Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy.
Ras proto-oncogenes play a central role in cell proliferation by the regulation of signal transduction pathways from receptors of the outer cell membrane to the nucleus via the activation of transcription factors. Wild-type Ras cycles between the activated GTP-bound and the inactivated GDP-bound state, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-1 protein kinase. Mutations of ras resulting in the loss of the intrinsic GTPase activity result in autonomous proliferation. Mutated Ras is found in a variety of human tumors. Therefore, monitoring of GTP-loaded conformation of Ras related proteins could be utilised in cancer diagnosis. To develop a fluorescence based bioassay we have coupled the gene for the N-terminal Ras binding domain (RBD) of Raf-1 protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently investigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of fluorescently labeled molecules. Molecular interactions increase the molecular weight and influence the diffusion time of RBDGFP. FCS diffusion value of the recombinant protein was in coincidence with the molecular weight of the construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transition times indicated the interaction of RBDGFP with its corresponding antibody. Suboptimal binding of the fusion protein to activated Ras, how ever, resulted in a modest influence on the diffusion value. Taken together our rational design and construct shows the way for a ready characterisation of novel GFP-connected fusion proteins employing FCS.
Trier
U
U
Institut für Pharmazie, Freie Universität Berlin, Germany.
Olah
Z
Z
Kleuser
B
B
Schäfer-Korting
M
M
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; D049452:Green Fluorescent Proteins; C048139:Coomassie blue; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)