Expression of P1-450 and P3-450 DNA coding sequences as enzymatically active cytochromes P-450 in mammalian cells.
Two cDNA clones representing the mRNA coding sequences for mouse cytochromes P1-450 and P3-450 were inserted into the thymidine kinase gene of the wild-type vaccinia virus under the control of the vaccinia virus promoter. Murine and human cells infected with each of the resulting infectious recombinant viruses efficiently expressed their respective P-450 proteins. The newly synthesized protein products are translocated into the microsomes, and their characterization by immunochemical analysis indicates that the sizes of the polypeptides expressed were indistinguishable from their cytochrome P-450 counterparts found in mammalian liver microsomes. Functional analysis of each of the proteins by spectral and enzymatic analysis indicates that the expressed proteins have incorporated heme, and the holoenzymes displayed catalytic activities characteristic of their respective cytochrome P-450 enzymes. Thus, this system can be used to produce properly processed and catalytically active P-450 gene products in a wide variety of cells. The remarkable fidelity of expression and processing of these enzymes suggests that the vaccinia virus recombinants can be used for a wide variety of studies, including analysis of the effects of defined mutations produced in vitro, and directly correlate the structure/activity relationships of the cytochrome P-450 enzymes.
D000818:Animals; D001483:Base Sequence; D002248:Carbon Monoxide / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004251:DNA Transposable Elements; D005796:Genes; D005814:Genes, Viral; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D002248:Carbon Monoxide; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013937:Thymidine Kinase