Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life.
Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 microM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/10(6) cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [35S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.
Barmada
S
S
Department of Pharmacology, Oregon Health Sciences University, Portland 97206.
Kienle
E
E
Koop
D R
DR
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D006207:Half-Life; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis*; D010957:Plasmids; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate