Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.
Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland.
Nat Protoc. 2017 Oct;12(10):2189-2214. doi: 10.1038/nprot.2017.091. Epub 2017 Sep 21.
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).
许多基于生物学和化学的方法已经被开发出来，用于设计高度活跃和选择性的蛋白酶底物和探针。然而，很难找到真正针对任何给定蛋白酶的底物序列，因为不同的蛋白酶在底物特异性上可能有很大的重叠。在某些情况下，使用含有非天然氨基酸的肽库可以获得更好的酶选择性，例如混合组合底物库（HyCoSuL），它同时使用天然和非天然氨基酸。HyCoSuL 是一种四肽组合文库，包含 P4-P2 位置的氨基酸混合物、P1 位置的固定氨基酸和占据 P1'位置的 ACC（7-氨基-4-羧甲基香豆素）荧光标记物。一旦肽被蛋白酶识别和切割，ACC 就会被释放并产生可读的荧光信号。在这里，我们描述了 HyCoSuL 用于人类 Caspases 和 Legumain 的合成和筛选。我们还讨论了这种方法的可能修改和适应，使其成为开发针对各种蛋白水解酶的高度活跃和选择性试剂的有用工具。该方案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 偏好性的筛选，以及（iii）配备 AOMK（酰氧甲基酮）反应基团和生物素标记物的优化活性探针的合成，以便于检测。从文库设计开始，整个方案可以在 4-8 周内完成（HyCoSuL 合成：3-5 周；每种酶的 HyCoSuL 筛选：4-8 天；基于活性的探针合成：1-2 周）。
