Bimolecular Fluorescence Complementation to Visualize Protein–Protein Interactions in Human Cells Based on Gateway Cloning Technology

Abstract

Bimolecular fluorescence complementation (BiFC) is a powerful and sensitive tool to discover new protein–protein interactions (PPIs). It enables visualization and localization of protein–protein interactions (PPIs) in living cells. The idea behind BiFC is to split a fluorescent protein, for example yellow fluorescent protein (YFP), into two parts that are unable to emit fluorescent signal on their own. Therefore, in order to regain fluorescence the split protein fragments must establish close proximity. This is accomplished by fusing the split fragments to proteins that are postulated to interact, and expressing them in living cells. Subsequently, detection of fluorescence indicates interaction of given proteins. Since complementation is practically irreversible it can capture weak and transient interactions. Using suitable vectors for human protein expression, thus avoiding viral cell transfection, we introduced Gateway-based cloning features to the BiFC system, thereby enabling time efficient vector construction in order to maximize the full potential of the BiFC approach to investigate many protein–protein interactions in a high-throughput fashion. This protocol explains steps in a typical protein–protein interaction survey, from the vector selection, cell transfection, and visualization of the fluorescent signal.

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