Expression, purification, and characterization of the human receptor activator of NF-kappa B ligand (RANKL) extracellular domain

Receptor activator of NF-kappa B ligand (RANKL) is a type II transmembrane protein, found on osteoblasts which functions as a major determinant of ost eoclast differentiation and activation. RANKL mediates bone homeostasis thr ough binding to the cognate ligand on osteoclasts, RANK, and a soluble deco y receptor, osteoprotegerin (OPC;). We designed a construct encoding the ex tracellular domain of human RANKL that conformed to reports of native proce ssing. To encourage folding and posttranslational modification of a normall y membrane-inserted moiety, we expressed the RANKL truncate as a secreted p rotein using the signal sequence from OPG in a Trichoplusia ni cell line us ing a baculovirus expression vector. RANKL was purified by a three-step pro cess including an OPG-Fe affinity column. SDS-PAGE and mass spectral analys is indicated that the protein was >99% pure and glycosylated. Circular dich roism spectra revealed that the protein exhibited structural elements simil ar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentr ifugation analyses established that our protein existed as a trimer. We con clude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members. (C) 2000 A cademic Press.