Receptor‐Specific Large‐Scale Purification of Cholera Toxin on Silica Beads Derivatized with LysoGM1 Ganglioside

Abstract

1 A receptor-specific affinity chromatographic method for large-scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, G_M1, is hydrolysed to lysoG_M1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE-dextran has been adsorbed and cross-linked before coupling. Columns of these Spherosil-DEAE-dextran-lysoG_M1 beads, in contrast to particles derivatized with lysoG_A1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8). 2 The toxin-binding capacity was directly proportional to the amount of lysoG_M1 in the column: 2.3 mg/μmol lysoG_M1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83–107%). 3 The affinity-purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G-100. 4 Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000-l cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.