Eukaryotic expression，purification and activity identification of human His-GRK2 recombinant protein / 安徽医科大学学报

ABSTRACT

Objective@#To construct a human G protein-coupled receptor kinase 2 ( GRK2) eukaryotic expression system．@*Methods@#The primers were designed ，and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2 (full-length) gene as the template．The His-GRK2 target gene was connected to the pcDNA3．1EGFP eukaryotic expression vector． The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells．48 h later，the expression of GRK2 protein was detected by Western blot，and the GRK2 protein was purified by nickel chelated magnetic bead method．The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot，and the activity of GRK2 protein was detected by His pull down. @*Results @#The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed．Western blot analysis showed that the molecular weight of GRK2 protein was about 80 ku，indicating that GRK2 protein was successfully expressed in HEK 293T cells (t = 6. 433，P = 0. 003) ．GRK2 protein was purified by nickel chelated magnetic beads．His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4 (EP4) ，suggesting that GRK2 protein had biological activity (t = 13. 5，P = 0. 000 2) ．@*Conclusion@#The pcDNA3．1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed．The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.