Expression In E.coli And Biological Activity Analysis Of Extracellular Domain Of Human Osteoclast Inhibitory Lectin

Osteoclasst inhibitory lectin (OCIL), expressed by the osteoblast lineage and stomal cells, is a 270 amino acid type-Ⅱ transmembrane protein with a 113-amino acid C-type lectin motif in the extracellular domain. Like OPG, OCIL is a powerful inhibitor of osteoclastogenesis, but the precise mechanism of OCIL action remains to be clarified. Thus the preparation of recombinant fusion protein of OCIL is of great importance for further studies.Based on the codon preference and degeneration, we changed the codons of extracellular domain of OCIL into those preferred by E.coli coding for the same amino acid sequence with the purpose to enhance the expression of recombinant protein. pMAL-c2x, a regularly used expression vector, encodes a fusion tag of maltose binding protein (MBP), which can facilitate soluble expression of exogenous proteins and purification by binding specifically to amylose through hydrogen bonds. 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 56 kD on SDS-PAGE gel. The fusion protein could be recognized by anti-MBP monoclonal antibody usingn Western-blotting analysis. After optimization of condition, the cells achieved its best production of the recombinant protein at 30°C at 0.25mM IPTG. The caculated yield of the fusion protein purified through Amylose affinity chromotography was about 24.30 mg/L. We then investigated the biological action of the fusion protein by analyzing its effect on osteoclast formation. In brief, MBP-OCIL with different concentration was added into murine bone marrow cell culture in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nucleic factor kB ligand (RANKL). The results showed thattartrate-resistant acid phosphatase (TRACP)-stained osteoclast like cells (OLCs) formed from M-CSF plus RANKL treated cultures. When the recombinant fusion protein was added to the culture, OLC formation was significantly blocked in a concentration-dependent manner, suggestiong that the recombinant OCIL expressed from E.coli was biologically active.