FRET多肽，天然底物和受体
肉毒杆菌神经毒素和炭疽致命毒素的强毒性是由于与毒素相关的锌依赖蛋白水解活性。这种酶活性的测量为检测毒素提供了一种潜在的敏感和直接的手段，并提供了一种使用高通量筛选识别潜在毒素抑制剂的方法。一种高效的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团，在另一端包含一个合适的显色受体基团。荧光最初是由供体/受体对之间的分子内能量转移猝灭的。FRET底物被适当的酶切割释放荧光团，充分的荧光恢复。荧光强度的增加与酶的存在量成正比。通过记录荧光强度随时间的增加，可以连续监测酶活性。当裂解发生时，相对荧光单位(RFU)的变化可以从使用校准肽生成的标准曲线转换为n摩尔的裂解底物，该校准肽是只包含n端附着荧光团的裂解底物。对于A型肉毒杆菌神经毒素，Control FRET肽底物不被神经毒素切割，但包含序列中所有剩余的非特异性位点，可用于筛选底物在复杂基质中可能发生的背景切割。 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.用于肉毒毒素和破伤风毒素的多肽、原生基质和受体
List Labs提供炭疽酶致死因子(LF)的两种FRET肽底物。这些LF底物可用于筛选具有治疗潜力的LF抑制剂。此外，还设计了一种荧光标记的LF底物，对血清和血浆等复杂基质中的LF具有高度特异性。在炭疽感染早期，致命因子水平较高。这种肽底物允许一种高度敏感、快速和特异性的检测方法来确定感染。快速诊断和开始治疗对疾病的成功治疗至关重要。LF肽底物及相关产品见下表。用于炭疽毒素，致命因子的多肽
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