title: High-yield expression of a catalytically active membrane-bound protein: human P450 oxidoreductase.
abstract: P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins.
authors.affiliation: Department of Pediatrics, University of California, HSE-1401, 513 Parnassus Avenue, San Francisco, San Francisco, California 94143-0978, USA.
authors.forename: Duanpen
authors.initials: D
authors.lastname: Sandee
authors.forename: Walter L
authors.initials: WL
authors.lastname: Miller
mesh_terms: D002846:Chromatography, Affinity; D045304:Cytochromes c / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D045304:Cytochromes c; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D005998:Glycine