Team:Georgia State/project/overview


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   The primary purpose of our project is to modify the class of pGAPZα expression vectors according to iGEM standards to allow for expression and purification of proteins from an inducible yeast system. The glyceraldehyde-3-phosphate dehydrogenase (pGAP) promoter shuttle vector has been used in the methylotrophic yeast, Pichia pastoris, to express high levels of recombinant proteins (Waterham et al., 1997). The pGAPZα expression system allows for methanol-inducible expression of proteins with appropriate eukaryotic post-translational modifications. Additionally, the pGAPZα expression system allows for controlled secretion of a protein, making the purification of a protein relatively simple.

   Using the pGAPZα expression system, the GSU iGEM team has been working to express and purify mambalgin, a protein component of the venom of Dendroaspis polylepis, better known as the Black Mamba. The mambalgin peptide is a powerful analgesic that directly blocks pain transmission in the peripheral nervous system (Diochot et al, 2012) by targeting acid-sensing ion channels within nociceptors beneath the epidermis . Because mambalgin acts on pain receptors within the skin rather than on opioid receptors in the brain, this peptide has great potential as a medication for pain treatment that is non-addicting and non-habit forming. Furthermore, recombinant purification of mambalgin could assist in developing anti-venom without the attendant risk of harvesting venom directly from snakes.

   By the end of this competition period, we hope to have modified pGAPzα A, B, and C expression vectors to allow for easy insertion of any iGEM BioBricks in frame with the secretion factor and myc epitope and His tags which will allow for inducible production, eukaryotic processing, and simplified purification of any desired proteins. Our ultimate goal is to use this system to express and purify mambalgin, which may then be developed as a potent analgesic and as a potential antigen to produce black mamba antivenom.