Heterologous expression, purification, and properties of human cytochrome P450 27C1

Arch Biochem Biophys. 2006 Jan 1;445(1):138-46. doi: 10.1016/j.abb.2005.11.002. Epub 2005 Nov 28.


Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.

Publication types

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

MeSH terms

  • Adrenodoxin / chemistry
  • Amino Acid Sequence
  • Calcifediol / chemistry
  • Catalysis
  • Cholecalciferol / chemistry
  • Cholesterol / chemistry
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Cytochrome P450 Family 27
  • Escherichia coli / metabolism
  • Ferredoxin-NADP Reductase / chemistry
  • Humans
  • Mass Spectrometry
  • Mitochondria / metabolism
  • Molecular Sequence Data
  • Mutation
  • Organ Specificity
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • RNA, Messenger / biosynthesis
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification


  • Peptide Fragments
  • RNA, Messenger
  • Recombinant Proteins
  • Adrenodoxin
  • Cholecalciferol
  • Cytochrome P-450 Enzyme System
  • Cholesterol
  • CYP27C1 protein, human
  • Cytochrome P450 Family 27
  • Ferredoxin-NADP Reductase
  • Calcifediol