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 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.
Sydor
J R
JR
Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Dortmund, Germany.
Seidel
R P
RP
Goody
R S
RS
Engelhard
M
M
D001665:Binding Sites; D002474:Cell-Free System; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018631:ras Proteins / Q000378:metabolism*
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins