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

It has previously been shown that the transient kinetics of the interaction between the Res-binding domain of c-Raf-1 and the proto-oncoprotein Ras ca n be followed by stopped-flow measurements using the 2',3'-(N-methylanthran iloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodipho sphate-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 a mounts for biochemical and biophysical investigations, The results obtained describe the first example of a cell-free synthesized protein which has be en used for stopped-flow measurements to determine the transient kinetics o f protein-protein interactions with an effector, (C) 1999 Federation of Eur opean Biochemical Societies.