Purification and characterisation of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction.
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 greater than 240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with beta-mercaptoethanol.
Microbial Pathogenicity Research Group, Clinical Research Centre, Harrow, Middlesex.
S
S
Kamiya
P J
PJ
Reed
S P
SP
Borriello
D000818:Animals; D001427:Bacterial Toxins; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000254:growth & development*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000302:isolation & purification* / Q000494:pharmacology; D006386:Hemagglutination Tests; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D013954:Thyroglobulin; D014709:Vero Cells
D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D013954:Thyroglobulin