Revealing the formation, distribution, and dynamics of PPIs in situ in living cells can deepen our understanding of how cells coordinate their multiple components to fulfill their activities. Here we improved the method of BiFC to detect and visual PPIs in living cells. Compared with other methods, BiFC is superior in the following aspects. First, BiFC does not require structural information of interacting proteins, the addition of dye or fluorophore staining, etc. Merely the complementation of two fluorophore segments (N-terminal and C-terminal ) will produce the fluorescence and reveal the information about the interaction of the proteins . In addition to the detection of PPIs, it also describes the spatial localization of interactions at the subcellular level, i.e., where the functional contact between two proteins takes place in a cell. Third, BiFC does not rely on energy transfer between fluorophores as seen in FRET and BRET assays but it can be detected by employing conventional fluorescence microscopy. And fourth, the intensity of the fluorescence signal is proportional to the strength of protein-protein interactions.
In our study, we introduced TagBFP2 into BiFC assay for the first time. Based on stereochemical structure of fluorescent proteins, we speculated that splitting at the disorder loop region would scarcely impact on their native structure and physiology. Hence, we tried above-mentioned three sites of TagBFP2 in this study. The results proved our hypothesis. As for other fluorescent proteins, it is necessary to conduct more experiments to estimate our conjecture. Between the interest protein and non-fluorescent fragment, we designed a flexible linker (2x GGGGS), which would avoid interplay between the two components. Compared with BiFC based on EYFP, TagBFP2 exhibited the higher signal-to-noise ratio, which help us distinguish the PPIs induced fluorescence from the noise signal.
Despite the advantages of BiFC assay, it could not be ignored that the reconstitution of BiFC complexes is irreversible . In some cases, this property offers a great advantage to detect low affinity and transient PPIs by facilitating the readout of complex formation, which is not readily achieved by other methods . Nevertheless, the persistent complexes hinder us to study the dynamics of signaling networks. In other words, BiFC assay cannot be used for function analysis. Furthermore, fluorescence would arise occasionally even though the fused protein pair did not interact with each other, which led to the background signal in the BiFC assay. Further research on the folding and dynamics of bimolecular complex formed by the two non-fluorescent fragments could help us solve these problems. At present, it is strongly recommended to introduce an appropriate negative control, such as one of the partners being deleted or mutated in the interacting region, to exclude the false-positive results in this assay.
The BiFC assay has become one of the most common approaches for visualizing PPIs in living cells. It is proved in this article that the BiFC assay is a reliable tool for detection of PPIs with appropriate negative controls. Mutational engineering of full-length GFP family members has produced proteins with an astounding range of photophysical and photochemical characteristics. Various fluorescence proteins are adapted for new purpose thanks to their own characteristics. Recently, the BiFC assay was introduced into experiments on animals for visualizing PPIs . For extending the applications of the BiFC assay, more fluorescent proteins should be taken into research to evaluate their relative merits.