Identification Of Proteins Interacting With PIF1 And The Preliminary Analysis Of The Mechanism

Objective: There are potential interactions between OTUD5,CNDBP1,CAP1 and PIF1.By means of experiment at the cellular level and in vitro to verify which of them, OTUD5,CNDBP1,CAP1, interact with PIF1. To provide a new access point and provide experimental evidence for further study of the functions of PIF1 helicase and its regulation mechanism.Methods: The plasmid p CMV-Myc was used as a vector to construct the eukaryotic recombinant plasmids of OTUD5, CCNDBP1 and CAP1. The recombinant proteins were connected to the Myc tag.By Using plasmid p CMV-Tag2 b as a vector, the full-length PIF1 protein eukaryotic expression recombinant plasmid is constructed, and its C-terminal was tagged with Flag. The all plasmids were expressed in 293 cells and He La cells.Using plasmid pGEX-4T-1 as a vector, we constructed the prokaryotic expression plasmids of OTUD5, CCNDBP1 and CAP1. The recombinant proteins were connected to the GST tag. The full-length PIF1 protein, C-terminal helicase motif(PIF1C) and N terminal PINT domain(PIF1N)prokaryotic recombinant plasmid recombinant plasmids were preserved by our laboratory. These plasmids based on p ET-15 b and marked with His Tag. All of these prokaryotic recombinant plasmids were induced expressingly in BL21 Escherichia coli petent cell.Based on the realization of eukaryotic overexpression of recombinant plasmids,the eukaryotic expression recombinant plasmids were divided into three groups and were co-transfected into 293 cell, respectively. The first group was composed by OTUD5 and PIF1, the second group was composed by CCNDBP1 and PIF1, the third group was composed by CAP1 and PIF1. The two kinds of protein in each group has been expressed in cell.Then, through the CO-IP experiment to verify the interaction between proteins. The cotransfection experiments was repeated for the immunofluorescence experiments.Then, the co-localization of protein was observed by laser scanning confocal microscope to further determine the interactions between proteins.Based on the realization of Prokaryotic expression of recombinant plasmids,Using the GST Pull-down experiment to test the existence of a direct interaction between the three kinds of protein and PIF1 protein and its truncated protein.Results: 1. CO-IP experiments were performed with Flag tag antibody and Myc tag antibody. After Western Blot testing, OTUD5 and CCNDBP1 can be obtained the appropriate bands at the expected location, CAP1 did not get the target band.. 2. Using Flag tag antibody and Myc tag antibody for immunofluorescence assay, OTUD5 and CCNDBP1 with the full-length PIF1 and N terminal truncated protein were colocalized, CAP1 protein and PIF1 protein or its N terminal truncated protein has no colocalization.3. The GST Pull-down experimental results show that OTUD5, CCNDBP1 and PIF1 showed the right band,but CAP1 protein was no target bands.Conclusion: CO-IP experimental results show that there is interaction between PIF1 protein and OTUD5 or CCNDBP1.Immunofluorescence experiments proved that CO-IP experimental results, PIF1 and OTUD5 or CCNDBP1 have cellular colocalization. Experimental results of the GST Pull-down shows that there is direct interaction between PIF1 and OTUD5 or CCNDBP1.