The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae.
Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds. In the present study, we have expressed human cytochrome P450IA1 in yeast cells. A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter. The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22. Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450. In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm. Control yeast cells transformed with pMA91 showed no cytochrome P450. Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1. A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91. When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained. The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.
Ching
M S
MS
University Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, U.K.
Lennard
M S
MS
Tucker
G T
GT
Woods
H F
HF
Kelly
D E
DE
Kelly
S L
SL
D002461:Cell Line, Transformed; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008861:Microsomes / Q000201:enzymology; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D010735:Phosphoglycerate Kinase