High-yield expression of a catalytically active membrane-bound protein: human P450 oxidoreductase

Endocrinology. 2011 Jul;152(7):2904-8. doi: 10.1210/en.2011-0230. Epub 2011 May 17.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, Affinity
  • Cytochromes c / metabolism
  • Escherichia coli / metabolism
  • Glycine / metabolism
  • Histidine / genetics
  • Histidine / metabolism
  • Humans
  • Kinetics
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism*
  • NADPH-Ferrihemoprotein Reductase / chemistry
  • NADPH-Ferrihemoprotein Reductase / genetics*
  • NADPH-Ferrihemoprotein Reductase / isolation & purification
  • NADPH-Ferrihemoprotein Reductase / metabolism*
  • Oligopeptides / chemistry
  • Oligopeptides / genetics
  • Oligopeptides / metabolism
  • Oxidation-Reduction
  • Protein Engineering / methods*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Steroid 17-alpha-Hydroxylase / metabolism


  • His-His-His-His-His-His
  • Membrane Proteins
  • Oligopeptides
  • Recombinant Proteins
  • Histidine
  • Cytochromes c
  • CYP17A1 protein, human
  • Steroid 17-alpha-Hydroxylase
  • NADPH-Ferrihemoprotein Reductase
  • Glycine