Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.
Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.
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DOI：
10.1007/s10969-009-9072-0
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发表时间：
2009-12
期刊：
影响因子：
--
通讯作者：
Inouye, Masayori
中科院分区：
文献类型：
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作者：
Mao, Lili;Tang, Yuefeng;Vaiphei, S Thangminlal;Shimazu, Tsutomu;Kim, Sung-Gun;Mani, Rajeswari;Fakhoury, Elias;White, Eileen;Montelione, Gaetano T;Inouye, Masayori
在单一蛋白生产（SPP）方法中，所有E.通过MazF（一种ACA特异性mRNA干扰酶）的诱导消除大肠杆菌细胞mRNA。当在MazF诱导的细胞中诱导膜蛋白的mRNA时，E.将大肠杆菌转化到仅产生靶向膜蛋白的生物反应器中。在这里，我们证明了三个原核内膜蛋白，两个原核外膜蛋白，和一个人病毒膜蛋白可以产生在非常高的水平，并组装在适当的膜组分。浓缩SPP（cSPP）系统用于在高达150倍的浓缩培养物中选择性地产生用于NMR研究的同位素富集的膜蛋白，而不影响蛋白质产量，为同位素节省了99%以上的成本。作为一种新的应用程序的cSPP系统的膜蛋白的研究之前，纯化，我们还证明，第一次，快速洗涤剂筛选微线圈NMR和良好的分辨NMR光谱的几个目标的完整的膜蛋白未经纯化。
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.