Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

von Berg, Laura; Stern, Daniel; Weisemann, Jasmin; Rummel, Andreas; Dorner, Martin Bernhard; Dorner, Brigitte Gertrud

doi:10.3390/toxins11100588

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Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

¹,

Martin Bernhard Dorner

and

Brigitte Gertrud Dorner

^1,*

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany

Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Author to whom correspondence should be addressed.

Toxins 2019, 11(10), 588; https://doi.org/10.3390/toxins11100588

Received: 31 July 2019 / Revised: 1 October 2019 / Accepted: 9 October 2019 / Published: 11 October 2019

(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)

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Abstract

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn²⁺, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn²⁺, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays. View Full-Text

Keywords: botulinum neurotoxins; detection; substrate cleavage; taguchi design-of-experiments; SNARE

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von Berg, L.; Stern, D.; Weisemann, J.; Rummel, A.; Dorner, M.B.; Dorner, B.G. Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments. Toxins 2019, 11, 588. https://doi.org/10.3390/toxins11100588

AMA Style

von Berg L, Stern D, Weisemann J, Rummel A, Dorner MB, Dorner BG. Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments. Toxins. 2019; 11(10):588. https://doi.org/10.3390/toxins11100588

Chicago/Turabian Style

von Berg, Laura, Daniel Stern, Jasmin Weisemann, Andreas Rummel, Martin B. Dorner, and Brigitte G. Dorner. 2019. "Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments" Toxins 11, no. 10: 588. https://doi.org/10.3390/toxins11100588

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Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

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