The expression,purification and characterization of metallo-?-lactamases(IMP-1)
- VernacularTitle:金属?-内酰胺酶(IMP-1)的表达,纯化和鉴定
- Author:
Shijia DING
- Publication Type:Journal Article
- Keywords:
Metallo-?-lactamases;
IMP-1;
Matrix assisted laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF-MS)
- From:
Journal of Chongqing Medical University
1986;0(04):-
- CountryChina
- Language:Chinese
-
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.
