Fluorogen Activating Protein鈥揂ffibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

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• 作者：Yi Wang ; Cheryl A. Telmer ; Brigitte F. Schmidt ; Josef D. Franke ; Stephan Ort ; Donna J. Arndt-Jovin ; Marcel P. Bruchez
• 刊名：Bioconjugate Chemistry
• 出版年：2015
• 出版时间：January 21, 2015
• 年：2015
• 卷：26
• 期：1
• 页码：137-144
• 全文大小：375K
• ISSN：1520-4812

文摘

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 malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody Z_EGFR: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鈥揳ffibody 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 K_d values were observed with the double-Z_EGFR: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.