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    • 5. 发明申请
    • SCREENING PROCESS FOR THE DETECTION AND CHARACTERIZATION OF PROTEIN-PROTEIN-INTERACTIONS IN VIVO BY FLUORESCENCE CROSS CORRELATION SPECTROSCOPY
    • 通过荧光检测和表征蛋白质 - 蛋白质相互作用的筛选过程交叉相关光谱
    • WO2006037622A1
    • 2006-04-13
    • PCT/EP2005/010714
    • 2005-10-05
    • DKFZ DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES ÖFFENTLICHEN RECHTSBAUDENDISTEL, NinaLANGOWSKI, Jörg
    • BAUDENDISTEL, NinaLANGOWSKI, Jörg
    • G01N33/58G01N33/542
    • G01N33/583G01N33/542G01N2333/43595
    • In a first aspect, the invention concerns a screening process for the detection of protein­protein-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 (FCC S) 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.
    • 在第一方面,本发明涉及用于检测体内蛋白质蛋白相互作用的筛选方法,包括以下步骤:a)表达包含与第一自身荧光蛋白融合并表达第二融合蛋白的第一蛋白质的第一融合蛋白 包括与细胞中的第二自发荧光蛋白融合的第二蛋白质,其中所述融合蛋白的第一和第二自发荧光蛋白在其可检测的光物理参数中的至少一个彼此不同,所述可见光物理参数包括不同的发射波长,不同的荧光寿命 以及自发荧光蛋白的发射光的不同极化,b)通过检测步骤a的细胞中第一和第二自发荧光蛋白的重合发生的荧光信号来鉴定显示与第一蛋白质的蛋白质 - 蛋白质相互作用的第二蛋白质 ),通过测量个体的强度 第一和第二自发荧光蛋白的荧光信号,其中第一和第二自发荧光蛋白的荧光信号通过它们的不同的发射波长,它们的不同的荧光寿命时间或不同的荧光寿命 这两种自发荧光蛋白发出的光。 在第二方面,本发明涉及在至少一种可检测的光物理参数中彼此不同的两种单独的自体荧光蛋白用于通过体内荧光互相关光谱法(FCC S)检测蛋白质 - 蛋白质相互作用的用途。 因此,在两个个体自发荧光蛋白之间不同的至少一个可检测的光物理参数是不同的发射波长,不同的荧光寿命或不同的极化。