Eukaryotic expression，purification and activity identification of human His-GRK2 recombinant protein
10.19405/j.cnki.issn1000-1492.2023.02.001
- Author:
Liping Jiang
1
;
Luying Chen
1
;
Jiajie Kuai
1
;
Fengling Wang
1
;
Hao Li
1
;
Yanling Guan
1
;
Yang Ma
1
;
Chenchen Han
1
;
Wei Wei
1
Author Information
1. Institute of Clinical Pharmacology，Anhui Medical University， Key Laboratory of Anti-inflammatory and Immune Medicine，Ministry of Education， Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine，Hefei 230032
- Publication Type:Journal Article
- Keywords:
G protein coupled receptor kinase 2;
HEK 293T cells;
eukaryotic expression;
protein purification;
activity identification
- From:
Acta Universitatis Medicinalis Anhui
2023;58(2):179-184
- CountryChina
- Language:Chinese
-
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.
- Full text:2024071322163688019人源GRK2的真核表达、纯化及活性检测_蒋励萍.pdf
