SUBSTRATE PHAGE - SELECTION OF PROTEASE SUBSTRATES BY MONOVALENT PHAGE DISPLAY
SUBSTRATE PHAGE - SELECTION OF PROTEASE SUBSTRATES BY MONOVALENT PHAGE DISPLAY
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DOI：
10.1126/science.8493554
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发表时间：
1993-05-21
期刊：
影响因子：
56.9
通讯作者：
WELLS, JA
中科院分区：
文献类型：
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作者：
MATTHEWS, DJ;WELLS, JA
本文描述了一种用于在大约10（7）种可能的序列中鉴定良好的蛋白酶底物的方法。构建融合蛋白文库，其含有用于结合亲和支持物的氨基末端结构域，随后是随机化的蛋白酶底物序列和M13基因III的羧基末端结构域。每种融合蛋白在丝状噬菌粒颗粒（底物噬菌体）上展示为单拷贝。然后将噬菌体结合到亲和支持物上并用目的蛋白酶处理。释放具有良好蛋白酶底物的噬菌体，而具有抵抗蛋白水解的底物的噬菌体保持结合。经过几轮的结合、蛋白水解和噬菌粒增殖后，鉴定了两种不同蛋白酶（枯草杆菌蛋白酶的变体和因子X（a））的敏感和抗性底物序列。该技术也可用于研究各种翻译后修饰的序列特异性。
A method is described here for identifying good protease substrates among approximately 10(7) possible sequences. A library of fusion proteins was constructed containing an amino-terminal domain used to bind to an affinity support, followed by a randomized protease substrate sequence and the carboxyl-terminal domain of M13 gene III. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). Phage were then bound to an affinity support and treated with the protease of interest. Phage with good protease substrates were released, whereas phage with substrates that resisted proteolysis remained bound. After several rounds of binding, proteolysis, and phagemid propagation, sensitive and resistant substrate sequences were identified for two different proteases, a variant of subtilisin and factor X(a). The technique may also be useful for studying the sequence specificity of a variety of posttranslational modifications.