Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy
View Patent ↗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 k cat /K m 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 modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn 2+ to a Zn 2+ -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.
1. An isolated nucleic acid molecule comprising the sequence set forth as nucleotides 9–1337 of SEQ ID NO:4.
2. The nucleic acid molecule of claim 1 , wherein the nucleic acid sequence is operably linked to at least one expression control sequence.
3. The nucleic acid molecule of claim 2 , wherein the expression control sequence comprises a promoter.
4. The nucleic acid molecule of claim 2 , wherein the expression control sequence comprises an enhancer.
5. An expression vector comprising the nucleic acid molecule of claim 1 .
6. A recombinant host cell comprising the expression vector of claim 5 .
7. The recombinant host cell of claim 6 , wherein the cell is selected from the group consisting of a gram negative bacteria, yeast, and a cell of a mammalian cell line.
8. The host cell of claim 7 , wherein the cell is an Escherichia coli cell.
9. The host cell of claim 7 , wherein the cell is a Pichia pastoris cell.
10. The recombinant host cell of claim 6 , wherein the host cell expresses a protein comprising a botulinum neurotoxin light chain serotype A.
11. The recombinant host cell of claim 10 , wherein the botulinum neurotoxin light chain serotype A comprises more than about 5% of the total cellular protein by weight.