PRODUCTION OF AN EXPRESSION SYSTEM FOR A SYNAPTOBREVIN FRAGMENT TO MONITOR CLEAVAGE BY BOTULINUM NEUROTOXIN-B

Recombinant DNA techniques were used to develop an expression system f or a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesi cles of nerve terminals and is a substrate for four of the seven serot ypes of botulinum neurotoxin (BoNT). The DNA for the recombinant pepti de was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, design ated as TSB-51, is intended for use in a cell-free assay to test poten tial inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with t he ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light c hain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (similar to 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as in dicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. T he concentration of free Zn2+ bad a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly de pressed by the naturally occurring metalloprotease inhibitor phosphora midon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the di scovery of effective BoNT inhibitors.