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Abstract
Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The neverland (NVD) family genes encode the 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 Escherichia coli BL21 (DE3). This bottleneck can be solved through refolding by urea or the introduction of a maltose-binding protein (MBP) tag at the N-terminus. Through further research on purification after the introduction of a MBP tag at the N-terminus, the 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 the large-scale manufacture of NVD protein for further research. The structural model contained the Rieske [2Fe–2S] domain and non-heme iron-binding motif, which were similar to 3-ketosteroid 9 α-hydroxylase.
Highlights
Sterol derivatives mediate a wide range of growth, development, and evolution in most living species (Thummel and Chory, 2002)
The refolded His_CeNVD was further purified by an Ni-NTA affinity chromatography (Figure 2C), and the fraction was further analyzed through a gel filtration chromatography, and a single peak was detected at 280 nm
Our study provides a methodological approximation for the purification of soluble Rieske domain-containing oxygenase expressed in E. coli
Summary
Sterol derivatives mediate a wide range of growth, development, and evolution in most living species (Thummel and Chory, 2002). The MBP was advantageous to the soluble expression and purification of CeNVD in E. coli, and the purification multiple-step purification method was necessary to obtain highly purified recombinant CeNVD. We added arginine and urea to refold the protein with an extra redox system to improve the refolding yield, such as reduced and oxidized glutathione (GSH and GSSG) (Chen et al, 2016).
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