Expression, purification and bioactivity characterization of extracellular domain of murine osteoprotegerin ligand.
Osteoprotegerin ligand (OPGL) is a key regulator of formation and activation of osteoclasts. In the present study, the cDNA encoding the extracellular domain of murine OPGL (sOPGL) was synthesized by RT-PCR and cloned into fusion expression vector pET-42a(+) in a certain strategy on purpose that the fusion tag could be completely removed by factor Xa from the expressed fusion protein without any vector-encoded sequence left. Induced with IPTG, the recombinant E. Coli cells produced a 47 kD protein in high level that could be recognized, through Western blotting analysis, by the antibody against OPGL. The expressed products were purified through Glutathione-sepharose 4B affinity chromatography. Along with the fusion molecule, a protein about 30 kD was also specifically bound to the resin. The 30 kD molecule could be recognized by polyclonal antibody against GST-IGF-1, but not by antibody against OPGL. It suggested that the 30 kD molecule was derived from the degradation of the fusion protein. After the cleavage with factor Xa and further purification, the fusion tag was removed and the recombinant sOPGL was obtained. Finally, we confirmed that the recombinant sOPGL could promote osteoclast formation from mouse bone marrow cells in a dose dependent manner.
Key Laboratory of Hormone and Development of the Public Health Ministry, Tianjin Medical University Hospital, Tianjin Institute of Endocrinology, Tianjin 300052, China. bliwang@163.net
Bao-Li
BL
Wang
Ming-Cai
MC
Qiu
Gang
G
Guo
Dong-Chun
DC
Liang
Jing-Yu
JY
Zhang
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse