Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3′ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

中文翻译：

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通过光编码的硒代半胱氨酸的遗传密码扩展产生重组哺乳动物硒代蛋白。

硒蛋白含有氨基酸硒代半胱氨酸（Sec），存在于生活的所有领域。人们对许多硒蛋白的功能了解甚少，部分原因是难以生产用于细胞生物学评估的重组硒蛋白。内源性哺乳动物硒蛋白是通过非规范的翻译机制产生的，该机制需要抑制UGA终止密码子和mRNA 3'非翻译区中的Sec插入序列（SECIS）元件。在这里，重组硒蛋白通过遗传密码扩展在哺乳动物细胞中产生，从而避免了对SECIS元素和硒可用性的需求。工程化的正交ê。大肠杆菌亮氨酰-tRNA合成酶/ tRNA对用于在UAG琥珀色终止密码子处掺入光笼Sec（DMNB-Sec）。DMNB-Sec已成功整合到GFP中，并且未通过活细胞辐照而解笼。此外，DMNB-Sec用于生成天然硒蛋白蛋氨酸-R-亚砜还原酶B1（MsrB1）。重要的是，MsrB1被显示为解开后具有催化活性，构成了在哺乳动物系统中首次使用遗传密码扩展来生成功能性硒蛋白。将Sec直接特异地引入哺乳动物细胞并暂时调节硒蛋白活性的能力将有助于表征哺乳动物硒蛋白的功能。