Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-02-02
2003-06-17
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S006120, C435S025000, C435S058000, C435S069100
Reexamination Certificate
active
06579693
ABSTRACT:
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:
diagnosing in vitro the formation of toxic or mutagenic metabolites by the human hepatic metabolism of natural or artificial xenobiotic molecules (pollutants, drugs or additives). This diagnosis is of prime importance for developing new pharmaceutical molecules,
identifying and destroying toxic or pollutant molecules from the environment, and
producing metabolites.
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:
the N-terminal sequences of the proteins which are expressed do not have to be modified (as is the case with expression in bacteria)
reasonable quantities of heterologous cytochrome P450 are obtained for various biochemical and structural studies,
a system of associated enzymes already exists in the organism.
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-cytochrome P450 reductase and the yeast cytochrome b5 with their human homologues. This is all the more surprising since simultaneous disruption of these two genes was known to be lethal in yeast and, until now, it has not been possible to obtain a viable strain in which these two genes are deleted.
In the strains which are claimed, the yeast cytochrome P450 reductase and/or the yeast cytochrome b5 have been replaced with their human homologue(s). This, very advantageously, makes it possible to create a system which is very similar to hepatic cells, given the fact that the whole multienzyme system is then of the same nature.
This novel system makes it possible to study the effect of the nature of the redox partners of the P450 cytochromes which are expressed as well as the stoichiometries which are required to obtain cytochrome P450 activities which are comparable to those which exist in liver.
The invention therefore relates, initially, to a genetically modified yeast strain which is characterized in that:
Firstly, the genes encoding the endogenous cytochrome b5 and the endogenous NADPH-cytochrome P450 reductase have been inactivated,
Secondly, it comprises a nucleic acid which encodes human NADPH-cytochrome P450 reductase,
Thirdly, it comprises a nucleic acid which encodes human cytochrome b5.
The nucleic acids which are used for integrating genes encoding human cytochrome b5 and human reductase into the strain are preferably cDNAs. The cDNAs containing the totality of the sequences encoding these two proteins have been isolated and sequenced (in the case of human reductase, see S. Yamano et al. Mol. Pharmacol. 1989 Vol. 36: 83-8, and, in the case of human cytochrome b5, see M. Miyata et al. Pharmacol. Res. 1989 Vol. 21: 513-20).
Very preferably, also, the selected yeast is
Saccharomyces cerevisiae.
Within the meaning of the present invention, an inactivated gene is understood as being a gene which has been rendered incapable of encoding its natural protein. The inability of the said genes to encode their natural proteins can be manifested either by the production of a protein which is inactive due to structural or conformational alterations, or by the absence of production, or by production of the natural protein at an attenuated level.
Various methods can be used to inactivate the native genes:
total or partial deletion of the gene. Deletion is understood as being any removal of the gene under consideration. This removal can be of a part of the region encoding the protein and/or of all or part of the transcription promoter region,
one or more point mutations in the gene. The mutations can be obtained by treatment with chemical mutagenic agents (such as alkylating, bialkylating or intercalating agents) or with physical mutagenic agents (X, gamma or ultraviolet rays), or by means of site-directed mutagenesis,
a mutational insertion due to the action of restriction enzymes, which interrupt the reading frame of the gene and inactivate the latter, and/or,
a gene disruption, for example in accordance with the protocol initially described by Rothstein [Meth. Enzymol. (1983)202]. In this case, the integrity of the coding sequence, will be disrupted in order to enable the wild-type sequence to be replaced, by means of homologous recombination, with a sequence which encodes the corresponding human protein.
According to the present invention, preference is given to using the method of gene disruption, as described below.
Various solutions are conceivable for transforming the claimed strains with a view to causing them to express the human enzymes according to the invention; on the one hand, it is po
Bellamine Aouatef
Delorme Frederic
Perret Alain
Pompon Denis
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner LLP
Loeb Bronwen M.
Yucel Remy
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