WO2002020789A1 - Gcn4-derived expression of heterologous coding sequences - Google Patents

Abstract

The invention provides an expression cassette comprising: a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor or a functional derivative or fragment thereof; a promoter operably linked upstream to said regulatory element; a heterologous coding sequence downstream to said regulatory element; and a termination signal operably linked downstream to said heterologous sequence; Optionally, the expression cassette further comprises an operably linked selectable marker. The expression system according to this invention provides high level expression of heterologous proteins or peptides which can be regulated at the translational level as well as at the transcriptional level.

Description

GCN4-DERIVED EXPRESSION OF HETEROLOGOUS CODING SEQUENCES

FIELD OF THE INVENTION

The present invention relates to an expression cassette comprising sequences that promote transcription, sequences that promote translation, a heterologous coding sequence, a termination signal and optionally a selectable marker as herein after defined, and to the different uses of said expression cassette.

BACKGROUND OF THE INVENTION

Expression of proteins in microorganisms is a powerful tool for basic research as well as for various medical and industrial needs. Most advanced expression systems have been developed in prokaryotes, in particular in the bacterium Eschericia coli [Gold L., Meth. Enzy ol. 185:11-14 (1990); Mattanovish D. et al, Ann. N. Y. Acad. Sci. USA 782:182-190 (1996)]. In this organism, a foreign protein could be expressed very efficiently, reaching a level up to 10% to 30% of the total protein content of the culture [Gold L. (1990) ibid.; Mattanovish D. et al., (1996) ibid.; and Gao B. et al., Gene 176:269-272 (1996)]. However, most types of bacteria, including E. coli, are pathogens and proteins expressed in them must be well purified and assayed in rigorous and expensive quality control systems. Therefore, although powerful, the bacterial systems are disadvantageous in many cases [Walsh G. and Headon D.R., Protein Biotechnology, John Wiley & Sons Ltd., West Sussex England. (1994)].

Methods of expression of heterologous proteins in eukaryotic cells have also been developed. For many reasons expression in eukaryotic cell systems is advantageous over that in prokaryotic cells, since, most proteins (endogenous or heterologous) obtained by such systems undergo accurate post-translational processing which result in the production of biologically active forms of these products [Walsh, G and Headon D R. (1994) ibid.; Romanos M.A. et al. Yeast 8:423-488 (1992)]. However, although expressed, only low levels of protein are obtained in mammalian cell systems. In addition, as indicated, there is a high risk of viral infection when using these expression systems, thus requiring expensive diagnostic tests to ensure the safety in using proteins obtained therefrom.

An attractive alternative, for an expression system is of the yeast, and in particular, the baker's yeast Saccharomyces cereυisiae. As with other eukaryotic systems, most foreign proteins expressed in this eukaryote are accurately processed post-translationally and are biologically active. In addition, as a food organism, yeast is highly acceptable for the production of pharmaceutical proteins, thus reducing the cost of manufacturing such proteins. Yet, despite the great potential of yeast as a protein factory, this organism is not widely used. The major reason for this may be the fact that the level of expression obtained in yeast is usually much lower than that obtained in bacteria [Romanos M.A. et al. (1992) ibid.].

To date, only few yeast expression vectors are available. All vectors (similarly to the bacterial and mammalian vectors) contain strong promoters (usually a promoter of glycolitic genes for example, GPD1, ADH1, PGKl). In addition, they contain the 2μ origin of replication which allows several plasmid replications during one round of cell cycle [Schneider J.C. and Guarente L. Meth. Enzymol. 194:373-388 (1991); Schena M. et al. Meth. Enzymol. 194:389-398 (1991); Rose A.B. and Broach J.R., Meth. Enzymol. 185:234-279 (1990)]. As a result, these plasmids are present in 60-100 copies per cell. Some of the vectors also contain an inducible promoter, the most widely used is the GALl-10 promoter [Schena M. et al. (1991) ibid.]. None of the currently used vectors (in yeast or any other system) harbors an efficient and regulated element to control translation.

