Characterization of LC-HCC fusion protein of botulinum neurotoxin type A.
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.
Biotechnology Division, Defence Research & Development Establishment, Gwalior, India.
Manglesh Kumar
MK
Singh
Ram Kumar
RK
Dhaked
Padma
P
Singh
Pallavi
P
Gupta
Lokendra
L
Singh
D000595:Amino Acid Sequence; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005732:Gangliosides / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007114:Immunization; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000201:enzymology / Q000378:metabolism
D005732:Gangliosides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019274:Botulinum Toxins, Type A