Engineering the elongation factor Tu for efficient selenoprotein synthesis.
Engineering the elongation factor Tu for efficient selenoprotein synthesis.
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DOI：
10.1093/nar/gku691
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发表时间：
2014-09
影响因子：
14.9
通讯作者：
Englert M
中科院分区：
文献类型：
--
作者：
Haruna K;Alkazemi MH;Liu Y;Söll D;Englert M
通过用专门的延伸因子（细菌中的SelB）和RNA结构信号（SECIS元件）重新编码UGA蛋白石密码子，硒代半胱氨酸（Sec）自然共翻译掺入蛋白质中。我们最近开发了一种无SECIS的硒蛋白合成系统，该系统位点特异性地使用UAG琥珀密码子插入取决于延伸因子Tu的Sec（EF-Tu）。在这里，我们描述了工程EF-Tu改善硒蛋白的合成。通过表达人蛋白O 6-烷基鸟嘌呤-DNA烷基转移酶（hAGT），将其活性位点半胱氨酸密码子替换为UAG琥珀密码子，建立了Sec特异性选择系统。所形成的hAGT硒蛋白修复由甲基化剂N-甲基-N′-硝基-N-亚硝基胍引起的DNA损伤，从而使大肠杆菌能够在该诱变剂存在下生长。创建EF-Tu文库，其中指定氨基酸结合口袋的密码子被随机化。进行选择以增强Sec掺入hAGT;所得EF-Tu变体在文库成员内含有高度保守的氨基酸变化。用EF-Sel 1改进的UTu系统将UAG特异性Sec掺入的效率提高到> 90%，并且还使硒蛋白生产的产量加倍。
Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.
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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：
2.9
作者：
Lothrop, Adam P.;Snider, Gregg W.;Ruggles, Erik L.;Hondal, Robert J.
通讯作者：
Hondal, Robert J.
影响因子：
56.9
作者：
Kryukov, GV;Castellano, S;Gladyshev, VN
通讯作者：
Gladyshev, VN
DOI：
10.1073/pnas.1102128108
发表时间：
2011-03-29
影响因子：
11.1
作者：
Schrader, Jared M.;Chapman, Stephen J.;Uhlenbeck, Olke C.
通讯作者：
Uhlenbeck, Olke C.
影响因子：
64.8
作者：
LEINFELDER, W;ZEHELEIN, E;BOCK, A
通讯作者：
BOCK, A