Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxinogenic mutants.

Cholera toxin was obtained in pure form by fractionation on two phosphocellulose columns successively. Cholera toxin and choleragenoid were quantitatively and selectively adsorbed to the first column in 10 mM phosphate buffer, pH 7.0, and were subsequently eluted with buffer of high ionic strength. The toxin was then separated from choleragenoid on the second column by chromatography at pH 8.3. The toxin obtained was highly active and pure as judged by electrophoresis, isoelectric focusing, and various immunological and chemical tests. Pure choleragenoid was by-product of the procedure. The A1 chain of the toxin was obtained in pure form by treating phosphocellulose-bound toxin with urea and a reducing agent. The anionic A1 peptide was thereby released, leaving a complex of the B and A2 chains (A25B) bound to the resin. The latter was then eluted and further purified to obtain nontoxic antigen. The overall yields of cholera toxin and choleragenoid were increased two- to threefold by the use of hypertoxinogenic mutants of Vibrio cholerae.