title: An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.
abstract: Protein-protein interactions (PPIs) play crucial roles in various biological processes. Among biochemical, genetic, and imaging approaches that have been used for the study of PPIs, visualization of PPIs in living cells is the key to understanding their cellular functions. The bimolecular fluorescence complementation (BiFC) assay represents one of these imaging tools for direct visualization of PPIs in living cells. The BiFC assay is based on the structural complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein when they are fused to a pair of interacting proteins. Although over 10 different fluorescent proteins have been used for BiFC assays, the two nonfluorescent fragments from all of these fluorescent proteins can spontaneously self-assemble, which contributes to background fluorescence and decreases the signal-to-noise (S/N) ratio in the BiFC assay. Here we report the identification of a mutation, I152L, that can specifically reduce self-assembly and decrease background fluorescence in a Venus-based BiFC system. This mutation allows a 4-fold increase in the S/N ratio of the BiFC assay in living cells. This improved Venus-based BiFC system will facilitate PPI studies in various biological research fields.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA. yutakakodama@psc.riken.jp
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kodama
authors.forename: Chang-Deng
authors.initials: CD
authors.lastname: Hu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; C054562:yellow fluorescent protein, Bacteria