FRET肽，天然底物和受体
肉毒杆菌神经毒素和炭疽致死毒素的有效毒性都是由于与毒素相关的锌依赖性蛋白水解活性所致。该酶活性的测量既可以为检测毒素的潜在敏感和直接手段，以及一种使用高吞吐量筛选鉴定潜在的毒素抑制剂的方法。一种高效监测酶活性的方法是基于荧光共振能量转移（FRET）底物的使用。这些荧光肽在一端包含一个供体荧光基，另一端含有合适的成色受体组。荧光最初是通过供体/受体对之间的分子内能转移来淬灭的。通过适当的酶释放荧光团和全荧光的裂解，恢复了荧光团。荧光强度的增加与存在的酶量成正比。可以通过记录随时间的记录荧光强度的增加来连续监测酶促活性。相对荧光单元（RFU）的变化可以通过使用校准肽产生的标准曲线转换为裂解底物的Nmoles，该曲线是仅包含N末端连接的荧光团的切割的底物。对于A型肉毒杆菌毒素A型，对控制的FRET肽底物不会被神经毒素裂解，但在序列中包含所有剩余的非特异性位点，可用于筛选可以在复杂矩阵中发生的底物的背景裂解。 There are seven immunologically distinct botulinum neurotoxins, designated serotypes A-G. Each neurotoxin cleaves one of two target proteins critical for synaptic vesicle fusion to the plasma membrane and neurotransmitter release, either SNAP-25 or Synaptobrevin. The latter is also called VAMP-2. Types A, C and E specifically bind and selectively cleave SNAP-25 while types B, D, F and G cleave Synaptobrevin. Type C also cleaves Syntaxin. The FRET peptides are based on the sequences of these native substrates. The enzymatic activity of these toxins can also be measured using the appropriate native substrate and monitoring the generation of the cleaved product on SDS-PAGE gel electrophoresis. The synaptic vesicle glycoprotein 2c (SV2c) has been shown to be the protein receptor for Botulinum neurotoxin, Type A (BoNT/A) and the luminal domain loop is the specific location of BoNT/A binding. This 124-amino acid domain of SV2c is available as a GST fusion protein. Substrates, both FRET peptide and native, available from List Labs for use with Botulinum toxins are shown in the table below. Also shown is the protein receptor for BoNT/A.
分销定位器