Optimizing the purification of wild type and mutant recombinant phospholipase D: An approach to producing a Corynebacterium pseudotuberculosis vaccine
The overall goal of the lab is to characterize the equine immune response to the disease, ‘pigeon fever’, caused by the gram-positive bacteria C. pseudotuberculosis. Mainly prevalent in warmer climates, but with increasing geographic range, the disease manifests in three forms: external abscesses, internal abscesses, and ulcerative lymphangitis. My project centers on the main exotoxin, phospholipase D (PLD), that plays a role in pathogenesis of the disease. Using a recombinant PLD gene in the plasmid, pTrcHis, expressed in E. coli, the protein is harvested and purified. For vaccine development, we have previously used formalin inactivated PLD. However, a lot of PLD is lost in the process of inactivation. With the mutant PLD, we have a non-toxic version of the protein that we expect to elicit an immune response in the mice. However, it has proven difficult to obtain significant yields of this mutant PLD. The major focus of this summer project was to increase the expression and recovery of this mutant PLD. Using various cell culture and harvesting conditions, we sought to improve the yield. By the end of the summer, protein levels were raised to over 150 μg/mL. The next step is to use the mutant PLD in a mouse model to test its ability to induce an immune response to PLD and provide protection from C. pseudotuberculosis infection
Patwardhan, Utsav, "Optimizing the purification of wild type and mutant recombinant phospholipase D: An approach to producing a Corynebacterium pseudotuberculosis vaccine" (2012). URC Student Scholarship.
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Roberta Pollock, Karen Molinder
Fletcher-Jones Chemistry and Biochemistry Research Endowment