Fig. 1
From: Elucidating the activation mechanism of botulinum neurotoxin a: role of α-clostripain and NTNH
Assay development for the BoNT/A-activating protease. (A) BoNT/A is expressed by C. botulinum A as a single polypeptide of ~ 150 kDa with low toxicity. The activating protease cleaves the single chain toxin at a specific site to produce the activated di-chain toxin, which consists of light (~ 50-kDa, LC) and heavy (~ 100-kDa, HC) chains connected by a disulfide bond. (B) Recombinant toxin-simulating substrate for the activating protease. The substrate is non-toxic and includes the catalytic (LC) and translocation (HN) domains of BoNT/A, but lacks the receptor-binding domain (HC). The substrate contains an N-terminal His-tag and two mutations in the catalytic domain (E224Q and H227Y) for further reduction of its toxicity. (C) SDS-PAGE analysis for evaluating the suitability of the assay to detect the activating protease. Lane 1–MW marker; lane 2–LCHN; lane 3–LCHN following incubation with C. botulinum A culture supernatant under non-reducing conditions; lane 4—LCHN following incubation with C. botulinum A culture supernatant under reducing conditions; lane 5—C. botulinum A culture supernatant.
