Expression, characterization and biological activity analysis of recombinant human bone morphogenetic protein 2 in CHO cells.
- Author:
Dao-Yong ZHANG
1
;
Shuang YANG
;
Shu-Jun LÜ
;
Ji-Dong YAN
;
Tian-Hui ZHU
Author Information
1. Laboratory of Medical Molecular Genetics, School of Medicine, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
Alkaline Phosphatase;
biosynthesis;
Animals;
Blotting, Western;
Bone Morphogenetic Protein 2;
biosynthesis;
chemistry;
isolation & purification;
pharmacology;
CHO Cells;
Cell Line;
Cricetinae;
Cricetulus;
Enzyme Induction;
drug effects;
Gene Expression;
Genetic Vectors;
genetics;
Humans;
Mice;
Recombinant Proteins;
biosynthesis;
chemistry;
isolation & purification;
pharmacology;
Solubility
- From:
Chinese Journal of Biotechnology
2006;22(6):968-972
- CountryChina
- Language:Chinese
-
Abstract:
Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
