Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

Arch Biochem Biophys. 2004 Aug 1;428(1):99-108. doi: 10.1016/


Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Delta 46A1 Delta and H Delta 46A1 Delta, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Cholesterol 24-Hydroxylase
  • Enzyme Activation
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Humans
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Engineering / methods*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Steroid Hydroxylases / biosynthesis*
  • Steroid Hydroxylases / chemistry*
  • Steroid Hydroxylases / genetics
  • Steroid Hydroxylases / isolation & purification
  • Structure-Activity Relationship


  • Recombinant Proteins
  • Steroid Hydroxylases
  • Cholesterol 24-Hydroxylase