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rdf:type |
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lifeskim:mentions |
umls-concept:C0009015,
umls-concept:C0017262,
umls-concept:C0027934,
umls-concept:C0185117,
umls-concept:C0332307,
umls-concept:C1167322,
umls-concept:C1167622,
umls-concept:C1420192,
umls-concept:C1514562,
umls-concept:C1880389,
umls-concept:C1883204,
umls-concept:C1883221,
umls-concept:C1998793,
umls-concept:C2266866,
umls-concept:C2752508,
umls-concept:C2911684
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pubmed:issue |
1
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pubmed:dateCreated |
2004-2-9
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pubmed:abstractText |
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.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
1046-5928
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
34
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
8-16
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pubmed:dateRevised |
2010-11-18
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pubmed:meshHeading |
pubmed-meshheading:14766296-Animals,
pubmed-meshheading:14766296-Binding Sites,
pubmed-meshheading:14766296-Blotting, Western,
pubmed-meshheading:14766296-Botulinum Toxins,
pubmed-meshheading:14766296-Chromatography, Affinity,
pubmed-meshheading:14766296-Cloning, Molecular,
pubmed-meshheading:14766296-Clostridium botulinum,
pubmed-meshheading:14766296-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:14766296-Enzyme-Linked Immunosorbent Assay,
pubmed-meshheading:14766296-Escherichia coli,
pubmed-meshheading:14766296-Gangliosides,
pubmed-meshheading:14766296-Gene Expression,
pubmed-meshheading:14766296-Histidine,
pubmed-meshheading:14766296-Immunoblotting,
pubmed-meshheading:14766296-Isoelectric Focusing,
pubmed-meshheading:14766296-Membrane Proteins,
pubmed-meshheading:14766296-Oligopeptides,
pubmed-meshheading:14766296-Peptide Fragments,
pubmed-meshheading:14766296-Plasmids,
pubmed-meshheading:14766296-Polymerase Chain Reaction,
pubmed-meshheading:14766296-Protein Binding,
pubmed-meshheading:14766296-Protein Folding,
pubmed-meshheading:14766296-Rats,
pubmed-meshheading:14766296-Recombinant Proteins
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pubmed:year |
2004
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pubmed:articleTitle |
Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain.
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pubmed:affiliation |
Department of Chemistry and Biochemistry, and The School for Marine Science and Technology, University of Massachusetts-Dartmouth, North Dartmouth, MA 02747, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, Non-P.H.S.,
Research Support, Non-U.S. Gov't
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