Expression and purification of recombinant human Cosmc extracellular domain in Sf-9 insect cells
10.3969/j.issn.1000-484X.2016.10.021
- VernacularTitle:人Cosmc胞外段蛋白在昆虫细胞Sf-9中的表达与纯化研究
- Author:
Qiuling XIE
;
Yue TAO
;
Xiaoqin ZHANG
;
Xi MO
;
Yanliang JIN
- Publication Type:Journal Article
- Keywords:
Human Cosmc extracellular domain;
Insect cells;
Baculovirus expression system ( BEVS);
Expression and purifi-cation
- From:
Chinese Journal of Immunology
2016;32(10):1507-1511,1518
- CountryChina
- Language:Chinese
-
Abstract:
Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
