Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.
Toxins (Basel)
; 11(10)2019 10 11.
Article
em En
| MEDLINE
| ID: mdl-31614566
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.
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Texto completo:
1
Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Toxinas Botulínicas
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Proteína 25 Associada a Sinaptossoma
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Proteína 2 Associada à Membrana da Vesícula
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Toxins (Basel)
Ano de publicação:
2019
Tipo de documento:
Article