title: Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A.
abstract: Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding-translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. The complete sequence of BoNT/A consists of 1296 amino acid residues. The beta-trefoil domain for BoNT/A to which gangliosides bind starts at Ser 1092 and this fragment represents the C-half of the C-terminus of the heavy chain (C-quarter HC or HCQ). The recombinant HCQ DNA was successfully cloned into an expression vector (pET15b), which was used to transform Escherichia coli strain BL21-Star (DE3) for expression. Expression of HCQ was obtained by an extended post-induction time of 15 h at 30 degrees C. The recombinant histidine tagged HCQ protein was isolated and purified by nickel affinity gel column chromatography and its molecular weight was verified by gel electrophoresis. The HCQ was positively identified by antibodies raised against BoNT/A employing immunological dot-blot and Western blot assays. HCQ was shown to bind with synaptotagmin (a known BoNT/A receptor) and gangliosides, indicating that the expressed and purified HCQ protein retains a functionally active conformation.
authors.affiliation: Department of Chemistry and Biochemistry, The Botulinum Research Center, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
authors.forename: Sapna
authors.initials: S
authors.lastname: Sharma
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005732:Gangliosides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050857:Synaptotagmins / Q000378:metabolism
chemical_list: D005732:Gangliosides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D050857:Synaptotagmins; D019274:Botulinum Toxins, Type A