Molecular farming

Green Factory
INTRODUCTION

The large-scale production of recombinant proteins in living cells or

organisms; frequently applied to the use of crop plants.

Utilizes plants biosynthetic capacity including the ability to use the

sun (photosynthesis) and/or very simple media to support
significant biomass and protein accumulation.

low-cost production

high quality and bioactive recombinant protein

WHY PLANTS AS BIOREACTOR ????????????????????????????

Adapted from The production of recombinant pharmaceutical proteins in plants

Christou et al; doi:10.1038/nrg1177
Adapted from Xu J, et al, Green factory: Plants as bioproduction platforms for
recombinant proteins, Biotechnol Adv (2011),
doi:10.1016/j.biotechadv.2011.08.020
 Classes of Recombinant protein being produced:

Adapted from Xu J, et al, Green factory: Plants as bioproduction platforms for

recombinant proteins, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.020
 Plant expression system

Whole plants-stable Whole plants-transient In-vitro culture Aquatic

expression systems expression systems system plants

Leaf based Plant cell suspension

Duckweed
Seed based Hairy root

Moss Micro Algae

 Comparison of Different expression system

Adapted from Xu J, et al, Green factory: Plants as bioproduction platforms for

recombinant proteins, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.020
 Comparison of Different expression system-Advantages & Challenges

Adapted from Xu J, et al, Green factory: Plants as bioproduction platforms for

recombinant proteins, Biotechnol Adv (2011), doi:10.1016/j.biotechadv.2011.08.020
 Genetic aspects of molecular farming in plants

Gene constructs:

Expression-construct design must optimize all stages of gene

expression, from transcription to protein stability.

For high-level transcription, the two most important elements are

the promoter and the polyadenylation site.
CaMV 35S promoter (Dicots)
maize ubiquitin-1 promoter (Monocots)

Tissue-specific promoters-increased stability of the protein and

the avoidance of protein accumulation in vegetative organs

Inducible promoter systems-restrict transgene expression on

a temporal basis. For example, the mechanical gene activation
(MeGA)system uses a tomato hydroxy-3-methylglutaryl CoA
reductase 2 (HMGR2) promoter,which is inducible by mechanical
stress.
Subcellular targeting-affects the interlinked processes of folding,
assembly and post-translational modification.

Proteins are targeted to the secretory pathway through the inclusion of

an N-terminal SIGNAL PEPTIDE in the expression construct

The oxidizing environment of the endoplasmic reticulum (ER), the lack

of proteases and the abundance of MOLECULAR CHAPERONES are
important factors for correct protein folding and assembly.

Transformation methods.
1.AGROBACTERIUM-MEDIATED TRANSFORMATION and
2.Particle bombardment

Post-translational modification.-
The protein synthesis pathway is conserved among plants and animals.
However some minor differences occur in PTM, As changes in glycan
structure which could potentially change the activity, Biodistribution
and longevity of recombinant proteins compared with native forms.
Plant-expression hosts:

Tobacco production systems-

Advantages: mature technology for gene transfer and expression,
high biomass yield, potential for rapid scale-up, and the availability of
large-scale infrastructure for processing.
Disadvantages: Instability of the product.

Cereals and legumes.

Advantages: long-term storage at ambient temperatures, high biomass
yield, ease of transformation in vitro manipulation, and ease of scale-up.

It has been shown that antibodies that are expressed in seeds remain
stable for at least three years at room temperature with no detectable
loss of activity.
Disadvantages: Food-feed shortage.

Fruit and vegetables:

Advantages: can be consumed raw or partially processed.
Bananas have been considered as hosts for the production of
recombinant vaccines, as they are widely grown in the countries and
can be consumed raw or as a puree by both adults and children
Transgenic rice as a vehicle for the production of the
industrial enzyme Transglutaminase
M.I. Claparols1, L. Bassie2,, B. Miro2, S. DelDuca3, J. Rodriguez Montesinos2,
P. Christou2, D. Serafini-Fracassini3 & T. Capell2,

Transglutaminase play important role in protein modification and cross-

linking.

The widespread use of transglutaminases has increased the demand for

an inexpensive, efficient and safe source of recombinant enzyme.

Transgenic rice plants engineered with a rat prostate transglutaminase

(rTGp), driven by the strong constitutive maize-1 ubiquitin promoter
and its first intron, were shown to express the recombinant enzyme at
themRNA and protein levels.

Most prevalent application of TGs is in the food industry, where the

enzyme is used to improve the texture of meat, fish, bread and dairy
products.
Genetic construct: Ubi:rTGp.

The 2004-bp ORF rat prostatic TG from pBluescript.

subcloned into the EcoRI site of pAL76 which contains the maize-1
ubiquitin promoter and first intron, and the nos transcriptional
termination.

Transformation Strategy

Mature rice embryos (var. EYI 105) were co-bombardedwith plasmid

Ubi:rTGp and a plasmid containing the hygromycin phosphotransferase
(hpt) gene as a selectable marker.
It corresponds to the presence of
2 kb fragment of rTGp ORF.
 Northern Analysis:
Confers full length
transgene integration

western blot:
using mouse monoclonal specific
anti-TG IgG antibodies (Ab-3
Neomarker).

A band of ca. 60 kDa was

detected in rice
leaf extracts.
Transglutaminase activity in rice
leaves.

(A) By the incorporation of 3H-

putrescine into N ,N-dimethylcaseine
(DMC) by crude cell extracts derived
from rice leaf tissue from different
transgenic lines and wild type plants.

(B) By the biotin-labelled cadaverine-

incorporation assay to DMC, by crude
cell extracts derived from rice leaf
tissue from different transgenic lines
and wild type plants.

Values are meanSE for control lines

(n=3) and meanSE in transgenic
lines (n=3).

Significance of data is as follows:

P<0.001; 0.05>P>0.01.
Conclusion:

From results it can be inferred that transgenic rice plants could be a

viable alternative to mammalian and prokaryotic expression system
for the production of TG (transglutaminase)

TG is activated only in the presence of Ca++ which confirms its

calcium dependent catalytic activity.

Once activated TG can catalyze a number of reactions leading to

post translation modification of protein through acyl-transfer
reaction.

No changes in cellular acid-soluble free and free insoluble

conjugates polyamines were measured in the transgenic rice plants
that expressed the recombinant enzyme.
Biosafety and regulatory issues

General Public concern

Accumulation of various protein in high concentration may affect
the host plants.

Biological active products from plants may elicit physiological

response in humans and animals.

Specific concerns
(i) co-mingling with food/feed crops and the resultant
economic risks to farmers and food industry,

(ii) transgenes spread by pollen, seed or fruit dispersal,

(iii) unintended exposures to nontarget organisms, particularly

birds, insects and soil microorganisms and,

(iv) horizontal gene transfer by asexual means

Problem of co-mingling and contamination of the food/feed chain

Using non-food and non-feed crops such as tobacco, lemna,

Arabidopsis, microalgae or mosses.

In the case of monoclonal antibodies and oral vaccines, strict

physical agronomic confinement and containment strategies are
being applied to avoid co-mingling.

The problem of gene spread and unintended exposures

Use of closed isolated containment facilities, such as greenhouses,

glasshouses, hydroponics and plant cell suspension cultures.

Use of biological containment, such as self pollinating species,

chloroplast transformation, male-sterile transgenic plants, sexually
incompatible Crop, production of non-germinating seeds,

Cytoplasmic male sterility, engineered parthenocarpy, and apomixis.