Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments
Abstract
:1. Introduction
2. Results
2.1. Experimental Design of Taguchi Experiments to Optimize Buffer Composition for BoNT/A–F Substrate Cleavage
2.2. Buffer Composition for Optimal Cleavage Varies between Different BoNT Serotypes
2.3. ANOM and ANOVA Reveal Optimal Buffer Composition and Impact on Cleavage Efficiency
2.4. Optimized Buffers Enhance BoNT Substrate Cleavage Compared to Reference Buffers
2.5. Design of a Consensus Buffer for all Serotypes
3. Discussion
3.1. The Taguchi DoE Method Enables Identification of Optimal Cleavage Conditions for each Serotype
3.2. Previously Published Results could be Confirmed by the Taguchi DoE Analysis
4. Conclusions
5. Materials and Methods
5.1. Chemicals and Toxins
5.2. Expression and Purification of Full-Length SNAP-25 and VAMP-2
5.3. Design of Taguchi Experiments
5.4. Analysis of Substrate Cleavage by SDS-PAGE
5.5. Statistical Analysis of Taguchi Experiments
5.6. Validation of Optimized Buffer Performance
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Exp. No. | L9-Array 1: Buffer Composition | L9-Array 2: Buffer Additives | |||||
---|---|---|---|---|---|---|---|
pH | ZnCl2 [µM] | DTT [mM] | NaCl [mM] | BSA [mg/mL] | TMAO [M] | Tween 20 [%] | |
1 | 6.5 | 10 | 1 | 0 | 0 | 0 | 0 |
2 | 6.5 | 50 | 5 | 20 | 0 | 0.75 | 0.5 |
3 | 6.5 | 250 | 25 | 100 | 0 | 1.5 | 1.0 |
4 | 7.0 | 10 | 5 | 100 | 0.2 | 0 | 0.5 |
5 | 7.0 | 50 | 25 | 0 | 0.2 | 0.75 | 1.0 |
6 | 7.0 | 250 | 1 | 20 | 0.2 | 1.5 | 0 |
7 | 7.5 | 10 | 25 | 20 | 1.0 | 0 | 1.0 |
8 | 7.5 | 50 | 1 | 100 | 1.0 | 0.75 | 0 |
9 | 7.5 | 250 | 5 | 0 | 1.0 | 1.5 | 0.5 |
BoNT/A | BoNT/B | BoNT/C | BoNT/D | BoNT/E | BoNT/F | |
---|---|---|---|---|---|---|
pH | 7.5 (10%) | 7 (3%) | 6.5 (22%) | 7.5 (10%) | 7 (7%) | 7.5 (14%) |
ZnCl2 [µM] a | 50 (6%) | 50 (18%) | 250 (56%) | 10 (51%) | 50 (52%) | 50 (21%) |
DTT [mM] a | 25 (61%) | 25 (52%) | 5 (21%) | 5 (9%) | 25 (36%) | 25 (58%) |
NaCl [mM] | 0 (23%) | 0 (27%) | 100 (2%)b | 0 (30%) | 100 (5%)b | 0 (7%) |
BSA [mg/mL] | 0 (15%) | 0.2 (0%) | 1 (25%) | 0.2 (1%) | 0.2 (8%) | 0 (32%) |
TMAO [M] a | 0.75 (76%) | 0.75 (57%) | 0.75 (70%) | 0 (93%) | 0 (77%) | 0.75 (60%) |
Tween 20 [%] | 1 (5%) | 1 (37%) | 0.5 (5%) | 0 (3%) | 1 (7%) | 0 (6%) |
Control c | n.a. (4%) | n.a. (6%) | n.a. (0%) | n.a. (3%) | n.a. (8%) | n.a. (3%) |
Toxin Concentration for 50% Cleavage [nM] a | ||||
---|---|---|---|---|
Serotype | Evans Buffer | Jones Buffer | Optimized Buffer | Consensus Buffer |
BoNT/A | 0.62 ± 0.05 | 0.18 ± 0.01 | 0.11 ± 0.01 | 0.24 ± 0.01 |
BoNT/B | 193 ± 31.6 | 23.8 ± 2.29 | 5.7 ± 0.77 | 10.5 ± 1.18 |
BoNT/C | 4717 ± 1202 | 537 ± 37.4 | 2.6 ± 0.2 | 53 ± 9.3 |
BoNT/D | 14.2 ± 1.0 | 11.9 ± 1.2 | 13.2 ± 1.1 | 24.6 ± 1.3 |
BoNT/E | 12.5 ± 1.2 | 3.6 ± 0.26 | 2.25 ± 0.15 | 15.6 ± 1.1 |
BoNT/F | 0.85 ± 0.08 | 0.44 ± 0,05 | 0.21 ± 0.02 | 0.18 ± 0.02 |
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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
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 Stylevon Berg, Laura, Daniel Stern, Jasmin Weisemann, Andreas Rummel, Martin Bernhard Dorner, and Brigitte Gertrud 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