Molecular farming is an experimental application of biotechnology to modify crops in order to produce proteins and chemicals for medicinal and commercial interests. The vast majority in the developing world cannot afford the high cost of therapeutics produced by existing methods. We not only need to produce new therapeutics but also need to produce cheaper versions of the existing ones. Molecular farming could offer a viable option for this growing need for biopharmaceuticals. Part of the thesis deals with investigating ways to produce DesB30 form of human insulin in transgenic tobacco. The human insulin was synthesized in vitro as strep-tag II-mini-insulin fusion protein. Expression of mini-insulin by transgenic tobacco was confirmed by RT-PCR, western blotting and ELISA. However, sufficient levels of purified insulin could not be obtained to carry out further functional assays. Strategies for increasing the yield of insulin by transgenic tobacco are discussed and further increases in yield would need to be developed for this to become a viable and cost effective source of this important pharmaceutical. The second part of the thesis describes the production of a recombinant microbial polysaccharide in tobacco. Seven type 2 pneumococcal polysaccharide biosynthetic genes were expressed in a single tobacco plant, utilizing the plant Kex2 (Kexin protease 2) like protease system for multiple gene expression. Expression of these genes in transgenic tobacco was confirmed by RT-PCR and western blotting. Correct processing of the expressed proteins by the Kex2 protease system was confirmed. However, In planta production of type 2 polysaccharide could not be confirmed mainly as a result of high background from the wild type plant polysaccharide extracts. Strategies to overcome these issues are described. The usefulness of Kex2 protease system for multiple gene expression and metabolic pathways engineering is also discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:551765 |
| Date | January 2011 |
| Creators | Ahmad, Kafeel |
| Contributors | Whitelam, Garry |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/10241 |
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