Expression, Purification, Refolding and Characterization of A Neverland Protein from Caenorhabditis Elegans

Abstract Background: Steroid hormones serving as vital compounds are required for a variety of organisms’ development and metabolism. The neverland (NVD) family genes encode conserved Rieske-type oxygenases, which are accountable for the dehydrogenation during the synthesis and regulation of steroid hormones. However, the His-tagged NVD protein from Caenorhabditis elegans expresses as inclusion bodies in E. coil BL21(DE3). This neck bottle can be solved through refolding by urea or introduction of maltose-binding protein (MBP) tag at N-terminus. Results: Further research on purification after introduction of maltose-binding protein (MBP) tag at N-terminus, CD measurement and fluorescence-based thermal shift assay indicated that MBP was favorable for the NVD proteins’ solubility and stability, which may be beneficial for large-scale manufacture of NVD protein for further research. The structural model contained Rieske [2Fe-2S] domain and non-heme iron-binding motif, which were similar with 3-ketosteroid 9 α-hydroxylase.Conclusions: The successful introduction of maltose-binding protein (MBP) tag at N-terminus could increase the soluble expression of CeNVD, is advantageous for further purification and improvement of thermostability. Our study provides a persuasive case for soluble expression of Rieske-domain oxygenase in E. coli.