Transgenic Plants in Production of Enzymes, Therapeutic Protein and Antibodies
Within the last decade plants genetic engineering has become a cornerstone of the expanding biotechnology revolution. The current achievements and growth of plant biotechnology research are a direct result of the initiative and effort put forth by numerous laboratories worldwide. Since the first report of successful gene transfer into plants genome in the early 1980's ( Bevan et al 1983, Facaley et al 1983, Herrera Estrella et al 1983, Zambeyski et al 1983). From the time of its inauguration up to the present, plant tissue culture has witnessed a steady development of new techniques for the identification and isolation of potentially important plant gene, an improvement of methods for manipulation and transfer of a gene into plant cells, and development of better techniques for the recovery of transformed cells and regeneration of transgenic plants.
Using these new and new technical approaches it has been possible to generate transgenic plants with variety of improved agronomic and good quality traits. Transgenic plants resistant to pests, diseases and herbicides, tolerance to environmental stress (drought, temperature and salinity), improved nutritional value, composition, flavor and storage ability are being generated in an increasing number of agronomically important crop species. Gene transfer is also being used to selectively modify the metabolic pathway of plants allowing the recovery of transgenic lines with altered lipids, carbohydrates and protein for the production of novel products useful in the agricultural, chemical, and pharmaceuticals industries.
The first transgenic plant was raised in 1980's. Today more than (find) crops species can be genetically manipulated using available Agrobacterium tumefaciens or a variety of free DNA delivery transformation systems. Some examples of plant in which transgenic varieties have been developed are Alfalfa, apple, Arabidopsis, asparagus, rye brassica rapa, cabbage, cauliflower, carrot and soybean. The view of many scientists, plants is extremely attractive system for the large scale production of biologically important molecules such as peptides, proteins, and enzymes compared to other industrial sale production systems plant offers numerous biological and economical advantages.
Production of Alpha amylase
Alpha amylase breaks up the long starch molecules by cutting the chain of glucose molecules internally. It's used for processing maize starch in production of high fructose syrup and alcohol (ethanol) for fructose. It is used to production of wines, juices and in production of bread and low calories beer. Currently the industrial requirement for this enzyme is met by bacterial fermentation which gives a low cost product. However the enzyme occurs naturally in plants but the amount produced in a normal plant are low. Much higher level of expression has been achieved by using the gene for a bacterial enzyme. Tobacco protoplasts were transformed with an expression vector containing a sequence encoding mature alpha amylase from Bacillus Licheniformis. Transformed protoplast showed high level of alpha amylase expression and could be employed for processing starch. Phytase containing transgenic seed have been produced as a novel feed addictive for improved phosphorus utilization. Phytic acid is the main storage form of phosphorous in many seeds, but in this form it's a poor nutrient for monogastric animals. Its availability increases by addition of phytase, which releases phosphate from the substrate phytate. A phytase gene from Aspergillus Niger has been engineered into seeds providing a stable and convenient packaging of enzyme that is directly applicable in animal feed.
Production of Cellulose Enzyme
Some crops are grown for food white others are grown to produce consumer products but a special group of transgenic potato plant now is doing both at once. Researchers at the department of Energy Pacific Northwest National Laboratory have developed a specialized capability to control genes that are transplanted into a plant. The experimental potatoes have sprouted valuable enzymes in the wines while the tubers can be used as food. These transgenic plants have been modified to produce cellulose enzyme in the foliage. The cellulose producing genes were isolated from bacterial and fungal organisms. Cellulose is an enzyme to break down plant materials and is used in food processing to ethanol production.
Production of Therapeutic Protein
Until recently pharmaceutical used for the treatment of diseases have been largely depended on the production of relatively small organic molecules synthesized by microbes or by organic chemistry. These include most antibiotics, analgesic, hormones and other pharmaceuticals. Increasingly attention has focused on large and more complex molecules as therapeutic agents. Proteins are large molecules composed of long chain of subunits called amino acids. Information stored in DNA directs the protein synthesizing machinery of cell to produce the specific protein required for its structure and metabolism. Since protein plays a critical role in cell biology, they have been many potential therapeutic uses in preventing and curing diseases and disorders. While short peptides chains can be synthesized chemically, large proteins are best produced by living cells. The DNA that encodes the inserted instructions for producing the desired protein is inserted into a cell and as the cells grows they synthesize the protein, which is subsequently harvested and purified. Transgenic plants also have an advantage over genetically engineered microorganism for the production of some proteins. Since plants cells like their animal counterpart are Eukaryotic In nature they are able to produce mammalian proteins that contain appropriate post translational modification is often required for protein or enzyme function.
