title: Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.
abstract: A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Gallo
authors.affiliation: Molecular Biosensor and Imaging Center, Pittsburgh, PA 15213, USA.
authors.forename: Avin C
authors.initials: AC
authors.lastname: Snyder
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA Molecular Biosensor and Imaging Center, Pittsburgh, PA 15213, USA jarvik@cmu.edu.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jarvik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D057127:Single-Chain Antibodies
keywords: FAP; biosensor; fluorescence; fluorogen; scFv
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