An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A
An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A
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用于分选酶 A 底物筛选的酶介导蛋白质片段互补测定
DOI：
10.1016/j.bbrc.2017.03.016
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发表时间：
2017
影响因子：
3.1
通讯作者：
Zhang Wei
中科院分区：
文献类型：
--
作者：
Li Ning;Yu Zheng;Ji Qun;Sun Jingying;Liu Xiao;Du Mingjuan;Zhang Wei
酶介导的蛋白质缀合由于其显着的位点选择性和受酶性质影响的温和反应条件最近受到了极大的关注。在已报道的各种酶中，来自金黄色葡萄球菌的分选酶 A (SaSrtA) 由于其选择性和良好的应用而成为最受欢迎的酶。位置扫描已广泛应用于了解酶底物特异性，但肽底物化学合成和分析方法（HPLC、LC-ESI-MS）的低通量一直是完全解码酶底物谱的主要障碍。我们开发了一种简单的高通量底物分析方法，通过改良的蛋白质片段互补测定 (PCA) 揭示 SaSrtA 7M（一种广泛使用的高活性肽连接酶）的新底物。生成了一个针对 SaSrtA 7M 识别的 LPATG 基序的小型文库，并针对携带 N 末端甘氨酸的蛋白质进行筛选。使用这种方法，我们已经确认了该酶的所有目前已知的底物，并且还鉴定了一些以前未知的具有不同活性的底物。该方法提供了一种简单、快速和高灵敏度的方法来以高通量方式确定肽连接酶的底物谱。
Enzyme-mediated protein conjugation has gained great attention recently due to the remarkable site-selectivity and mild reaction condition affected by the nature of enzyme. Among all sorts of enzymes reported, sortase A fromStaphylococcus aureus(SaSrtA) is the most popular enzyme due to its selectivity and well-demonstrated applications. Position scanning has been widely applied to understand enzyme substrate specificity, but the low throughput of chemical synthesis of peptide substrates and analytical methods (HPLC, LC-ESI-MS) have been the major hurdle to fully decode enzyme substrate profile. We have developed a simple high-throughput substrate profiling method to reveal novel substrates of SaSrtA 7M, a widely used hyperactive peptide ligase, by modified protein-fragment complementation assay (PCA). A small library targeting the LPATG motif recognized by SaSrtA 7M was generated and screened against proteins carrying N-terminal glycine. Using this method, we have confirmed all currently known substrates of the enzyme, and moreover identified some previously unknown substrates with varying activities. The method provides an easy, fast and highly-sensitive way to determine substrate profile of a peptide ligase in a high-throughput manner.