Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene.
To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into 'leaky' E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, Ala(-4)/Ala(-3)-Pro(-2)-Gly and Ala(-4)-Ala-3/Pro(-2)-Gly-1, of pre-SII. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN' was almost the same as that of authentic SSI.
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins