title: Cloning, high-level expression, single-step purification, and binding activity of His6-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 approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) 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 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-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.
authors.affiliation: Department of Chemistry and Biochemistry, and The School for Marine Science and Technology, University of Massachusetts-Dartmouth, North Dartmouth, MA 02747, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics*; D019274:Botulinum Toxins, Type A; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005732:Gangliosides / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000235:genetics; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005732:Gangliosides; C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D006639:Histidine; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A