...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Nucleic acids research >Site‐specific incorporation of an unnatural amino acid into proteins in mammalian cells
【24h】

Site‐specific incorporation of an unnatural amino acid into proteins in mammalian cells

机译：将非天然氨基酸定点整合到哺乳动物细胞中的蛋白质中

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

A suppressor tRNATyr and mutant tyrosyl‐tRNA synthetase (TyrRS) pair was developed to incorporate 3‐iodo‐l‐tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNATyr gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNATyr, which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3‐iodo‐l‐tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNATyr, while 3‐iodo‐l‐tyrosine was supplied in the growth medium. 3‐Iodo‐l‐tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of 95%. Finally, we demonstrated conditional 3‐iodo‐l‐tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline‐regulated promoter.
机译：开发了抑制性tRNA Tyr 和突变型酪氨酰-tRNA合成酶（TyrRS）对，将3-碘-1-酪氨酸掺入哺乳动物细胞的蛋白质中。首先，将大肠杆菌抑制基因tRNA Tyr 基因在D臂的三个位置突变，以产生用于表达的内部启动子。但是，该tRNA与相关的TyrRS一起在哺乳动物细胞中未表现出抑制活性。然后，我们发现琥珀酸抑制可通过天然含有启动子序列的大肠杆菌TyrRS和嗜热脂肪芽孢杆菌抑制子tRNA Tyr 的异源对发生。此外，当从基因簇表达抑制性tRNA时，这种抑制的效率显着提高，其中tRNA基因在相同方向上串联重复9次。为了掺入3-碘-1-酪氨酸，我们在哺乳动物细胞中表达了我们最近创建的特异大肠杆菌TyrRS变体TyrRS（V37C195），以及嗜热脂肪芽孢杆菌抑制剂tRNA Tyr ，而在生长培养基中提供了3-碘-1-酪氨酸。因此，将3-碘-l-酪氨酸掺入蛋白质的琥珀色位置，占有率> 95％。最后，我们证明了有条件的3-碘-1-酪氨酸掺入，受四环素调节启动子的TyrRS（V37C195）基因的可诱导表达调节。

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