Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli
Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli
复制标题
DOI：
10.1016/j.pep.2009.04.011
复制
发表时间：
2009-10-01
影响因子：
1.6
通讯作者：
Tsuji, Takao
中科院分区：
文献类型：
--
作者：
Arimitsu, Hideyuki;Tsukamoto, Kentaro;Tsuji, Takao
使用新型表达系统在大肠杆菌中过量产生霍乱毒素 (CT) B 亚基 (CTB)。通过将编码全长 CTB 的基因和源自 CTB 或产肠毒素大肠杆菌不耐热肠毒素 B 亚基 (LTB) 的 Shine-Dalgarno (SD) 序列插入到 pBluescript SK(+) 载体的 lacZ α 基因片段中构建表达质粒。用每种质粒转化大肠杆菌菌株MV1184，然后在含有林可霉素的CAYE肉汤中培养。从每种细胞提取物中纯化重组 CTB (rCTB)。 rCTB在两种转化子中过量产生，没有明显毒性，并且在结构和生物学上与从霍乱弧菌纯化的CT相同，表明原始的SD和CTB信号序列也足以在大肠杆菌中表达rCTB。林可霉素诱导的 rCTB 表达通过 lac 启动子突变而受到抑制，表明林可霉素影响乳糖操纵子。基于这些发现，我们构建了包含野生型 CT 操纵子的质粒，并使用与 rCTB 相同的程序成功过量产生了 CT (rCT)。尽管 rCT 具有完整的 A 亚基，但氨基末端 modi。 rCT 的 A 和 B 亚基的阳离子和生物学特性与 CT 相同。这些结果表明，这种新型 rCTB 过表达系统也可用于生成野生型和突变型 CT 蛋白，这将有助于进一步研究 CT 的特征，例如粘膜佐剂活性。 (C) 2009 Elsevier Inc. 保留所有权利。
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 lacZ alpha 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 modi. cations 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. (C) 2009 Elsevier Inc. All rights reserved.