Screening process for the detection and characterization of protein-protein-interactions in vivo by fluorescence cross correlation spectroscopy

摘要

In a first aspect, the invention concerns a screening process for the detection of protein-protein-interactions in vivo comprising the following steps:a) expressing a first fusion protein comprising a first protein that is fused with a first autofluorescent protein and expressing a second fusion protein comprising a second protein that is fused with a second autofluorescent protein in a cell, wherein the first and the second autofluorescent proteins of the fusion proteins differ from one another in at least one of their detectable photophysical parameters comprising different emission wavelengthes, different fluorescence life times and different polarisations of the emitted light of the autofluorescent proteins,b) identifying the second protein that shows a protein-protein-interaction with the first protein by detecting coincidently occurring fluorescence signals of the first and the second autofluorescent proteins in the cells of step a), by measuring the intensities of the individual fluorescence signals of the first and of the second autofluorescent proteins, wherein the fluorescence signals of the first and of the second autofluorescent protein are differentiated from each other either by their different emission wavelengthes, by their different fluorescence life times or by the different polarization of the light emitted by those two autofluorescent proteins.;In a second aspect, the invention concerns the use of two individual autofluorescent proteins, which differ from each another in at least one detectable photophysical parameter, for detecting protein-protein interactions by fluorescence cross correlation spectroscopy (FCCS) in vivo. Hereby, the at least one detectable photophysical parameter which differs between the two individual autofluorescent proteins are either different emission wavelengthes, different fluorescence life times or different polarisations.