Site-Specific Incorporation of Selenocysteine Using an Expanded Genetic Code and Palladium-Mediated Chemical Deprotection.
Site-Specific Incorporation of Selenocysteine Using an Expanded Genetic Code and Palladium-Mediated Chemical Deprotection.
复制标题
DOI：
10.1021/jacs.8b04603
复制
发表时间：
2018-07-18
影响因子：
15
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Liu J;Zheng F;Cheng R;Li S;Rozovsky S;Wang Q;Wang L
含有第 21 个氨基酸硒代半胱氨酸 (Sec) 的硒蛋白存在于生命的所有三个王国中，并在人类健康和发育中发挥着重要作用。 Sec 独特的低 pKa、高反应性和氧化还原特性也为蛋白质修饰和工程提供了独特的途径。然而，天然的 Sec 掺入需要专用于 Sec 且具有物种依赖性的特殊翻译机制，这使得重组制备具有高 Sec 特异性的硒蛋白具有挑战性。因此，一半的人类硒蛋白的功能仍不清楚，基于 Sec 的蛋白质操作受到极大阻碍。在这里，我们报告了一种新的通用方法，能够将 Sec 定点掺入大肠杆菌中的蛋白质中。正交的 tRNAPyl-ASecRS 进化为响应琥珀密码子而特异性掺入 Se-烯丙基硒代半胱氨酸 (ASec)，并且掺入的 ASec 在与蛋白质和细胞相容的温和条件下通过钯介导的裂解高效转化为 Sec。这种方法完全消除了天然的 Sec 专用因子，因此无论 Sec 位置和物种来源如何，都可以制备出具有高 Sec 特异性和酶活性的各种硒蛋白，如人硫氧还蛋白和谷胱甘肽过氧化物酶 1 的制备所示。在活的大肠杆菌细胞表面也证明了在存在 Cys 的情况下的 Sec 选择性标记。 tRNAPyl-ASecRS 对进一步用于哺乳动物细胞中以掺入 ASec，ASec 在纤维素中通过钯催化剂转化为 Sec。这种稳健且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位点特异性引入 Sec 来进行一般蛋白质的工程。
Selenoproteins containing the 21st amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low pKa, 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 remain 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 tRNAPyl-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 tRNAPyl-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.
登录
查看更多内容
影响因子：
--
作者：
Gonzalez-Flores JN;Shetty SP;Dubey A;Copeland PR
通讯作者：
Copeland PR
影响因子：
16.6
作者：
Aldag, Caroline;Broecker, Markus J.;Hohn, Michael J.;Prat, Laure;Hammond, Gifty;Plummer, Abigail;Soell, Dieter
通讯作者：
Soell, Dieter
影响因子：
2.1
作者：
Harris, Katharine M.;Flemer, Stevenson, Jr.;Hondal, Robert J.
通讯作者：
Hondal, Robert J.
DOI：
10.1073/pnas.0800800105
发表时间：
2008-08-26
影响因子：
11.1
作者：
Hofer, Thomas;Thomas, Joshua D.;Rader, Christoph
通讯作者：
Rader, Christoph
影响因子：
8.4
作者：
Dery S;Reddy PS;Dery L;Mousa R;Dardashti RN;Metanis N
通讯作者：
Metanis N