Document Detail


Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile.
MedLine Citation:
PMID:  20433927     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Amit Sundriyal; April K Roberts; Roger Ling; Joanna McGlashan; Clifford C Shone; K Ravi Acharya
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-04-28
Journal Detail:
Title:  Protein expression and purification     Volume:  74     ISSN:  1096-0279     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-08-23     Completed Date:  2010-12-07     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  42-8     Citation Subset:  IM    
Copyright Information:
Copyright 2010 Elsevier Inc. All rights reserved.
Affiliation:
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
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MeSH Terms
Descriptor/Qualifier:
Actins / metabolism*
Animals
Bacterial Toxins / genetics,  isolation & purification*,  metabolism*
Cell Survival
Cercopithecus aethiops
Chymotrypsin / metabolism
Clostridium Infections / metabolism*
Clostridium difficile / genetics,  metabolism*,  pathogenicity
Cytotoxins / genetics,  isolation & purification,  metabolism*
Escherichia coli / genetics
Gene Expression
Host-Pathogen Interactions
Humans
Vero Cells
Grant Support
ID/Acronym/Agency:
//Biotechnology and Biological Sciences Research Council
Chemical
Reg. No./Substance:
0/Actins; 0/Bacterial Toxins; 0/Cytotoxins; EC 3.4.21.1/Chymotrypsin

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