Stabilization of a degradable protein by its overexpression in Escherichia coli.
Synthesis of proteins in Escherichia coli using recombinant DNA methodology has become an important tool for isolating and studying proteins. However, the E. coli protein degradation systems can interfere with the expression of cloned genes. To examine the effect of protein degradation, we have cloned the X90 allele of the E. coli lacZ gene. The X90 allele, an ochre mutant, codes for beta-galactosidase lacking approx. 12 amino acids from the carboxyl terminus. The X90 protein is rapidly degraded in wild-type E. coli. Randomly sheared DNA fragments from lambda placZ-X90 were inserted into the EcoRI site of the plasmid pOP203-UV5-3, a derivative of pMB9 containing the lactose operator-promoter region. Recombinant plasmids that carry the lacZ-X90 gene were identified by the Lac+ phenotype of their transformants in an ochre-suppressor-containing host and the Lac- phenotype in Su degrees or supE hosts. One recombinant plasmid, p41, with an insert of 7.6 kb codes for the synthesis of the X90 promoter at a quantity equal to or greater than 50% of the total cellular protein of several strains. In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates. The surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.
D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D007763:Lac Operon; D009154:Mutation; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase