Dual-fluorescent bacterial two-hybrid system for quantitative Protein–Protein interaction measurement via flow cytometry

Characterization of protein–protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement the binding strength remains challenging. Building upon classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that relative reporter protein expression (RRPE), defined as level normalized to interacting protein, an intrinsic characteristic associated with between two proteins. In this study, inserted fluorescent tdTomato in chromosome by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) tag one proteins, which can be further labeled membrane-permeable biarsenical dye. The combined use TC-tag offers rapid high-throughput analysis levels both proteins at single-cell multicolor flow cytometry, simplifies PPI. as-developed RRPE-tdTomato-TC-BACTH approach was three demanding applications. First, affinities could correctly ranked discriminating interaction strengths tenfold difference or same order magnitude. We demonstrate method sensitive enough discriminate small 1.4-fold. Moreover, residues involved PPI easily mapped ranked. Lastly, inhibitors rapidly screened. • Rapid quantification developed protein-protein based on BACTH system. Simultaneously read-out RRPE measurement. Sensitive affinity Provides fast screening ranking key PPIs. Validated usage inhibitor identification.