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. 2019 Jun 17;11(6):348. doi: 10.3390/toxins11060348

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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In vitro autoprocessing of TcdB. The in vitro processing of TcdB_VPI (VPI), TcdB_NAP1 (NAP1), and TcdB_NAP1v (NAP1v) was evaluated in the presence of 10 mM of the co-factor inositol hexakisphosphate (InsP6). The reactions were incubated at 37°C for the indicated times, and toxins or toxin fragments were then detected by Coomassie Blue staining. The enzymatic activity was determined by the presence of full-length (FL) toxin (1–2366), which decreased over time, and processed toxin (544–2366, PT), which increased over time. Shown in (A) is one representative SDS-PAGE gel from three independent experiments; (B) The bands corresponding to the FL toxin and the PT were quantified by densitometry. The autoprocessing activity of each toxin was determined by comparing the relative amounts of both forms of the toxin, and the percentage obtained was plotted using nonlinear regression. Error bars represent means ± SD of three independent experiments.