Research Of TNFRSF11B(OPG) Function Domain Of Homo Sapiens

The discoveries of osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) were significant breakthroughs that rapidly expanded the understanding of bone remodeling. A major paradox in bone biology was that most of the hormones, cytokines and growth factors that regulated osteoclast activity had cognate receptors on osteoblasts rather than osteoclasts. An unknown osteoblast-derived factor was invoked to explain the response to pro-resorptive stimuli. This factor was shown to be RANKL, a tumor necrosis factor (TNF) family member that is essential for osteoclast formation, function and survival. An agent so indispensable for bone resorption would be expected to have a counterregulatory partner, and OPG satisfied that criterion. OPG is a soluble decoy receptor from the TNF receptor family with a simple mechanism of action that does not involve any direct signaling activity. OPG binds to RANKL and prevents RANKL from binding and activating receptor activator of nuclear factor-kB (RANK). RANK is another member of the TNF receptor family that is present on osteoclasts and osteoclast precursors. This triad of proteins OPG/RANKL/RANK has been shown in genetic and pharmacology studies to have a critical role in the regulation of osteoclasts and bone resorption. Although other hormones and cytokines are able to influence osteoclasts in various ways, RANK/ RANKL signaling is indispensable for the existence andactivity of osteoclasts.This study aims to analysis the second structure of the N terminal 24-106 peptide of Tnbfrsf11b (Tumor Necrosis Factor Receptor Superfamilie members 11b),after bing successfully prokaryotic expressed and purified.Analysis the relative sequence basing on the published sequences of Homo Sapiens Tnfrsf11b by BLAST bio-software . Then constructed genes by PCR and cloned genes into the BamHⅠ,EcoRⅠrestriction sites of the GST fusion expression vector pGEX-6P-1, named as pGEX-6P-1-Tnfrsf11b, in which there was a PreScission Protease cleavage site for the fusion protein. Expressed fusion GST- Tnfrsf11b protein in E.coli strain BL21(DE3) at 16℃with 0.1 mmol/L IPTG for 25-30 hours. The supernatants lysed by sonication and clarified by centrifugation were passed over Glutathione SepharoseTM 4B column for purifying. The GST fusion proteins were cleaved by PreScission Protease and then purified by Superdex75 filter. Lastly the soluble highly purified protein was analysised by the circular dichroism (CD).The fused protein was successfully expressed in E.coli strain BL21(DE3); After purified, high quality CRD domains of Tnbfrsf11b N terminal was obtained. The circular dichroism showed that the soluble highly purified protein had richβ-sheet. Richβ-sheet is in favor of the interactions between proteins, Which is important to the function. This research lay a solid foundation for the further research of the structure and function of Tnfrsf11b N-terminal CRD domain of Homo.