plants as bio reactors nisha g v

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Plants asBioreactors

NISHA G V

3RD SEMESTERMSc BIOTECHNOLOGY

11

04-10-2011


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MOLECULARFARMING

Is a method used to integrate a foreign gene intoplants for the production of commercial products

Two types

Medical: includes pharmaceutical products

Non-Medical: includes Industrial Enzymes and

Polymers

Industrial enzymes: laccase in transgenicmaize, avidin in maize

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The first synthesis of a pharmaceuticallyrelevant protein was human growth

hormone in transgenic tobacco plants in1986

In 1989 the first antibody was expressedin tobacco which showed that plants couldassemble complex glycoprotein withseveral subunits

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WHY PLANTS?

v Plants are very flexible

v Crop plants can synthesize a wide variety of proteinsthat are free of mammalian toxins and pathogens

v Crop plants produce large amounts of biomass at lowcost and require limited facilities

v Well suited for the production of safe low-costtherapeutic proteins

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HOW IS IT DONE?

Genetic enhancement is used to introduceand 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 plant66


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Target protein can be expressed in a singleplant tissue or during a specific

developmental phase of the plants growthcycle

Thus the production of any target protein77


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PHARMACEUTICALPROTEINS

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STRATEGIES FOR PROTEIN

PRODUCTION STABLE EXPRESSION Strong, constitutive promoter (35S)

Bulk production of soluble proteins in leaves But yields may low

Targeted gene expression

Higher yields Storage organs helps to maintain biological

integrity

TRANSIENT EXPRESSION 99


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HIRUDIN PRODUCTION INBrassica napus

Anticoagulant peptide of 7 kDa found in salivaryglands of the leech Hirudo medicinalis

Inhibitor of thrombin

Low immunogenicity, not require endogenous

cofactors for activity and not interfere with otherblood proteins

Expressed in Brassica napus 1010


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An endoprotease Factor Xa cleavage site wasengineered between the C- terminus end of theoleosin and N- terminus end of the Hirudin

Protein produced in oil bodies

Purification

centrifugation of oil bodies

treat them with endoprotease Factor Xa

Re-centrifugation

Hirudin will be present in aqueous phase1111


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Glcocerebrosidase Glucocerebroside into glucose and ceremide

as aged red blood cells are removed by body

Gauchiers disease Carole Cramer at Crop Tech Development

corporation at Virginia developed transgenic

tobacco

Human Serum Albumin

Using 35S promoter in tobacco 1313


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Plant Transformation

The plant leaf disc is dippedin a solution of bacteria. Thebacterial "Trojan Horse"infects the edges of the leafdisc and in the processintegrates thepharmaceutical protein goneinto the plant genome

After infection the discs areplaced on selection media

that a flows only plant cellsthat carry the protein gene tosurvive and regenerate intoplantlets. After about sixweeks on selection media, alarge number of plantletsthat carry the pharmaceuticalprotein gene are visible at1414


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The plantlets areremoved from theleaf disc and placedin clear plasticboxes that containmedia that allowsthem to form roots

The rooted plantletsare placed in potsand plants are

allowed to growand produce seed.This seed can thenbe used for largescale production of

the pharmaceuticalrotein1515


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Plants can be used to produce monoclonalantibodies

Tobacco, corn, potatoes, soy, alfalfa, 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

Plantibodies

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AVICIDIN IgG produced in corn plants

showed anticancer activity

side effects so product withdrawn38C13

ScFV based on idiotype of malignant Blymphocyte in mouse lymphoma cell line

for non Hodgkin lymphoma

CaroRX

IgG/A in transgenic tobacco expression 4

separate transgene 1919


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Full size monoclonal antibodies recently

produced in transgenic plants

2020


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Abs expressed in transgenicplants

Variable Light chain

Variable Heavychain

Recombinant hinge region

Constantdomains

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Production Costs for

AntibodiesProduction

costs

cost in

$ /

gr

amhybridomas

1000

transgenicanimals

100

transgenic

plants

1

0Source: Daniell et al. (2001) TIPS 6,219-226

E. coli &

yeast

Tr. animals and

animal cells

Transgenic

plants2222


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Comparison of Mammalian and Plant-producedAntibodies

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, terminal sugar different plus

xylose & fucose antigenicity & clearance: apparently identical

(shorter half-life)

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Transgenic scFv expressing tobacco plants(in the background) and control plants (in theforeground) 14 days after infection withdifferent plant viruses

Source: AIPlanta Institute 2424


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Plant produced Vaccines

Measles virus haemagglutinin Porcine TGEV Tetanus toxin

Vacinia virus B5 Allergy vaccines HPV HIV gp41 and HIV-suppressing protein in

spinach, Human vaccine for hepatitis B in potato Newcastle disease virus Rotavirus VP7

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PLANT DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES


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PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES

DISEASES VACCINES PLANT ANTIBODY PLANT

RespiratorySyncytial

Tobacco

Hepatitis B Lettuce

HIV Spinach

Rabies SpinachTobacco

Tobacco

Anthrax TobaccoTomato

Diphtheria Tobacco

SARS TobaccoTomato

Smallpox TobaccoTomato

HUMAN DISEASES VACCINES ANTIBODY

Colorectal Cancer

Epithelial Tumors(EGF receptor)

PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF CANCER

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PMP Development - Highlights

Series of plant-derived vaccines from Arizona StateUniversity 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, approved Feb

2006

Heberbiovac (Cuba) approved antibody for HepB

vaccine purification 2727


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EDIBLE VACCINES

Concept introduced by Charles Arntzen of TexasUniversity, USA

Hiatt and coworkers in 1989

First model system was tobacco Now produced in banana, tomato and potato

How????

vaccines present in food shown to induceimmune response

Leave behind the memory cells

High mucosal immunity 2828


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EDIBLE VACCINES FOR HUMANDISEASES

First published report on cell surfaceadhesion protein SpaA in transgenictobacco plants

Arnitzens group developed tobaccoplants producing hepatitis B

HBsAg linked to 35S promoter

0.01% of soluble protein

Assembled to VLPs 2929


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METABOLIC ENGINEERING OFCARBOHYDRATE

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v Starch

v Chemical feed stocks: cereals, potatoes

v Alternation of ADP glucose pyrophosphorylase

v This enzyme in E.coli was mutated to alter itsallosteric properties and transferred to potato

v Amylose to amylopectin ratio

v Food industry: GBSS1 down regulation produceamylose free starch

v Feed stock: SBE A and B down re ulation3232


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v Cyclodextrin

v

Used in medical industry as solubilization ofpharmaceuticals

v Made of seven membered ring ofglucopyranose

v Transgenic Potato tuber produced bytransferring glycosyl tranferase gene fromKlebsiella pneumoniae by fusing gene to plastidtargetting sequence driven by patatin gene

promoterv Polyfructans

v Synthesized and stored in cellular vacuoles

v

Expressed chichory fructosyl transferase inonion 3333


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BIOPLASTICS

Form of plastics derived from plantsources such as sweet potatoes,sugarcane, hemp oil, soy bean oil andcorn starch

Environmental friendly

Biodegradable

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COMPOSITIONPlant Oil Starch Cellulose

Corn Sugarcane Potato

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Classification ofBioplastics

Starch based plastics: Bioplasticsproduced from classical chemicalsynthesis from bio based monomers

Polylactic Acid (PLA) plastics:Bioplastics produced directly bynatural or genetically modifiedorganisms

Polyamides 11: Polycaprolactones

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Thermoset polymers

Soy polyurethanes

Unsaturated polyester resins

Thermoplastic polymers

Polylactic acid

Modified starch polymers

Thermoplastic polyesters

Bio-polyolefins

Polyhydroxy alkonates (PHA)

Cellulose ethers 3737


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Enzymes are used to break starch into glucose, which isfermented to lactic acid

This lactic acid is polymerized and converted into a plastic called

polylactic acid, which can be used in the manufacture of productsafter bein heated and sha ed

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Bioplastic Production in

Switchgrass Plants

Metabolix, Inc. recently-completed greenhouse

trials, switchgrass plants engineered usingMetabolix multi-gene expression technologyproduced significant amounts of PHA bioplasticsin leaf tissues

This result is the first expression of a newfunctional multi-gene pathway in switchgrass

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Spring waterbottle madefrom bioplastic

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Applications

Packaging

shopping bags

Trays and containersCatering products

Disposable crockery and cutlery

Gardening

flower pots

Medical Products4242

Bi l ti d ti i


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Bioplastics production inIndia

Jammu and Kashmir will be the first state in India tohave a fully dedicated Bioplastics productmanufacturing facility with an installed capacity ofabout 960 metric tones per year

TheJ&K Agro Industries Ltd is going for a joint venturewith EARTHSOUL for the manufacture of 100 per centbio-degradable and compostable products. This would

be Indias first integrated biopolymer facility

The facility would be manufacturing flower pots andtrays for floriculture, carry bags for all shopping

applications, outer packaging material for foodstuff4343
http://www.jkagro.com/http://www.earthsoulindia.com/http://www.earthsoulindia.com/http://www.jkagro.com/


