The Preliminary Study On The Interaction Between CLIC1 And β4GalTⅡ Protein

Objective To intensively study the potential biological functions of CLIC1 gene in mammalian cells,we made use of yeast two hybrid system to screen CLIC1 binding protein beta 1, 4- galactosyltransferase Ⅱ(β4Gal TⅡ), and then we constructed the relevant β4Gal TⅡ expression vector(CLIC1 related vector had been constructed before).With the investigation of immunofluorescence microscopy, GST- pulldown and co-immunoprecipitation assay,we tried to jointly explore whether there existed interaction between the two proteins being expressed.Methods The full-length sequences of wild-type β4Gal TⅡwere amplified and the amplified sequences were inserted into eukaryotic yeast expression vector p GADT7 and p GBKT7.After that, immediately following two types of hybrid plasmids being contransformed by the AH109 strain using the small-scale yeast transformation protocol, the cotransformation mixtures were plated on SD/-Leu/-Trp/-His/3-AT/ X-α-gal solid medium for screening to detect whether β4Gal TⅡwas the binding protein of CLIC1.Then sp ecific primers with FLAG-tag were designed for β4Gal TⅡgene. The genes encoding for β4Gal TⅡwere amplified directly by PCR technique using c DNA as template. The amplified genes were directed cloned into eukaryotic expression vector pc DNA3.1(+) and p CDGFP to construct recombinant plasmids. After recombinant plasmids identified by restriction enzyme digestion and DNA sequencing, the recombinant plasmids of β4-Gal TⅡ and CLIC1 with different tags were transfected into COS7 cells with Lipofect-2000® Reagent. Their single localization changes were observed by fluorescence micr oroscopy.Then the plasmids of pc DNA3.1-β4Gal TⅡ-FLAG and p CDGFP-CLIC1, p C-DGFP-β4Gal TⅡand pc DNA3.1-CLIC1-FLAG with interchangeable tags were cotransfected into COS7 cells respectively to investigate their colocalizations in mammalian cells. After that, GST and GST-CLIC1 were transformed into BL21 competent cells to prepare the fusion proteins. Plasmids of β4Gal TⅡ with different tags were transiently transfected into HEK 293 T cells. GST-pull down assay was performed to determine the interaction between CLIC1 and β4Gal TⅡ in vitro. Eventually, co-immunoprecipitation assay was performed with the contransfection of pc DNA3.1-β4Gal TⅡ-FLAG and p CD GFP-CLIC1, p CDGFP-β4Gal TⅡand pc DNA3.1-CLIC1-FLAG with interchangeable tags to define the interaction between β4Gal TⅡand CLIC1 in vivo.Results Recombined expression vectors of pc DNA3.1-β4Gal TⅡ-FLAG、p CDGFP-β4Gal TⅡ、p GADT7-β4Gal TⅡand p GBKT7-β4Gal TⅡwere successfully constructed and confirmed by both restriction enzyme digestion and DNA sequencing.The results observed by the fluorescence microscope demonstrated that both pc DNA3.1-β4Gal TⅡ-FLAG and p CDGFP-β4Gal T proteins in COS7 cells were predominantly detected in Ⅱthe perinuclear spot and showed obvious signs of massive dying, and they were not distributed in the nucleus evidently. CLIC1 protein was found distributed mainly in the nuclei and nuclear membrane, partly in the cytoplasm. The localization of the both proteins did not change due to their altered tags. Immunofluorescence images of CLIC1 protein and β4Gal TⅡ protein did not appear to be colocalized in COS7 cells. The yeast two-hybrid assay manifested that there was some β-galactosidase activity between the two simultaneously co-expressed protiens-CLIC1 protein and β4Gal TⅡprotein, which suggested that the two proteins may interact with each other in yeast cells. However the subsequent GST-pull down assay verified that CLIC1 protein could not pull down β4Gal T Ⅱprotein in the in vitro condition.Moreover, co-immunoprecipitation assay further demonstrated that CLIC1 protein could not co-precipitate β4Gal T Ⅱprotein in HEK 293 T cells.Conclusion The result reflected by yeast two-hybrid assay confirmed to be false positive. Indirect immunofluorescence, GST-pull down and co-immunoprecipitation assays validated that CLIC1 protein could not do interaction with β4Gal T protein in Ⅱvivo or in vitro.Objective To construct two different types of mutants(point mutant, middle fragment deletion mutant) by overlap extension PCR, then they were transfected into COS7 cells to investigate the localization, respectively.Methods Specific primers with FLAG-tag were designed for DEPP gene 34 amino acid point S�'A mutant and N-terminal 31-34 amino acid(PPPSP) deletion mutant. The gene encoding DEPP(S34A) and DEPP(△PPPSP) were amplified directly by overlap extension PCR using recombinant plasmid GFP-DEPP as a template. Then the amplified genes were directed cloned into eukaryotic expression vector pc DNA3.1 to construct recombinant plasmids. After mutants identified by restriction enzyme digestion and DNA sequencing, both of the recombinant plasmids were transfected into COS7 cells cells with Lipofect2000 Reagent. The localization of the recombinant proteins were examined by the the confocal laser scanning microscope.Results The point mutant pc DNA3.1-FLAG-DEPP(S34A)and middle fragment deletion mutant pc DNA3.1-FLAG-DEPP(△PPPSP) were successfully constructed and expressed in eukaryotic cells. The results which observed by confocal laser scanning microscope demonstrated that FLAG-DEPP(S34A)and FLAG-DEPP(△PPPSP) proteins were predominantly located in perinuclear. These proteins were also diffusely diffusely distributed in the cytoplasm.Conclusion Construction of eukaryotic expression recombinant plasmids of DEPP gene S34 A point mutation and PPPSP deletion mutants provide the basis for further function studies of DEPP and their interaction with other proteins.