Citation

Purpose of Study:The purpose of this study was to express and purify the luminal domain loop of human SV2c containing dual His6tags, one each on the C-and N-terminals. This region, residues 454-579, has been identified as the protein receptor specific for botulinum neurotoxin type A (BoNT/A) binding. This construct, when attached to, for example, nickel coated beads, has the potential to form a loop similar to the native structure and increase the binding affinity of BoNT/A for in vitroassays. Methods Used:The His6-SV2c-His6 was expressed as a GST fusion protein to increase solubility during isolation and purification. Purified His6-SV2c-His6 was attached to magnetic nickel coated beads using various ratios of beads to protein. Each set of coated beads was exposed to varying amounts of BoNT/A in buffer, washed, and incubated with our FRET peptide SNAPtide, Prod #520. Generation of cleaved peptide was monitored using reverse phase HPLC with fluorescence detection. Summary of Results: The His6-SV2c-His6 was purified in a soluble form and linked to magnetic nickel coated beads. Various buffer conditions and binding conditions were tested to optimize the binding and limit the non specific binding of toxin to the beads. We are able to detect cleavage of SNAPtideafter exposure to as little as 5pg of BoNT/A. Conclusions: Our study shows the ability to purify a soluble form of the SV2c BoNT/A binding domain and its utility in the capture and detection of BoNT/A. This is a key step in establishing an in vitropotency assay. This assay can also be used to screen for inhibitors that interfere in the binding of BoNT/A to the SV2c protein receptor.