Blog
About

13
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A.

      Protein Expression and Purification

      metabolism, Synaptotagmins, isolation & purification, genetics, chemistry, Recombinant Proteins, Protein Conformation, Peptide Fragments, Gangliosides, Cloning, Molecular, Botulinum Toxins, Type A

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Related collections

          Author and article information

          Journal
          16143544
          10.1016/j.pep.2005.07.020

          Comments

          Comment on this article