Fluorogen Activating Protein - Affibody Probes: Modular, No-wash Measurement of Epidermal Growth Factor Receptors.

Bioconjugate chemistry

PubMedID: 25490520

Wang Y, Telmer CA, Schmidt BF, Franke JD, Ort S, Arndt-Jovin DJ, Bruchez MP. Fluorogen Activating Protein - Affibody Probes: Modular, No-wash Measurement of Epidermal Growth Factor Receptors. Bioconjug Chem. 2014;.
Fluorescent affinity reagents are essential for dynamic live cell imaging studies. Antibodies are the conventional reagents in endogenous protein labeling, but the utility of the conjugate is limited by the conjugated dye and new conjugates must be prepared for different purposes. Recently, we developed a new technology based on noncovalent binding and almost instantaneous activation of a series of fluorogenic dyes by a single genetically encoded protein tag. Here we present the characterization of a new platform for affinity probes, in which a protein domain (FAP dL5**) capable of binding to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the EGFR binding affibody ZEGFR:1907, resulting in a recombinant and fluorogenic labeling reagent. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP-affibody fusions produced thousands-fold fluorescence enhancements, with high binding affinity and fast association rate. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nM surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. We have shown that this probe can not only be used in tracing surface receptors trafficking under stimulation, but also the sequential addition of the probe and dyes allowed the differentiation between surface and endocytotic pools of receptors to reveal the dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes for labeling of endogenous proteins on living cells.