Knowledge Bank

The 7 serotypes of Botulinum neurotoxin (BoNT) are some of the most lethal substances known. The Center for Disease Control (CDC) regards them as Category A Tier 1 agents due to their lethal dose for a 70 kg human of approximately 0.09-0.15 ug when given intravenously or intramuscularly and 0.7-0.9 ug when inhaled, and their tendency to stay in the body for long periods of time (3-6 months).1 making the potential for their use as a weapon of mass destruction extremely unnerving.1 The high level of toxicity and ease of production associated with BoNTs makes their potential for use as a biological warfare agent unnerving.

Standard prophylactic approaches for protection, such as vaccination, are not ideal as BoNTs also have applications as a therapeutic. The current treatments that exist for BoNT poisoning involves supportive care, mechanical ventilation, and the administration of antitoxins that are only effective when the toxin is still in the circulatory system and has not entered the nerve cell.2 The current treatments are impractical in the case of a widespread attack, and they are limited to the time between exposure and entry into the nerve cells, therefore new therapeutics that are capable of inhibiting BoNTs after nerve cell entry are needed.

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.2 A variety of conditions were manipulated to determine optimal protein expression: 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) and TEV protease were used to purify the protein based on the IMAC's affinity to the 6xHis-tag engineered on the protein and the protein's TEV cleavage site. Additional methods of purification such as Size Exclusion Chromatography (SEC) and anionic 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 most optimal conditions were in 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.9 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 potency of novel BoNT/A-LC inhibitors compared to a known inhibitor.