一种用于酶的定向进化和功能克隆的方法。
A method for directed evolution and functional cloning of enzymes.
作者信息
Pedersen H, Hölder S, Sutherlin D P, Schwitter U, King D S, Schultz P G
机构信息
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
出版信息
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10523-8. doi: 10.1073/pnas.95.18.10523.
A general scheme is described for the in vitro evolution 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.
描述了一种在生物可扩增系统中进行蛋白质催化剂体外进化的通用方案。底物通过柔性连接子共价且位点特异性地连接到丝状噬菌体的pIII外壳蛋白上，该噬菌体也展示催化剂。底物向产物的分子内转化为从突变体文库中选择活性催化剂提供了基础，可通过从固体支持物上释放或附着来实现。该方法已以葡萄球菌核酸酶作为模型进行开发。给出了影响选择效率的因素分析，结果表明展示葡萄球菌核酸酶的噬菌体可在一步中从展示非催化蛋白的噬菌体样集合中富集100倍。此外，这种方法应允许基于其催化特定反应（例如糖基转移、序列特异性蛋白水解或磷酸化、聚合等）的能力而不是其与已知酶的序列或结构同源性来功能克隆天然酶。
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