Molecular farming utilizes plants' ability to produce large amounts of biomass and proteins using sunlight and simple media. Plants are effective bioreactors for producing recombinant pharmaceutical proteins due to their low-cost production and ability to perform post-translational modifications. Common plant systems for protein expression include tobacco, cereals, fruits and vegetables. Safety concerns include cross-contamination of food/feed and unintended exposure, which can be addressed through physical and biological containment methods.
Molecular farming utilizes plants' ability to produce large amounts of biomass and proteins using sunlight and simple media. Plants are effective bioreactors for producing recombinant pharmaceutical proteins due to their low-cost production and ability to perform post-translational modifications. Common plant systems for protein expression include tobacco, cereals, fruits and vegetables. Safety concerns include cross-contamination of food/feed and unintended exposure, which can be addressed through physical and biological containment methods.
Molecular farming utilizes plants' ability to produce large amounts of biomass and proteins using sunlight and simple media. Plants are effective bioreactors for producing recombinant pharmaceutical proteins due to their low-cost production and ability to perform post-translational modifications. Common plant systems for protein expression include tobacco, cereals, fruits and vegetables. Safety concerns include cross-contamination of food/feed and unintended exposure, which can be addressed through physical and biological containment methods.
Molecular farming utilizes plants' ability to produce large amounts of biomass and proteins using sunlight and simple media. Plants are effective bioreactors for producing recombinant pharmaceutical proteins due to their low-cost production and ability to perform post-translational modifications. Common plant systems for protein expression include tobacco, cereals, fruits and vegetables. Safety concerns include cross-contamination of food/feed and unintended exposure, which can be addressed through physical and biological containment methods.
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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
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.
