EXPRESSION AND PURIFICATION OF PROPERLY FOLDED PSEUDO-NITZSCHIA PRENYLTRANSFERASES: A PRECURSOR TO DOMOIC ACID
Michael Flores, Thomas Savage.
California State University Sacramento, Sacramento, CA.
Domoic acid (DA) is a neurotoxin synthesized by the marine diatom Pseudo-nitzchia australis, which accumulates in fish, causing harm to humans and high mortality rates in other vertebrates. DA accumulates in high concentration during algal blooms, which may be influenced by anthropogenic factors. It is undetermined what and how environmental signals stimulate DA biosynthesis. Geranyl diphosphate has been identified as a precursor to DA. It is determined that DA biosynthetic enzymes that catalyze the formation of geranyl diphosphate are homologous to enzymes (e.g., prenyltransferases) catalyzing similar reactions. Here, we are trying to identify conditions that allow for proper folding and functional testing of E. coli-expressed prenyltransferases. Transformed E. coli were cultured, inoculated, then induced using IPTG (isopropyl-β-d-thiogalactopyranoside). Cells were then isolated and lysed using lysozyme and sonication. Proteins were obtained by centrifugation. Ni-agarose affinity chromatography was used to isolate the expressed protein. SDS-PAGE analysis was used to confirm the isolation of the properly folded protein. Functional testing of the prenyltransferase can help determine geranyl diphosphate production during the biosynthesis of DA. Determining the biosynthetic pathway of DA production will help us to understand what environmental factors influence the production of DA.