A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli

Mol Microbiol. 1989 Dec;3(12):1813-7. doi: 10.1111/j.1365-2958.1989.tb00167.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Cell Membrane / enzymology
  • Drug Resistance, Microbial / genetics
  • Escherichia coli / genetics*
  • Gene Expression
  • Genes, Bacterial
  • Membrane Proteins / genetics*
  • Mutation
  • Plasmids*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • beta-Lactamases / biosynthesis
  • beta-Lactamases / genetics*

Substances

  • Membrane Proteins
  • Recombinant Fusion Proteins
  • beta-Lactamases