Expression of human cytochrome P4502B6 in Escherichia coli: Characterization of catalytic activity and expression levels in human liver

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achi eved following supplementation of the expression medium with chloramphenico l, The recombinant protein was purified using Ni2+-nitrilotriacetate chroma tography and was characterized with regard to its spectral properties and c atalytic activities toward typical P450 substrates. The purified recombinan t protein was also used to raise polyclonal antibodies in rabbits. Examinat ion of a panel of human liver microsomal preparations revealed expression o f P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presenc e of a protease-sensitive site located 126 residues away from the N-terminu s. The identity of the cleavage boundary was verified by protein sequence a nalysis. Cleavage of P450 2B6 at that site results in the presence of a low er molecular weight fragment of approximately 35 kDa in purified preparatio ns. An immunoreactive peptide of a similar molecular weight was consistentl y observed in some but not all human liver microsomal preparations suggesti ng cleavage at the same site. Examination of catalytic activities of the pu rified reconstituted protein indicated the potential utility of (S)-mepheny toin N-demethylation and testosterone 16 beta-hydroxylation as markers for P450 2B6. (C) 2000 Academic Press.