Expression and Purification of Botulinum Neurotoxin Type ‘A’ Toxin Domain in E. coli and its Application in Detection

Abstract

Botulism is a neuroparalytic disease caused by botulinum toxins type A-G. It has been listed as category ‘A’ biowarfare agent by CDC due to its extreme toxicity.. In the present study, we developed a simple method for the pro-duction and purification of recombinant toxin domain of BoNT type ‘A’ from Escherichia coli and established its application in BoNT detection. The BoNT/A LC gene was cloned in pET28b+ vector and expressed in E.coli. The recom-binant BoNT/A LC protein expression was achieved at 25°C with the induc-tion of 0.5mM IPTG at 16 h. The recombinant protein was purified under native conditions with more than 98% purity using simple affinity chroma-tography and confirmed by Mass spectrometry and Western blot analysis. Generated polyclonal antibodies and the toxin domain elicited a strong im-mune response in mice and rabbit. Optimized the Sandwich ELISA system resulted with the sensitivity of 7.5 ng/ml for the detection of BoNT/A. The limit of detection (LOD) in different food matrices were studied through spik-ing studies using rBoNT/A LC protein as antigen and achieved a detection limit ranging from7.5 ng/ml to 250 ng/ml. The developed method has the potential for the industrial production towards its application in detection, development of candidate vaccine molecule and production of neutralizing antibodies. Current study explored for detection of BoNT/A.