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Plants asBioreactors
NISHA G V
3RD SEMESTERMSc BIOTECHNOLOGY
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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
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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
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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
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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
Ri k d C
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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
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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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