Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative...

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.