New stable-isotope labeling methods for structural studies

1. 蛋白质稳定同位素标记无细胞蛋白生产方法的改进：采用批处理法，大肠杆菌无细胞蛋白合成体系的产率在数小时内提高到0.3 mg/mL左右的反应混合物，采用改进的批处理体系进一步提高到透析法10-20小时的反应混合物的产率约为6 mg/mL。采用透析法，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标记酪氨酸的琥珀色抑制tRNA，我们在批处理系统中合成了位点特异性标记的蛋白质。通过制备[Ile/Leu/Val-^1H/^<13>C, Phe/Tyr-^1H, u-^2H, u-^< 15 >N]蛋白，分析了30kda蛋白的全局折叠。合成了RLNAs[^<15>N]-、[^<13>C、^<15>N]-、[^2H（30-70%）、^<13 b> C]和[^2H（50%）、^<13>C、^<15>N] ntp的稳定同位素标记，用于T7 RNA聚合酶转录，制备均匀标记的RNA。通过随机2H标记，可以很好地观察到大rna的核磁共振信号。另一方面，[5-^2H]尿嘧啶和[3-^<15>N]尿嘧啶在特定位置被掺入。这种位点定向标记被发现对多尿嘧啶rna与蛋白质络合的NMR研究非常有用。此外，通过将[^<l3>C,^<l5>N]pGp与未标记的RNA片段连接，制备了位点特异性标记的更长的RNA。

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.