Botulinum neurotoxins, the most potent of all toxins, induce lethalneuromuscular paralysis by inhibiting exocytosis at the neuromuscularjunction. The light chains (LC) of these dichain neurotoxins are a new classof zinc-endopeptidases that specifically cleave the synaptosomal proteins,SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates tothe construction, expression, purification, and use of synthetic orrecombinant botulinum neutoroxin genes. For example, a synthetic gene for theLC of the botulinum neurotoxin serotype A (BoNT/A) was constructed andoverexpressed in Escherichia coli. The gene product was purified frominclusion bodies. The methods of the invention can provide 1.1 g of the LC perliter of culture. The LC product was stable in solution at 4 ~C for at least 6months. This rBoNT/A LC was proteolytically active, specifically cleaving theGlu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reportedcleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km washigher than that reported for the native BoNT/A dichain. Treating the rBoNT/ALC with mercuric compounds completely abolished its activity, most probably bymodifying the cysteine-164 residue located in the vicinity of the active site.About 70 % activity of the LC was restored by adding Zn2+-free, apo-LCpreparation. The LC was nontoxic to mice and failed to elicit neutralizingepitope(s) when the animals were vaccinated with this protein. In addition,injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependentplasma membrane resealing.
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