Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded noncanonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei ( Mma PylRS/PylT) being the most active and versatile to date. We found a pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that this PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its noncognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

中文翻译：

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Methanomethylophilus alvus Mx1201为哺乳动物细胞中相互正交的焦磷酸赖氨酰tRNA /氨酰基-tRNA合成酶对提供了基础。

通过终止密码子抑制进行的遗传密码扩展是一种功能强大的技术，可通过定点编码的非规范氨基酸（ncAAs）在哺乳动物细胞中工程化蛋白质。当前的方法依赖于在哺乳动物细胞中正交的很少可用的tRNA /氨基酰基-tRNA合成酶对，迄今为止，来自马氏甲烷八叠球菌的吡咯基tRNA /氨基酰基-tRNA合成酶对（Mma PylRS / PylT）是最活跃和最通用的。我们发现来自人类肠道古细菌Methanomethylophilus alvus Mx1201（Mx1201 PylRS / PylT）的吡咯基tRNA /氨基酰基-tRNA合成酶对在哺乳动物细胞中是活跃且正交的。我们表明，该PylRS酶可以被工程化以扩展其ncAA底物谱。我们发现由于两对的进化距离较大，Mx1201 PylRS / PylT与Mma PylRS / PylT部分正交。通过Mx1201 PylT的合理突变，我们取消了其与Mma PylRS的非同源相互作用，从而创建了两个相互正交的PylRS / PylT对。结合在同一个单元格中，我们表明这两个对可以响应于两个不同的终止密码子，选择性地引入两个不同的ncAA。我们的工作扩展了用于哺乳动物细胞遗传密码扩展的相互正交工具的功能，并为细胞生物学和蛋白质生产的高级体内蛋白质工程应用提供了基础。