University of California, San Francisco , Department of Pharmaceutical Chemistry , 555 Mission Bay Boulevard South , San Francisco , California 94158 , United States.
Hangzhou Research Institute of Technical Institute of Physics and Chemistry , Chinese Academy of Sciences , Hangzhou 310018 , China.
J Am Chem Soc. 2018 Jul 18;140(28):8807-8816. doi: 10.1021/jacs.8b04603. Epub 2018 Jul 9.
Selenoproteins containing the 21 amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low p K, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNA-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNA-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.
含 21 个氨基酸硒代半胱氨酸 (Sec) 的硒蛋白存在于生命的三个领域，在人类健康和发展中发挥着重要作用。Sec 的独特低 pK 值、高反应性和氧化还原特性还为蛋白质修饰和工程提供了独特的途径。然而，天然 Sec 的掺入需要专门针对 Sec 的特殊翻译机制，而且依赖于物种，这使得用高 Sec 特异性重组制备硒蛋白具有挑战性。因此，一半人类硒蛋白的功能仍然不清楚，Sec 为基础的蛋白质操作受到了极大的阻碍。在这里，我们报告了一种在大肠杆菌中实现蛋白质中 Sec 定点掺入的新通用方法。一种正交的 tRNA-ASecRS 被进化为专门识别 amber 密码子并掺入 Se-allyl selenocysteine (ASec)，掺入的 Asec 可以在温和条件下通过钯介导的切割高效转化为 Sec，该条件与蛋白质和细胞兼容。这种方法完全排除了天然的 Sec 专用因子，因此可以用高 Sec 特异性和酶活性制备各种硒蛋白，无论硒蛋白的位置和物种来源如何，如通过制备人硫氧还蛋白和谷胱甘肽过氧化物酶 1 得到了证明。还在活大肠杆菌细胞表面证明了 Cys 存在下的 Sec 选择性标记。该 tRNA-ASecRS 对还进一步用于哺乳动物细胞中掺入 ASec，钯催化剂在细胞内将其转化为 Sec。这种强大而通用的方法应该极大地促进各种天然硒蛋白的研究以及通过 Sec 定点引入来一般地工程化蛋白质。