The strategy used with- yeast vectors in order to increase expression is a combination of high transcription rate with the presence of many copies of the vector per cell. Cells harboring those vectors must be grown on synthetic media under selective conditions to maintain the plasmids in the cell. These conditions are usually not optimal. When grown in laboratory flasks these cultures enter stationary phase at low concentrations and the quantities of proteins obtained are usually low. When grown in industrial fermentors, high cell concentrations could be obtained via special treatments and long and expensive incubation time.

The present invention aims at the development of an expression vector utilizing regulatory elements of the GCN4 gene. GCN4 encodes a transcription activator, which induces transcription of coding sequences of biosynthetic enzymes [Hinnebusch A.G., Microbiol. Rev. 52:248-273 (1988); Hinnebusch A.G. In Broach J.R. et al., Eds. The Molecular and Cellular Biology of the Yeast Saccharomyces Gene Expression. Cold Spring Harbor Laboratory. NY pp 319-414 (1992); Hinnebusch A.G., J. Biol. Chem.272:21661-21664 (1997)]. Under optimal growth conditions on media supplemented with amino acids and pyrimidine and purine bases, GCN4 is not expressed. When cells are starved for amino acids, Gcn4 expression is dramatically induced, and consequently, transcription of biosynthetic genes commences. Expression of GCN4 is regulated mainly at the translational level. The mRNA levels of GCN4 are high under most growth conditions, but nder optimal growth conditions, translation rate of this mRNA is extremely low and GCN4 protein is barely detectable. This minimal translation rate is maintained by 4 short open reading f ames located in the 5' non-coding leader of the message (4 upstream ORFs) [Hinnebusch, A.G. (1997) ibid.; Dever, T.E. et al. Cell 68:585-596 (1992)]. Ribosomes recognize the most upstream ORF and begin translation. Yet, they dissociate from the mRNA at the stop codon and re-initiate translation at another short ORF. In most cases ribosomes do not reach the 5th ORF which is the GCN4 coding sequence [Hinnebusch A.G. (1997) ibid.]. This prevention of GCN4 translation depends on the high rate of re-initiation. Under starvation for amino acids the rate of re-initiation is reduced to a minimum because the activity ofinitiation factor 2μ (eIF2) is inhibited. Inhibition of eIF2 is obtained through its phosphorylation, catalyzed by the Gcn2 Mnase. This inhibition halts most translation activity in the cell, an obvious cellular protecting response to amino acid starvation. Although translation in the cell is generally ceased under these conditions, the GCN4 mRNA is preferably translated. This seems to be a logical response since the Gcn4 protein is required for induction of amino acids biosynthesis. In the laboratory, a ino acid starvation could be easily mimicked by treatment of yeast cells with inhibitors of biosynthetic enzymes. The most widely used compound is 3-amino-l,2,4-triazole (3-AT), a competitive inhibitor of His3. Cells combat the competitive inhibition effect simply by producing more His3. Indeed, the rate of GCN4 translation (leading to HIS3 transcription) dramatically increases in response to 3-AT treatment [Hinnebusch A.G. (1988) ibid.; Hill D.E. et al. Science 234:451-457].

The sequence which resides upstream to the GCN4 coding sequence (including the 5'UTR and the promoter) was shown to govern transcription and translation of GCN4/β-galactosidase (β-GAL) chimeric gene [Hinnebusch A.G. PNAS USA 81:6442-6446 (1984); Thireos G. et al, PNAS USA 81:5096-5100 (1984); Hinnebusch A.G. Mol. Cell. Biol. 5:2349-2360 (1985)]. As translation of GCN4 (and of the GCN4/β-GAL chimeric gene) increases when eIF2 (is phosphorylated and its activity decreases, it was recently suggested to use the GNC4/β-GAL gene as a marker which monitors the level of eIF2α phosphorylation and activity [Dever T.E. Methods 11:403-417 (1997)].

The inventors have previously described preliminary results with production of HSA using a GCN4 derived expression system |Mimram A., et al., BioTechniques 28:552-560 (2000)].

