Recombinant Production of Human Microsomal Cytochrome P450 2D6 in the Methylotrophic Yeast Pichia pastoris

摘要

Microsomal cytochrome P450 monooxygenases of groups 1-3 are mainly expressed in the liver and play a crucial role in phase 1 reactions of xenobiotic metabolism.The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed with control of the methanol-inducible AOX1 promoter.The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa.CPR activity was detected by conversion of cytochrome c by using isolated microsomes.Nearly all of the recombinant CYP was composed of the active holoenzyme,as confirmed by reduced CO difference spectra,which showed a single peak at 450 nm.Only by coexpression of human CPR and CYP was CYP2D6 activity obtained.Microsomes containing human CPR and CYP2D6 converted different substrates,such as 3-cyano-7-ethoxycoumarin,parathion and dextrometorphan.The kinetic parameters of dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems and human microsomes.The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6,as expression of CYP2D6 without human CPR did not result in any CYP activity.These recombinant strains provide a novel,easy-to-handie and cheap source for the biochemical characterisation of single microsomal cytochromes,as well as their allelic variants.