Cell‐free production and stable‐isotope labeling of milligram quantities of proteins

FEBS Letters

Tsutsui, Michio³  Kigawa, Takanori²  Yokoyama, Shigeyuki²  Yabuki, Takashi²  Shibata, Takehiko¹  Ito, Yutaka¹  Yoshida, Yasuhiko² 

[1] Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan;Cellular Signaling Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan;Central Laboratory, Amersham Japan, 2802-1 Hiratsuka, Shiroi, Inba, Chiba 270-1402, Japan

关键词: Cell-free protein synthesis;    In vitro translation;    Chloramphenicol acetyltransferase;    Ras;    Dialysis;    Nuclear magnetic resonance;    AK;    acetyl kinase;    AP;    acetyl phosphate;    CAT;    chloramphenicol acetyltransferase;    CK;    creatine kinase;    CP;    creatine phosphate;    DTT;    dithiothreitol;    MWCO;    molecular weight cut off;    PEG;    polyethylene glycol;    PEP;    phosphoenolpyruvate;    PK;    pyruvate kinase;   

DOI  :  10.1016/S0014-5793(98)01620-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have improved the productivity of an Escherichia coli cell-free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable-isotope labeling of a protein with ¹³C/¹⁵N-labeled amino acids for NMR spectroscopy was achieved by this method.

【 授权许可】

Unknown