Characterization of LC-HCC fusion protein of botulinum neurotoxin type A

• Published in: Protein and peptide letters Vol. 18; no. 3; p. 295
• Main Authors: Singh, Manglesh Kumar, Dhaked, Ram Kumar, Singh, Padma, Gupta, Pallavi, Singh, Lokendra
• Format: Journal Article
• Language: English
• Published: Netherlands 01.03.2011
• Subjects: Amino Acid Sequence; Animals; Botulinum Toxins, Type A - chemistry; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - immunology; Botulinum Toxins, Type A - metabolism; Catalytic Domain; Cloning, Molecular; Clostridium botulinum - enzymology; Clostridium botulinum - genetics; Escherichia coli - genetics; Female; Gangliosides - metabolism; Genetic Vectors - genetics; Immunization; Mice; Molecular Sequence Data; Neutralization Tests; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Synaptic Vesicles - enzymology; Synaptic Vesicles - metabolism
• ISSN: 1875-5305
• DOI: 10.2174/092986611794578314

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 H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). 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- H(CC) 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-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.