Expression, Purification And Function Of Osteoprotrgerin

Osteoprotrgerin (OPG), also termed Osteocalstogenesis inhibitory factor(OCIF), is a novel secreted protein involved in the regulation of bone resorption. It is reported that OPG acts as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis and hypercalcemic diseases. As a newly found protein, OPG is on the focus because of its research significance and medical foreground. Presently the study of the structure-function relationship of OPG is mainly using the recombiant protein from CHO cells, however the lower level and high costs limit its further research. So in this study, we expressed OPG in different system and tried to find an ideal system for mass-production of proper bioactivity recombinant osteoprotegerin.The TNFR domain of OPG, which is involved in the inhibition of formation and activity of osteoclasts, was amplified by PCR and named as OPG-T. In order to investigate the feasibility of using E.coli to produce recombinant osteoprotegerin, OPG, OPG-372 and OPG-T were inserted into multiple cloning site of PET-28a. The recombinant plasimid was transferred into E.coli BL21 to express recombinant protein. The results indicated that the expression of OPG and OPG-372 were not desirable. and the the expression level of OPG-T was high. It was found that the product of OPG-T existed in the inclusion body. The inclusion body was solublized, refolded and purified by affinity chromatography. High specificity Polyclonal antibodies were obtained from the serum of rabbit immunized with purified recombinant protein. Mice were used to determine the hypocalcemic effect of the recombinant protein, the results showed that the recombinant protein expressed in E.coli had the some bioactivity.Because the bioactivity of recombinant OPG from E.coli is not desirable, the OPG and mutated osteoprotegerin (OPG-372) gene were inserted into the baculovirus tranfer vector pBacPAK8, and the recombinant plasmid was co-transfected with lineared DNA of Bm-BacPAK6 virus in BmN cells, then homologous recombinationwas occured inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG and OPG-372 was expressed in culture cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that two recombinant protein were expressed in BmN cells and silkworm larvae, 43 kDa OPG and 42 kDa OPG-372 were expressed in cells, and 55 kDa OPG and 46 kDa OPG-372 in larvae, respectively. It was also found that homodimer of OPG was expressed in silkworm larvae.rh-OPG and rh-OPG-372 were expressed in silkworm larvae and purified by Seize? Primary Mammalian Immunoprecipitation Kit. SDS-PAGE showed that there was only one band of rh-OPG-372, the mass was about 46 kDa. However two bands of rh-OPG, 55 kDa and 110 kDa respectively, were found on SDS-PAGE. after reducing, the band of 110 kDa was disappear, which indicated the 110 kDa rh-OPG was homodimer of 55 kDa OPG. With the purified recombinant protein, quantification was processed by ELISA. The results showed that the expression level is the highest on the day 5 in Bm cells, the expression level of rh-OPG-372 is 80 u g/2X 106cells and that of rh-OPG is 19 u g/2X 106cells. And the expression level is the highest on the day 6 in silkworm larvae, the expression level of rh-OPG-372 is llOu g/ml and that of rh-OPG is 120 u g/ml. The rh-OPG was run on SDS-PAGE and the band was excised for trysin digestion, then was analysed by Q TRAP LC/MS/MS, the results indicated the product purified from silkworm larvae was recombinant human osteoprotegerin.In order to investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant OPG and OPG-372 as drugs for the prevention and treatment of bone disease, mice were used to determine the effects of hypercalcemia, increasing trabecular bone area and bone protection. The results showed that abdominal cavity injection of 6 mg/kg recombinant protein was effective to decrease serum calcium concentration at 1.5 h, and injection of 3 mg/kg recombinant protein for a month significantly increased the percent trabecular bone area in the distal femurs of mice.