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Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity.
Nature Protocols ( IF 13.1 ) Pub Date : 2017-Oct-01 , DOI: 10.1038/nprot.2017.091
Marcin Poreba , Guy S Salvesen , Marcin Drag

Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

中文翻译:

HyCoSuL肽底物文库的合成以分析蛋白酶底物特异性。

已经开发出许多基于生物学和化学的方法来设计高活性和选择性的蛋白酶底物和探针。但是,很难找到对任何给定蛋白酶真正具有选择性的底物序列,因为不同的蛋白酶可以显示出底物特异性的大量重叠。在某些情况下,使用包含非天然氨基酸的肽库(例如同时使用天然和非天然氨基酸的杂合组合底物库(HyCoSuL))可以实现更好的酶选择性。HyCoSuL是四肽的组合文库,包含在P4-P2位置的氨基酸混合物,在P1位置的固定氨基酸和占据P1'位置的ACC(7-氨基-4-氨基甲酰基甲基香豆素)荧光标签。一旦肽被蛋白酶识别并裂解,ACC被释放并产生可读的荧光信号。在这里,我们描述了人类半胱氨酸蛋白酶和豆荚菌素的HyCoSuL的合成和筛选。我们还讨论了这种方法的可能修改和改编,使其成为开发适用于多种蛋白水解酶的高活性和选择性试剂的有用工具。该协议可分为三个主要部分:(i)荧光标记的HyCoSuL的固相合成,(ii)蛋白酶P4-P2偏好的筛选,以及(iii)配备AOMK的优化活性探针的合成(酰氧基甲基酮)反应基团和生物素标记物,易于检测。从文库设计开始,整个方案可在4-8周内完成(HyCoSuL合成:3-5周;每种酶的HyCoSuL筛选:4-8 d;基于活性的探针合成:
更新日期:2017-09-21
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