Optimizing the purification of wild type recombinant phospholipase D: Approaches to producing a C. pseudotuberculosis vaccine
Authors:Christopher Hino, Utsav Patwardhan, Nathan Young
- Karen Molinder, Adjunct Professor of Biology, Occidental College
- Roberta Pollock, Professor of Biology, Occidental College
Pigeon Fever is a major infectious equine disease caused by the gram-positive bacterium Corynebacterium pseudotuberculosis. Currently, there is no vaccine available for protecting the equine community. A major focus of our lab is the use of immunogenic proteins produced by C. pseudotuberculosis for the development of a vaccine. Phospholipase D (PLD), the major exotoxin secreted by Corynebacterium pseudotuberculosis, plays a role in the pathogenesis of the disease and is believed to be the major antigen in inducing immune responses to this bacterium. Results from our lab have shown that PLD is actually the major antigen recognized by infected horses. We hypothesized that PLD would be a viable vaccine component. Furthermore, a mutated variant of PLD, generated in lab, has additionally been hypothesized to be an equally effective vaccine component with fewer side effects, and lower cost. In order to attain a superior understanding of their expression and recovery, wildtype and mutant PLD were purified using affinity chromatography. Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis allowed us to visualize impurities in purified samples. Its concentration was additionally evaluated using a Bicinchoninic Acid assay. The current purification protocol yielded unsatisfactory results. Samples contained relatively high concentrations of impurities, and low concentrations of PLD (~200ug/ml). To demonstrate the feasibility of PLD as a vaccine component, the purification protocol was troubleshot. Washing conditions, sonication and induction times and resin bed volume were modified for optimization. Having attained ~4000ug/ml of PLD following optimization, we were able to confirm the viability of PLD for use in vaccine development. These experiments demonstrated the difficulties of purification. Balancing between yield and purity remains the chief challenge of PLD purification. While obtaining high yields is important for improving cost and time efficiency, purity is necessary for accurate characterization of PLD response in other assays and for use in vaccine preparation.