A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families

Title:A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families

Volume: 21 Issue: 12

Author(s): Eugenio Gallo, Sophia Wienbar, Avin C. Snyder, Kalin V. Vasilev, Bruce A. Armitage and Jonathan W. Jarvik

Affiliation:

Keywords: Biosensor, FAP, fluorescence, fluorogen, scFv.

Abstract: 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.

Cite this article as:

Gallo Eugenio, Wienbar Sophia, Snyder C. Avin, Vasilev V. Kalin, Armitage A. Bruce and Jarvik W. Jonathan, A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families, Protein & Peptide Letters 2014; 21 (12) . https://dx.doi.org/10.2174/0929866521666140616121800