High yield purification and characterization of engineered human P450 1A2 and generation of immuno-inhibitor antibodies.
P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2. To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested. The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration. This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420. P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases. The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems. Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.
Louërat-Oriou
B
B
Laboratoire d'Ingénierie des Protéines Membranaires, Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Flinois
J P
JP
Beaune
P H
PH
Pompon
D
D
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000302:isolation & purification*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000906:Antibodies; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D017931:DNA Primers; D011994:Recombinant Proteins; D019388:Cytochrome P-450 CYP1A2