Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases,Biochemistry

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Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases
Biochemistry ( IF 2.9 ) Pub Date : 2017-11-15 00:00:00 , DOI: 10.1021/acs.biochem.7b00952
Yunan Zheng ₁ , Raja Mukherjee ₁ , Melissa A. Chin ₁ , Peter Igo ₁ , Martin J. Gilgenast ₁ , Abhishek Chatterjee ₁

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Engineered aminoacyl-tRNA synthetase/tRNA pairs that enable site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells have emerged as powerful tools in chemical biology. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair is a promising candidate for ncAA mutagenesis in mammalian cells, but it has been engineered to charge only a limited set of ncAAs so far. Here we show that two highly polyspecific EcLeuRS mutants can efficiently charge a large array of useful ncAAs into proteins expressed in mammalian cells, while discriminating against the 20 canonical amino acids. When combined with an opal-suppressing pyrrolysyl pair, these EcLeuRS variants further enabled site-specific incorporation of different combinations of two distinct ncAAs into proteins expressed in mammalian cells.

中文翻译：

使用正交多特异性亮氨酰tRNA合成酶扩大哺乳动物细胞中单和双非氨基酸突变的范围。

工程化的氨酰基-tRNA合成酶/ tRNA对使得能够将非规范氨基酸（ncAAs）特异地掺入活细胞中的蛋白质中，这已成为化学生物学中的强大工具。该大肠杆菌来源的亮氨酰-tRNA合成酶（EcLeuRS）/ tRNA对是哺乳动物细胞中ncAA诱变的有前途的候选者，但到目前为止，它已被工程化为仅可充电有限的ncAA。在这里，我们显示了两个高度多特异性的EcLeuRS突变体可以有效地将大量有用的ncAA充入哺乳动物细胞中表达的蛋白质中，同时区分20个规范氨基酸。当与抑制蛋白石的吡咯基对结合时，这些EcLeuRS变体进一步实现了将两种不同ncAA的不同组合位点特异性掺入哺乳动物细胞表达的蛋白质中。

更新日期：2017-11-16

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