利用噬菌体肽库筛选胰凝乳蛋白酶的底物

The Selection of Chymotrypsin's Substrate from Phage Peptide Library

利用脱水胰凝乳蛋白酶 (保持天然酶的完整结合部位 ,但没有催化活性 )作为靶蛋白 ,在噬菌体肽库中钓取一些有结合活力噬菌体 ,再将得的噬菌体同固定化的天然酶一起保温 ,能够被天然酶水解的那部分噬菌体即为底物噬菌体 .利用 DNA测序技术测出筛得的短肽序列 ,分析序列保守性 ,发现 WR和 YF的组合具有很强的保守性 .合成相应的几个短肽 ,与天然酶作用 ,发现芳香族氨基酸与碱性氨基酸的组合较易被胰凝乳蛋白酶切割 .而且 ,当 P2 、P3 位置为侧链较小的氨基酸或碱性氨基酸时 ,更有利于水解的发生 .精氨酸无论处在任何位置 。

The anhydrase of chymotrypsin was prepared, which kept total chymotrypsin binding site without catalytic activity. The phages recognized by the anhydrase of chymotrypsin were screened from the general peptide library. The phages obtained together with the fixed chymotrypsin, were kept at 37℃. Substrate phages were cut and stayed in solution. After the substrate phages were enriched by four rounds of panning, the DNA sequences of the inserted peptides were analyzed. The peptides with conservative sequences were synthesized by the method of solid peptide synthesis. By determining the kinetic constant of these peptides, the most fitful cutting site of chymotrypsin was found. The peptides whose P 1 was W, P 2 and P 3 were small side chain amino acids or alkali amino acids and whose P′ 1 was R were cut most easily. Wherever R was, it would accelerate the hydrolysis. The success of finding the most fitful cutting site of chymotrypsin proved the usefulness and simpleness of the new substrate screening method. And it was the first time to find the conservatives of chymotrypsin substrate.