Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.4.24.69 (
botulinum neurotoxin
)
1,901
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
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, Zn
2+
, 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, Zn
2+
, 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.
...
PMID:Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments. 3161 66
