• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of structural and functional genomics >Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system
【24h】

Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system

机译：使用缩合单蛋白（cSPP）生产系统生产用于NMR研究的膜蛋白

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

摘要

In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.
机译：在单一蛋白质生产（SPP）方法中，通过诱导MazF（一种ACA特异性mRNA干扰酶）消除了所有大肠杆菌细胞mRNA。当在MazF诱导的细胞中诱导膜蛋白的mRNA被工程化为无ACA序列而不改变其氨基酸序列时，大肠杆菌转化为仅产生目标膜蛋白的生物反应器。在这里，我们证明了可以以非常高的水平产生三种原核内膜蛋白，两种原核外膜蛋白和一种人病毒膜蛋白，并以适当的膜级分组装。浓缩的SPP（cSPP）系统用于在多达150倍的浓缩培养物中选择性地生产富含同位素的膜蛋白，用于NMR研究，而不会影响蛋白质的收率，从而为同位素节省了99％以上的成本。作为纯化前用于研究膜蛋白的cSPP系统的一种新颖应用，我们还首次展示了通过微线圈NMR快速分离去污剂和良好分离的NMR光谱，可对几种无需纯化的靶向膜蛋白进行分离。

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