Go to navigation (press enter key)Menu
Completed Project

Optimizing the Purification of Lysophospholipase L2, a Membrane Associated Protein Derived from Escherichia coli K-12

Gabriel Elizalde Jr., Vassar College ’17 and Prof. Teresa A. Garrett
Lysophospholipase L2, also known as PldB, is a membrane-associated protein found in Escherichia coli K-12. Thoroughly purifying PldB is essential for characterization; however, previous attempts at purification have resulted in substantial loss of protein activity. A previously used purification procedure was modified to determine if alternate treatments and purification steps would lead to a improved recovery of protein activity. Crude PldB was obtained from E. coli cells containing a pET vector designed to over-express the pldB gene, which was modified to add a six-histidine to the N-terminus of PldB. PldB over-expressing E. coli cells were then physically lysed in a French Pressure cell to create a cell free extract. The PldB containing cell free extract was ultracentrifuged to isolate E. coli membrane fragments, which were then either treated with a pH 7.0 Tris/HCl or pH 7.0 HEPES/NaOH buffer. These two samples then underwent a salting/desalting procedure with 1M KCl salt to wash off PldB from the membrane fragments. The resulting fraction that contained partially purified PldB was subject to nickel-affinity chromatography to isolate PldB from other membrane proteins that were not histidine-tagged. The purity of the protein was analyzed by SDS-PAGE protein mini-gels. Enzyme activity was determined by reacting PldB containing protein fractions with 14C labeled 2-acyl lyso phosphatidylglycerol and analyzing the resulting product using thin layer chromatography. Preliminary results suggest that the buffer treatment of the PldB samples did impact on the purity or activity of the protein. This experiment also suggests that PldB purity increased when processed through nickel affinity chromatography in comparison to a salting/desalting-only treatment. These findings will be applied to future projects seeking to purify PldB mutants in order to characterize their substrate specificity with lipids present in E. coli.