Purification of Phospholipase D: Corynebacterium pseudotuberculosis ? Major Exotoxin
Corynebacterium pseudotuberculosis is a gram positive bacteria that poses a significant threat to large farm animals. Conspicuous external abscesses or fever and lethargy with possible internal abscesses indicate infection in horses. Developing a vaccine for C. pseudotuberculosis necessitates an understanding of the equine immune response to the infection. Thus, Dr. Pollock's research lab concentrates on gaining a greater knowledge of the equine immune response to Phospholipase D (PLD, the major exotoxin produced by C. pseudotuberculosis ). Because of PLD?s instrumental role in many of the methods employed in our lab to study the equine immune response to C. pseudotuberculosis , we need to purify and inactivate large amounts of PLD. My specific goal for the summer was to learn the protein purification and concentration procedures to isolate recombinant PLD. After isolating the Histidine tagged PLD on TALON Cobalt resin, we ran several samples from different steps of the purification process on SDS-PAGE to check the purity. Western blot analysis was also used to detect the presence of PLD in the samples. We then determined the concentration of our purified PLD with a Nanodrop spectrophotometer and also with BCA assays. Our initial results indicated a low yield of purified PLD, so much of my research focused on revising the purification protocols to optimize the purity and concentration of PLD. Most recently, we began employing the Slide-A-Lyzer Dialysis Cassette G2 instead of Amicon Ultra-15 Centrifugal filter devices to obtain a more concentrated PLD.
Barajas, Eliseo, " Purification of Phospholipase D: Corynebacterium pseudotuberculosis ? Major Exotoxin " (2010). URC Student Scholarship.
Howard Hughes Medical Institute Undergraduate Science Education Grant