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 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.
Urbain
J L
JL
College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.
Wittich
C M
CM
Campion
S R
SR
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*
D011994:Recombinant Proteins; D000667:Ampicillin; D001618:beta-Lactamases