Ca2+-independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli
Ca2+-independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli
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DOI：
10.1002/biot.201500012
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发表时间：
2015-09-01
影响因子：
4.7
通讯作者：
Nagamune, Teruyuki
中科院分区：
文献类型：
--
作者：
Hirakawa, Hidehiko;Ishikawa, Suguru;Nagamune, Teruyuki
金黄色葡萄球菌转肽酶，分选酶A (SrtA)，催化肽连接具有高底物特异性，是一种有用的工具，可以位点特异性地将蛋白/肽功能分子连接到靶蛋白上。然而，其强烈的Ca2+依赖性使得SrtA在低Ca2+浓度和Ca2+结合物质存在下难以使用。为了克服这个问题，我们设计了一个SrtA突变体，Ca2+独立表现出高的催化活性。该突变体（P94R/E105K/E108A/D160N/D165A/K190E/K196T）是由Ca2+结合位点和底物结合位点周围的已知突变组合而成，成功催化了大肠杆菌细胞质中的选择性蛋白-蛋白连接。活细胞中的选择性蛋白修饰是研究细胞事件和调节细胞功能的一种很有前途的方法。这种SrtA突变体可能被证明是一种多功能工具，通过将功能蛋白和化学修饰的肽结合到活细胞中，为感兴趣的蛋白添加新的功能，活细胞通常保持低Ca2+浓度。
A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca2+ dependency makes SrtA difficult for use under low Ca2+ concentrations and in the presence of Ca2+-binding substances. To overcome this problem, we designed a SrtA mutant that Ca2+-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca2+-binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca2+ concentrations.