期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 307, 期 3, 页码 584-588出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0006-291X(03)01234-8
关键词
cytotoxicity assay; endotoxin-free expression system; glucosyltransferase; pWH1520 vector
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.
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