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Plants as Bioreactors

NISHA G V 3RD SEMESTER MSc BIOTECHNOLOGY
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Is a method used to integrate a foreign gene into plants for the production of commercial products Two types Medical: includes pharmaceutical products Non-Medical: includes Industrial Enzymes and Polymers

MOLECULAR FARMING

Industrial enzymes: laccase in transgenic maize, avidin in maize
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The first synthesis of a pharmaceutically relevant protein was human growth hormone in transgenic tobacco plants in 1986 In 1989 the first antibody was expressed in tobacco which showed that plants could assemble complex glycoprotein with several subunits
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WHY PLANTS? v Plants are very flexible Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens Crop plants produce large amounts of biomass at low cost and require limited facilities Well suited for the therapeutic proteins 4/22/12 v v v production of safe 44 low-cost .

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HOW IS IT DONE? Ø Genetic enhancement is used to introduce and express genes coding for the high value proteins Through careful and skilled manipulation of genes Controlled expression of a specific gene which then expressed in a transgenic plant 4/22/12 66 Ø Ø .

Ø Target protein can be expressed in a single plant tissue or during a specific developmental phase of the plants growth cycle Thus the production of any target protein 77 Ø 4/22/12 .

PHARMACEUTICAL PROTEINS

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STRATEGIES FOR PROTEIN PRODUCTION
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STABLE EXPRESSION
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Strong, constitutive promoter (35S)
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Bulk production of soluble proteins in leaves But yields may low

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Targeted gene expression
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Higher yields Storage organs helps to maintain biological integrity
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TRANSIENT EXPRESSION

HIRUDIN PRODUCTION IN Brassica napus
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Anticoagulant peptide of 7 kDa found in salivary glands of the leech Hirudo medicinalis Inhibitor of thrombin Low immunogenicity, not require endogenous cofactors for activity and not interfere with other blood proteins Expressed in Brassica napus

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terminus end of the oleosin and N.Ø An endoprotease Factor Xa cleavage site was engineered between the C.terminus end of the Hirudin Protein produced in oil bodies Purification Ø Ø Ø centrifugation of oil bodies treat them with endoprotease Factor Xa Re-centrifugation will be present in aqueous 1111 phase Ø Ø Ø 4/22/12 Hirudin .

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Glcocerebrosidase § Glucocerebroside into glucose and ceremide as aged red blood cells are removed by body Gauchier’s disease Carole Cramer at Crop Tech Development corporation at Virginia developed transgenic tobacco § § Human Serum Albumin § Using 4/22/12 35S promoter in tobacco 1313 .

Plant Transformation Ø The plant leaf disc is dipped in a solution of bacteria. The bacterial "Trojan Horse" infects the edges of the leaf disc and in the process integrates the pharmaceutical protein gone into the plant genome After infection the discs are placed on selection media that a flows only plant cells that carry the protein gene to survive and regenerate into plantlets. After about six weeks on selection media. a large number of plantlets that carry the pharmaceutical 1414 protein gene are visible at Ø 4/22/12 .

This seed can then be used for large scale production of the 4/22/12 pharmaceutical protein Ø 1515 .Ø The plantlets are removed from the leaf disc and placed in clear plastic boxes that contain media that allows them to form roots The rooted plantlets are placed in pots and plants are allowed to grow and produce seed.

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corn. alfalfa. soy. rice Free from potential contamination of mammalian viruses GE Corn can produce up to 1 kg antibody/acre and can be stored at RT for up to 5 years 4/22/12 1717 Ø Ø Ø . potatoes.Plantibodies Ø Plants can be used to produce monoclonal antibodies Tobacco.

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AVICIDIN § IgG produced in corn plants showed anticancer activity side effects so product withdrawn ScFV based on idiotype of malignant B lymphocyte in mouse lymphoma cell line for non Hodgkin lymphoma § § 38C13 § § CaroRX IgG/A in transgenic tobacco expression 4 4/22/12 separate transgene 1919 § .

Full size monoclonal antibodies recently produced in transgenic plants 4/22/12 2020 .

Abs expressed in transgenic plants Constant domains Variable Light chain Variable Heavy chain 4/22/12 Recombinant hinge region 2121 .

coli & 4/22/12yeast Tr. 219-226 00 1 0 1 0 0 E. animals and animal cells Transgenic 2222plants .Production Costs for Production cost in gr Antibodies costs $/ am hybrido 10 mas transgenic animals transgenic plants Source: Daniell et al. (2001) TIPS 6.

terminal sugar different plus xylose & fucose antigenicity & clearance: (shorter half-life) apparently identical § 4/22/12 2323 .Comparison of Mammalian and Plant-produced Antibodies § § § § § § peptide sequence: identical correct cleavage of Ig-derived signal peptides kinetics & affinity: identical stability in seeds > 30 months antibody types: plant system more versatile (sIgA) post-translational processing: different § core glycan identical.

Transgenic scFv expressing tobacco plants (in the background) and control plants (in the foreground) 14 days after infection with different plant viruses 4/22/12 Source: AIPlanta Institute 2424 .

• Human vaccine for hepatitis B in potato • Newcastle disease virus • Rotavirus VP7 • • • • • • • 4/22/12 2525 .Plant produced Vaccines Measles virus haemagglutinin Porcine TGEV Tetanus toxin Vacinia virus B5 Allergy vaccines HPV HIV gp41 and HIV-suppressing protein in spinach.

PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES DISEASES Respiratory Syncytial Hepatitis B HIV Rabies Anthrax Diphtheria SARS Smallpox VACCINES         PLANT Tobacco Lettuce Spinach Spinach Tobacco Tobacco Tomato Tobacco Tobacco Tomato Tobacco Tomato  Tobacco ANTIBODY PLANT PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF CANCER HUMAN DISEASES Colorectal Cancer Epithelial Tumors (EGF receptor) VACCINES  ANTIBODY   4/22/12 2626 .

PMP Development . approved Feb 2006 Heberbiovac (Cuba) approved antibody for HepB vaccine purification 4/22/12 2727 § § § § § § .Highlights § Series of plant-derived vaccines from Arizona State University have completed clinical trials Prodigene has trialled two plant-derived vaccines LSBC pipeline of cancer vaccines prior to insolvency Guardian Bioscience coccidiosis vaccine. CFIA phase II ongoing Fraunhofer CMB. rabies vaccine trialled in humans DowAgro Newcastle disease vaccine.

tomato and potato How???? Ø Ø Ø Ø Ø vaccines present in food shown to induce immune response Leave behind the memory cells High mucosal immunity 2828 Ø 4/22/12 Ø . USA Hiatt and coworkers in 1989 First model system was tobacco Now produced in banana.EDIBLE VACCINES Ø Concept introduced by Charles Arntzen of Texas University.

01% of soluble protein to VLPs 2929 § Assembled 4/22/12 § .EDIBLE VACCINES FOR HUMAN DISEASES § First published report on cell surface adhesion protein SpaA in transgenic tobacco plants Arnitzens group developed tobacco plants producing hepatitis B § § HBsAg linked to 35S promoter 0.

Ø Advantages: Ø Administered Directly Ø no purification required Ø no hazards assoc. where needed most no transportation costs Ø Naturally stored 3030 4/22/12 . w/injections Ø Production Ø may be grown locally.

METABOLIC ENGINEERING OF CARBOHYDRATE 4/22/12 3131 .

potatoes Alternation of ADP glucose pyrophosphorylase This enzyme in E.coli was mutated to alter its allosteric properties and transferred to potato Amylose to amylopectin ratio v v v v Food industry: GBSS1 down regulation produce amylose free starch Feed stock: SBE A and B down regulation 3232 4/22/12 v .v Starch v Chemical feed stocks: cereals.

v Cyclodextrin v Used in medical industry as solubilization of pharmaceuticals Made of glucopyranose seven membered ring of v v Transgenic Potato tuber produced by transferring glycosyl tranferase gene from Klebsiella pneumoniae by fusing gene to plastid targetting sequence driven by patatin gene promoter Synthesized and stored in cellular vacuoles v Polyfructans v Expressed chichory fructosyl transferase in 4/22/12 3333 onion v .

sugarcane. hemp oil. soy bean oil and corn starch Environmental friendly Biodegradable 4/22/12 3434 Ø Ø .BIOPLASTICS Ø Form of plastics derived from plant sources such as sweet potatoes.

Plant Oil COMPOSITION Starch Cellulose Corn Sugarcane Potato 4/22/12 35 35 .

§ Starch based plastics: Bioplastics produced from classical chemical synthesis from bio based monomers § Polylactic Acid (PLA) plastics: Bioplastics produced directly by natural or genetically modified organisms § Polyamides 11: Polycaprolactones 4/22/12 36 36 Classification of Bioplastics .

Thermoset polymers • Soy polyurethanes • Unsaturated polyester resins Thermoplastic polymers • Polylactic acid • Modified starch polymers • Thermoplastic polyesters • Bio-polyolefins • Polyhydroxy alkonates (PHA) 4/22/12 • Cellulose ethers 3737 .

which can be used in the manufacture of products 4/22/12 3838 after being heated and shaped Ø .Enzymes are used to break starch into glucose. which is fermented to lactic acid Ø This lactic acid is polymerized and converted into a plastic called polylactic acid.

switchgrass plants engineered using Metabolix multi-gene expression technology produced significant amounts of PHA bioplastics in leaf tissues This result is the first expression of a new functional multi-gene pathway in switchgrass 4/22/12 3939 . recently-completed greenhouse trials. Inc.Bioplastic Production in Switchgrass Plants Metabolix.

Spring water bottle made from bioplastic 4/22/12 4040 .

COMPANIES 4/22/12 4141 .

Applications Packaging shopping bags Trays and containers Catering products Disposable crockery and cutlery Gardening flower pots 4/22/12 Medical Products 4242 .

carry bags for all shopping 4/22/12 4343 applications. outer packaging material for foodstuff .Bioplastics production in India Jammu and Kashmir will be the first state in India to have a fully dedicated Bioplastics product manufacturing facility with an installed capacity of about 960 metric tones per year The J&K Agro Industries Ltd is going for a joint venture with EARTHSOUL for the manufacture of 100 per cent bio-degradable and compostable products. This would be India’s first integrated biopolymer facility The facility would be manufacturing flower pots and trays for floriculture.

Industrial enzymes 4/22/12 4444 .

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Phytase § Pytate into inositol and organic phosphate High quantities in seeds used as feeds to pigs and poultry animals Eutrophication Transgenic plant seed with this enzyme 4646 § § § 4/22/12 .

Tobacco Natural defence protein Antiviral. antibacterial Natural protein sweetner 4747 . Maize Maize Rice USE Imunodiagnostic Skin sealent Scar treatment Cystic fibrosis Lysozyme Brazzein Aprotinin 4/22/12 California. Meristem therapeutics Ventria Ventria Large scale biological. France Texas California. Taxas Maize.PRODUCTS CLOSE TO MARKET PRODUCT Avidin collagen Lipase Lactoferrin COMPANY Prodigen Medicago. Rice Maize Maize. Prodigen Meristem therapeutics Ventria. Texas California France PLANTS Maize Alfa alfa. Prodigen LOCATION Texas Canada.

4848 § § § § .GENETIC APPROACH § Expression construct is optimized for high yield Promoter and polyadenylation site derived from 19S and 35S of CaMV required in Franssinic strategy Ubiquitin-1 promoters for monocots Intron in the 5’ untranslated region Tissue 4/22/12 specific promoters: Zein gene promoter.

N2 fixation) for rAbs 4949 4/22/12 . soyabean (Atm.CHOICE OF PLANT PRODUCTION SYSTEM v LEAFY CROPS v Tobacco for pharmaceutical products § Gene transfer technology for in vitro regeneration Gene expression and high biomass yield Phyllosecretion and rhizosecretion § § v Alfa alfa.

oil seed rape reduction due to secondary revenues 5050 cost 4/22/12 v . nutriceuticals and antibodies Potatoes for vaccine and tomatoes for rabies vaccine v v v FIBRE AND OIL CROPS v Flax. cotton.v FRUITS AND VEGETABLE CROPS v Edible organs can be consumed uncooked and unprocessed Ideal for rSubunit vaccines.

Secretary Abs § § § Stability and Safety § Only difference in post translational modification like glycan chain structure 4/22/12 5151 § .BENEFITS § § LIMITATIONS Environment Low cost and Large scale production contamination § Capital and running cost Food contamination are low § § supply Health safety concerns § Scalability PRODUCT AUTHENTICITY Plants safer than microbes Ability to fold and assemble complex proteins Serum proteins.

Current Challenges in Molecular Farming § § § § yield of recombinant proteins real quantitative comparison protein stability (proteases) post-translational modification and downstream processing trials & regulatory approval §extraction §clinical 4/22/12 5252 .

Regulatory Challenges § loci of transgene insertion properties and levels § expression § effects of the transgene on the expression of flanking endogenous genes line banking to ensure product consistency with animal excreta. pesticides. § master § contamination organic fertiliser § procedures for detection and removal of weeds and pests § cultivation 4/22/12 variables 5353 .

Roadmap Plants for the Future Plant production platforms - Health food and quality - - Amino acids Oil Starch Efficient agriculture - Plant protection - Vitamines Fatty acids Enzymes Bio-polymers Pigments Pharmaceutical products Fibers Bt technology Herbicide resistance Viruses Nematodes Fungi Insects Stress resistance - Cold Drought Salinization 199 7 4/22/12 200 5 201 5 202 5 5454 .

• Monoclonal antibodies • Recombinant proetins Produce PHAs in green tissue plants for fuel generation. (duckweed) Colds due to Rhinovirus: RhinoRx™ Drug-induced Alopecia: DoxoRx™ Rhizosecretion Biomass biorefinery based on switchgrass.Planet Biotechnology Dental Caries: CaroRx™ Current ‘Pharm’ Companies LEX System™ Lemna. 4/22/12 5555 .

Kentucky Tobacco Research and Development Center Trangenic tobacco PMPs and non-protein substances (flavors and fragrances. medicinals. and natural insecticides) Trangenic tobacco GeneWare® 4/22/12 5656 .

Ventria Bioscience Prodigene Transgenic corn Trypsin Aprotinin 4/22/12 Transgenic rice Lactoferrin Lysozyme 5757 .

.• Examples of Current Researchengineered Arabidopsis plants can Genetically sequester arsenic from the soil (Dhankher et al. 2002 Nature Biotechnology) • • Immunogenicity in human of an edible vaccine for hepatitis B (Thanavala et al. 2005 Vaccine) Plant based HIV-1 vaccine candidate: Tat protein produced in spinach (Karasev et al.. 2005 Vaccine) • • . PNAS) Expression of single-chain antibodies in transgenic plants (Galeffi et al. 2005 Vaccine) Plant-derived vaccines against diarrheal diseases 4/22/12 5858 (Tacket. 2005.

Risks and Concerns Environment contamination Gene flow via pollen Non-target species near field sites Food supply contamination Accident gene flow Health safety concerns Non-target organ responses Side-effects Allergenicity 4/22/12 5959 .

Regulatory System (existing regulations) Food safety Feed safety FD A EPA Determination of non-regulated status 4/22/12 Pesticide and herbicide registration 6060 .S.Field Testing -permits -notifications USD A U.

GFP Sterility Use male sterile plants and Terminator technology Easily detectable by addition of 'reporter genes' PCR markers (avoid antibiotic resistance markers) 1. 1. 4. Chloroplast expression system Increase yield Eliminates potential gene flow Technically difficult (Chlorogen Company) 5. Physical differences E. Complete disclosure of DNA sequences 4/22/12 6161 .Suggested Safeguards for ‘molecular farming’ 1.g. “purple” maize.

ppt Influencing Agriculture: host Molecular and ü ü ü Molecular farming in plants: expression technology. Twyman.REFERENCES ü PLANT MOLECULAR BIOTECHNOLOGY S. Stefan Schillberg. MAHESH Biotechnology U satyanarayana Biotechnologies Pharming. 4/22/12 6262 . Eva Stoger. ppf Paul Christou and Rainer Fischer systems Richard M.

A.pdf D.pdf Update on Agricultural Biotechnology Controversies Pharmaceutical Crops. THOMAS Molecular Farming: Plants as New Product Producers.pdf Allan Felsot Use of plant roots for phytoremediation and molecular farming.pdf 4/22/12 6363 Ø Ø Ø Ø .A. GOLDSTEIN and J.Ø Action FA0804: Molecular farming: plants as a production platform for high value proteins.pdf DOLORESSA GLEBA PNAS Biopharmaceuticals derived from genetically modified plants.

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