Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B

A new single-step purification method for Shiga toxin (Stx) was developed u sing receptor-mediated affinity chromatography, in which Gb3Cer (globotriao sylceramide) was conjugated to octyl Sepharose CL-4B as a carrier This meth od achieves high yield and high purity in a small column on which Gb3Cer ha s been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purif ied Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purifie d Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burki tt's lymphoma cells. This versatile purification method can be used to isol ate various types of natural as well as recombinant Stx, facilitating funda mental studies of human diseases caused by this toxin. (C) 2001 Academic Pr ess.