Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9.
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.
Boye
S L
SL
Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, SA, Australia.
Kerdpin
O
O
Elliot
D J
DJ
Miners
J O
JO
Kelly
L
L
McKinnon
R A
RA
Bhasker
C R
CR
Yoovathaworn
K
K
Birkett
D J
DJ
D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism; D000077786:Torsemide
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D009251:NADPH-Ferrihemoprotein Reductase; D000077786:Torsemide