Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli

• Published in: Protein expression and purification 2009-10, Vol.67 (2), p.96-103
• Main Authors: Arimitsu, Hideyuki, Tsukamoto, Kentaro, Ochi, Sadayuki, Sasaki, Keiko, Kato, Michio, Taniguchi, Koki, Oguma, Keiji, Tsuji, Takao
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; B subunit; Base Sequence; Cell Enlargement; CHO Cells; Cholera toxin; Cholera Toxin - biosynthesis; Cholera Toxin - genetics; Chromatography, Liquid; Cricetinae; Cricetulus; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Gangliosides - metabolism; Heat-labile enterotoxin; Kinetics; Lac Operon; Lactose operon; Lincomycin; Lincomycin - pharmacology; Molecular Sequence Data; Promoter Regions, Genetic; Protein Engineering - methods; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Shine-Dalgarno sequence; Vibrio cholerae
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2009.04.011

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 lacZα 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.