Simultaneous Purification of Enterotoxin A and C by Fast Protein Liquid Chromatography

A new method developed for simultaneous purification of enterotoxin A and C from Staphylococcus aureus strain L 350/1 consisted of chromatography on carboxymethyl (CM)-cellulose using a buffer of variable pH, gel filtration on Ultro gel, and fast protein liquid chromatography(FPLC) using a buffer of variable pH. The enterotoxin A and C were purified by three steps: batchwise adsorption from culture supernatant on Amberlite CG-50; chromatography on CM-cellulose using a buffer of constant pH and molarity; and gel filtration on Sephadex G-75. The purified enterotoxin appeared homogeneous by gel diffusion and polyacrylamide gel electrophoresis. Upon treatment with CM-cellulose using a elution of variable pH, enterotoxin A and C were so close that they were not separated completely. After elution from gels, the enterotoxins appeared as a single peak at the same position. Gel filtration gave a reaction of complete identity to enterotoxin A and C in Ouchterlony immunodiffusion. In FPLC using a CM-cellulose, enterotoxin A and C were simultaneously separated at pH 8.6 and 6.8. When each fraction was performed to gel immunodiffusion, at peak of enterotoxin A and C were not detected each other. In a method of elution by pH-gradient was to be more efficient as a simultaneous separation method in terms of speed, yields and simplicity. The purified toxin A and C were identical to type A and C reference enterotoxin on both disc electrophoresis and Ouchterlony gel diffusion.