Expression And Activity Analysis Of Polyhedrin-fused Osteoprotrgerin In E. Coli And Bombyx Mori Cells

A secreted glycoprotein that regulates bone resorption has been identified. The protein, termed osteoprotegerin (OPG), is a member of the TNF receptor superfamily. OPG can be used in the treatment of osteoporosis and hypercalcemic diseases. Presently, OPG is mainly expressed through CHO cells, but it's expression level is low. In this study we expressed OPG in different expression system and tried to find an ideal system for mass-production of bioactive OPG.To improve the expression level of OPG, a fusion protein Polh-OPG was expressed in E. coli expression system and Bombyx mori baculovirus expression system respectively in this study. Firstly, we used recombinant virus Bm-OPG as a template for PCR amplification to abtain OPG gene, and constructed plasmids pBacPAK8-OPG, pBacPAK8-Polh-OPG, pET-32a-OPG and pET-32a-Polh-OPG. Then, we compared the expression level between BL21-pET-32a-Polh-OPG and BL21-pET-32a-OPG under the same conditions. Results showed that the protein Polh-OPG expressed more than protein OPG significantly in E. coli. It was found that Polh-OPG existed in the form of inclusion body. The inclusion body was dissolved and purified by affinity chromatography. SDS-PAGE and Western blotting results showed that we obtained pure Polh-OPG. Mice were used to determine the hypocalcemic effect of the recombinant protein Polh-OPG. Results indicated that the protein Polh-OPG which expressed in E. coli could reduce the serum calcium concentration significantly in the mice, suggestting that Polh-OPG had certain bioactivity. Secondly, to analyze the expression level of fusion protein Polh-OPG in Bombyx mori cells, we constructed a transfer plasmid pFastBac HTb-Polh-OPG, and got the recombinant shuttle plasmid Bacmid-Polh-OPG with Bac-to-Bac expression system. Then, Bacmid-Polh-OPG was transfected into Bombyx mori cell line BmN to get the recombinant virus Bm-Polh-OPG. Western blotting analyze the fusion protein Polh-OPG and OPG. Results indicated that Polh had little effect on increasing the expression level of Polh-OPG in Bombyx mori baculovirus expression system. We used the same method to purify the inclusion body Polh-OPG by affinity chromatography, and got pure Polh-OPG. Experiments in mice showed that Polh-OPG expressed in Bombyx mori cells also had certain bioactivity.These results lay a foundation for further enhancing the expression of OPG.