Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein.
An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression in Escherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5' end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3' end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser-Thr. This fusion plasmid was expressed in E. coli and the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP-agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochrome c reduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase in E. coli enhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.
Parikh
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A
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Guengerich
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FP
D000818:Animals; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase