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A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli
Author(s) -
BroomeSmith J. K.,
Bowler L. D.,
Spratt B. G.
Publication year - 1989
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1989.tb00167.x
Subject(s) - periplasmic space , escherichia coli , biology , plasmid , mutant , fusion protein , gene , amp resistance , bacterial outer membrane , coding region , enterobacteriaceae , fusion gene , lac operon , signal peptide , biochemistry , recombinant dna
Summary The feasibility of using a β‐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 β‐lactamase was fused after the signal peptide and amino‐terminal 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 I β‐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 β‐lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.