Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus

Arch Biochem Biophys. 1997 Mar 1;339(1):107-14. doi: 10.1006/abbi.1996.9859.


A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor. A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3-20. This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coli and of serines for 24His and 25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels in E. coli, 800-1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E. coli, the subcellular distribution of P450 2C3d in E. coli is dependent on the ionic strength of the buffer used for cell disruption. In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 M NaCl or 0.5 M KPi, P450 2C3d was predominantly found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one. P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers. The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme. Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (> 8-mer). Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3. These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Aryl Hydrocarbon Hydroxylases*
  • Cell Compartmentation
  • Cell Membrane / chemistry
  • Cytochrome P-450 Enzyme System / chemistry*
  • Detergents
  • Escherichia coli
  • Genetic Engineering
  • Macromolecular Substances
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Solubility
  • Steroid 16-alpha-Hydroxylase*
  • Steroid Hydroxylases / chemistry*
  • Structure-Activity Relationship


  • Detergents
  • Macromolecular Substances
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Steroid 16-alpha-Hydroxylase