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

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 1350 nmol/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 D3, 1α- or 25-hydroxy vitamin D3, or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D3 and the 1α-hydroxylation of 25-hydroxy vitamin D3, respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D3, 1α- or 25-hydroxy vitamin D3, or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.