Purification of Clostridium difficile toxin A by affinity chromatography on immobilized thyroglobulin.
An efficient, single-step method for isolating highly purified toxin A from Clostridium difficile culture filtrates is described. The purification procedure was based on the affinity binding and release of toxin A to bovine thyroglobulin conjugated to agarose beads. The toxin strongly bound at 4 degrees C to the carbohydrate binding determinant Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence which occurs on bovine thyroglobulin. Toxin bound to thyroglobulin at 4 degrees C, allowing its separation from the culture filtrate and contaminating proteins during the purification scheme. The toxin was eluted by increasing the temperature to 37 degrees C. The toxin-binding capacity was related to the amount of thyroglobulin immobilized on the gel: an affinity column containing 15 mg of bovine thyroglobulin per ml of gel bound 0.53 mg of toxin A per ml of gel. The percent recovery of purified toxin ranged from 56 to 80% and was inversely related to the amount of thyroglobulin coupled to the gel. The affinity-purified toxin was homogeneous as judged by crossed immunoelectrophoresis and gradient polyacrylamide gel electrophoresis and was immunologically identical to toxin A purified by conventional methods as determined by immunodiffusion analysis. The biochemical, hemagglutinating, and toxic properties of the toxin were preserved after affinity chromatography and were comparable with those of toxin A purified by conventional methods.
D001427:Bacterial Toxins / Q000302:isolation & purification*; D001681:Biological Assay; D002846:Chromatography, Affinity; D003013:Clostridium / Q000032:analysis*; D003080:Cold Temperature; D003603:Cytotoxins / Q000302:isolation & purification; D004768:Enterotoxins / Q000302:isolation & purification; D013954:Thyroglobulin
D001427:Bacterial Toxins; D003603:Cytotoxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D013954:Thyroglobulin