Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy
US7227010B2 · kind B2 · utility
Assignee
Inventor
Key dates
| Filing date | Nov 6, 2001 |
| Grant date | Jun 5, 2007 |
| Priority date | — |
| Expiry date | Nov 6, 2021 |
Classification
- Technology area (CPC A)Human Necessities
- CPC primaryA61K39/00
- WIPO fieldPharmaceuticals
- WIPO sectorChemistry
Abstract
Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates to the construction, expression, purification, and use of synthetic or recombinant botulinum neutoroxin genes. For example, a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A) was constructed and overexpressed in Escherichia coli. The gene product was purified from inclusion bodies. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product was stable in solution at 4° C. for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modif…
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