A TdTomato-Based Reporter System For Quantitative Analysis Of Protein-Protein Interactions By Flow Cytometry

Protein-protein interactions are involved in every cellular process,and their study is crucial in revealing protein-protein interaction networks,confirming signal transduction pathway and identifying novel therapeutic targets.Bacterial adenylate cyclase two-hybrid(BACTH)system is a simple and fast genetic approach to analyze protein-protein interactions in vivo.In this system,association of the hybrid proteins restores synthesis of cAMP,which then triggers the expression of report genes.As the expression of the report gene as well as two-hybrid protein genes is under the control of lac promoter,the production of the two-hybrid proteins is promoted by the interaction behavor,and thus exaggerates the expression of the report gene.On the contrary,when there is no interaction between the two proteins,the expression level of the two-hybrid proteins is very low because of the lack of cAMP.As a result,the expression of the reporters is affected by both the binding strength of the interactions and the level of the interacting proteins expression.Therefore,quantitative measurement of protein interactions is not an easy endeavor due to the lack of understanding of how the strength of the interactions correlates with the level of reconstituted reporters.In addition,the population heterogeneity has been masked by the ensemble measurements using indicator LB plates or ?-galactosidase assays.Our group have reported a method for quantitative in vivo detection of the interactions by using relative reporter protein expression(RRPE),defined as the level of reporter expression normalized to that of the interacting protein in BACTH system.In the method,a multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the?-galactosidase reporter protein upon dual immunofluorescence staining.It was found that the RRPE,as an intrinsic characteristic associated with the binding affinity of protein pair,is a good index for quantifying the protein-protein interactions.However,the immunofluorescence staining is tedious for the fluorescent labeling of proteins in the cytoplasm,which requires fixing,permeabilizing,blocking,and labeling.The time-consuming sample handing limits its application for large-scale screening experiments.To advance this method for high-throughput identification of protein-protein interactions,we further developed the BACTH system by integrating tdTomato into the chromosome of the BACTH strain as the reporter for protein-protein interactions and using a tetracysteine(TC)peptide tag with a membrane permeable biarsenical dye(FlAsH)for the interacting protein detection.The improved assay could perform convenient and quantitative analysis of protein-protein interactions.The contents are summarized as follows:In chapter one,we make an overview of the methods to analyze the protein-protein interactions and the significance of single bacteria detection in BACTH system.The research plan and main contents of this dissertation are proposed.Chapter two introduces the establishment of a dual fluorescence detection for the expression of reporter protein and interacting protein in a tdTomato reporter based BACTH system.Using Pal and TolB as a model of interacting proteins,TC-tag was inserted between T25 domain and TolB protein to track the expression of the hybrid protein,and td Tomato gene was integrated into the chromosome of the BACTH strain as the reporter for protein-protein interactions.By optimizing the staining method,concentration of FlASH-EDT2 and HSFCM detection channels for dual fluorescence,simultaneous measurement of the expression of tdTomato reporter protein and interacting protein was achieved.Chapter three describes the establishment of an quantitative measurement of protein-protein interactions in the tdTomato reporter based BACTH system.For improvement of the fluorescence signal of TC-TolB,the two hybrid genes were placed on a high-copy plasmid to construct a single-plasmid suitable for the tandem coexpression of two hybrid genes,which increased the expression of interacting protein TC-TolB.By dual fluorescence detection for the expression of reporter protein(tdTomato)and interacting protein(TC-TolB)for bacterial samples cultivated from different colonies,it was identified that the relative reporter protein expression(RRPE)is constant and can be used to evaluate the interaction strength of protein pairs.To validate the potential of using RRPE for evaluating the strength of protein-protein interactions,five pairs of acid(En)and base(Kn)a-helices with various heptad repeats(n)and associated into coiled coils were constructed into the tdTomato-BACTH system.The measured RRPE exhibited a good correlation with the binding affinity.Therefore,by combining biarsenical-tetracysteine labeling system and fluorescent protein labeling technology,we developed a convenient and versatile method for the quantitative detection of protein-protein interactions.Chapter four demonstrates the application of the tdTomato reporter based BACTH system for identifying of determinant residues in protein-protein interactions.Because flow cytometry is capable of analyzing rare target cells in a larger heterogeneous cell population,it would be useful to replace the agar plating,and the target cells(transformant cultivated directly in liquid medium)could be directly analyzed by flow cytometry.Therefore,we established a rapid analysis method for protein-protein interactions based on tdTomato reporter.To examine whether the established methods could be used to investigate determinant residues in protein-protein interactions,we used TBE and TolB as a model of interacting proteins,and introduced point mutations at various positions in TBE.The rapid analysis method for protein-protein interactions based on tdTomato reporter was applied to-identify the key residues for interactions(mutations that abolish binding to TolB).The interactions between TolB and TBE mutants were further analyzed using quantitative measurement of protein-protein interactions established in Chapter three.The RRPE was measured to investigate the determinant residues in interactions between TolB and TBE mutations(mutations that weak binding to TolB).The results indicate that the tdTomato-BACTH methods provide efficient tools to investigate determinant residues in protein-protein interactions.In chapter five,the work of this paper is summarized and the follow-up research work is given.