title: Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli.
abstract: Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZalpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modifications and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity.
authors.affiliation: Department of Microbiology, Fujita Health University, School of Medicine, Toyoake, Aichi 470-1192, Japan. arimitsu@fujita-hu.ac.jp
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Arimitsu
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tsukamoto
authors.forename: Sadayuki
authors.initials: S
authors.lastname: Ochi
authors.forename: Keiko
authors.initials: K
authors.lastname: Sasaki
authors.forename: Michio
authors.initials: M
authors.lastname: Kato
authors.forename: Koki
authors.initials: K
authors.lastname: Taniguchi
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
authors.forename: Takao
authors.initials: T
authors.lastname: Tsuji
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D049108:Cell Enlargement; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005732:Gangliosides / Q000378:metabolism; D007700:Kinetics; D007763:Lac Operon; D008034:Lincomycin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D005732:Gangliosides; D011994:Recombinant Proteins; D002772:Cholera Toxin; D008034:Lincomycin