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Industrial enzymes

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Phytase

Pytate into inositol and organic

phosphate

High quantities in seeds used as feedsto pigs and poultry animals

Eutrophication

Transgenic plant seed with this enzyme

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PRODUCTS CLOSE


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PRODUCTS CLOSETO MARKET

PRODUCT COMPANY LOCATION PLANTS USE

Avidin Prodigen Texas Maize Imunodiagnostic

collagen Medicago,Prodigen

Canada,Texas

Alfa alfa,Maize

Skin sealent

Lipase Meristemtherapeutics

California Maize Scar treatment

Lactoferrin Ventria,Meristemtherapeutics

France Rice Cystic fibrosis

Lysozyme Ventria California,France

Maize,Rice

Natural defenceprotein

Brazzein Ventria Texas Maize Antiviral,antibacterial

Aprotinin Large scale

biological,Prodigen

California,

Taxas

Maize,

Tobacco

Natural protein

sweetner4747


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CHOICE OF PLANT


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CHOICE OF PLANT

PRODUCTION SYSTEM

v LEAFY CROPS

v Tobacco for pharmaceutical products

Gene transfer technology for in vitroregeneration

Gene expression and high biomass yield

Phyllosecretion and rhizosecretion

v Alfa alfa, soyabean (Atm. N2 fixation) forrAbs

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v

FRUITS AND VEGETABLE CROPSv Edible organs can be consumed uncooked and

unprocessed

v Ideal for rSubunit vaccines, nutriceuticals and

antibodies

v Potatoes for vaccine and tomatoes for rabiesvaccine

v FIBRE AND OIL CROPS

v Flax, cotton, oil seed rape

v

cost reduction due to secondary revenues5050

BENEFITS LIMITATIONS


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BENEFITS

Low cost and Large scale production

Capital and running cost are low

Scalability

Stability and Safety

PRODUCT AUTHENTICITY

Plants safer than microbes

Ability to fold and assemble complex proteins

Serum proteins, Secretary Abs

Only difference in post translational modification like glycan

chain structure

Environmentcontamination

Food supplycontamination

Health safety concerns

LIMITATIONS

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Current Challenges in Molecular Farming


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Current Challenges in Molecular Farming

yield of recombinant proteins

real quantitative comparison

protein stability (proteases)

post-translational modification

extraction and downstream processing

clinical trials & regulatory approval

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Regulatory Challenges

loci of transgene insertion

expression properties and levels

effects of the transgene on the expression of flanking

endogenous genes

master line banking to ensure product consistency

contamination with animal excreta, pesticides,

organic fertiliser

procedures for detection and removal of weeds andpests

cultivation variables

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Roadmap Plants for the Future


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Roadmap Plants for the Future

1997

2005

2015

2025

Efficientagriculture

-

Bt technology- Herbicide

resistance

Health food andquality

- Amino acids- Oil- Starch

Plant protection

- Viruses- Nematodes- Fungi- Insects

Plant production platforms- Vitamines- Fatty acids- Enzymes- Bio-polymers- Pigments- Pharmaceutical products- Fibers

Stress resistance

- Cold- Drought- Salinization

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C t Ph


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Rhizosecretion Monoclonal antibodies Recombinant proetins

LEX SystemLemna, (duckweed)

Dental Caries: CaroRxColds due to Rhinovirus:RhinoRx

Drug-induced Alopecia: DoxoRx

PlanetBiotechnology

Biomass biorefinerybased on switchgrass.Produce PHAs ingreen tissue plantsfor fuel generation.

Current PharmCompanies

5555


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Trangenic tobaccoPMPs and non-proteinsubstances (flavorsand fragrances,medicinals, andnatural insecticides)

Kentucky Tobacco

Research andDevelopment Center

TrangenictobaccoGeneWare

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Examples of Current


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Genetically engineered Arabidopsis plants can

sequester arsenic from the soil (Dhankher et al.2002 Nature Biotechnology)

Immunogenicity in human of an edible vaccine forhepatitis B (Thanavala et al., 2005. PNAS)

Examples of CurrentResearch

Expression of single-chain antibodies in transgenicplants (Galeffi et al., 2005 Vaccine)

Plant based HIV-1 vaccine candidate: Tat protein

produced in spinach (Karasev et al. 2005Vaccine)

Plant-derived vaccines against diarrhealdiseases

(Tacket. 2005 Vaccine) 5858

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Environment contaminationGene flow via pollenNon-target species near field sites

Food supply contaminationAccident gene flow

Health safety concerns

Non-target organ responsesSide-effectsAllergenicity

Risks and Concerns

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U S R l t


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U.S. RegulatorySystem (existing

regulations)

Field Testing-permits-notifications

Determination ofnon-regulatedstatus

Food safetyFeed safety

Pesticideandherbicide

registration

USD

A

FD

A

EPA

6060

Suggested Safeguards for


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1.

Physical differencesE.g. purple maize, GFP

1. SterilityUse male sterile plants and Terminator technology

1. Easily detectable by addition of 'reporter genes'PCR markers (avoid antibiotic resistance markers)

4. Chloroplast expression system

Increase yieldEliminates potential gene flowTechnically difficult (Chlorogen Company)

5. Complete disclosure of DNA sequences

Suggested Safeguards formolecular farming

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REFERENCES

PLANT MOLECULAR BIOTECHNOLOGY

S. MAHESH

Biotechnology

U satyanarayana

Biotechnologies Influencing Agriculture: MolecularPharming.ppt

Molecular farming in plants: host systems andexpression technology. ppf

Richard M. Twyman, Eva Stoger, Stefan Schillberg,

Paul Christou and Rainer Fischer

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Action FA0804: Molecular farming: plants as aproduction platform for high value proteins.pdf

Update on Agricultural BiotechnologyControversies

Pharmaceutical Crops.pdf Allan Felsot

Use of plant roots for phytoremediation andmolecular farming.pdf DOLORESSA GLEBA PNAS

Biopharmaceuticals derived from genetically

modified plants.pdf D.A. GOLDSTEIN and J.A.THOMAS

Molecular Farming: Plants as New ProductProducers.pdf

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plants as bio reactors nisha g v

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