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

Hideyuki Arimitsu, Kentaro Tsukamoto, Sadayuki Ochi, Keiko Sasaki, Michio Kato, Koki Taniguchi, Keiji Oguma, Takao Tsuji

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)96-103
Number of pages8
JournalProtein Expression and Purification
Volume67
Issue number2
DOIs
Publication statusPublished - 01-10-2009

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Lincomycin
Cholera Toxin
Escherichia coli
Lac Operon
Plasmids
Enterotoxigenic Escherichia coli
Vibrio cholerae
Enterotoxins
Operon
Protein Sorting Signals
stichoposide
Cell Extracts
Hot Temperature

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Arimitsu, Hideyuki ; Tsukamoto, Kentaro ; Ochi, Sadayuki ; Sasaki, Keiko ; Kato, Michio ; Taniguchi, Koki ; Oguma, Keiji ; Tsuji, Takao. / Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli. In: Protein Expression and Purification. 2009 ; Vol. 67, No. 2. pp. 96-103.
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Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli. / Arimitsu, Hideyuki; Tsukamoto, Kentaro; Ochi, Sadayuki; Sasaki, Keiko; Kato, Michio; Taniguchi, Koki; Oguma, Keiji; Tsuji, Takao.

In: Protein Expression and Purification, Vol. 67, No. 2, 01.10.2009, p. 96-103.

Research output: Contribution to journalArticle

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T1 - Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli

AU - Arimitsu, Hideyuki

AU - Tsukamoto, Kentaro

AU - Ochi, Sadayuki

AU - Sasaki, Keiko

AU - Kato, Michio

AU - Taniguchi, Koki

AU - Oguma, Keiji

AU - Tsuji, Takao

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