Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9

Abstract

1. 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. 2. 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 (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated. 3. 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 Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8. 4. Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- 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. 5. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.

Effect of Albumin on Human Liver Microsomal and Recombinant CYP1A2 Activities: Impact on In Vitro-In Vivo Extrapolation of Drug Clearance

“…Microsomes were prepared by differential centrifugation, as described previously (Bowalgaha et al, 2005). rCYP1A2 and rat NADPH cytochrome P450 oxidoreductase were coexpressed in Escherichia coli according to the general procedure of Boye et al (2004). The CYP1A2/NADPH cytochrome P450 oxidoreductase ratio was unity.…”

The “Albumin Effect” and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9

“…CYP2C9 and OxR membrane fractions were mixed on ice to provide a P450/OxR ratio of 1:5. A CYP2C9/OxR ratio of 1:5 has been shown previously to result in optimal CYP2C9 activity (Boye et al, 2004). CYP2C9 expression was determined by carbon monoxide difference spectroscopy, whereas the rate of cytochrome c reduction was used as a measure of the OxR activity of membrane fractions.…”

“…N-terminal modifications previously shown to promote high levels of bacterial expression of human P450s were made to the wild-type CYP2C9 cDNA as previously described (Boye et al, 2004). The CYP2C9 cDNA was modified for bacterial expression by replacing the second codon with GCT (codes for Ala), deleting codons 3 to 20 and adjusting codons 21 through 26 for bacterial codon bias.…”

“…pCW-CYP2C9 was transformed into DH5␣ Escherichia coli cells and colonies screened for the correct plasmid by restriction enzyme analysis. Plasmid DNA was purified and confirmed on both strands by sequencing (ABI Prism 3100).The cDNA coding for OxR consisted of the OmpA signal sequence fused upstream of the full-length native rat OxR sequence (Boye et al, 2004). The rOxR expression construct was generated using the bacterial plasmid, pACYC.…”

“…pCW-CYP2C9 and pACYC-rOxR were separately transformed into DH5␣ E. coli cells. Cells were cultured, and E. coli membrane fractions were separated using the method of Boye et al (2004). CYP2C9 and OxR membrane fractions were mixed on ice to provide a P450/OxR ratio of 1:5.…”