Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies

Abstract

Complex protein networks are involved in nearly all cellular processes. To uncover these vast networks of protein interactions, various high-throughput screening technologies have been developed. Over the last decade, bimolecular fluorescence complementation (BiFC) assay has been widely used to detect protein-protein interactions (PPIs) in living cells. This technique is based on the reconstitution of a fluorescent protein in vivo. Easy quantification of the BiFC signals allows effective cell-based high-throughput screenings for protein-binding partners and drugs that modulate PPIs. Recently, with the development of large screening libraries, BiFC has been effectively applied for genome-wide PPI studies and has uncovered novel protein interactions, providing new insight into protein functions. In this review, we describe the development of reagents and methods used for BiFC-based screens in yeast, plants, and mammalian cells. We also discuss the advantages and drawbacks of these methods and highlight the application of BiFC in large-scale studies.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

“…While many of these indicators are based on FRET, protein fragment complementation [84] is another popular approach. Protein fragment complementation requires two non-fluorescent FP fragments that do not self-associate on their own, but that can recombine to form a functional FP if brought into close proximity with the help of an interacting pair of proteins.…”

Detection of protein–protein interactions at the septin collar inSaccharomyces cerevisiaeusing a tripartite split-GFP system

“…Among the PCA approaches, bimolecular fluorescence complementation (BiFC) has gained rather wide acceptance (Magliery et al ., 2005; Kerppola, 2009; Ohashi and Mizuno, 2014; Miller et al ., 2015) because it can detect even weak or transient interactions for the reason that once two associating proteins bring the two halves of the fluorescent reporter protein together, reconstitution of the reporter stabilizes the complex. In addition, BiFC does not require cell fixation, cell lysis, or any special treatment with dyes or other reagents.…”

Techniques for the analysis of protein-protein interactions in vivo

“…Over the past decade, various laboratories have coupled BiFC analysis with flow cytometry for analysis, sorting (fluorescence-activated cell sorting), or both (Miller et al, 2015). Flow cytometry is a technique used to examine a population of particles or cells in free suspension, one by one, typically by collecting and analyzing fluorescence and scattered light.…”

“…According to Hu et al (2002), the split fluorescent protein fragments are estimated to assemble with a half-time of 60 s (Hu et al, 2002). Due to the irreversibility of BiFC, it has been proposed that BiFC might be able to capture an enzyme interacting with its substrate (Morell et al, 2007;Miller et al, 2015). In this case, one can expect very weak (infrequent) signals if we presume that the PCA is dominated by the POIs.…”