However, hitherto it was unknown whether the GCN4 upstream sequences (with no coding sequences) are capable of controlling transcription and translation of an heterologous gene at the translational level, in conjunction with another heterologous promoter.

Further, as gene expression is a complex multi-step process and problems can arise at numerous stages, from transcription through to protein stability, it is clear that the insertion of a foreign gene into an expression vector does not guarantee a high level or properly regulated expression of that foreign protein. It would be advantageous to have an expression system capable of regulated gene expression at the translation level as well as at the level of transcription. As indicated currently available expression systems do not provide efficient control of translation.

SUMMARY OF THE INVENTION

The inventors have now surprisingly found that GCN4 upstream sequences could be utilized for efficient and regulated expression of foreign genes by the expression cassette of the invention. The present invention provides an expression vector utilizing the 5'UTR of the GCN4 gene. This element, and variations or modifications thereof, is used to enhance translation of foreign coding sequences. This 5'UTR is used as a modular unit to function as a regulator of in combination with any promoter of yeast or of other organisms. The promoter of the GCN4 gene can be used as well.

The present invention relates to an expression cassette comprising: (a) an element capable of post-transcriptional regulation comprising the nucleotide sequence of the GCN4 transcription factor 5'UTR or a functional derivative or fragment thereof; (b) a promoter linked upstream to the sequence of the 5'UTR of GCN4 or a functional derivative thereof; (c) a heterologous coding sequence downstream to said regulatory element; and (c) a termination signal operably linked downstream to said heterologous sequence.

Optionally, the expression cassette further comprises an operably linked selectable marker. The regulatory element derived from the gene encoding the GCN4 transcription factor within the expression cassette of the present invention is preferably of the yeast's Saccharomyces cereυisiae, and more preferably comprises a sequence between -600 to -1 upstream to the GCN4 coding sequence, or a functional fragment thereof. This 5'UTR that can be used as a modular unit that increases translation and can function in combination with any promoter.

In another aspect the invention relates to sequences comprising at least-600 to -1 of the 5'UTR that can be used as a modular unit that increases translation and can function in combination with any promoter.

A preferred embodiment relates to the heterologous coding sequences within the expression cassette of the invention encode amino acid sequences, particularly those having a therapeutic -utility.

In a second aspect, the invention relates to an expression vector comprising the expression cassette of the invention.

Yet, the invention also relates to a method for the regulated production of a therapeutic protein or peptide in eukaryotic cells, which method comprises the steps of:- (a) providing eukaryotic cells; (b) transforming said eukaryotic cells with an expression vector comprising:- (i) a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor, preferably that of the yeast S. cereυisiae, ox a functional derivative or fragment thereof; (ϋ) a promoter, (iii) a heterologous coding sequence downstream to said regulatory element; (iv) a termination signal operably linked to said heterologous sequence; and (v) optionally, an operably linked selectable marker; (c) selecting from the cell population obtained in (b) those cells which harbor said expression vector and growing the same;and (d) inducing expression of said heterologous coding sequence in said cells under amino acid starvation conditions or under conditions which mimic said starvation to obtain a functionally active protein or peptide encoded by said heterologous coding sequence. The method of the invention optionally comprising the step of isolating said functionally active protein or peptide obtained in step (c) from said cells.

In an alternative embodiment according to the present invention derivatives of said expression vector may be expressed constitutively so that induction is not required. In additional alternative embodiments according to the present invention the promoter could be inducible by certain inducers including but not limited to galactose, cuprum and other inducers as are known in the art. In those embodiments using an inducible promoter the 5'UTR regulatory element of GCN4 may be inducible by starvation conditions or could be active constitutively.

Evidently, for efficient expression of the heterologous coding sequence, when necessary, the expression vector of the invention may further comprise at least one replication element.

