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New stable-isotope labeling methods for structural studies

用于结构研究的新稳定同位素标记方法

基本信息

批准号：
08558076
负责人：
YOKOYAMA Shigeyuki
金额：
$ 12.61万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

1. Stable-isotope labeling of proteinsImprovement of cell-free protein production method : The yield of Escherichia coli cell-free protein synthesis system has been improved to be about 0.3 mg/mL reaction mixture in a few hours for the batch method, and has further been increased to be about 6 mg/mL reaction mixture in 10-20 hours by using the improved batch system to the dialysis method. Using the dialysis method, we have synthesized uniformly ^<15>N or ^<13>C labeled proteins. On the other hand, by preparing an amber suppressor tRNA charged with ^<15>N or ^<13>C labeled tyrosine, we have synthesized site-specifically labeled proteins in the batch system. The global fold of a 30-kDa protein was analyzed by preparation of [Ile/Leu/Val-^1H/^<13>C, Phe/Tyr-^1H, u-^2H, u-^<l5>N]protein.2. Stable-isotope labeling of RLNAs[^<15>N]-, [^<13>C, ^<15>N]-, [^2H(30-70%), ^<13>C], and [^2H(50%), ^<13>C, ^<15>N]NTPs were synthesized, and used in T7 RNA polymerase transcription for preparation of uniformly labeled RNAs. By the random 2H labeling, NMR signals of large RNAs were well observed. On the other hand, [5-^2H]uridine and [3-^<15>N]uridine were incorporated in specific position(s). This site-directed labeling was found to be very useful for NMR stydies of polyuridine RNAs complexed with a protein. Furthermore, by ligation of [^<l3>C,^<l5>N]pGp with non-labeled RNA fragment(s), site-specifically labeled longer RNAs were prepared.

1.蛋白质的稳定同位素标记无细胞蛋白质生产方法的改进：大肠杆菌无细胞蛋白质合成系统的产率对于分批法在几小时内提高到约0.3 mg/mL反应混合物，并且通过使用改进的分批系统到透析法在10-20小时内进一步提高到约6 mg/mL反应混合物。利用透析法，我们合成了均匀的^<15>N或^<13>C标记蛋白。另一方面，通过制备带有^<15>N或^<13>C标记的酪氨酸的琥珀抑制tRNA，我们在分批系统中合成了位点特异性标记的蛋白质。通过制备[Ile/Leu/瓦尔-^1H/^<13>C，Phe/Tyr-^1H，u-^2 H，u-^<l5>N]蛋白来分析30-kDa蛋白的全局折叠。合成稳定同位素标记的RLNAs[^<15>N]-、[^<13>C，^<15>N]-、[^2H（30-70%）、^<13>C]和[^2H（50%）、^<13>C，^<15>N] NTP，并将其用于T7 RNA聚合酶转录以制备均匀标记的RNA。通过随机2 H标记，可以很好地观察到大RNA的NMR信号。另一方面，[5-^2H]尿苷和[3-^<15>N]尿苷掺入特定位置。这种定点标记被发现是非常有用的NMR研究的多聚尿苷RNA与蛋白质复合。此外，通过将[^<l3>C，^<l5>N]pGp与未标记的RNA片段连接，制备位点特异性标记的较长RNA。