Rapid method for altering bacterial ribosome-binding sequences for overexpression of proteins in Escherichia coli.
In an Escherichia coli expression system, two genes, one from an anaerobic intestinal bacterium and one from E. coli, were overexpressed following the alteration of ribosome-binding (Shine-Dalgarno) sequences. For both genes, the polymerase chain reaction (PCR) was used to modify the ribosome-binding sequence and, at the same time, provide restriction endonuclease sequences at each end of the gene. These restriction endonuclease sequences were used for inserting the DNA into the E. coli plasmid vector pGEM2, which has the T7 promoter upstream from its multiple cloning sites. Each chimeric plasmid, made by ligating the PCR product into pGEM2, was transformed into E. coli strain HMS174(DE3) which, when induced, produces T7 RNA polymerase for regulated overexpression. The gene isolated from the anaerobic intestinal bacterium, a 27-kDa polypeptide gene from Eubacterium sp. strain 12708, when expressed using this system, produced about one-third of the total cell protein as measured in Coomassie-stained protein gels and confirmed by Western blots with rabbit antibody. The E. coli enzyme, a 28.4-kDa tRNA methylation enzyme, was increased fivefold in activity of cell extracts over that of the best previous strain.
Roberts
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Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
Hylemon
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PB
Holmes
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D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005051:Eubacterium / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase