A method for directed evolution and functional cloning of enzymes.
A method for directed evolution and functional cloning of enzymes.
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一种酶的定向进化和功能克隆的方法。
DOI：
10.1073/pnas.95.18.10523
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发表时间：
1998
影响因子：
11.1
通讯作者：
Schultz,PG
中科院分区：
文献类型：
--
作者：
Pedersen,H;Hölder,S;Sutherlin,DP;Schwitter,U;King,DS;Schultz,PG
本文描述了在生物可降解系统中蛋白质催化剂的体外进化的一般方案。底物通过柔性系链共价且位点特异性地连接至也展示催化剂的丝状噬菌体的pIII外壳蛋白。底物到产物的分子内转化为从突变体库中选择活性催化剂提供了基础，无论是通过从固体载体释放还是附着到固体载体。这种方法已经开发的葡萄球菌核酸酶作为一个模型。影响选择效率的因素的分析，它表明，噬菌体展示葡萄球菌核酸酶可以富集100倍，在一个单一的步骤从一个类似的集成噬菌体展示非催化蛋白质的库。此外，这种方法应该允许人们基于天然酶催化特定反应的能力（例如，糖基转移、序列特异性蛋白水解或磷酸化、聚合等）而不是它们与已知酶的序列或结构同源性。
A general scheme is described for thein vitroevolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.