Method for the preparation of a protein by yeasts using an...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

Reexamination Certificate

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C435S320100, C435S254800, C435S255300, C435S285100, C536S023100, C536S023500, C536S024100, C536S024200

Reexamination Certificate

active

06444438

ABSTRACT:

The invention relates to inducible expression systems, to the corresponding transformed strains and to methods for obtaining them for producing proteins, in particular heterologous proteins, in yeasts.
The recombinant DNA technique enables genes for heterologous proteins to be expressed in yeasts. Thus, the construction of vectors containing yeast promoter sequences corresponding to the genes for glycolytic enzymes of
Saccharomyces cerevisiae,
such as 3-phosphoglycerate kinase (PGK), an alcohol dehydrogenase (ADH1) or glyceraldehyde-3-phosphate dehydrogenase (GPD), has enabled important proteins to be produced by fusion of their coding sequence to the yeast promoter, for example leukocyte interferon according to Hitzeman R. A. et al., Nature 293, 717-722 (1981) or hepatitis B surface antigen according to Bitter G. A. et al., Gene 263-274 (1984).
In many cases, it has been observed that the heterologous protein produced is toxic for the host cell, and leads to instability of the plasmids and the selection of cells which no longer express the protein in question. Methods enabling the expression of the gene for the protein to be repressed during the cellular growth phase, and then a high production of the protein to be induced during the final stage of culture, have been found using a promoter regulated by a change in carbon sources, for example the promoter of the galactokinase gene (GAL1) or the promoter of the uridinediphosphoglucose 4-epimerase gene (GAL10) which correspond to two of the four genes responsible for the utilization of galactose in yeast and whose expression is repressed by glucose and induced by galactose. Vectors comprising the GAL1 promoter linked to a heterologous gene have been described, enabling yeast to be grown in a medium containing glucose, and the protein then to be expressed when galactose is present in the medium. Yeast strains transformed with such vectors are described, for example, in British Patent Application 2,137,208, and these vectors, under the control of the GAL1 promoter, express in the presence of galactose proteins such as bovine growth hormone or prorenin. Most yeast promoters contain, upstream from TATA elements which mediate transcription initiation, elements which regulate transcription, “upstream activator sequences” (designated UAS), which represent the binding site of proteins which regulate transcription, such as GAL4 for the activation of the genes of galactose metabolism, these proteins themselves being under the control of their own promoters.
Different means have already been proposed for improving the efficiency of transcription of heterologous genes in yeasts. In particular, it has been proposed to use hybrid promoters which contain, upstream from the TATA element of a constitutive yeast promoter, an extrinsic UAS regulatory sequence, such as UASg which corresponds to the GAL1-GAL10 intergenic sequence and which is inserted, for example, upstream from the TATA element of a constitutive yeast promoter, such as GPD as described in Patent Application WO 86/00,680, or such as PGK as described in European Patent Application 258,067. The inserted UASg sequences enable the constitutive promoter to be successively repressed and then expressed by the carbon source used, such as glucose as a repressor and galactose as an inducer.
However, the use of promoters in general or of any hybrid promoter involves a control over totally repressed or induced levels, which is difficult to achieve because these levels involve a group of proteins which regulate transcription. Thus, the GAL4 gene activates transcription by the presence of galactose, which is directly or indirectly responsible for the dissociation of a complex between GAL4 and an antagonistic protein GAL80, while other proteins appear to be involved in the repression by glucose, such as GAL82 and GAL83 according to Yocum R.R. et al., MBC 4 1985-1998 (1984).
In addition to the drawbacks mentioned above, this type of system based on induction with galactose and/or repression with glucose is not very versatile; in effect, it cannot be used for yeasts which do not assimilate glucose and/or galactose and cannot be used in the case where one of these components constitute all or part of the carbon substrate of the culture medium.
It is hence advantageous to have available in yeasts inducing systems capable of being induced by products which are not in themselves necessary for the culturing of these cells, which is the case, for example, with hormones, in particular steroid hormones.
Thus, the present invention relates to a method for the preparation of a protein by yeasts, according to which:
yeast cells which contain the following are cultured:
a DNA sequence coding for the said protein under the control of elements providing for its expression in yeasts, the said elements comprising a transcription control sequence which is inducible by a complex formed by a receptor and a ligand,
a DNA sequence which is functional in yeast, coding for the said receptor, the receptor comprising two essential portions, one of which recognizes the ligand so as to form a complex with the said ligand and the other binds to the said transcription control sequence; the portion of the receptor which recognizes the ligand is preferably of higher eukaryotic origin, and the ligand is not necessary for the culturing of the cells but is capable of entering the said cells when added to the culture medium,
the said ligand is added to the culture medium at an appropriate time point for induction,
the synthesized protein is recovered.
The expression of proteins by yeasts using recombinant DNA techniques is considered to be well known to those versed in the art. A considerable number of publications have already described the preparation of proteins, in particular heterologous proteins, by means of yeasts, using expression vectors. These expression vectors contain, more often than not, apart from the sequence coding for the said protein, the elements controlling its expression in yeasts, that is to say, in general, a promoter and a terminator.
In most cases, these vectors are nonintegrative vectors, that is to say plasmids; they then contain an origin of replication which is efficacious in yeasts, in particular the origin of the 2&mgr; plasmid or an ars sequence peculiar to the said yeast.
These nonintegrative expression vectors contain, in addition, elements enabling provision to be made for their maintenance in the cells, either using as a selection marker a gene for resistance or alternatively an element complementing an auxotrophy of the host strain, URA3 or LEU2 for example.
In some cases, and in order to overcome the drawbacks linked to the use of self-replicating vectors, it has been possible to develop vectors providing for the integration of the sequences in question at chromosomal level. This type of vector enables a more stable strain to be obtained, but the amplification in respect of expression is sometimes smaller than with a nonintegrative plasmid.
Integration vectors contain, more often than not, at least one sequence homologous with a chromosomal sequence which will provide for exchange and integration.
It must be understood that, since the present invention relates essentially to the induction of transcription, the expression vectors which will be used can be of either the integrative or the nonintegrative type.
Although the method in question is more especially intended for the preparation of heterologous protein, it is possible to use it for expressing yeast proteins, in particular in the case of systems providing for the hyperexpression of a gene which can lead, in some cases to cell death before the optimum biomass is obtained.
“Heterologous protein” is understood to mean a protein foreign to the host cell which expresses it, that is to say different in origin from that of the host cell which, according to the invention, is a yeast. The protein can be of bacterial origin, for example
Escherichia coli
beta-galactosidase, or of higher éukaryotic origin, for example of human origin, such as, fo

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