Rekombinant hODF ekstrasellüler domain'in moleküler klonlanması ve üretimi

Human osteoclast differentiation factor (hODF) is a membrane protein expressed by osteoblasts. Its proteolytic cleavage releases a truncated ecto-domain composed of its extracellular domain between amino acids 143-317. hODF extracellular domain, binds to and activates its receptor RANK on osteoclast membrane. Activated RANK triggers an intracellular signaling pathway which leads to activation and nuclear localization of NF-KB and transcription of osteoclastogenic genes. This eventually results in differentiation and activation of osteoclasts, eliciting a bone remodeling response. To study osteoclastogenesis in vivo and in vitro requires a significant amount of hODF protein which can be used in downstream experiments such as RAW264.7 osteoclastogenesis assay and in vitro binding assays. Therefore, we decided to express and purify recombinant hODF extracellular domain. Recombinant hODF DNA was generated from human cellular genomic DNA and cloned into several expression vectors by PCR based molecular cloning methods. After trial and optimization of different purification strategies, GST tagged hODF was expressed in bacterial cells and purified by glutathione–agarose beads. hODF was released from the beads by proteolytic cleavage by HRV3C protease and results were analyzed by SDS-PAGE. The protocol we developed enabled us to produce sub-milligram quantities of hODF with higher than %70 purity.