Expression And Bioactivity Study Of Human Osteoprotegerin

Bone is a dynamic tissue, the integrity and strength of bone depends upon bone remodeling, the permanent and controlled resorption of bone by osteoclasts and its subsequent replacement by bone forming osteoblasts. As we age or as a result of disease, imbalance between bone resorption and bone formation results in osteoporosis or osteopetrosis. In case of osteoporosis, Paget's disease, bone metastasis of cancer, and inflammation of bone associated with Rheumatoid arthritis or periodontal disease, bone resorption exceeds bone formation in favour of osteoclasts, and osteoclast is certain to be the target for treatment of these diseases.Osteoprotegerin (OPG, "protector of the bone"; also known as osteoclastogenesis inhibitory factor, OCIF) is a secreted protein which was found in 1997 and 1998 independently by several research groups. OPG is a member of the TNFR-family, it was initially synthesized as a 401 amino acid peptide, with a 21-amino acid signal peptide and a 380-amino acid mature peptide, which encodes a 44-kDa protein that is posttranslationally modified to a 55-kDa molecule through N-linked glycosylation. OPG is produced by osteoblastic stromal cells and inhibits osteoclast formation and activity, it functions as a soluble decoy receptor to RANKL and competes with RANK for RANKL binding. RANKL is produced by osteoblastic stromal cells and activited T cells, RANKL and M-CSF are crucial and necessary for osteoclast differentiation and activation. RANKL exhibits its activity on osteoclastprecursors through RANK which locates on the membrane of osteoclast and osteoclast precursors. A variety of local growth factors and cytokines as well as systemic peptide and steroid hormones, such as TNF-a TGF-b. 1, 25 (OH) 2D3, estrogen, BMP-2, PTH, PGE2 and glucocorticoid, regulate the differentiation of osteoclast precursor cells and the activity of mature osteoclast through the regulation of OPG/RANKL ratio. The rapidly evolving progress made in our understanding of OPG and RANKL, including their protein and gene organization, their expression and regulation, and their effects both in vitro and in vivo may lead to a new paradigm of osteoclast biology with major novel implications for bone metabolism and the pathogenesis of metabolic bone diseases.There are two aims of this study, one is to find a way to obtain functional OPG protein , the other aim is to evaluate the possibility of gene therapy for bone disease such as osteoprosis using OPG and BMP-2.To obtain the encoding gene of Human OPG is the basis of this whole study. Using the isolated total RNA from human osteosarcoma cell line MG63 stimulated by rhBMP-2 as a template, the cDNA encoding hOPG was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method. The PCR product was cloned into pUC19 and sequenced, and the OPG cDNAwas obtained successfully.To obtain OPG protein expressed in E.Coli, the OPG encoding region gene was inserted into prokaryotic expression vector pGEX-4T-1, transformed into competent E.Coli BL21 and induced by 0.1 mM IPTG. SDS-PAGE and Western blot indicated that the molecular weight of the OPG-GST fusion protein was about 66 kDa and could react specifically with anti-OPG antibody. After being purified by affinity chromatography on glutathione Sepharose 4B, the purity grade of the expressed recombinant protein is of 91.7%. The purified OPG protein can be used for production of OPG antibody or as a template for screening of OPG-binding protein.To study the expression of OPG in eukaryotic cells, the OPG encoding region gene was inserted into mammalian expression vector pcDNAS, then recombinant plasmid pcDNA3-OPG was transformed into C2C12 cell line and OPG overexpression cell line C2C12 was selected and confirmed by Western blot.In order to find a cheap and effective method to produce functional OPG protein, Yeast expression system was chosen. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and postt...