Genetic Incorporation of ϵ-N-Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.
Genetic Incorporation of ϵ-N-Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.
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DOI：
10.1002/cbic.202100218
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发表时间：
2021-08-03
期刊：
影响因子：
--
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Cao L;Liu J;Ghelichkhani F;Rozovsky S;Wang L
蛋白质翻译后修饰调节蛋白质的结构和功能。赖氨酸苯甲酰化是一种新型组蛋白标记，具有独特的生理相关性。为了构建具有位点特异性引入的这种修饰的蛋白质，我们通过改造嗜甲烷菌吡咯赖氨酰-tRNA合成酶，生成了正交的tRNAPyl-MaBzKRS对，从而能够在大肠杆菌和哺乳动物细胞中高效地将ε-N-苯甲酰赖氨酸（BzK）基因整合到蛋白质中。两种类型的 MaBzKRS 被鉴定为利用位于氨基酸结合袋不同位置的突变来掺入 BzK。这些 MaBzKRS 体积小且表达量高，为研究赖氨酸苯甲酰化的生物效应提供了广泛的用途。两种 M. alvus PylRS 突变体经过工程改造，可高效地将 ε-N-苯甲酰赖氨酸（一种新发现的组蛋白翻译后修饰）基因编码为大肠杆菌和哺乳动物细胞中的蛋白质。 M. alvus PylRS 活性位点（粉色和黄色）不同位置的突变能够对相同氨基酸底物（灰色）实现高特异性。
Protein post-translational modifications regulate protein structure and function. Lysine benzoylation is a new type of histone mark with unique physiological relevance. To construct proteins with this modification site-specifically introduced, here we generated orthogonal tRNAPyl-MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of ε-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation. Two M. alvus PylRS mutants were engineered to genetically encode ε-N-benzoyllysine, a newly discovered histone posttranslational modification, into proteins in E. coli and mammalian cells with high efficiency. Mutations at different locations in M. alvus PylRS active site (pink and yellow) were able to achieve high specificity for the same amino acid substrate (grey).
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使用扩展的遗传密码将磷酸酪氨酸的特定于位点掺入。
影响因子：
14.8
作者：
Hoppmann C;Wong A;Yang B;Li S;Hunter T;Shokat KM;Wang L
通讯作者：
Wang L
影响因子：
15
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Wang L
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4
作者：
Lee, Yan-Jiun;Wu, Bo;Raymond, Jeffrey E.;Zeng, Yu;Fang, Xinqiang;Wooley, Karen L.;Liu, Wenshe R.
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Liu, Wenshe R.
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64.5
作者：
Simon, Matthew D.;Chu, Feixia;Shokat, Kevan M.
通讯作者：
Shokat, Kevan M.
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15
作者：
Liu J;Cheng R;Van Eps N;Wang N;Morizumi T;Ou WL;Klauser PC;Rozovsky S;Ernst OP;Wang L
通讯作者：
Wang L