Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes.
Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes.
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DOI：
10.1016/j.chembiol.2018.07.002
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发表时间：
2018-10-18
影响因子：
8.6
通讯作者：
Chatterjee A
中科院分区：
文献类型：
--
作者：
Italia JS;Latour C;Wrobel CJJ;Chatterjee A
细菌来源的酪氨酸-tRNA合成酶(TyrRS)/tRNA对在15年前首次用于真核细胞的非天然氨基酸（Uaa）诱变。它为真核生物中大量有用的waas的遗传编码提供了一个理想的平台。然而，到目前为止，这一对已经被设计成只能为一小部分无人机充电。这一对的uaa选择性变体的开发受到了与基于酵母的定向进化平台相关的技术挑战的限制，该平台目前需要改变其底物特异性。在这里，我们克服了这一限制，使其在一种新的大肠杆菌菌株（ATMY）中定向进化，其中内源性TyrRS/tRNA对在功能上被古细菌对应物取代。基于大肠杆菌的简单选择系统使这对基因的快速工程能够开发出选择性地将各种waas（包括对硼苯丙氨酸）结合到哺乳动物细胞和大肠杆菌ATMY菌株中表达的蛋白质中的变体。
The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15 years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity. Here we overcome this limitation by enabling its directed evolution in a novel strain of E. coli (ATMY), where the endogenous TyrRS/tRNA pair has been functionally replaced with an archaeal counterpart. The facile E. coli-based selection system enabled rapid engineering of this pair to develop variants that selectively incorporate various Uaas, including p-boronophenylalanine, into proteins expressed in mammalian cells as well as in the ATMY strain of E. coli.
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影响因子：
15
作者：
Lee HS;Spraggon G;Schultz PG;Wang F
通讯作者：
Wang F
影响因子：
15
作者：
Chen ZJ;Tian Z;Kallio K;Oleson AL;Ji A;Borchardt D;Jiang DE;Remington SJ;Ai HW
通讯作者：
Ai HW
DOI：
10.1073/pnas.1309584110
发表时间：
2013-07-16
影响因子：
11.1
作者：
Chatterjee, Abhishek;Xiao, Han;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
15
作者：
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通讯作者：
Ai, Hui-wang
影响因子：
14.9
作者：
Iraha F;Oki K;Kobayashi T;Ohno S;Yokogawa T;Nishikawa K;Yokoyama S;Sakamoto K
通讯作者：
Sakamoto K