title: Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative Reporter Protein Expression Measurement.
abstract: Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein-protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein-protein interaction, and compare interaction strength of different pairs.
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
authors.forename: Lina
authors.initials: L
authors.lastname: Wu
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
authors.forename: Tian
authors.initials: T
authors.lastname: Luan
authors.affiliation: Laboratory of Macromolecular System Engineering, Institute of Microbiology of the Mediterranean, Aix-Marseille Université and Centre National de la Recherche Scientifique , Marseille 13402, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Bouveret
authors.affiliation: The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.
authors.forename: Xiaomei
authors.identifier: 0000-0002-7482-6863
authors.initials: X
authors.lastname: Yan
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D008856:Microscopy, Fluorescence; D060066:Protein Interaction Maps; D059010:Single-Cell Analysis; D055672:Static Electricity; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001616:beta-Galactosidase; D000262:Adenylyl Cyclases