[Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product].
Zhonghua Wai Ke Za Zhi
; 41(9): 641-5, 2003 Sep.
Article
en Zh
| MEDLINE
| ID: mdl-14680558
ABSTRACT
OBJECTIVE:
To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro.METHODS:
Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products.RESULTS:
The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L.CONCLUSIONS:
Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes de Fusión
/
Glicoproteínas
/
Receptores Citoplasmáticos y Nucleares
/
Escherichia coli
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
Zh
Revista:
Zhonghua Wai Ke Za Zhi
Año:
2003
Tipo del documento:
Article
País de afiliación:
China