Abstract
Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in E. coli. We optimized the codon usage of mRANKL sequence to a preferred set of codons for E. coli changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in E. coli. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD600, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.
Highlights
Receptor activator of nuclear factor (NF)- B ligand (RANKL), a member of tumor necrosis factor (TNF) superfamily, is preferentially expressed on osteoblast and stromal lineage cells whereas its receptor RANK is preferentially expressed on osteoclast lineage cells [1,2,3]
The biological activity of RANKL is balanced by its physiological decoy receptor, osteoprotegerin (OPG) that competes with RANK for RANKL and dictates the quantity of bone resorbed [12,13,14]
MRANKL was codon optimized while maintaining the integrity of the native amino acid structure (Table S1)
Summary
Receptor activator of nuclear factor (NF)- B ligand (RANKL), a member of tumor necrosis factor (TNF) superfamily, is preferentially expressed on osteoblast and stromal lineage cells whereas its receptor RANK is preferentially expressed on osteoclast lineage cells [1,2,3]. RANKL is produced as a type II transmembrane protein on these cells, and cleaved into an extracellular soluble form by specific metalloproteinases [4,5,6,7]. The latter form has high similarity to TNF-related apoptosis inducing ligand (TRAIL), FasL (TNF-related ligand) and TNF itself [8]. The essential physiological roles of RANKL-RANK have been elucidated through several in vitro and in vivo studies. The elucidation of the signaling pathway mediated by OPG, RANK and RANKL provided a major breakthrough that clarified the role played by RANKL in osteoclast biology [15]
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