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Volume 30, Issue 1

Purification and Characterisation of Toxin A by Bovine Thyroglobulin Affinity Chromatography and Dissociation in Denaturing Conditions with or Without Reduction Free

Abstract

Summary

Highly purified toxin A of 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.

  • Received:
  • Accepted:
  • Published Online:
© J. MED. MICROBIOL. 1989
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1989-09-01
2024-04-25
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Purification and Characterisation of Clostridium Difficile Toxin A by Bovine Thyroglobulin Affinity Chromatography and Dissociation in Denaturing Conditions with or Without Reduction
J. Med. Microbiol. 30, 69 (1989); https://doi.org/10.1099/00222615-30-1-69
/content/journal/jmm/10.1099/00222615-30-1-69
/content/journal/jmm/10.1099/00222615-30-1-69
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