Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae.
Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors. The relative efficiency of the phosphoglycerate kinase and GAL10-CYC1 promoters to direct the P-450 P1 mRNA synthesis was determined. The level of protein synthesis was found to be dependent on the amount of the 5'-noncoding sequence of the original cDNA removed during the construction. Yeast-synthesised P-450 P1 was found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities. The amount of P-450 reached up to 0.6% of the microsomal protein level. A nucleotide sequence coding for a chimeric enzyme in which 40 N-terminal codons of P-450 P1 were replaced by 36 N-terminal codons of P-450 P3 was constructed and expressed in yeast. The resulting protein retained full P-450 P1 activity and was produced with a similar efficiency suggesting that the P-450 N-terminal sequence is not involved in structures critical for the substrate specificities of the P1 isoenzyme.
D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System