Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a similar to 150 kDa protein, consisting of a binding/translocating heavy chain (HC; 100 kDa) and a toxifying light chain (LC; 50 kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (HiS(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni2+ affinity column by removing P-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98%, pure as assessed by SDS-polyacrylamide gel. (HiS(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay. (C) 2003 Elsevier Inc. All rights reserved.