Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system

被引：52

作者：

Burger, S ^{[1
]}

Tatge, H ^{[1
]}

Hofmann, F ^{[1
]}

Genth, H ^{[1
]}

Just, I ^{[1
]}

Gerhard, R ^{[1
]}

机构：

[1] Hannover Med Sch, Inst Toxicol, Hannover, Germany

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2003年 / 307卷 / 03期

关键词：

cytotoxicity assay; endotoxin-free expression system; glucosyltransferase; pWH1520; vector;

D O I：

10.1016/S0006-291X(03)01234-8

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B. We used the Bacillus megaterium expression system for expression of recombinant toxin A. The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector. The three parts were sequentially fused to the complete gene. The holotoxin gene was ligated into the expression vector pWH1520. This vector was modified to generate a toxin with a C-terminally located His-tag. Gene expression in the B. megaterium system resulted in an approximate 300 kDa protein, which was identified by specific antibody as toxin A. Recombinant, His-tagged toxin A was purified by Ni2+ as well as thyroglobulin affinity chromatography. Characterization of the recombinant toxin A showed identical cytotoxicity and in vitro-glucosyltransferase activity as the native toxin A from C. difficile. (C) 2003 Elsevier Inc. All rights reserved.

引用

页码：584 / 588

页数：5

共 19 条

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