[Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product].
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.
Liu
Ji-zhong
JZ
Institute of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Hu
Yun-yu
YY
Ji
Zong-ling
ZL
Chen
Su-min
SM
D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology
D006023:Glycoproteins; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C506030:TNFRSF11B protein, human; C506031:Tnfrsf11b protein, mouse