Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae.
We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.
Bligh
H F
HF
Institute of Cell and Molecular Biology, Edinburgh University, U.K.
Wolf
C R
CR
Smith
G
G
Beggs
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases