Building A Novel Pichia Yeast To Secreted Express Recombined Human Osteoprotegerin And Associated Studies

Bone is a dynamic tissue that is morphogenized and maintained by continuous formation and resorption. An imbalance between bone formation and resorption causes such metabolic bone diseases as osteopetrosis and osteoporosis. Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. Bone remodeling and bone loss are controlled by a balance between the tumor necrosis factor family molecule receptor activator of nuclear factor-KB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG). OPG is a key factor acting as a negative regulator against osteoclasts' diferantiation and activation, through competitively inhibiting RANKL binding to its membrane receptor -Receptor Activator of Nuclear Factor-KB (RANK). The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation osteoclast differentiation and function. OPG is a member of TNFR superfamily, which active site is its domain 1-4, and a secretory glycoprotein. The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide OPG for the pre-clinical experiments, we are working on the high-throughput expression of human proteins. Therefore, cDNAs of OPG 22-201 aas and IgG1-Fc are cloned for secretory expression. The resulting fusion proteins carry affinity tags (IgG1-Fc) at the N- and C-terminus for the immunological detection and chromatographic purification of protein. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 0.2g/L culturevolume or higher.To assess the effect of different dosages of rhOPG-Fc on the differentiation and activation of osteoclasts, RAW264-7 cell line was employed as preosteoclast coculture with mouse osteoblasts for osteoclastogenesis. The results of in vitro experiments showed that rhOPG-Fc has different effects on preosteoclasts and osteoblasts at different dosages. At 2 × 10-6~2×10~8g/ml, rhOPG-Fc could disrupted differentiation and activation of osteoclasts and proliferation of preosteoclasts, and at 2 × 10-4g/ml or higher, has a toxic effect on bone cells and stomral cells from giant cell tumor of bone. The bone mass of ovariectomied rats that were abdominal injected rhOPG-Fc 5mg/kg/d × 14d did not decrease significantly 3 months after administration. These findings led us to conclude that the effect of rhOPG-Fc on anti-bone resorption is similar to OPG full-length protein.