Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs.
Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs.
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DOI：
10.1038/s41557-018-0052-5
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发表时间：
2018-08
期刊：
影响因子：
21.8
通讯作者：
Chin JW
中科院分区：
文献类型：
--
作者：
Willis JCW;Chin JW
将不同的非典型氨基酸（ncAA）遗传编码到细胞中合成的蛋白质中需要相互正交的氨酰-tRNA合成酶（阿尔斯）/tRNA对。吡咯赖氨酰tRNA合成酶/PyltRNA对来自M. Mazei（Mm）已被工程化以掺入不同的ncAA，并且通常被认为是用于遗传密码扩增的理想对。然而，发现共享MmPylRS/MmPyltRNA对的优点并且与内源性阿尔斯/tRNA对和MmPylRS/MmPyltRNA对正交的新阿尔斯/tRNA对已被证明具有挑战性。在这里，我们证明了几个ΔNPylRS/PyltRNACUA对，其中PylRS缺乏N-末端结构域，是活性的，正交的，并有效地掺入大肠杆菌中的ncAA。杆菌我们创建了与MmPylRS/MmPyltRNA对相互正交的新PylRS/PyltRNA对，并表明将MmPylRS的ncAA特异性重新编程的突变移植到新PylRS中重新编程其底物特异性。最后，我们表明，不同的PylRS/PyltRNA衍生对可以在同一细胞中发挥作用，解码不同的密码子，并纳入不同的ncAA。
Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from M. mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in E. coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally we show that distinct PylRS/PyltRNA derived pairs can function in the same cell, decode distinct codons, and incorporate distinct ncAAs.
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影响因子：
4.8
作者：
Jiang, Ruisheng;Krzycki, Joseph A.
通讯作者：
Krzycki, Joseph A.
影响因子：
48
作者：
Zhang MS;Brunner SF;Huguenin-Dezot N;Liang AD;Schmied WH;Rogerson DT;Chin JW
通讯作者：
Chin JW
影响因子：
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作者：
Yanagisawa, Tatsuo;Ishii, Ryohei;Yokoyama, Shigeyuki
通讯作者：
Yokoyama, Shigeyuki
影响因子：
14.8
作者：
Suzuki T;Miller C;Guo LT;Ho JML;Bryson DI;Wang YS;Liu DR;Söll D
通讯作者：
Söll D
影响因子：
8.4
作者：
Dumas A;Lercher L;Spicer CD;Davis BG
通讯作者：
Davis BG