Abstract
BackgroundReceptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer.ResultsThe coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P. pastoris. The expression of RANK-N protein was confirmed using SDS-PAGE with Coomassie Brilliant Blue stain and western blotting. Recombinant RANK-N protein was purified by a multistep process including ultrafiltration (UF), Sephadex G-50 size-exclusion chromatography and Q-Sepharose Fast Flow ion exchange chromatography, which resulted in a purity >95%. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Furthermore, using a patient-derived xenograft of human colon cancer, we found that the recombinant RANK-N protein can inhibit the growth of colorectal cancer.ConclusionsThe results show that a simple system to express and purify functional RANK-N protein has been developed. This work has thus laid a foundation for further research and clinical applications of RANK-N protein in treating bone-related diseases or even colorectal cancer.
Highlights
Receptor activator of nuclear factorκB (NF-κB) ligand (RANKL)/RANK signaling essentially functions within the skeletal system, participating in osteoclastogenesis and bone resorption
We showed that RANK-N interacted with RANKL, suppressed the downstream signaling by RANK activation in vitro, and inhibited tumor growth in a patient-derived xenograft (PDX) model in vivo
Construction of the expression system and expression and characterization of RANK-N The RANK-N gene fragment encoding RANK-N, a part of DNA fragment of the RANK gene (85–639 bp), was codon optimized for expression in Pichia pastoris and cloned into the expression vector pPIC9K cut with XhoI and NotI
Summary
Receptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, participating in osteoclastogenesis and bone resorption. This signaling pathway has been shown to influence tumor progression as well as the development and function of the immune system. RANK is constitutively expressed in multiple organs, including skeletal muscle, thymus and skin It is strongly induced on osteoclast precursors by macrophage colony-. The RANKL/RANK system was originally described by different groups as being critical for T-cell activation, dendritic cell survival [1, 8], and bone homeostasis by the regulation of the differentiation of osteoclasts (OCs) [9, 10]. The RANK-N protein we obtained may be useful for the potential application in treatment of bone-related diseases or even colorectal cancer
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