Bioproduction of Human Enzymes
Transgenic plants have significant potential in the Bioproduction of complex human therapeutic protein due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification and low cost of biomass production . Tobacco has been used as an initial transgenic system because Agrobacterium mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale up and development of new "health position" uses for tobacco has significant regional support. Human protein C (hpc) a highly processed serum protease of the coagulation / anticoagulation cascade was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian system tobacco synthesized hpc appears to undergo multiple proteolytic cleavage, disulfide bond formation and N linked glycosylation. Although tobacco derived hpc has not yet been tested for all post transcriptional modification or for enzymatic activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. Crop technology researchers have also produced the human lysosomal enzyme Glucocerebrosidase (hGC) in transgenic tobacco. The glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's diseases. Regular intravenous administration of modified glucocerbrosidase derived from human placenta has proven highly effective in reducing diseases manifestation in patient's with gauchers diseases. However the enzyme is expensive making it a dramatic model for evaluating the potential of plants to provide a safe, low cost source of bioreactive human enzyme. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross reactivity with hGC antibodies. hGC production of 1 mg/gram fresh weight of leaf tissue had been attained in crude extracts. Studies have provided a strong support for the utilization of tobacco for high level production of active hgc for purification and eventually therapeutic use at potentially much reduced costs. Plants have been tested as production system for a range of therapeutic protein to be used directly in food or after purification. Expression in plants of milk protein such as lactoferrin and beta casein may contribute the therapeutic value of these proteins to other food products.
Production of Vaccines from Plant
The aim of vaccination is to prevent infectious diseases. It can be considered as one ol' the most successful breakthrough of this century in the medical field. Mainly vaccination involves mimicking an infection in such a way that the specific natural defense mechanism of the host against the pathogen gets activated hence the host remains free of the disease that normally results from such infection. Vaccination is also referred to as active immunization', since host immune system is 'activated' to respond to infection through humoral and cellular responses, resulting in acquired immunity against the particular pathogen. Most soluble, non-viable antigens are subject to degradation in the gastrointestinal tract due to acidic and proteolytic environment of the gut. So by expressing the antigen in plant cell can be considered as a means of bioencapsulating protein for oral delivery. Plant cell wall consisting of cellulose, proteins and pectin will serve as barrier to enzymatic degradation, cell membrane and internal organelle membranes provide further protection. The first report on the use of plants for the production of edible vaccine appeared in the form of a patent application by Curtiss and Cardineau-3. They expressed the Streptococcus mutans surface protein antigen A (SpaA) in tobacco. Since then various antigenic determinants against viral and bacterial pathogens have been expressed in transgenic plants.
Transgenic plants have been used for the production of antibodies directed against dental caries, rheumatoid arthritis, cholera, E. coli diarrhea, malaria, certain cancers, Norwalk virus, HIV, rhinovirus, influenza, hepatitis B virus, and herpes simplex virus. Some of these have demonstrated preventative or therapeutic value and are currently in clinical trials. The most advanced product to date is an anti Streptococcus mutant's secretory antibody for the prevention of dental caries that is currently in Phase II clinical trials. Plants offer the only viable, large scale production system for this antibody (Gavilondo and Larrick 2000, Larrick and Thomas 2001).
Larrick, J. W., and D. W. Thomas. 2001. Producing proteins in transgenic plants and animals. Cur. Opinion in Biotech. 12: 411-418.
Sundstrom, F. J., J. Williams, A. Van Deynze, and K. J. Bradford. 2002. Identity preservation of agricultural commodities. Oakland: University of California Division of Agriculture and Natural Resources, Publication 8077.
Dus Santos M J, Wigdorovitz A, Trono K, Rios R D, Franzone P M et al, A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants, Vaccine, 20 (2002) 1141-1147. Vaquero C, Sack M, Schuster F, Finnern R, Drossard J et al, A carcinoembryonic antigen-specific diabody produced in tobacco, FASEB J, 16 (2002) 408-410.
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