Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2006-05-02
2006-05-02
Minnifield, N. M. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069300, C435S070100, C435S071100, C435S071200, C435S842000, C435S252300, C435S252330, C435S254230, C435S252100, C435S320100, C536S023100, C536S023400, C536S023700, C536S024100
Reexamination Certificate
active
07037680
ABSTRACT:
Botulinumneurotoxins, 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 recombinantbotulinumneutoroxin genes. For example, a synthetic gene for the LC of thebotulinumneurotoxin serotype A (BoNT/A) was constructed and overexpressed inEscherichia 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/Kmwas 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 modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing.
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Jensen Melody
Smith Leonard A.
Arwine Elizabeth
Minnifield N. M.
The United States of America as represented by the Secretary of
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