Related Experiment Videos
In vitro measurement of beta-lactamase-catalyzed ampicillin hydrolysis by recombinant Escherichia coli extracts using
J L Urbain1, C M Wittich, S R Campion
1College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.
Abstract:
We report a rapid and simple protocol for measuring the beta-lactamase activity from recombinant Escherichia coli cells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the beta-lactamase TEM-1. The hydrolytic enzyme was extracted from the E. coli periplasm and the beta-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography. Under saturating conditions the in vitro assay was linear as a function of both incubation time and enzyme concentration. Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation.