Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster

Abstract

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequnently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC-MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.