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Characterization of LC-HCC Fusion Protein of Botulinum Neurotoxin Type A

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Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with HCC (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an Nterminally six-histidine-tagged fusion protein (rBoNT/A LC-HCC). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- HCC in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-HCC was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-HCC protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

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Keywords: BoNT/A; LC- HCC subunit; endopeptidase; ganglioside; synaptic vesicles

Document Type: Research Article

Publication date: 01 March 2011

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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