AUTHOR=Mao Shuhong , Song Zhan , Wu Mian , Wang Xiaorui , Lu Fuping , Qin Hui-Min 

TITLE=Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.593041

DOI=10.3389/fbioe.2020.593041

ISSN=2296-4185

ABSTRACT=<p>Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The <italic>neverland</italic> (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 <italic>Caenorhabditis elegans</italic> expresses as inclusion bodies in <italic>Escherichia coli</italic> BL21 (DE3). This bottleneck can be solved through refolding by urea or the introduction of a maltose-binding protein (MBP) tag at the <italic>N</italic>-terminus. Through further research on purification after the introduction of a MBP tag at the <italic>N</italic>-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.</p>