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Show COLLEGE OF SCIENCE UNDERGRADUATE RESEARCH ABSTRACTS Archana Murugesan Martin P. Horvath 68 BIOCHEMISTRY OF CONE SNAIL TOXIN ACTIVATION Archana Murugesan (Martin P. Horvath) Department of Biology University of Utah The cone snail makes venom composed of over 100 active peptides that it utilizes for defense and to paralyze fish when hunting. Several of the individual peptides have the potential to become medicine for treatment of pain, depression, seizures, and neurological disorders such as Alzheimer's disease. The production of each toxin involves several steps beginning with translating RNA to protein. This protein when it is first made is much bigger than the final toxin because it contains a signal peptide that directs the protein to be secreted and a pro-peptide segment that helps with folding of the toxin. The signal peptide is removed upon translocation into the ER. In the final step of toxin activation, mature toxin is released from the propeptide segment by action of a protease. In an ongoing effort to understand h o w cone snails make their venom components, our main objective is to purify and characterize the Astacin-like protease that cleaves the pro-peptide sequence to release the active and mature toxin. To make the protease, the gene was moved from snail into bacteria, since bacteria can make the protease faster compared with extracting the venom directly from a cone snail. To turn the gene on in bacteria and produce proteins, a chemical inducer (IPTG) was added to cultures. Initial tests showed that most of the bacteria m a d e protein, and that a large fraction of this protein is insoluble and therefore not usable. I have been testing conditions to improve yield of the soluble, folded and hopefully functional protein. I varied conditions such as time of IPTG induction and temperature of culture growth and then measured the amount of protein made with SDS-polyacrylamide gel electrophoresis. To further optimize yield of the Astacin-like protease, I varied the salt concentration, pH, availability of metal ions, and additives such as glycine in the extraction buffers. Once the optimal conditions have been found, I will purify and characterize the Astacin-like protease to test if this is the candidate thought to be responsible for the final proteolysis event necessary for activating toxins. VENOM DUCT OF C. geographus Conus Geographus Salivary gland PtMryfn Esophagi SDS-page gel showing a) inactive propeptide d) active Astacin-like peptide c) control using NR1 subunit 10 11 12 13 14 15 16 |
