Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States.
ACS Chem Biol. 2020 Jun 19;15(6):1535-1540. doi: 10.1021/acschembio.0c00147. Epub 2020 May 5.
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 . 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.
硒蛋白含有氨基酸硒代半胱氨酸（Sec），存在于所有生命领域。许多硒蛋白的功能还不太了解，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一种非典型的翻译机制产生的，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，通过遗传密码扩展在哺乳动物细胞中产生重组硒蛋白，从而避免了 SECIS 元件和硒可用性的要求。一种工程化的正交. 亮氨酰-tRNA 合成酶/tRNA 对用于在 UAG 琥珀终止密码子处掺入光笼 Sec（DMNB-Sec）。DMNB-Sec 成功掺入 GFP 并通过照射活细胞去笼。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，去笼后证明 MsrB1 具有催化活性，这是首次在哺乳动物系统中使用遗传密码扩展来产生功能性硒蛋白。在哺乳动物细胞中直接定点引入 Sec 的能力以及对硒蛋白活性的时间调节将有助于研究哺乳动物硒蛋白的功能。
