Cloning, high-level expression, single-step purification, and binding activity of His 6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain

Abstract

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a ∼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 β-mercaptoethanol, the protein was purified and refolded in a single step on Ni 2+ affinity column by removing β-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.