Determination of the binding affinity between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) utilizing Gaussia luciferase in living cells

The interaction between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) regulates osteoclast differentiation and bone resorption. Although their binding affinity has been analyzed using purified proteins in biochemical assays, including surface plasmon resonance, measurements under living-cell conditions remain limited. In this study, a bioluminescence-based assay using Gaussia luciferase (GLase/GLuc) was adapted to quantify the binding affinity between OPG and RANKL in living cells. OPG and OPG–GLase fusion proteins (OPG-GLase) were purified from conditioned media of 293T cells. Osteoclast differentiation assays were performed to determine whether OPG-GLase retained the biological activity of OPG. Purified proteins were then incubated with CHO–K1 cells expressing RANKL, and saturation binding assays were performed to calculate dissociation constants (Kd). The same assay was performed using pgsD-677 cells, a CHO–K1 mutant line that lacks heparan sulfate proteoglycan synthesis. Immunofluorescence and blocking experiments confirmed that OPG-GLase specifically binds to RANKL on the surface of CHO–K1 cells. OPG-GLase retained the ability to inhibit osteoclast differentiation to a similar extent as OPG. Saturation binding assays and non-linear regression analysis yielded a Kd of 11.0 nM in CHO–K1 cells. A parallel assay using pgsD-677 cells produced a comparable Kd of 12.6 nM, indicating that heparan sulfate proteoglycans contributed minimally to OPG–RANKL affinity under the experimental conditions used in this study. This bioluminescence-based method provides a simple and accessible platform for evaluating interactions between soluble and membrane-bound proteins in living cells, with potential application in the development of drugs targeting the RANKL–RANK–OPG axis. • OPG–RANKL binding affinity in living cells is quantified using Gaussia luciferase. • OPG-GLase retains equivalent inhibitory activity to OPG in osteoclast differentiation. • Kd of OPG–RANKL in living cells is 11.0 nM, matching values from SPR studies. • Heparan sulfate minimally influences Kd of OPG–RANKL binding in living cells. • GLase assay offers a versatile live-cell platform to measure ligand–receptor affinity.