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Open Access Highly Accessed Methodology article

Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium

Guilin Yang12, Boping Zhou2, Jufang Wang3, Xiangyun He1, Xingmin Sun1, Weijia Nie1, Saul Tzipori1 and Hanping Feng1*

Author Affiliations

1 Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA

2 Institute of Hepatology, Shenzhen East Lake Hospital, Shenzhen518020, PR China

3 The Department of Biochemical Engineering, School of Bioscience and Biotechnology, South China University of Technology (SCUT), Guangzhou 510006, PR China

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BMC Microbiology 2008, 8:192  doi:10.1186/1471-2180-8-192

Published: 6 November 2008

Abstract

Background

Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.

Results

The toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB) were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination.

Conclusion

We have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.