Cloning And Expression Of CDNA Sequence Encoding Human Osteoprotegerin

Osteoprotegerin ( OPG ), also known as osteoclastogenesis inhibitory factor ( OCIF ), is a secreted glycoprotein. Its cDNA encodes a 401 amino acid propeptide, of which the 21 amino acid signal peptide is cleaved leaving a 380 amino acid mature protein. Its N-terminal region is essential for its function in osteoclast differention and activation. Receptor activator of NF- k. B ligand ( RANKL ), also called osteoclast differentiation factor ( ODF ), is mainly expressed on stromal cells and osteoblasts. RANKL activates osteoclast differentiation, stimulates osteoclast activation and keeps osteoclast survival. These effects occur when RANKL binds to its receptor, RANK, which is present on osteoclast precursors and mature osteoclasts, leading to bone resorption. OPG functions as a soluble decoy receptor for RANKL by competing with RANK and can inhibit the effects of RANKL, thereby preventing osteoclast development and subsequent bone resorption. OPG, RANKL and RANK represent a novel cytokine network and act as key regulators of osteoclast biology and bone metabolism. The abnormalities of OPG / RANKL / RANK system have been detected in various benign and malignant metabolic bone diseases characterized by abnormal osteoclast function. By contrast, OPG administration was found to abolish these abnormalities. OPG plays a pivotal role in bone metabilism regulation by inhibiting RANKL-mediated effects. Recombinant OPG may provide a therapeutic tool for osteoporosis, rheumatoid arthritis and other erosive bone diseases. It has been reported that the bioactivity of OPG is dependent on the presence of the N-terminal D1-D4 domain. In the present study, we cloned and expressed the cDNA sequence encoding the N-terminal D1-D4 domain andmature peptide of human osteoprotegerin.It has been noted in our laboratory that the N-terminal D1-D4 domain region is encoded by only two exons, exon 2 and exon 3. In the first part of present study, we amplified the sequence coding for this region by using human genomic DNA as template to amplify the two exons respectively and then linking them through a so-called overlap-extension method. The amplified coding sequence was inserted into two prokaryocytic expression vectors, pET42a and pQE30, respectively. The procedures are described as follows. (1) Using expression vector pET42a: The amplified coding sequence was followed by connecting them to a sequence in the vector pET42a between factor Xa and Bgl II sites before the Multiple Cloning Site(MCS) through another overlap extension PCR. Then the chimeric molecule was inserted into expression vector pET42a , and then the recombinant plasmid was transformed into E.coli BL21(DE3) for expression. Induced with IPTG, the expressed fusion protein existed mainly in the form of insoluble inclusion bodies. SDS-PAGE and Western blotting were performed to identify the expressed fusion protein. After optimization of condition, the fusion protein accounted for 20% of total cellular proteins. The inclusion bodies were dissolved and then refolded, followed by purification by Glutathione Sepharose 4B affmity-chromatography. Then the purified protein was cleaved by factor Xa while the recombinant protein was obtained. The yield of the recombinant protein was 34mg/L medium. (2) Using expression vector pQE30: The amplified coding sequence was added with 2xHis sequence at 5' end, followed by connection to a sequence in the vector pQE30 before the 6xHis sequence through another overlap extension PCR. Then the chimeric molecule was inserted into expression vector pQE30, and then the recombinant plasmid was transformed into E.coli M15[pREP4] forexpression. The expressed 8xHis fusion protein, about 21kD in molecular weight, existed mainly in the form of insoluble inclusion bodies on SDS-PAGE gel. It could be recognized, through western blotting analysis, by the polyclonal antibody against hOPG. After optimizing the condition, the fusion protein accounted for 15.6% of total cellular proteins. The inclusion bodies were dissolved, followed by purification using one...