f Purification and Characterisation of Clostridium Difficile Toxin A by Bovine Thyroglobulin Affinity Chromatography and Dissociation in Denaturing Conditions with or Without Reduction
- Authors: S. Kamiya, P. J. Reed, S. P. Borriello*
- *Correspondence should be sent to Dr S. P. Borriello.
- J. Med. Microbiol., September 1989 30: 69-77, doi: 10.1099/00222615-30-1-69
- Subject: Articles
- Published Online:
Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of >240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with β-mercaptoethanol.
© 1989 Pathological Society of Great Britain and Ireland | Published by the Microbiology Society
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