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    • 2. 发明申请
    • PROTEIN-PROTEIN INTERACTION DETECTION SYSTEM USING FLUORESCENT PROTEIN MICRODOMAINS
    • 荧光蛋白微球的蛋白质 - 蛋白质相互作用检测系统
    • WO2006062882A2
    • 2006-06-15
    • PCT/US2005/043874
    • 2005-12-03
    • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA LOS ALAMOS NATIONAL LABORATORY
    • WALDO, Geoffrey, S.CABANTOUS, Stephanie
    • C12Q1/00
    • C40B30/04G01N33/5005G01N33/5008G01N33/542G01N33/582G01N33/6845
    • The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (β-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (β-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GPF amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.
    • 本发明提供基于荧光和发色蛋白质的工程化片段的蛋白质标记和相互作用检测系统,其需要融合的相互作用多肽来驱动片段的缔合,并且进一步是可溶的和稳定的,并且不 改变它们所融合的多肽的溶解度。 在一个实施方案中,测试蛋白X与GFP的十六个氨基酸片段(β-链10,氨基酸198-214)融合,工程化为不干扰融合蛋白溶解性。 第二种测试蛋白Y与GFP的16个氨基酸片段(β-链11,氨基酸215-230)融合,工程化为不干扰融合蛋白的溶解性。 当X和Y相互作用时,它们使GFP链接近,并通过与由GPF氨基酸1-198(链1-9)组成的第三个GFP片段互补来检测。 当GFP链10和11通过蛋白质X和Y的相互作用结合在一起时,它们与GFP链1-9自发缔合,导致结构互补,折叠和伴随的GFP荧光。