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RESEARCH PRODUCT

A Protein-Interaction Array Inside a Living Cell

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description

Cell phenotype is determined by protein network states that are maintained by the dynamics of multiple protein interactions.1 Fluorescence microscopy approaches that measure protein interactions in individual cells, such as by Forster resonant energy transfer (FRET), are limited by the spectral separation of fluorophores and thus are most suitable to analyze a single protein interaction in a given cell. However, analysis of correlations between multiple protein interactions is required to uncover the interdependence of protein reactions in dynamic signal networks. Available protein-array technologies enable the parallel analysis of interacting proteins from cell extracts, however, they can only provide a single snapshot of dynamic interaction networks. Moreover, because of the high level of variance from cell to cell in protein expression levels and reaction state, cell extracts only provide an average measure of protein interaction states and therefore the detection of the relations between proteins is blurred. As an intermediate step, a visual immunoprecipitation assay was developed that allowed direct observation of multiple, dynamic protein interactions on immobilized, distinguishable beads in cell extracts.2 A microstructuring approach allowed for analysis of the interaction of one naturally occurring receptor type with one of its interaction partners inside cells.3 To analyze multiple protein interactions inside a single living cell, multiple receptors must be arranged in a defined pattern to distinguish their identity. Herein, we developed a general strategy to generate protein arrays with multiple arbitrary bait proteins by way of artificial-receptor constructs at sub-cellular feature size and applied this technology to simultaneously measure two-protein interaction kinetics inside an individual living cell.