A fluorescence-based binding assay for proteins using the cell surface as a sensing platform

Abstract

Protein–protein interaction (PPI) analysis is very important for elucidating the functions of proteins because many proteins execute their functions in living cells by interacting with one another. In PPI analysis, methods using the sensor chips are widely employed to obtain quantitative data. However, these methods require that the target proteins be immobilized on the sensor chips, and the immobilization processes can affect the binding of the target proteins to their binding partners. In the present work, we propose a PPI analysis system in which the surface of the living cells is utilized as a sensing platform. In our approach, the target protein is displayed on the cell surface by expressing it as a fusion protein with a membrane protein, and the PPI analysis is then conducted by applying its binding partner labeled with a fluorescent dye to the cell surface. We have constructed a model of this binding analysis system using the interaction between biotin protein ligase (BPL) and biotin carboxyl carrier protein (BCCP), where BCCP was displayed on the cell surface and BPL labeled with fluorescein was applied to the cell surface. Here, a red fluorescent protein, mApple, was attached to the C-terminus of the fusion protein of BCCP with a membrane protein. We evaluated the binding level of the labeled BPL by using the intensity ratios of fluorescence from fluorescein to that from mApple. We found that the binding level of the labeled BPL was stably evaluated at least across 60 min observation period and estimated the binding dissociation constant between BPL and BCCP by equilibrium analysis to be 0.33 ± 0.05 μM.

Graphical abstract