Microsomal P450 2C3 Is Expressed as a Soluble Dimer inEscherichia coliFollowing Modifications of Its N-terminus

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 for2Asp to facilitate expression inEscherichia coliand of serines for24His and25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels inE. coli,800–1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression inE. coli,the subcellular distribution of P450 2C3d inE. coliis 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 1mNaCl or 0.5mKPi, P450 2C3d was predominately 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.