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Abstract
Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared.
Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (A3-20 and 17a-leader) of the individual P450s were additionally investigated.
Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the A3-20 construct for CYP2C9 and with the 17a-presequence construct for CYP2C8.
Using torsemide as substrate, all methods gave catalytically active systems with Km values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the A3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17a- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes.
Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.
This article was downloaded by: [Collard, Jonathan][informa internal users]
On: 30 Ju148180_755239199_713767701ne 2010
Access details: Access Details: [subscription number 755239602]
Publisher Informa Healthcare
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London WlT 3JH, UK
This article was downloaded by: [Collard, Jonathan][informa internal users]
On: 30 Ju148180_755239199_713767701ne 2010
Access details: Access Details: [subscription number 755239602]
Publisher Informa Healthcare
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London WlT 3JH, UK