The affinity of human RANK binding to its ligand RANKL.

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re- modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombi- nant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was mea- sured with surface plasmon resonance technology and KD value is about 1.09 � 10 � 10 M.