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

Biochem Biophys Res Commun. 2003 Aug 1;307(3):584-8. doi: 10.1016/s0006-291x(03)01234-8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacillus megaterium / genetics*
  • Bacterial Toxins / genetics*
  • Bacterial Toxins / metabolism
  • Bacterial Toxins / toxicity
  • Cell Line
  • Enterotoxins / genetics*
  • Enterotoxins / metabolism
  • Enterotoxins / toxicity
  • Gene Expression
  • Genetic Vectors
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / toxicity


  • Bacterial Toxins
  • Enterotoxins
  • Recombinant Proteins
  • tcdA protein, Clostridium difficile