IN-VITRO MEASUREMENT OF BETA-LACTAMASE-CATALYZED AMPICILLIN HYDROLYSIS BY RECOMBINANT ESCHERICHIA-COLI EXTRACTS USING QUANTITATIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

We report a rapid and simple protocol for measuring the beta-lactamase activity from recombinant Escherichia coil cells transformed with any of the common plasmid vectors that provide ampicillin resistance thro ugh constitutive expression and periplasmic localization of the beta-l actamase TEM-1. The hydrolytic enzyme was extracted hom the E. coil pe riplasm and the beta-lactamase activity determined by measuring conver sion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography. Under saturating conditions th e in vitro assay was linear as a function of both incubation time and enzyme concentration. Application of this assay to investigate TEM-1 e xpression, from two different protein expression vector systems, demon strated the potential importance of this assay in studies of recombina nt protein expression and translocation. (C) 1998 Academic Press.