title: Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system.
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.
authors.affiliation: Institute of Toxicology, Hanover Medical School, Hanover, Germany. gerhard.ralf@mh-hannover.de
authors.forename: Silke
authors.initials: S
authors.lastname: Burger
authors.forename: Helma
authors.initials: H
authors.lastname: Tatge
authors.forename: Fred
authors.initials: F
authors.lastname: Hofmann
authors.forename: Harald
authors.initials: H
authors.lastname: Genth
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
mesh_terms: D000818:Animals; D001410:Bacillus megaterium / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D002460:Cell Line; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D015870:Gene Expression; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile