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Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives
Yutaka Kodama1 and Chang-Deng Hu2
1Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan
2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, USA
BioTechniques, Vol. 53, No. 5, November 2012, pp. 285–298
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Over the past decade, bimolecular fluorescence complementation (BiFC) has emerged as a key technique to visualize protein-protein interactions in a variety of model organisms. The BiFC assay is based on reconstitution of an intact fluorescent protein when two complementary non-fluorescent fragments are brought together by a pair of interacting proteins. While the originally reported BiFC method has enabled the study of many protein-protein interactions, increasing demands to visualize protein-protein interactions under various physiological conditions have not only prompted a series of recent BiFC technology improvements, but also stimulated interest in developing completely new approaches. Here we review current BiFC technology, focusing on the development and improvement of BiFC systems, the understanding of split sites in fluorescent proteins, and enhancements in the signal-to-noise ratio. In addition, we provide perspectives on possible future improvements of the technique.

The dawn of BiFC technology

Protein-protein interactions (PPIs) play important roles in various biological processes. In the post-genome era, numerous PPI networks have been identified in various organisms, such as humans, worms, yeast, and plants (1-4). To verify these PPIs identified from genome-wide studies, numerous methods, including canonical yeast two-hybrid assay, in vitro pull-down assay, in vivo immunoprecipitation assay, fluorescence resonance energy transfer (FRET) assay, bioluminescence resonance energy transfer (BRET) assay, and bimolecular fluorescence complementation (BiFC) assay, have been used. Among these methods, the fluorescent protein-based BiFC assay has become widely accepted over the past decade.

The BiFC assay is based on structural complementation between two non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein. Fluorescent proteins, e.g., green fluorescent protein (GFP), consist of 11 antiparallel β-strands forming a β-barrel, with an α-helix inside and several short helical structures (5) (Figure 1, A and B). The chromophore, located in the α-helix within the β-barrel structure, is chemically formed by three residues (6) (Figure 1, A and B). For BiFC analysis, several studies have demonstrated that fluorescent proteins can be split at a loop or within a β-stand (Figure 1, A and B). The two resulting non-fluorescent fragments can then be fused to proteins of interest that may interact (Figure 1C). If the proteins do interact, the non-fluorescent fragments are brought into close proximity and reconstitute an intact fluorescent protein that can be imaged using any fluorescence microscope (Figure 1C). Acquired images can then be used for quantitative analysis. The fluorescence complementation of GFP was initially demonstrated in vitro and in Escherichia coli by using two antiparallel leucine zippers fused to the two non-fluorescent fragments of a split GFP (7). Subsequently, an enhanced yellow fluorescent protein (EYFP)-based BiFC assay was successfully developed for visualizing protein interactions between the basic leucine zipper (bZIP) and NF-κB family of proteins in living mammalian cells (8). This study uncovered interesting localization patterns for several bZIP dimers and demonstrated crosstalk between Fos and Jun proteins and NF-κB family proteins (8). These results stimulated more widespread interest in applying BiFC technology to the visualization of PPIs in mammalian cells, bacteria, worms and plants (9).

Various fluorescence complementation-based technologies also have been developed to visualize molecular events involving more than two interacting proteins (10). The availability of these fluorescence complementation-based assays has enabled visualization of protein aggregation, protein folding, protein topology, conformational change, multiple protein complexes (multicolor BiFC), and ternary and tetramer complexes (BiFC-FRET and BiFC-BRET) in an unprecedented manner (10). In the meantime, several laboratories continue to add new fluorescent proteins to the BiFC toolbox. In this review, we will introduce recent improvements of BiFC technology and provide our thoughts on future development.

New fluorescent proteins added to the BiFC toolbox

Many fluorescent proteins possessing distinct spectral and physicochemical properties have been discovered or developed since 1994 (11-14). Among these, 15 fluorescent proteins have been found to work with fluorescence complementation assays (Table 1).


The development and subsequent refinement of BiFC-supportive fluorescent proteins can be broadly classified into three stages (Figure 2). From 2000–2003, GFP from the jellyfish Aequorea victoria and its variants (EGFP, EBFP, ECFP, EYFP) were initially employed for BiFC (Figure 2; Table 1). The use of spectral variants was largely driven by the desire to perform multicolor analysis (15-20). These early results using GFP and its spectral variants provided proof of principle that most, if not all, fluorescent proteins could be used for BiFC assays (Figure 2, stage 1). Following the initial developments with GFP, it was realized that the sensitivity of GFP and its variants to the environment prohibited further application of BiFC under physiological conditions. Hence, a search for fluorescent proteins that would yield a bright signal under physiological culture conditions was initiated (Figure 2, stage 2). During this time (2004–2008), fluorescent proteins such as frGFP, Citrine, Venus, and Cerulean were demonstrated to be BiFC competent (21, 22, Table 1). The similar development of brighter BiFC systems has continued to date, e.g., sfGFP- and GFP-S65T-based BiFC systems (23, 24) (Table 1), owing to the availability of improved fluorescent proteins engineered by several labs (25-30). Starting in 2006, researchers began exploring the use of fluorescent proteins with unique spectral and physicochemical properties for BiFC analysis (Figure 2, stage 3). These BiFC-supportive fluorescent proteins provide optical tools for visualizing PPIs in living animals. For example, several red fluorescent proteins have been demonstrated to support BiFC analysis, including red fluorescent protein (RFP) variants from Discosoma sp., i.e., monomeric RFP (mRFP), mCherry, DsRed monomer, and mKate (20, 31-33) (Table 1). These longer wavelength RFP variants enable visualization of PPIs in deep tissues. Given that some of these fluorescent proteins have been used in whole body imaging, it is likely that PPIs can be visualized in small animals using RFP-based BiFC systems.

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