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Abstract:
Fluorescence is essential for dynamic live cell
imaging, and affinity reagents are required for quantification of
endogenous proteins. Various fluorescent dyes can report on
different aspects of biological trafficking, but must be
independently conjugated to affinity reagents and characterized
for specific biological readouts. Here we present the
characterization of a new modular platform for small anti-
EGFR affinity probes for studying rapid changes in receptor
pools. A protein domain (FAP dL5**) that binds to malachitegreen
(MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact
EGFR binding affibody ZEGFR:1907. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules. In vitro
fluorescence assays demonstrated that the binding of these dyes to the FAP−affibody fusions produced thousand-fold
fluorescence enhancements, with high binding affinity and fast association rates. Flow cytometry assays and fluorescence
microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and
low nanomolar surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting
fluorogens (dyedrons) significantly improved the detected fluorescence signal. Altering the order of addition of the ligand, probe,
and dyes allowed differentiation between surface and endocytotic pools of receptors to reveal the rapid dynamics of endocytic
trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that
label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.
