Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag
2
조회
0
다운로드
3
인용
0
참고문헌
키워드
초록
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. Cytochrome c and 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.
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. Cytochrome c and 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.
참고문헌 (20)
- Paine MJI, "Cytochrome P450: Structure, Mechanism, and Biochemistry", pp.115~148 (2005), Plenum Press
- Kim D, "Functional expression and characterization of CYP51 from dandruffcausing Malassezia globosa", FEMS Yeast Res, 11, pp.80~87 (2011)
- Hwayoun Lee, "Functional Expression and Characterization of Recombinant NADPHP450 Reductase from Malassezia globosa", Journal of Microbiology and Biotechnology, 22(1), pp.141~146 (2012), 한국미생물·생명공학회
- Porter TD, "Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains", Proc. Natl. Acad. Sci. USA, 82, pp.973~977 (1985)
- Shen AL, "Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis", J. Biol. Chem, 264, pp.7584~7589 (1989)
- Hanna IH, "Role of the alanine at position 363 of cytochrome P450 2B2in influencing the NADPH- and hydroperoxide-supported activities", Arch. Biochem. Biophys, 350, pp.324~332 (1998)
- Guengerich FP, "Purification of cytochromes P450. Rat and human hepatic forms", Methods Mol. Biol, 107, pp.35~53 (1998)
- Wilks A, "Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1with human cytochrome P450 reductase", Biochemistry, 34, pp.4421~4427 (1995)
- Guengerich FP, "Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase", Nat. Protoc, 4, pp.1245~1251 (2009)
- Seon-Ha Park, "The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase", Biomolecules & Therapeutics, 20(6), pp.562~568 (2012), 한국응용약물학회
- Park HG, "Heterologous expression and characterization of the sterol 14alpha-demethylase CYP51F1from Candida albicans", Arch. Biochem. Biophys, 509, pp.9~15 (2011)
- Vermilion JL, "Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase", J. Biol. Chem, 256, pp.266~277 (1981)
- Kim D, "Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding", Arch. Biochem. Biophys, 432, pp.102~108 (2004)
- Guengerich FP, "Principles and Methods of Toxicology", pp.1625~1687 (2001), Taylor &Francis
- 최승혜, "Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11", Biomolecules & Therapeutics, 21(6), pp.487~492 (2013), 한국응용약물학회
- Kim D, "Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11", Biochemistry, 53, pp.6161~6172 (2014)
- Vermilion JL, "Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states", J. Biol. Chem, 253, pp.2694~2704 (1978)
- Park HG, "Candida albicans NADPH-P450 reductase: expression, purification, and characterization of recombinant protein", Biochem. Biophys. Res. Commun, 396, pp.534~538 (2010)
- Kim D, "Functional expression and characterization of cytochrome P450 52A21 from Candida albicans", Arch. Biochem. Biophys, 464, pp.213~220 (2007)
- Han S, "Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A", Arch. Biochem. Biophys, 575, pp.1~7 (2015)