Please use this identifier to cite or link to this item: http://dx.doi.org/10.18419/opus-823
Authors: Dietrich, Matthias
Grundmann, Lisa
Kurr, Katja
Valinotto, Laura
Richter, Tanja
Schmid, Rolf D.
Lange, Stefan
Title: Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris
Issue Date: 2005
metadata.ubs.publikation.typ: Preprint
metadata.ubs.publikation.source: ChemBioChem 6 (2005), S. 2014-2022. URL http://dx.doi.org./10.1002/cbic.200500200
URI: http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-26630
http://elib.uni-stuttgart.de/handle/11682/840
http://dx.doi.org/10.18419/opus-823
Abstract: Microsomal cytochrome P450 monooxygenases of the groups 1 – 3 are mainly expressed in liver and play a crucial role in phase 1 reactions of the xenobiotics metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed under control of the methanol inducible AOX1 promotor. 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 using isolated microsomes. Nearly all the recombinant CYP is composed of the active holo-enzyme as confirmed by reduced CO-difference spectra which showed a single peak at 450 nm. Only by co-expression of human CPR and CYP, CYP2D6 activity was obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion, and dextrometorphan. The kinetic parameters of the dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems as well as from 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-handle and cheap source for the biochemical characterization of single microsomal cytochromes as well as their allelic variants.
Appears in Collections:03 Fakultät Chemie

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