chromophore, and the resulting signal can be determined by calculating the enhanced acceptor emission and the reduced donor
emission [1, 2]. An alternative method for the study of in vivo
PPIs is the protein-fragment complementation assay (PCA),
which entails at least two-part split bimolecular fragments that
enable direct visualization of a reporter activity correlated with
complex formation [3]. The reporters comprise enzymes, such as
dihydroorotate reductase, β-galactosidase, and the firefly and Gaussia luciferases, and the fluorescent proteins (FPs) referred as bimolecular fluorescence complementation (BiFC) or “split-FP”
technology [4–6]. The ease of carrying out BiFC experiments
using a fluorescence microscope with confocal or epi-illumination
reinforces the merit of this method.
In principle, the 11-stranded β-barrel of FP or its variants are
split into the N- and C-terminal halves at different positions, each
fused to a protein of interest, allowing to test the binary interaction
of protein pairs by reconstitution of a functional fluorophore
[5, 7]. In other words, unlike split-enzyme assays, the implementation of BiFC is relatively simple by measuring the fluorescence
without the need of externally added substrate and extensive calibration [5, 7]. However, because of the intrinsic properties of
BiFC—the slow and nearly irreversible formation of the reconstituted fluorophore, BiFC is poor in real-time detection [1, 6]. Moreover, high expression of two nonfluorescent fragments of a FP can
lead to spontaneous co-folding of split-proteins moieties and thus
fluorescence reconstitution, which accumulates over time and
increases background signals [8, 9]. Nevertheless, BiFC offers a
great advantage to detect low affinity and/or transient protein
complexes by facilitating the readout of complex formation,
which is not readily achieved by other methods [1, 6].
Compared with the two GFP variants, peak-shift GFP (sGFP or
GFP5; S65T) and enhanced GFP (EGFP; F64L/S65T) [10] that
are commonly used in the plant science community, superfolder
GFP (spGFP) exhibits enhanced folding and maturation efficiency,
tolerance of circular permutation and resistance to oxidizing environment, but is only applied in a few plant studies [11–14]. Of
note, sfGFP was shown to be brighter than sGFP and EGFP in
transfected Arabidopsis protoplasts [12]. Likewise, the fluorescence
intensity of sfGFP-based BiFC was stronger than the other four
split-GFP variants tested [12], revealing an intriguing potential of
using split-sfGFP in analyzing PPIs in planta. By introducing
several point mutations into sfGFP, Cabantous et al. developed a
tripartite split-sfGFP method to overcome the poor folding and
background fluorescence signals arising from the self-assembly of
bipartite BiFC, thus enhancing the signal-to-noise ratio [15]. In
the tripartite split-sfGFP system, the GFP β-strand 10 (S10,
20 amino acids) and the β-strand 11 (S11, 19 amino acids) fragments are each fused to the N- and the C-terminal of the protein
324
Tzu-Yin Liu
emission [1, 2]. An alternative method for the study of in vivo
PPIs is the protein-fragment complementation assay (PCA),
which entails at least two-part split bimolecular fragments that
enable direct visualization of a reporter activity correlated with
complex formation [3]. The reporters comprise enzymes, such as
dihydroorotate reductase, β-galactosidase, and the firefly and Gaussia luciferases, and the fluorescent proteins (FPs) referred as bimolecular fluorescence complementation (BiFC) or “split-FP”
technology [4–6]. The ease of carrying out BiFC experiments
using a fluorescence microscope with confocal or epi-illumination
reinforces the merit of this method.
In principle, the 11-stranded β-barrel of FP or its variants are
split into the N- and C-terminal halves at different positions, each
fused to a protein of interest, allowing to test the binary interaction
of protein pairs by reconstitution of a functional fluorophore
[5, 7]. In other words, unlike split-enzyme assays, the implementation of BiFC is relatively simple by measuring the fluorescence
without the need of externally added substrate and extensive calibration [5, 7]. However, because of the intrinsic properties of
BiFC—the slow and nearly irreversible formation of the reconstituted fluorophore, BiFC is poor in real-time detection [1, 6]. Moreover, high expression of two nonfluorescent fragments of a FP can
lead to spontaneous co-folding of split-proteins moieties and thus
fluorescence reconstitution, which accumulates over time and
increases background signals [8, 9]. Nevertheless, BiFC offers a
great advantage to detect low affinity and/or transient protein
complexes by facilitating the readout of complex formation,
which is not readily achieved by other methods [1, 6].
Compared with the two GFP variants, peak-shift GFP (sGFP or
GFP5; S65T) and enhanced GFP (EGFP; F64L/S65T) [10] that
are commonly used in the plant science community, superfolder
GFP (spGFP) exhibits enhanced folding and maturation efficiency,
tolerance of circular permutation and resistance to oxidizing environment, but is only applied in a few plant studies [11–14]. Of
note, sfGFP was shown to be brighter than sGFP and EGFP in
transfected Arabidopsis protoplasts [12]. Likewise, the fluorescence
intensity of sfGFP-based BiFC was stronger than the other four
split-GFP variants tested [12], revealing an intriguing potential of
using split-sfGFP in analyzing PPIs in planta. By introducing
several point mutations into sfGFP, Cabantous et al. developed a
tripartite split-sfGFP method to overcome the poor folding and
background fluorescence signals arising from the self-assembly of
bipartite BiFC, thus enhancing the signal-to-noise ratio [15]. In
the tripartite split-sfGFP system, the GFP β-strand 10 (S10,
20 amino acids) and the β-strand 11 (S11, 19 amino acids) fragments are each fused to the N- and the C-terminal of the protein
324
Tzu-Yin Liu