Expression Of RANKL And The Affinity Analysis Between RANKL And RANK

RANKL is an important member of TNF family, and RANK is its receptor. The interaction between RANKL and RANK plays an important role in bone rebuilding, rheumatoid arthritis, bone metastasis and breast development progress. In the present work, we analyzed the affinity between RANKL and RANK at both molecular and cellular level, among which the later was of more physiological significance.Firstly, mouse RANKL and RANK genes were cloned into prokaryotic expression vector by standard genetic engineering procedures, and the proteins were expressed in E.coli cells, purified through affinity chromatography, size-exclusion chromatography and protein refolding procedures. Then pull-down assay, protein shift assay were performed to prove that these two proteins could bind to each other. Formation of complex between RANKL and RANK was analyzed by size-exclusion chromatography and protein crosslinking assay. The biological function of RANKL, which could block the differentiation of RAW264.7 cells was confrimed.Next, affinity between RANKL and RNAK in solution format was analyzed through BIAcore, with a dissociation coefficient of 1.09e-10M. Then ATTANA CELL 200 was used to measure the dissociation coefficient between RANKL and its receptor on cell membrane of RAW264.7 cells, the KD was 1.8e-8M. This is the first glimpse of the affinity between RANKL and its receptor on cell membrane, which is more authentic and of more physiological significance.