Chemical complementation: A reaction-independent genetic assay for enzyme catalysis
Chemical complementation: A reaction-independent genetic assay for enzyme catalysis
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DOI：
10.1073/pnas.262420099
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发表时间：
2002-12-24
影响因子：
11.1
通讯作者：
Cornish, VW
中科院分区：
文献类型：
--
作者：
Baker, K;Bleczinski, C;Cornish, VW
已经开发了一种用于酶活性的高通量测定法，其是反应独立的。在该测定中，使用小分子酵母三杂交系统将酶催化与体内报告基因的转录联系起来。在这里，我们证明了这种方法的可行性，通过使用一个充分研究的酶催化反应，头孢菌素水解的阴沟肠杆菌P99头孢菌素酶（β-内酰胺水解酶，EC 3.5.2.6）。我们表明，三杂交系统可以用来读出头孢菌素酶活性在体内作为一个lacZ报告基因的转录水平的变化，野生型头孢菌素酶可以从一个池中分离的非活性突变体通过使用lacZ屏幕。该检测方法的设计使其可以应用于不同的化学反应，而不改变三杂交系统的组成部分。蛋白质功能的非反应依赖性高通量分析应该是蛋白质工程和酶学，药物发现和蛋白质组学的有力工具。
A high-throughput assay for enzyme activity has been developed that is reaction independent. In this assay, a small-molecule yeast three-hybrid system is used to link enzyme catalysis to transcription of a reporter gene in vivo. Here we demonstrate the feasibility of this approach by using a well-studied enzyme-catalyzed reaction, cephalosporin hydrolysis by the Enterobacter cloacae P99 cephalosporinase (beta-lactam hydrolase, EC 3.5.2.6). We show that the three-hybrid system can be used to read out cephalosporinase activity in vivo as a change in the level of transcription of a lacZ reporter gene and that the wild-type cephalosporinase can be isolated from a pool of inactive mutants by using a lacZ screen. The assay has been designed so that it can be applied to different chemical reactions without changing the components of the three-hybrid system. A reaction-independent high-throughput assay for protein function should be a powerful tool for protein engineering and enzymology, drug discovery, and proteomics.