Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2.
An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.
Hull
A K
AK
Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts, 02215, USA.
Celenza
J L
JL
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C089817:cytochrome P450TYR; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase