Claims
- 1. A chimeric enzyme gene which codes for a monooxygenase having both monooxygenase activity derived from cytochrome P- 450 and reductase ability derived from NADPH-cytochrome P-450 reductase, said gene comprising DNA coding for a single protein in which the soluble region of the NADPH-cytochrome P-450 reductase is operably linked to the C-terminal of the cytochrome P-450 such that the DNA can be transcribed and translated to form a single protein having both monooxygenase activity and reductase ability.
- 2. A gene according to claim 1 which codes for an enzyme specified by the amino acid sequences shown in FIGS. 2, 3, 4, 5 or 6.
- 3. A yeast expression plasmid which contains a chimeric enzyme gene coding for monooxygenase having both monooxygenase activity derived from cytochrome P-450 and reductase ability derived from NADPH-cytochrome P-450 reductase, said gene comprising DNA coding for a single protein in which the soluble region of the NADPH-cytochrome P-450 reductase is operably linked to the C-terminal of the cytochrome P-450 such that the DNA can be transcribed and translated to form a single protein having both monooxygenase activity and reductase ability, said plasmid being capable of being stably maintained in a yeast strain whereby the yeast strain can be transformed with said plasmid and caused to produce said single protein.
- 4. A plasmid according to claim 3 which is yeast expression plasmid pAMP19, pALP1, pALP17, pALP25 or pALP4.
- 5. A transformed yeast strain carrying a yeast expression plasmid which contains a chimeric enzyme gene coding for a monooxygenase having both monooxygenase activity derived from cytochrome P-450 and reductase ability derived from NADPH-cytochrome P-450 reductase, said gene comprising DNA coding for a single protein in which the soluble region of the NADPH-cytochrome P-450 reductase is operably linked to the C-terminal of the cytochrome P-450 such that the DNA can be transcribed and translated to form a single protein having both monooxygenase activity and reductase ability, said yeast being transformed with said DNA such that the yeast can make said single protein.
- 6. A yeast strain according to claim 5 which is Saccharomyces cerevisiae strain AH22 (pAMP19), AH22 (pALP1), AH22 (pALP17), AH22 (pALP25) or AH22 (pALP4).
- 7. A gene according to claim 1 wherein said cytochrome P-450 is rat liver cytochrome P-450 and said NADPH-cytochrome P-450 reductase is rat liver NADPH-cytochrome P-450 reductase.
- 8. A process for producing monooxygenase which has both monooxygenase activity derived from cytochrome P-450 and reductase ability derived from NADPH-cytochrome P-450 reductase, said process comprising (a) operably linking a first DNA which codes for the soluble region of NADPH-cytochrome P-450 reductase and a second DNA which codes for monooxygenase from the C-terminal of cytochrome P-450 such that the first and second DNA can be transcribed and translated to form a single protein having both monooxygenase activity and reductase ability; (b) transforming a yeast strain with said operably linked first and second DNA; and (c) growing the yeast strain whereby it produces said single protein.
- 9. A transformed yeast strain comprising a yeast expression plasmid containing a gene coding for soluble NADPH-cytochrome P-450 reductase having an amino acid sequence as shown in FIG. 7 but not coding for an amino acid sequence by which native rat liver reductase can bind to a microsomal membrane, such that said yeast strain produces said soluble NADPH-cytochrome P-450 reductase without binding said reductase to a microsomal membrane.
Priority Claims (1)
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61-187713 |
Aug 1986 |
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Parent Case Info
This is a continuation of application(s) Ser. No. 07/786,307filed Nov. 1, 1991, (now abandoned) which is a Divisional of application Ser. No. 07/500,220 filed Mar. 27, 1990 (now U.S. Pat. No.: 5,114,852 Issued May 19, 1992) which is a continuation of application Ser. No.: 07/081,647, filed Aug. 4, 1987, (now abandoned).
Non-Patent Literature Citations (3)
Entry |
Yabusaki, Y., et al. (1988) DNA 7(10), 701-711. |
Porter, T. D., et al. (1985) Proc. Natl. Acad. Sci., USA 82, 972-977. |
Black, S. D., et al. (1982) J. Biol. Chem. 257(10), 5929-5938. |
Divisions (1)
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500220 |
Mar 1990 |
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Continuations (2)
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786307 |
Nov 1991 |
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Parent |
81647 |
Aug 1987 |
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