Cell‐free synthesis of the Ras‐binding domain of c‐Raf‐1: binding studies to fluorescently labelled H‐Ras

FEBS Letters

Seidel, Ralf P¹  Goody, Roger S¹  Sydor, Jens R¹  Engelhard, Martin¹ 

[1] Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Postfach 50024, 44202 Dortmund, Germany

关键词: Cell-free expression;    Transient kinetics;    Ras-binding domain of c-Raf-1;    H-Ras;    Fluorescence;    GppNHp;    guanyl-5′-yl-imidodiphosphate;    mant;    2′;    3′-(N-methylanthraniloyl);    a fluorophore attached at the 2′- or 3′-position;    RBD;    Ras-binding domain of c-Raf-1;    RBD/H;    RBD with an additional His-tag at the C-terminus;    PVDF;    polyvinylidene difluoride;   

DOI  :  10.1016/S0014-5793(99)00633-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2′,3′-(N-methylanthraniloyl) fluorescence of 2′,3′-(N-methylanthraniloyl) guanyl-5′-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni²⁺ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.

【 授权许可】

Unknown