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.

Fluorogen-Activating Proteins: Next-Generation Fluorescence Probes for Biological Research

“…The added expression of the same scFvs from the same gene construct resulted in the linear amplification of the fluorescence signal, thereby enhancing signal brightness. 102 A similar tandem assembly strategy annexed two different fluorogen-activating scFvs for cellsurface expression. This resulted in alternate color detection of FAP-tagged proteins, or simultaneous multicolor detection when both different-colored fluorogens were presented.…”

“…102 For cases where different FAP/fluorogen complexes share fluorescence spectra overlap, tandem FAP scFv constructs of these proteins result in high efficiency energy transfer between the tandem donor scFv/fluorogen and the acceptor scFv/ fluorogen complexes. 102 This engineering strategy expanded the Stokes shift of donor scFv/fluorogen into longer wavelengths. 102 A limitation of tandem scFv constructs, however, is the restriction in size with each incremental scFv addition per gene construct.…”

“…102 This engineering strategy expanded the Stokes shift of donor scFv/fluorogen into longer wavelengths. 102 A limitation of tandem scFv constructs, however, is the restriction in size with each incremental scFv addition per gene construct. Additionally, the incremental addition of scFvs may lead into incorrect expression, folding, and/or localization of a target cellular protein to be studied.…”

“…An example includes the genetic assembly of the same FAP scFvs annexed via short linkers for cell-surface expression. The added expression of the same scFvs from the same gene construct resulted in the linear amplification of the fluorescence signal, thereby enhancing signal brightness . A similar tandem assembly strategy annexed two different fluorogen-activating scFvs for cell-surface expression.…”

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

“…Some breakthroughs occurred in the covalent bio-conjugate field, where the same target ligand for capture may be chemically coupled with unique color fluorophores, a very similar approach to using commercially labeled antibodies for labeling cells (Chen et al, 2007;Kosaka et al, 2009;Vivero-Pol et al, 2005;Lee et al, 2010;Liu et al, 2014;Uttamapinant et al, 2010;Wombacher et al, 2010;Yao et al, 2012). Likewise, other groups have utilized bio-conjugate platforms based on tandem dye interactions that have resulted in fluorescence resonance energy transfer (FRET), a donor-acceptor approach that amplifies the Stokes shift of a molecule resulting in fluorescence emissions at longer wavelengths (Brun et al, 2009(Brun et al, , 2011Gallo et al, 2015;Pham et al, 2015;Rajapakse et al, 2010;Robers et al, 2009;Saunders et al, 2014;Yushchenko et al, 2012;Zürn et al, 2010). As a result, we find that current methods prove lacking in multi-color detection and real-time signal modulation.…”