Expression, Purification And Blocking Activity Of Extra-cellular Domain Of Human RANK In Pichia Pastoris

The role of the receptor activator of nuclear factor-κB ligand(RANKL)/RANK system is well characterized within bone, where RANKL/RANK signaling mediates osteoclastogenesis and bone resorption.However, this signaling has also been shown to influence biologic processes beyond the skeletal system, including in the immune system and in cancer. RANKL/RANK signaling is important in lymph-node development, lymphocyte differentiation, dendritic cell survival, T-cell activation, and the establishment of T cell central tolerance. Therefore, the blockade of the interaction between RANKL and RANK provides a new therapeutic approach to prevent bone diseases and immune mechanisms of bone-related diseases, to treat cancers that develop in the skeleton,to break the central immune tolerance for reducing tumor-specific effector T cell. In this study, the extra-cellular domain of human RANK(RANK-N) coding sequence, created with Pichia Pastoris favored codons, was cloned into pPIC9 K vector, and the recombinant plasmid was transformed into Pichia Pastoris. The expression of RANK-N protein was confirmed by SDS-PAGE, Coomassie brilliant blue stain and western blotting. Recombinant RANK-N protein was purified by a multiple step process including ultrafiltration(UF), Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange chromatography.The purity of the protein was more than >95%. SRP analysis revealed the interaction of RANK-N protein with RANKL. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Moreover, we also found the recombinant RANK-N protein can inhibit colon cancer cell growth by using patient derived xenograft of human colon cancer. Therefore, we established a simple but efficient system to express and purify RANK-N, laid a foundation for future mass production and application.