• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Traceless Splicing Enabled by Substrate-Induced Activation of the Nostoc punctiforme Npu DnaE Intein after Mutation of a Catalytic Cysteine to Serine
【24h】

Traceless Splicing Enabled by Substrate-Induced Activation of the Nostoc punctiforme Npu DnaE Intein after Mutation of a Catalytic Cysteine to Serine

机译：催化半胱氨酸突变为丝氨酸后，通过基质诱导点状点蛋白Npu DnaE内含蛋白的激活实现了无痕剪接。

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

Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. (C) 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.
机译：内含肽可自催化裂解出前体蛋白，同时将周围的内含肽片段与天然肽键连接。人们对这些分子奇迹给予了极大的关注，希望能够理解和利用它们的化学特性进行新的生物化学转化，包括偶联合成或生物来源的肽并控制蛋白质功能。尽管有大量强大的应用程序，但由于我们对它们的特异性的理解（定义为允许剪接的侧翼序列）以及将蛋白插入目标蛋白的挑战仍然存在局限，因此仍然难以使用蛋白。在C-蛋白半胱氨酸亲核试剂（Cys + 1）突变为丝氨酸或苏氨酸后，我们检查了常用的点状点虫Npu DnaE内含肽。先前的研究表明，将Cys + 1突变为Ser + 1后，Npu DnaE内含肽的比率和/或剪接产率降低。在这项研究中，遗传选择鉴定出具有Ser + 1的蛋白外显子序列，使Npu DnaE内蛋白与野生型Cys + 1内蛋白相比剪接率仅降低了5倍，而内蛋白本身却没有突变以激活Ser +1为亲核试剂。插入内含肽后，有效连接了三种不同的蛋白质，两侧插入了选定的序列。然后，我们使用这种选择的特异性，在靶向酶中，在仅与所选C-extein序列相似的一级序列所预测的位置上实现了无痕剪接。这项研究强调了Npu DnaE内含肽潜在的催化潜力，以剪接替代亲核试剂，并通过增加插入位点的选择，使内含肽具有更广泛的用途。 （C）2014 MRC分子生物学实验室。由Elsevier Ltd.发布

著录项

相似文献

  • 外文文献
  • 中文文献
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