Construction of Eukaryotic Expression Vector of TP53BP2 Gene and Its Expression,Purification and Activity Identification in Human Embryonic Kidney Expi293F Cells
10.3969/j.issn.1671-7414.2024.06.002
- VernacularTitle:TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定
- Author:
Quanwei LI
1
;
Minghui GAO
;
Puxin KOU
;
Mengyin CHAI
;
Ying SHI
;
Xiaoni LIU
Author Information
1. 北京肝病研究所/首都医科大学附属北京佑安医院,北京 100069
- Keywords:
tumor suppressor p53-binding protein 2;
human full-length TP53BP2 eukaryotic expression;
human embryonic kidney cell Expi293F;
transient transfection;
protein purification;
activity identification
- From:
Journal of Modern Laboratory Medicine
2024;39(6):11-17
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant eukaryotic expression vector of human tumor suppressor p53-binding protein 2(TP53BP2)and transfect human embryonic kidney Expi293F cells.High-purity recombinant human full-length TP53BP2 protein was obtained and its biological activity was identified.Methods The TP53BP2 gene sequence was queried on the UniProt website,and the Expi293F expression system was optimized.The TP53BP2 gene was connected to pcDNA3.1(+)-P2A-eGFP vector by homologous recombination,and identified by double enzyme digestion and sequencing.Transect pcDNA3.1(+)-P2A-eGFP-TP53BP2 plasmid into Expi293F cells of Polyethylenimine(PEI),observe the transfection efficiency with a fluorescence microscope,collected cells from the experiment group and control group.The expression level of TP53BP2 recombinant protein was detected by Western blot(WB).Protein was purified by His label purification kit and Superdex 20010/300 GL chromatographic column.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The purified recombinant protein was identified by SDS-PAGE.Combining recombinant human full-length TP53BP2 protein with p65 protein was investigated for Co-immunoprecipitation(Co-IP)precipitation.Recombinant human full-length TP53BP2 protein was co-localized with p65 protein by Immunofluorescence(IF).The surface plasmon resonance(SPR)technique was used to detect the interaction between purified recombinant human full-length TP53BP2 protein and TP53BP2 antibody.Results The recombinant plasmid pcDNA3.1(+)-P2A-eGFP-TP53BP2 was successfully constructed by sequencing and double digestion.The fluorescence microscopy results showed that the transfection efficiency was about 60%.WB showed that the TP53BP2 protein was overexpressed in Expi293F cells,which proved that transfection was successful.SDS-PAGE results showed that the purity of the purified recombinant protein was above 90%,which proved that the purification was successful.Co-IP results showed that the TP53BP2 could interact with p65 protein.The results of IF showed that His tag protein,TP53BP2 protein,and p65 protein were co-located,indicating the interaction between the three proteins.SPR results showed that the purified TP53BP2 recombinant protein had good binding activity with the TP53BP2 antibody.These results all prove that the recombinant human full-length TP53BP2 protein has biological activity.Conclusion The eukaryotic expression vector of TP53BP2 gene was successfully constructed and the recombinant full-length human TP53BP2 protein with biological activity was successfully expressed in human embryonic kidney Expi293F cells.It lays a foundation for further study on the structure and function of TP53BP2.
