title: In vitro measurement of beta-lactamase-catalyzed ampicillin hydrolysis by recombinant Escherichia coli extracts using quantitative high-performance liquid chromatography.
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.
authors.affiliation: College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Urbain
authors.forename: C M
authors.initials: CM
authors.lastname: Wittich
authors.forename: S R
authors.initials: SR
authors.lastname: Campion
mesh_terms: D000667:Ampicillin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000667:Ampicillin; D001618:beta-Lactamases