title: A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli.
abstract: The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.
authors.affiliation: Microbial Genetics Groups, School of Biological Sciences, University of Sussex, Falmer, Brighton, UK.
authors.forename: J K
authors.initials: JK
authors.lastname: Broome-Smith
authors.forename: L D
authors.initials: LD
authors.lastname: Bowler
authors.forename: B G
authors.initials: BG
authors.lastname: Spratt
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases