Quantitative Analysis Of Protein-protein Interaction In BACTH System Using Flow Cytometry

Protein-protein interactions(PPIs)are fundamental to virtually every aspect of life processes.Characterizing the interacting partners of a protein is important in the understanding of protein function,deciphering biochemical networks,discovering novel drug targets,and developing diagnostic and therapeutic assays.Current approaches for PPI study in vitro include isothermal titration calorimetry,surface plasmon resonance,fluorescence polarization and so on.Compared with these methods,in vivo approaches,such as yeast two-hybrid(Y2H),protein-fragment complementation assay,and synthetic lethality,not only eliminate the cumbersome protein purification and labeling procedures but also measure interactions at their native status.Among these methods,yeast two-hybrid system is the most frequently used method.For the study of the interaction of two proteins of interest.one protein is fused to the DNA-binding domain(DBD)of Gal4(the bait)and the other protein is fused with the transcription-activating domain(AD)of Gal4(the prey).The bait fusion binds upstream activating sequences(UAS)of the reporter gene such as lacZ and His.Association of the interacting proteins bring the Gal4 AD to the reporter gene,followed by recruitment of the basal transcriptional machinery,which establishes the gene transcription that can be detected by following analysis.Although numerous protein-protein interactions have been identified by the Y2H system,the false-positive and false-negative rate of the system is relatively high,and the fusion proteins must be targeted to the nucleus.Bacterial two-hybrid based on the reconstitution of adenylate cyclase activity in E.coli(BACTH)is an ideal alternative for Y2H.It is an easy and reliable method with few reported case of false positives or false negatives.Moreover,studying the interactions of membrane proteins was made possible as the BACTH system does not require the hybrid proteins to be located in the nucleus as Y2H.However,both the Y2H and BATCH methods for PPI detection suffer from some shortcomings.First,lack of quantitation capability renders these methods unsuitable for measuring the affinity between two interacting proteins,nor for the affinity comparison of different PPIs.Second,the widely-existed population heterogeneity has been masked by the ensemble measurements using coventional reporter plate or colorimetric assays.Flow cytometry is a powerful technique for the quantitative and multiparameter measurement of single mammalian or plant cells at high speed.Combined with the BACTH system with flow cytometry,a new method for the quantitative analysis of PPIs was developed in this dissertation.The contents are summarized as follows:In chapter one,the methods for study of PPI are systematically reviewed including the BACTH system.Flow cytometry is introduced in detail followed by a discussion for the significance of PPIs analysis at the single-cell level.The research plan and main contents of this dissertation are proposed.Chapter two describes the construction of the two-tag BACTH system.Because in the BACTH system,the transcription and expression level of reporter protein is determined not only by the binding affinities between the interacting proteins,but also by the cytoplasmic concentrations of the proteins themselves.It is valuable to assess protein expression level side by side with protein-protein interaction measurement.Therefore,His-tag and Flag-tag were inserted between T25 or T18 domain and the hybrid proteins,respectively.Pal and TolB,two important proteins for bacterial outer membrane integrity were chosen as a model of interacting proteins.The constructed plasmids were cotransformed into reporter bacteria E.coli BTH101.The PPI was detected on a reporter plate or by colorimetric assays.Besides,the expression of target proteins was detected via Western blot using Flag/His-tag specific antibodies.These experiments demonstrate the successful construction of the BACTH system.Chapter three describes the development of flow cytometric approach for the detection of bacterial cytoplasmic protein.The membrane of Gram-negative bacteria like E.coli is impermeable for most of the molecules with large molecular weight.It is necessary to permeabilize bacteria before cytoplasmic protein labeling.The optimation of experimental conditions was carried out on a laboratory-built high sensitivity flow cytometer(HSFCM)by taking advantage of its high sensitivity.C12FDG was used as a fluorescent substrate of ?-gal to stain permeabilized bacteria for the detection of PPI on HSFCM at single-bacterium level.Employing immunofluorescent labeling,the expression level of interacting proteins can be measured by flow cytometry through labeling the epitope tags fused to the hybrid proteins.Due to the leakage of the hydrolysate of C12FDG after permeabilization and enzyme activity decreasing caused by fixation,immunofluorescent staining was chosen to detect ?-gal.At last,the Flag/His-tag fused with interacting protein pair and the reporter protein ?-gal of protein-protein interaction were immunofluorescently labeled and analyzed on the flow cytometer simultaneously.Chapter four describes the quantitative measurement of PPI in the BACTH system using flow cytometry.Bistability phenomenon was observed in the BACTH system via flow cytometric analysis that there existed two distinct populations when bacteria were immunofluorescently stained.Neither the reporter ?-gal nor the interacting proteins were detected in one population of the bacteria.The ratio of the bacterial population with protein expressed varied with the cultivation time for the same coloby and with different individual colonies.Two parameter detection was conduced for both the interacting protein(His-TolB)and the reporter protein(?-gal)at the single-cell level for bacterial samples collected at different cultivation time and from different individual colonies.It was identified that the relative reporter protein expression(RRPE),which is defined as the normalized ?-gal expression to that of the interacting protein,is constant and can be used to evaluate the interaction strength of protein pairs.To validate the applicability of RRPE in the measurement of protein interaction affinity,two mutated forms of TolB,which renders lower binding affinity to Pal,were constructed and compared with the wild type TolB using the BACTH systems.Chapter five describes the applications of the flow cytomteric-based BACTH system for the quantitative measurement of PPIs.To investigate the potential of using RREP measured in the BACTH system for the strength evaluation of protein-protein interactions,five pairs of acid(En)and base(Kn)?-helices with various heptad repeats(n)and associate into coiled coils were constructed into the BACTH system.The measured RRPE and the affinity reported in literature yields a good correlation.The as-developed flow cytometric-based BACTH system not only allows rapid in vivo detection of protein interaction in single cells but also provides relative affinity assessment of the protein interaction.In addition,using TolB and Pal as a model,the effect of PPI inhibitor on reporter protein expression was examined.Due to the direct detection of the expression level for both the reporter protein and the interacting protein,the decrease of ?-gal activity caused by other effect,such as protein expression inhibition,can be excluded.Thus,the as-developed method can be used for the convenient and high-throughput inhibitor screening for PPIs.In chapter six,the contents of present thesis are summarized and the future prospects of the research are given.