Identification Of Rab40b And Rab40c Interacting Proteins

Objective:To identify the Rab40 b and Rab40 c interacting proteins in dendritic cell with a novel tandem affinity purification strategy based on a new eXact-6×His affinity tag.To illustrate the biological function of Rab40 b and Rab40 c protein complex with bioinformatic analysis and verification of protein interaction.Background:Dendritic cells are professional antigen presenting cell,orchestrate complex membrane trafficking events in immunological process.Rab GTPase stand out as crucial regulators for systemic integration of complex trafficking of intracellular membranes.There are more than 70 Rab GTPaes in mammalian cells,participating in specific vesicle trafficking pathway.Xenopus homolog of Rab40(XRab40)plays a crucial role in the regulation of the noncanonical Wnt pathway,and the Wnt pathway have critical effect in the process of dendritic cell differentiation.Hence,the research of Rab40 b subfamaily is a good way to illustrate the link between Wnt pathway and dendritic cells' function.Protein carry out biological function by the formation of protein complex,protein protein interaction is important methods to illustrate the function of protein.All the technologies relate to protein protein interaction have their own advantages and disadvantages,TAP-MS(tandem affinity purification-mass spectrometry)which have wide application in this area also have some limitation.Hence,the research of Rab40 b and Rab40 c interacting proteins make much sence to understanding the role of Rab40b/c in the DCs' biological function,and its make us illustrate the mechanism of Wnt pathway regulate DCs' differentiation.Method:1.Construction of Rab40 b and Rab40 c expression plasmid.Amplify the specific DNA fragment of Rab40 b and Rab40 c by PCR,then insert the fragment into expression vector Fu GW-eXact-6×His-EYFP through molecular cloning technology.Align DNA sequence of recombinant plasmid Fu GW-eXact-6×His-EYFPRab40 b and FuGW-e Xact-6×His-EYFP-Rab40 b by DNA sequencing.Amplify the specific DNA fragment of Tag RFP,then get recombinant plasmid FUGW-e Xact-6×His-tag RFPRab40 b by the methods of digestion and ligation.2.Construction of stable dendritic cell line express infusion proteinsTransfect plasmid FuGW-e Xact-6×His-EYFP-Rab40 b,Fu GW-eXact-6×His-EYFPRab40 c and FUGW-e Xact-6×His-tag RFP-Rab40 b into 293 FT cell line by Ca3(PO4)2 Tricalcium phosphate precipitation,then we can harvest HIV lentivirus to infect DC2.4 cell line.To achieve the cell line which stable express infusion protein e Xact-6×HisEYFP-Rab40 b,eXact-6×His-EYFP-Rab40 c and e Xact-6×His-tag RFP-Rab40 b by flow cytometry sorting.3.Identification and bioinfoermatic anlysis of Rab40 b and Rab40 c interacting proteins based on a novel tandem affinity purification.To get Rab40 b and Rab40 c protein complex through two sequential affinity purification using a new e Xact-6×His purification tag.Compartment of the protein complex identified with HPLC-ESI-MS,using a software similar to Comp PASS to separate highconfidence interacting proteins from the protein complex.Analyze interacting proteins' function via gene ontology analysis of biological process.4.To verify the interaction protein complexAccording to bioinformatic annlysis results and article reported,we choose some proteins to verify the interaction via co-immunoprecipitation.Observe co-localization between Rab40 b and Rab40 c through Laser scanning confocal microscopy.Conclusion:1.DC2.4 cell lines which stable express infusion protein eXact-6×His-EYFP-Rab40 b,e Xact-6×His-EYFP-Rab40 c and e Xact-6×His-tag RFPRab40 b are construted successfully.2.26 Rab40 b high-confidence interacting proteins and 16 Rab40 c interacting proteins are identified with a novel tandem affinity purification-mass spectrometry platform.3.Elongin B,ElonginC,Cullin5 interact with Rab40 b and Rab40 c,they form protein complex to participate in the ubiquitination pathway.It's a basis to illustrate the function of Rab40 b and Rab40 c in the biological process.