Open AccessReceptor‐Specific Large‐Scale Purification of Cholera Toxin on Silica Beads Derivatized with LysoG M1 Ganglioside
Author(s) -
TAYOT JeanLouis,
HOLMGREN Jan,
SVENNERHOLM Lars,
LINDBLAD Marianne,
TARDY Michel
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05060.x
Subject(s) - chromatography , vibrio cholerae , cholera toxin , toxin , chemistry , sephadex , size exclusion chromatography , ganglioside , elution , affinity chromatography , biochemistry , enzyme , microbiology and biotechnology , bacteria , biology , genetics
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.