We describe a procedure for the affinity purification of Shiga toxin I subunit B (SLTB) using a commercial galabiose-agarose resin. Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54. SDS-PAGE of the affinity purified SUB showed one band of 8 kDa MW. SUB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose-agarose resin could isolate roughly I mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost. (C) 2004 Elsevier Inc. All rights reserved.