Fluorogen‐activating‐proteins as universal affinity biosensors for immunodetection

Abstract

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching.

Fluorogen-activating proteins: beyond classical fluorescent proteins

“…However, in all cases introduced above, the FAP was expressed from a recombinant gene that encoded a protein fusion between the FAP and the protein of interest. This approach results in two significant setbacks 61 : (1) time and labor regarding quality control and generation of each recombinant protein, and (2) artificial protein expression from a nonnative promoter, typically altering protein regulation and abundance in the cell. Therefore, careful upstream studies of their toxicity and their influence on cellular processes is still required.…”

A Bifunctional Converter: Fluorescein Quenching scFv/Fluorogen Activating Protein for Photostability and Improved Signal to Noise in Fluorescence Experiments

“…17 Compared to these approaches, an advantage of the FITC-E2–dL5 converter is that FITC-labeled antibodies are typically conjugated with multiple FITC molecules per protein, allowing more monovalent FITC-E2–dL5 and fluorogen molecules to bind without promoting cross-linking or aggregation (e.g., streptavidin or bivalent antibodies). This is an approach to increase the signal beyond that seen with a one MG to one protein approach in a FAP-fused protein.…”

“…Alternate approaches to secondary labeling systems involving fluorogens include genetic addition of fluorogen activating protein directly to protein G or streptavidin to label primary antibodies or biotinylated proteins with fluorogens. 17 Compared to these approaches, an advantage of the FITC-E2–dL5 converter is that FITC-labeled antibodies are typically conjugated with multiple FITC molecules per protein, allowing more monovalent FITC-E2–dL5 and fluorogen molecules to bind without promoting cross-linking or aggregation (e.g., streptavidin or bivalent antibodies). This is an approach to increase the signal beyond that seen with a one MG to one protein approach in a FAP-fused protein.…”

Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation

“…A given FAP can activate the fluorescence of multiple chemically-related fluorogens, each with distinct biochemical and spectral properties 4,[10][11][12][13][14][15] . The dL5**/MG pair used here, for instance, emits light in the far red spectrum where tissue autofluorescence and phototoxicity are low 1,5 .…”