Optimization of Expression and Purification of Full-Length and Truncated Botulinum Neurotoxin A Light Chain (BoNT/A-LC) for Assay Development
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Date
2023-05
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The Ohio State University
Abstract
The research conducted focused on the protein expression and purification of BoNT serotype A Light Chain (BoNT/A-LC), the portion of the BoNT protein specifically involved in SNAP-25 cleavage, in both a truncated and full-length form.1 A variety of conditions were manipulated to determine optimal protein expression conditions: including time after IPTG induction, growth temperature, E. coli strain, and media type. Immobilized metal affinity chromatography (IMAC) eluted using Fast Protein Liquid Chromatography (FPLC) were used to purify the protein based on the affinity of the 6xHis-tag engineered into the protein and then the purified protein was cleaved using TEV protease to remove the 6xHis-tag. Additional methods of purification such as Size Exclusion Chromatography (SEC) and anion exchange were used to further purify the protein. The optimization and purification experiments were analyzed using SDS-PAGE, and fractions corresponding to peaks on the FPLC chromatogram. The empirically determined optimal conditions for expression were BL21(DE3) cells grown in terrific broth (TB) for 22 hours following 1 mM IPTG induction at OD600 of 0.6 with shaking at 20 °C. The pure protein
will be used to develop a Fluorescence Resonance Energy Transfer (FRET) Assay that measures BoNT/A-LC cleavage of the SNAP-25 complex using a fluorescence plate reader to obtain the relative fluorescence unit (RFU) values at 470 nm and 527 nm.2 The detection of BoNT/A-LC activity will be calculated by the decrease in the emission ratio (RFU 527/RFU 470) compared to the control wells. This assay will allow for the quantification of the inhibitory potency of novel BoNT/A-LC inhibitors compared to a known inhibitor.