title: Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.
abstract: NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP+in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni2+-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochromecand nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
authors.forename: Hyoung-Goo
authors.initials: HG
authors.lastname: Park
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
authors.forename: Young-Ran
authors.initials: YR
authors.lastname: Lim
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
authors.forename: Songhee
authors.initials: S
authors.lastname: Han
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
authors.forename: Dabin
authors.initials: D
authors.lastname: Jeong
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea.
authors.forename: Donghak
authors.initials: D
authors.lastname: Kim
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D009580:Nitroblue Tetrazolium; D006639:Histidine; D009249:NADP; D045304:Cytochromes c; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase
keywords: NADPH; NADPH-P450 reductase; Ni2+-affinity chromatography; cytochrome P450