Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system |
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Authors: | Burger Silke Tatge Helma Hofmann Fred Genth Harald Just Ingo Gerhard Ralf |
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Institution: | Institute of Toxicology, Hanover Medical School, Hanover, Germany. gerhard.ralf@mh-hannover.de |
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Abstract: | 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 Ni(2+) 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. |
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Keywords: | Cytotoxicity assay Endotoxin-free expression system Glucosyltransferase pWH1520 vector |
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