Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1998-05-04
2000-09-12
Yucel, Remy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435 6, 435 29, 43525411, 43525421, 4353201, 536 231, 536 232, 536 235, 536 241, C12Q 126
Patent
active
061176494
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel yeast strains which express cytochrome P450 activity, and to their use. It relates, in particular, to yeast strains which are able to produce a system of human cytochrome P450 enzymes, and to the plasmids which are used for constructing these strains.
The P450 cytochromes constitute a superfamily of membrane enzymes. These enzymes are monooxygenases which are involved, more specifically, in the metabolism of xenobiotics and drugs.
They are used, in particular, for: human hepatic metabolism of natural or artificial xenobiotic molecules (pollutants, drugs or additives). This diagnosis is of prime importance for developing new pharmaceutical molecules, environment, and
Because of their involvement, at one and the same time, in these detoxification processes and these toxicity phenomena, these proteins have been studied intensively (Guenguerich, 1988).
Nevertheless, these studies have rapidly come up against difficulties such as that of studying individual forms of P450 cytochromes. Heterologous expression systems have therefore been developed to overcome these problems.
The use of mammalian cells as hosts for heterologous expression has been developed since 1986 (Zuber et al., 1986). While these systems have the advantage of being closely related to hepatic cells (main location of the P450 cytochromes), they unfortunately suffer from low levels of expression.
While prokaryotic hosts, such as bacteria, admittedly enable substantial quantities of correctly folded cytochrome P450 to be obtained (Barnes et al., 1991), modifications of the 5'-terminal expressed part of the DNA, which cannot be circumvented, are observed with this type of host (Doehmer & Greim, 1992).
On the other hand, it is very particularly advantageous to choose eukaryotic hosts of the yeast type: this organism makes it possible to achieve conditions which are similar to those of human hepatic cells and gives rise to a high level of protein expression. Furthermore, yeast possesses, in endogenous form, all the enzymic machinery which is required for expressing membrane proteins of the cytochrome P450 type and their associated enzymes; thus, yeast has available a cytochrome b5 and an NADPH-cytochrome P450 reductase, i.e. two enzymes whose presence is required for cytochrome P450 to function.
Yeast therefore offers an advantageous solution to the different problems (Oeda K. et al., 1985; Pompon, 1988), since, with this organism: be modified (as is the case with expression in bacteria) various biochemical and structural studies,
Yeasts which have been specially studied for expressing heterologous proteins and which may in particular be mentioned are Kluyveromyces, Pichia, Hansenula, Candida and Saccharomyces, whose genome structures are well known. Various systems for expressing cytochrome P450 in yeasts have been described in the literature.
In strains termed first generation strains, P450 cytochromes have been expressed from plasmids and use the NADPH-cytochrome P450 reductase and the cytochrome b5 which are endogenous to yeast as electron donors (Pompon, 1988; Cullin & Pompon, 1988).
A first improvement of this system gave rise to strains termed second generation strains in which yeast cytochrome P450 reductase was overexpressed (under the control of the GAL10-CYC1 promoter) and a human cytochrome b5 was coexpressed (patent WO93/02200 and Truan et al., 1993). These strains thus made it possible to obtain recombinant cytochrome P450 enzymic activities which were from 5 to 60 times greater in the isoform than in the starting strain.
Nevertheless, the existing systems are not entirely satisfactory: either they do not enable adequate expression of the proteins to be obtained, or the proteins which are obtained are not sufficiently similar to the human system.
The specific objective of the present invention is to propose a third strain generation which does not suffer from the abovementioned drawbacks. Unexpectedly, the Applicant demonstrated that it was possible simultaneously to replace both the yeast NADPH-cy
REFERENCES:
Guengerich et al., Oxidation of Dihydropyridine Calcium Channel Blockers and Analogues by Human Liver Cytochrome P-450 IIIA4, J. Med. Chem. 34: 1838-1844 (1991).
Renaud et al., Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme., Eur. J. Biochem. 194:L 889-896 (1990).
Truan et al., Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5, Gene 125: 49-55 (1993).
Yamano et al., Human NADPH-P450 Oxidoreductase: Complementary DNA Cloning, Sequence and Vaccinia Virus-Mediated Expression and Localization of the CYPOR Gene to Chromosome 7, Molecular Pharmacology 35: 83-88 (1989).
Miyata et al., Isolation and Characterization of Human Liver Cytochrome b5 cDNA, Pharmacological Research 21(5): 513-520 (1989).
Urban et al., Xenobiotic metabolism in humanized yeast: engineered yeast cells producing human NADPH-cytochrome P-450 reductase, cytochrome b5, epoxide hydrolase and P-450s., Biochemical Society Transactions 21: 1028-1034 (1993).
Urban et al. Biochimie. vol. 72, pp. 463-472, 1990.
Peyronneau et al. Eur. J. Biochem. vol. 207, pp. 109-116, 1992.
Eugster et al. Toxicology. vol. 82, pp. 61-73, 1993.
Bellamine Aouatef
Delorme Frederic
Perret Alain
Pompon Denis
Centre National de la Recherche
Rhone-Poulenc Rorer S.A.
Yucel Remy
LandOfFree
Genetically engineered yeast strains does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Genetically engineered yeast strains, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Genetically engineered yeast strains will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-94583