The expression,purification and characterization of metallo-?-lactamases(IMP-1) / 重庆医科大学学报

ABSTRACT

Objective:To establish the method for the expression,purification and characterization of metallo-?-lactamases(IMP-1) and to explore the influence of experimental conditions on its separation and purification.Methods:pET9a/d-IMP-1 plasmid was transformed into E.coli competent BL21(DE3) cell and inoculated in LB medium.The supernatant were applied to SP Sepharose Fast Flow column,following by Sephadex G-75 column,and the purified enzymes were identified by SDS-PAGE and MALDI-TOF-MS and their activity was determined using Nitrocefin as substrate.Results:The purified enzyme showed high catalytic activity in the hydrolysis of most antibiotics.Its Michaelis constant K_m9.28?mol/L,and molecular weight Mr equalled 25111.9(determined by MALDI-TOF-MS).Conclusion:The method was established for the expression,purification and characterization of metallo-?-lactamases(IMP-1),which can be applicable to other metallo-?-lactamases.