Research On MCherry Based Bimolecular Fluorescence Complementation (BiFC)

Monitoring protein-protein interactions in living cells is of crucial importance for understanding the dynamics and mechanism of biological processes. So far, many technologies have been developed and utilized in detection of protein-protein interactions in vivo, such as yeast two hybrid, fluorescence resonance energy transfer, protein fragments complementation, and so on. These technologies, however, have their own advantages and disadvantages.In recent years, one new developed technology, bimolecular fluorescence complementation (BiFC), has been widely used in detecction of protein-protein interactions in vivo. This technology relies on the reconstruction of a fluorescent protein from its two non-fluorescent fragments when they are brought together and complement each other because of the association or interaction between proteins fused to each fragment. The BiFC assay is simple, rapid, sensitive, visualizable and localizable. However, the optical spectra of existing BiFC systems are somewhat short (≤570 nm). In this study, a long optical spectrum BiFC system was developed based on the second generation monomer red fluorescent protein, mCherry, for its long excitation and emission wavelengths (587/610 nm), and thus expanded the spectra of BiFC systems.According to its crystal structure, mCherry was split between amino acids 136/137,159/160 and 174/175 respectively and produced three pairs of peptide fragments. The pair of peptide fragments dissected at position 159/160 could produce BiFC signals selected by EGFPs because of its weak interaction, thus mCherry based BiFC system was developed. The applicability and reliability of the mCherry based BiFC system was investigated using SV40 lager T antigen (LTag) and human p53 protein, which are known for their strong interaction. The results demonstrated that mCherry based BiFC system act perfectly in detecting protein-protein interactions in living cells.By combined use of the mCherry based red BiFC system with a Venus based yellow BiFC system, the interaction between LTag and p53 as well as the interaction between sp100 and promyelocytic leukemia protein (PML), were detected simultaneously in living cells. The brilliant redness, short maturation time, and the long excitation and emission wavelengths (587/610 nm) of mCherry make the new BiFC system an excellent candidate for analyzing protein–protein interactions in living cells and for studying multiple protein–protein interactions when coupled with other BiFC systems.