Gallo Eugenio, Wienbar Sophia, Snyder Avin C, Vasilev Kalin V, Armitage Bruce A, Jarvik Jonathan W
Carnegie Mellon University, 4400 Fifth Ave., MI 257, Pittsburgh, Pennsylvania, 15213, USA.
Protein Pept Lett. 2014;21(12):1289-94.
Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.
当前生物蛋白质发现领域的进展采用了荧光检测的双组分方法，其中发色团和支架是解偶联的。一种这样的技术，称为荧光激活蛋白（FAPs），由针对小分子有机化合物（荧光原）筛选出的单链可变片段（scFvs）组成，这些小分子在溶液中无荧光，但与scFv结合时具有高荧光性。在特殊情况下，一个scFv可能会激活来自单一化学家族的类似荧光原。在本报告中，我们鉴定了一种对来自两个结构不同家族的多种荧光原具有荧光活性的scFv生物传感器。体外分析显示在亚微摩尔范围内存在高度选择性的scFv-配体相互作用。此外，每个scFv-荧光原复合物都具有独特的激发和发射光谱，这使得生物传感器具有更宽的检测限。进一步分析表明，无论化学家族如何，配体激活都发生在一个共同的scFv结合区域，该区域具有灵活性，但对荧光原结合具有选择性。作为哺乳动物细胞表面的蛋白质报告分子，scFv显示出明亮的信号检测和最小的背景。此外，当与G蛋白偶联受体标记时，我们通过多色荧光追踪观察到激动剂依赖性信号传导，导致蛋白质从细胞表面转运至内体。总之，本报告揭示了一种具有高亲和力配体和多通道荧光检测特性的非经典scFv生物传感器，从而为细胞蛋白质发现提供了更多机会。
