...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Nucleic Acids Research >Engineering the elongation factor Tu for efficient selenoprotein synthesis
【24h】

Engineering the elongation factor Tu for efficient selenoprotein synthesis

机译：工程化延伸因子Tu以有效合成硒蛋白

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

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expression of human protein O-6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon.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-Nnitrosoguanidine, 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.
机译：通过用特殊的延伸因子（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，可以将硒代半胱氨酸（Sec）自然共翻译地掺入蛋白质中。我们最近开发了一种无SECIS的硒蛋白合成系统，该系统可根据延伸因子Tu（EF-Tu）使用UAG琥珀色密码子插入位点Sec。在这里，我们描述了改进的硒蛋白合成的EF-Tu工程。通过表达人蛋白O-6-烷基鸟嘌呤-DNA烷基转移酶（hAGT）建立了Sec特异性选择系统，其中活性位点半胱氨酸密码子已被UAG琥珀色密码子替代。形成的hAGT硒蛋白修复了甲基化剂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这种诱变剂的存在下生长。创建了一个EF-Tu文库，其中指定氨基酸结合口袋的密码子是随机的。进行选择以增强Sec并入hAGT;所得的EF-Tu变体在文库成员中包含高度保守的氨基酸变化。改进的带有EF-Sel1的UTu系统将UAG特异性Sec掺入的效率提高到90％以上，并且硒蛋白产量也翻了一番。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号