Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli.
A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned. The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed. The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2('),5(')-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column. The average yield was 47mg per liter of culture medium. The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN. The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1. However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450. Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain. With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts. This expression system will be useful for mechanistic as well as structural studies of CPR.
Hayashi
Shunsuke
S
Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
Omata
Yoshiaki
Y
Sakamoto
Hiroshi
H
Hara
Takayuki
T
Noguchi
Masato
M
D000818:Animals; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry; D008099:Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006419:Heme Oxygenase (Decyclizing); D009251:NADPH-Ferrihemoprotein Reductase