Vasilev Kalin V, Gallo Eugenio, Shank Nathaniel, Jarvik Jonathan W
Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, Pennsylvania 15213, USA.
Comb Chem High Throughput Screen. 2016;19(5):392-9. doi: 10.2174/1386207319666160408150320.
We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.
我们描述了一种用于报告活细胞培养中细胞表面蛋白之间接近程度的新型生物传感器系统。该生物传感器利用了最近开发的荧光原激活蛋白（FAP），这种蛋白只有在与原本无荧光的荧光原分子结合时才会发出荧光。为了证明该方法的可行性，将两种重组雷帕霉素结合蛋白表达为HeLa细胞中的单次跨膜质膜蛋白；其中一种蛋白（scAvd-FRB）带有细胞外抗生物素蛋白标签；另一种（HL1-TO1-FKBP）带有细胞外FAP。用一种由生物素通过聚乙二醇接头连接到二甲基吲哚红（DIR）荧光原组成的膜不可渗透二价配体（生物素-PEG2000-DIR）孵育细胞，从而将荧光原与scAvd-FRB融合蛋白相连。添加雷帕霉素可促进FKBP-FRB二聚化，从而使FAP与相连的荧光原紧密接近，导致DIR荧光显著增加。我们将这种新的接近度检测方法称为TEFLA，即 tethered fluorogen assay（相连荧光原检测法）。
