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

ISSN: 1875-5305 (Online)
ISSN: 0929-8665 (Print)


Volume 21, 12 Issues, 2014


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Protein & Peptide Letters

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

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

Affiliation: Carnegie Mellon University 4400 Fifth Ave., MI 257 Pittsburgh, Pennsylvania, 15213

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 non-canonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery


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Article Details

Volume: 21
First Page: 1
Page Count: 1
DOI: 10.2174/0929866521666140616121800
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