Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Amit Sundriyal, April Roberts, Roger Ling, Joanna McGlashan, Clifford Shone, K. Ravi Acharya

    Research output: Contribution to journalArticlepeer-review

    32 Citations (Scopus)

    Abstract

    Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

    Original languageEnglish
    Pages (from-to)42-48
    Number of pages7
    JournalProtein Expression and Purification
    Volume74
    Issue number1
    DOIs
    Publication statusPublished - Nov 2010

    Bibliographical note

    Funding Information:
    K.R.A. wishes to acknowledge the Royal Society (UK) for an Industry Fellowship. This research was supported by a joint postgraduate studentship from the Health Protection Agency and BBSRC (UK) to A.S. We thank Abigail Davies for preparing Fig. 1 B.

    Keywords

    • Binary toxin
    • C. difficile
    • CDTa
    • CDTb
    • Cytotoxicity
    • Purification

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