Functional properties of a rainbow trout CYP3A27 expressed by recombinant baculovirus in insect cells.
Cytochrome p450 3A27 (CYP3A27) is highly expressed in liver and intestine of rainbow trout (Oncorhynchus mykiss). In many animal species, the intestine and liver are responsible for the first-pass metabolism of a wide range of xenobiotics. To help determine its physiological role, the catalytic capabilities of CYP3A27 protein were examined. An open reading frame of CYP3A27 in pFastBac donor plasmid was transferred to the baculovirus genome (bacmid DNA) through Tn7 site-specific transposition in DH10Bac competent cells. The CYP3A27 cDNA was positioned under the control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus. The recombinant baculovirus containing a full-length CYP3A27 cDNA (Bv-3A27) was then transfected into Spodoptera frugiperda (Sf9) insect cells for overexpression of CYP3A27 protein. The expressed CYP3A27 protein (714 pmol/mg total protein) exhibited a maximum CO-reduced spectrum at 450 nm at 72 h postinfection after addition of 1 micro g/ml exogenous hemin. The expressed CYP3A27 protein comigrated with the purified trout LMC5 cytochrome p450 (p450) and was recognized by anti-p450 LMC5 IgG on Western blot analysis. The expressed CYP3A27 protein was reconstituted with human NADPH-cytochrome p450 reductase and cytochrome b(5). The reconstitution system showed catalytic activities for the 6 beta-, 2 beta-, and 16 beta-hydroxylation of testosterone at 1.428, 0.043, 0.034 nmol/min/nmol CYP3A27, respectively, and the dehydrogenation of nifedipine at 50 pmol/min/nmol CYP3A27. The present results demonstrated that the baculovirus system is useful for the production of the functional aquatic CYP3A form and that CYP3A27 has the capability to metabolize steroid hormone as reported for mammalian CYP3A forms.
Lee
Su-Jun
SJ
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331, USA.
Buhler
Donald R
DR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000235:genetics; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D051544:Cytochrome P-450 CYP3A; D004789:Enzyme Activation / Q000235:genetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D017686:Oncorhynchus mykiss; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D014162:Transfection
D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating