low levels of Phe
Possible treatment: metabolism of Phe
PAH multienzyme complex, requiring cofactor
Phe ammonia lyase (PAL) converts Phe [too] vs PAH
Stable and does not require cofactor
To test concept, yPAL cloned and overexpressed in E. coli
Preclinical studies (2003) with mice decient in PAL
See lower plasma levels of Phe when PAL injected or
administered as oral encapsulated enzyme

Smart therapies and treatment[s]: Should we?

Traditional treatment: diagnosis at birth or prenatal test

Controlled semi-synthetic diet with low levels of Phe
Possible treatment: metabolism of Phe
PAH multienzyme complex, requiring cofactor

ELSI: Should we, just because we can?
JGelsinger, 18yo (Sept 99)
Ornithine transcarbamylase deciency, X-linked genetic disease
Cannot metabolize ammonia from protein catabolism
Usually fatal at birth, but this case was not inherited
Sporadic and enough cells normal to allow life with diet and meds
First Gene Therapy-associated death in US
Third childgene therapy in France developed cancer as a result

Treatments for digestive tract diseases

Ulcerative colitis, Crohn disease

Diseases of intestinal tract
~1/ 500-1,000
Ulcerative colitis- associated with excess type 2 T-helper cell cytokines, including Il-4 and -5
Crohn disease- associated with excess type 1 T-helper cell cytokines, including TNF-, IFN-, IL-2

http://digestive.niddk.nih.gov/ddiseases/pubs/crohns/index.htm

Smart therapies: Treatment with live secreting bacteria

Ulcerative colitis- associated with excess type 2 T-helper cell cytokines, including IL-4 and -5
Treatment: 1) antibodies against TNF-a, to lower levels of cytokines and 2) targeting IL-10
IL-10 modulates regulatory T-cells, that control inammatory responses to intestinal Ag
Delivery is through injections directly or rectal enemas
Alternative strategy: produce and deliver by intestinal bacteria
L. lactis to synthesize and secrete IL-10
Mice fed water laced with dextran sulfate +/- recombinant L. lactis
Positive effect- Proof of Principle
However, these mouse models not identical to disease in humans
Concern: recombinant bacteria released into environment

Treatment with live secreting bacteria

Concern: recombinant bacteria released into environment

Method: synthetic human interleukin-10 gene replaces L. lactis thymidylate synthetase gene [thyA]
Essential for bacterial growth
Entire construct recombined into chromosome
Produces human interleukin-10 and grows well if thymidine or thymine is provided
Tested in pig [digestive tract ~ human]
Extremely unlikely L. lactis will pick up thyA from environment
2009, clinical trials with 10 Crohns patients
Bacteria isolated in feces are thyA-

Smart therapies: Treatment of obesity

30% North Americans and 20% Europeans are overweight

10s of $B spent on weight reduction schemes
Leptin: 16 kDa protein hormone, regulates Energy uptake/expediture
Appetite and metabolism; satiety- appetite control
1950 Jackson Lab: mutant obese mice, massively obese and excessively voracious
Several strains homozygous for single-gene mutations
Two classes: ob/ob, leptin mutations and db/db, leptin receptor mutations
ob/ob mice + injected leptin => lose excess fat and return to normal body weight
1995 leptin gene, EDGreenJMFriedman Genome Research 5:5-12
Acts on hypothalamus of brain http://en.wikipedia.org/wiki/File:Fatmouse.jpg

http://en.wikipedia.org/wiki/Leptin

Smart therapies: Treatment of obesity

30% North Americans and 20% Europeans are overweight

10s of $B spent on weight reduction schemes
Treatment with recombinant leptin can reduce food intake and correct
metabolic perturbations in ob/ob mice
Can help human congenital leptin deciency
Subcutaneously, not particular effective unless serum conc 20-30x higher than normal
Blood-brain barrier
Method: intranasal delivery of leptin
Leptin is synthesized as precursor molecule with 21-amino acid signal peptide
E. coli product insoluble -> solubilize
Time-consuming, inefcient and expensive
Nisin promoter and deliver using L. lactis
No inclusion body and secreted
Intranasal administration signicantly reduced food intake and body weight in obese mice

Therapeutic agents from

Transgenic animals, Molecular biology, Genomics

Post-traumatic stress (PTS) disorder

Treating a mental disorder?
Once poorly understood and
Little-known mental health problem
Better known as war vets seek treatment
Rate of 1 per 7 back from deployment
Many myths
1- psychological
biologically-based
2- military combat is top cause
car accidents is top cause
Long-term effects if not treated
Can develop other problems, eg addictions
Treatment can include therapy and medication

http://www.usatoday.com/news/health/2008-10-26-PTSD-main_N.htm

Post-traumatic stress (PTS) disorder

USAToday, Oct 27, 2008
Many myths
1- psychological
biologically-based
2- military combat is top cause
car accidents is top cause
Long-term effects if not treated
With PTSD, an average of 12 years
before seeking treatment after accident
2.5M in US injured per year in car accidents
Can develop other problems, eg addictions
Treatment can include therapy and medication
SSRI-class antidepressants
(selective serotonin reuptake inhibitor)

http://www.usatoday.com/news/health/2008-10-26-PTSD-main_N.htm

Proteins and memory, and mice

XCaoJZTsien, et al. Neuron Oct08

Memory is separated into four stages
Acquisition, consolidation, storage and retrieval
Specic proteins play roles in these phases
One memory molecule is
Calcium/calmodulin-dependent protein kinase II (CaMKII)
Plays key role in brain cell communication; linked to learning and memory
Created transgenic mice with inducible promoter hooked to CaMKII
Examined transgenic mice for retrieval of short-term and long-term fear memories
and novel object recognition memory
(fear= mild electric shock and cat odor causes mice to freeze in the context
of a specic environment)

The Scientist, Oct 22, 2008

Memory, molecular biology and cinema

NPR 4/13/10 Hippocampus: memory and lingering feelings

Emotions outlast the memories that drive them; JFeinsteinDTranel PNAS pPub Apr10
Alzheimers patients, visits/moods, post-visit: no memory but depressed or elated feeling remains

Protein and memory,

and mice

XCaoJZTsien, et al. Neuron Oct08
Transgenic with CaMKII activity that
can be inducibly and reversibly switched off
and on within minutes by injection of a
genetically sensitized inhibitor, NM-PP1 ->
Transient overexpression of CaMKII
at time of recall can lead to rapid erasure
of a memory that is being retrieved, leaving
other memories intact
Also memories formed in the past hour
Effect persists for a month after
Applications include PTS treatment and
Understanding memory circuits in the brain
Earlier work on memory and protein

Protein and memory, and mice

YPTangJZTsien, et al. Nature Sept99

1999 Doogie mouse- smart transgenic mouse
with enhanced learning and memory abilities
NR2B gene, encoding NMDA receptor
Two chemicals needed to trigger this nerve cell receptor
Mice with extra NR2B genes have heightened NMDA receptor activity
Adding a single gene to fertilized eggs
Assay by environment containing two objects, then removed one
Observe how long spent at new object versus old: test memory
Assay with environment and electric shock: test learning
Assay with pool and hidden ramp: test spatial intelligence
Application: Gene therapy for dementia

Earlier work, limiting expression of NR2B could impair ability to learn and remember
Deletion of the gene in certain brain regions resulted in mice that were considerably less intelligent

http://www.sciam.com/article.cfm?id=making-smart-mice
wiki

Protein and memory, and mice

http://www.princeton.edu/pr/pictures/other/smartmouse/index.html

Chemistry, sex, and mice

Serotonin controls sexual preference in mice [Liu, Jiang, Kim, et al., March 2011 Nature]
1) Males bred not receptive to serotonin lose preference for females
Mount and emit mating call
2) Different mutation, lacking tryptophan hydroxylase 2 gene (precursor to serotonin)
Could restore preference by injecting serotonin into brain
Mouse sexual preference linked to smell
Humans: limited evidence for altered responses to selective serotonin uptake inhibitors (SSRIs)
Psychoactive drugs that increase or decrease serotonin function =>
sexual arousal, impulsivity and aggression
http://www.bbc.co.uk/news/health-12825688

http://www.nature.com/jp/journal/v25/n9/g_tab/7211352f1.html
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature09822.html

The Brain

Phineas P. Gage 1823-1848-1860

Left frontal lobe destroyed
Effects on personality and behavior
Friends said no longer Gage
First case to suggest specic brain damage and personality and behavior

http://en.wikipedia.org/wiki/Phineas_Gage

The Brain: mapping behavior to physical location

Damage to hippocampus interferes with new memory storage

Also, use of language, recognize familiar faces, count and read

http://www.headinjury.com/brainmap.htm

The Brain: mapping behavior to physical location

Frontal lobes

http://www.headinjury.com/brainmap.htm

The Brain: mapping behavior to physical location

Temporal lobes

http://www.headinjury.com/brainmap.htm

The Brain: mapping behavior to physical location

Parietal lobes

http://www.headinjury.com/brainmap.htm

The Brain: mapping behavior to physical location

Occipital lobes

http://www.headinjury.com/brainmap.htm

Genomics: Genetic vs epigenetic- unusual symptoms/syndromes (new understandings)

10/28/06 Scott Adams. Rare example of recovery- largely but not totally
Spasmodic Dysphonia, mysterious disease in which parts of the brain
controlling speech shut down or go haywire
30,000 Americans, typically in 40s and 50s
Phenotype: Typically unable to converse in normal voice, but under
different circumstances, immediately after sneezing or laughing,
can speak in exaggerated falsetto or baritone, or while reciting poetry
Off-label drug use: Botox

Genomics: Genetic vs epigenetic- unusual symptoms/syndromes (new understandings)

Diane Rehm, Spasmodic Dysphonia, diagnosed in 1998

Cause unknown
May run in families (inherited)
Chrom 9 region/gene affecting spasms of the vocal cords
Also spasms in eyes, arms, legs and mouth
Also may have multiple dystonias, movement disorders
Some cases, following upper respiratory infection, injury to larynx, overuse of voice, or stress