Expression Of The Extracellular Domain Of Human Osteostat In E. Coli And Its Bioactivity Analysis

Osteostat was initially identified as a member of the human TNF ligand superfamily through high throughput sequencing of human cDNAs.They called TNF superfamily 18 (TNFSF18). Hydrophilicity analysis of the full-length cDNA clone predicts for a 177-amino acid typeⅡtransmembrane protein.Recently one study show that Osteostat is a strong negative regulator of osteoclastogenesis. It suppresses the differentiation of myeloid precursor cells into osteoclasts. but the precise mechanism of Osteostat action remains to be clarified.Osteostat the predicted protein comprises of a short intracellular domain.a hydrophobic transmembrane domain, an extracellular domain with two potential glycosylation sites near the extracellular C-terminal region, which is the active region to via inhibition of RANK expression in the monocytic cells.Thus we adopt engineered method to get the express protein in E.coli and detect its bioactivity in HEK293.Based on the codon preference and degeneration, we changed the codons of extracellular domain of Osteostat into those preferred by E.coli coding for the same amino acid sequence by overlapping extension PCR with the purpose to enhance the expression of recombinant protein. pQE-30Xa, a regularly used expression vector, encodes a fusion tag of 6 X His purification, Which can be seperated with Ni2+-IDA resin affinity chromatography via the bond between histidine and Ni2+. After rinsing the mixed other kinds of protein, we can wash the aimed protein from resin. By Ni2+-IDA resin affinity chromatography we can purify the aimed protein produced in batches.Induced with IPTG, the cells produced a protein which accounted for about 15% of the total soluble protein in cell lysates with a molecular weight of about 16 kD on SDS-PAGE gel. The fusion protein could be recognized by anti-His polyclonal antibody usingn Western-blotting analysis. After optimization of condition, the cells achieved its best production of the recombinant protein at 37℃at 0.5mM IPTG. The caculated yield of the fusion protein purified through Ni2+-IDA affinity chromotography was about 20.18mg/L.Glucocorticoid-induced tumor necrosis factor receptor (GITR) is an emerging member of TNFRSF with crucial roles in immune regulation.A great of research manifest the binding of GITR by GITRL has many bio-activities, including cell's proliferation, differentiate, survival and so on. The research from abroad demonstrated GITR-GITRL interaction induce the NF-κB activation. Based on that result, we can use technique of report gene to detecte the bio-activation of osteostat recombination protain. First of all, we construct the expression vector of the human GITR coding gene and raise the HEK293 cell line which can espress GITR stable. The objective gene NF-κB encoding its transcription sequence and, as a enhancer its expression product can initiae the luciferase expression in pGL3-Promotor.The pcDNA3.1/hOsteostat and luc/NF-κB contransfected the cell model which could express GITR stable. It proved the luciferas expression pruduct significantly increased in 24 hours.Based on that observation we can see osteostat protain could bind GITR initiaing luciferase expression in that cell model. We can use that model to detect the recombination protain activity.In cotransfection cells,we simultaneously added in different doses of His-Osteostat recombinate fusion protain and the expression of luciferas is obviously raised using luciferas determination kit to detect,We can get the conclusion the recombination fusion protain His-Osteostat has bio-activity.