title: Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.
abstract: We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression.
authors: The Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors: B
authors: B
authors: Miroux
authors: J E
authors: JE
authors: Walker
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics*; D024101:Mitochondrial Proteins; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics; D013045:Species Specificity; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C066909:2-oxoglutarate-malate carrier protein; D007544:Isopropyl Thiogalactoside; D000667:Ampicillin