Growth and Purification of the Pseudomonas putida HMG-CoA Reductase Enzyme
Faculty Sponsor
Jeff Watson, Gonzaga University
Research Project Abstract
This research project attempted to grow and purify soluble Pseudomonas putida HMGR (PpHMGR) enzyme for the purpose of experimentation on its hypothesized morpheein protein behavior. Inclusion body formation has been the primary limiting factor in the growth and purification of soluble PpHMGR. Variation on incubation temperature, IPTG concentration, and growth time were explored in the growth phase, and imidazole concentration, presence of DTT, and presence of TCEP were explored in the purification phase. PpHMGR was grown in an E. coli host, cell lysis was performed by sonication, and subsequent purification was performed by nickel column elution FPLC. The purified product was characterized by FPLC, gel electrophoresis, and enzyme kinetics to determine the reduction of inclusion body formation and presence of soluble PpHMGR.
Session Number
RS15
Location
Weyerhaeuser 303
Abstract Number
RS15-d
Growth and Purification of the Pseudomonas putida HMG-CoA Reductase Enzyme
Weyerhaeuser 303
This research project attempted to grow and purify soluble Pseudomonas putida HMGR (PpHMGR) enzyme for the purpose of experimentation on its hypothesized morpheein protein behavior. Inclusion body formation has been the primary limiting factor in the growth and purification of soluble PpHMGR. Variation on incubation temperature, IPTG concentration, and growth time were explored in the growth phase, and imidazole concentration, presence of DTT, and presence of TCEP were explored in the purification phase. PpHMGR was grown in an E. coli host, cell lysis was performed by sonication, and subsequent purification was performed by nickel column elution FPLC. The purified product was characterized by FPLC, gel electrophoresis, and enzyme kinetics to determine the reduction of inclusion body formation and presence of soluble PpHMGR.
