caa a22 4500 475743938 CHVBK 20180406123507.0 cr unu---uuuuu 170329e20000801xx s 000 0 eng 10.1023/A:1026549431380 doi (NATIONALLICENCE)springer-10.1023/A:1026549431380 Light Chain of Botulinum A Neurotoxin Expressed as an Inclusion Body from a Synthetic Gene Is Catalytically and Functionally Active [Elektronische Daten] [S. Ahmed, Leonard Smith] 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. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L 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 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. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form. Plenum Publishing Corporation, 2000 Botulinum neurotoxin nationallicence zinc-endopeptidase nationallicence light chain nationallicence apo-light chain nationallicence exocytosis nationallicence inclusion body nationallicence synthetic gene nationallicence Ahmed S. Department of Immunology and Molecular Biology, Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 21702, Fort Detrick, Maryland aut Smith Leonard Department of Immunology and Molecular Biology, Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 21702, Fort Detrick, Maryland aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 475-487 0277-8033 19:6<475 2000 19 10930 https://doi.org/10.1023/A:1026549431380 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026549431380 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ahmed S. Department of Immunology and Molecular Biology, Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 21702, Fort Detrick, Maryland aut NATIONALLICENCE 700 1- Smith Leonard Department of Immunology and Molecular Biology, Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 21702, Fort Detrick, Maryland aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 475-487 0277-8033 19:6<475 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer