Purification of enterotoxigenic Escherichia Coli heat labile toxin (Part 1)

ABSTRACT

Purification of Escherichia Coli heat labile toxin (LT) was done from 9 litres of culture. The cells were suspended in 0.01M Tris (hydroxymethyl) amino-methane (Tris)-hydrochloride buffer (pH 8.6) containing 0.9 percent sodium chloride. The suspension was sonicated. The supernatant was then treated with ammonium sulphate and disolved in TEAN buffer and dialysed. This crude LT was then passed through A5M Biogel and collected by D-galactose and concentrated to about 3 ml. It was then passed through Sephacryl S-200 column and again concentrated by Amicon PM 10 membrane and used as purified LT. Separation of subunits were carried out by using 6M urea solution in 0.1M propionic acid (pH 4.0). Every steps of fractions were checked by Ouchterlony double gel diffusion test using anti cholera sera and by CHO cell assay. Protein assay was done and specificity of the protein was also checked by SDS polyacrylamide slab gel electrophoresis.