| Co-Investigator(Kenkyū-buntansha) |
ITO Yutaka RIKEN (The Institute of Physical and Chemical Research) Laboratory of Cellular a, 遺伝生化学研究室, 研究員 (80261147)
KIGAWA Takanori RIKEN (The Institute of Physical and Chemical Research) Cellular Signaling Labor, 細胞情報伝達研究室, 研究員 (20270598)
KAWAI Gota Chiba Institute of Technology, Department of Industrial Chemistry, Associate Pro, 工学部, 助教授 (70211860)
MUTO Yutaka The University of Tokyo, Graduate School of Science, Lecturer, 大学院・理学系研究科, 講師 (30192769)
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| Research Abstract |
1. Stable-isotope labeling of proteins
Improvement 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.
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