Protein-Protein Interaction Detection Technology
Protein-Protein Interaction Detection in Living Cells using Fluoppi
Fluoppi (Fluorescent based technology detecting Protein-Protein Interaction) is a novel technology to detect Protein-Protein Interaction (PPI) in living cells with a high signal to noise ratio. The result is a bright, clearly analyzable image. Fluoppi detects PPI as the absence or presence of fluorescent foci during inhibition or induction, respectively. The Fluoppi advantage is the ease of the construction of the PPI detection system with no need for optimizing the linker.
Fluoppi is a tag technology. Tetramer fluorescent protein (FP-tag) and Assembly helper tag (Ash-tag) are genetically fused to Protein X and Y, respectively. For the FP-tag, tetrameric fluorescent protein (e.g Azami Green (hAG)) can be used. In our in vitro Fluorescence correlation spectroscopy analysis, Ash-tag behaved as tetramer to octomer in diluted solution. Before protein X and Y interact, most of the molecule are disseminated. When protein X and Y interact, these proteins associate each other to form detectable foci.
Fluoppi tags are able to work in both N and C terminal fusion. Plasmid vectors of Fluoppi consist of CMV promoter, Fluoppi tags, flexible peptide linker, Multiple Cloning Site (MCS) and Neomycin resistant gene. First, proteins X & Y of interest are fused to FP-tag and Ash-tag respectively. Expression level of tagged protein and foci formation efficiency depend on tag and tag location Formation of foci is reversible so that they can be dissociated and the fluorescent signal will spread over the cell by PPI inhibitors, and vice versa by PPI inducers.
|Excit./Emiss.Maxima (nm)||Extinction coefficient (M-1cm-1)||Fluorescence quantum yield||pH sensitivity|
|AG||492/505||72,300 (492 nm)||0.67||pKa=5.0|
1. Target gene amplification & restriction enzyme cut
2. Ligation & sequencing
Because fluorescent signal of Fluoppi is very high, conventional fluorescence microscopy can be used to image the cell. If the proteins interact with each other upon expression, fluorescent foci will be detected. Formation of foci is reversible so that they can be dissociated and the fluorescent signal will spread over the cell by PPI inhibitors, and vice versa by PPI inducer.
PPI modulator [Induction]
PPI modulator [Inhibition]
Time course of foci dissociation after adding Nutlin-3 was analyzed. Y axis represents fluorescent intensity of foci.
Koyano F, et al., Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature. 510, 162-166 (2014)