Objective:To establish the method for the expression

purification and characterization of metallo-β-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_m9.28μmol/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-β-lactamases(IMP-1)

which can be applicable to other metallo-β-lactamases.