In a further aspect, the invention relates to a therapeutic protein or peptide produced by the method of the invention and to eukaryotic cells transformed with the expression cassette or vector of the invention, said cells being capable of expressing a heterologous coding sequence comprised therein, to obtain said therapeutic protein or peptide. ^' .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Schematic description of the GCN4 gene and the primers used for cloning the sequence comprising the promoter and the post-transcriptional regulatory elements of the 5'UTR. Open reading frames are shown as boxes and primers as arrows. Abbreviations: Lo=Long, Mid= Medium, Sh=Short and Prim is primer.

Figure 2 The sequence of the GCN4 elements used in the invention. The sequence covers the DNA that resides from -1 to -2000 base pairs upstream of the GCN4 coding sequence. The sequences encoding the 4 upstream ORFs are underlined. The transcription initiation site (starting point of 5'UTR) is marked by an asterisk.

Figure 3A-3C The basic pGES vector family containing the HSA cDNA. Fig. 3A shows the integrative vector pGES306-HSA, Fig. 3B shows the centromeric vector pGES316-HSA and^' ig. 3C shows the multicopy vector pGES426-HSA. Abbreviations: Bac ori is a bacterial origin of rephcation, Prom = promoter and Term = terminator.

Figure 4 Induction and expression of HSA by pGES vectors containing either the long regulatory element (Long) or the middle sized regulatory element (Mid), both containing the GCN4 promoter and post-transcriptional regulatory elements of the 5'UTR. Western blot analysis using anti-HSA antibodies was performed on protein extracts prepared from cultures (BJ2168 cells) harboring either pGES306-L-HSA (Long) or pGES306-M-HSA (Mid.), 8 and 10 hours after addition of 3-AT. Abbreviations: Lo = Long, Mid = Medium, Prom = Promoter and T = Time.

Figure 5A-5B Time course and dose response analysis of induction of HSA expression'. Fig. 5A shows a cultμre of BJ2168 cells harboring pGES426-L-HSA grown to logarithmic phase and than supplemented with 3-AT (20 mM). Samples were removed at indicated time points and subjected to western blot analysis. Fig. 5B shows a similar culture grown to logarithmic phase and then divided to six fractions, each fraction supplemented with the indicated concentrations of 3-AT. Cultures were further grown for 7 hours before collection and analysis by western blotting. Purified HSA (10 and 50 ng) were loaded on the right lane of each gel. Abbreviations: T + is time with.

Figure 6A-6C Expression of HSA from pGES vectors is induced at the translational level. A culture harboring the oGES306-L-HSA vector and culture harboring the control plasmid pRS306-S were grown to logarithmic phase when 3-AT was added. At the indicated time points samples were removed. Protein extracts and RNAs were prepared from each sample and analyzed by western blot and primer extension respectively. Fig. 6A shows the western blot analysis. Fig. 6B shows the primer extension analysis using GCN4 primer. Fig. 6C shows the primer extension analysis using HSA primer. Abbreviations: T + is time with.

Figure 7 pGES system is inducible in highly concentrated cultures. A culture harboring the pGES306-L-HSA was grown on YPD to OD600=3.3 when the media was replaced with SD(-HIS) supplemented with 40mM 3-AT. Samples were removed and analyzed by western blot as the indicated time points after induction. Purified HSA (50 ng) was loaded on the right lane of the gel. Abbreviations: T + is time with.

Figure 8 Various derivatives of the basic pGES family, illuminating the modularity of the system. Fig. 8A and 8B shows derivatives of pGES containing useful multicloning sites and different selectable markers. Fig. 8C shows a vector in which the 5'UTR of GCN4 was combined with another promoter (of GAL1). Fig. 8D shows a vector in which the four upstreams ORFs in the 5'UTR of GCN4 were either mutated or eliminated. Translation from this vector is not regulated, but rather constitutive. Fig. 8E shows a vector with GAL1 promoter 5'UTR of GCN4 with 4 upstream ORFs either mutated or eliminated.

Fig 8F and 8G show a pGES vector with sequences encoding a leader peptide (LP) and either haemaglutinin (HA) or polyhistidine (PolyH) tags as well as multicloning site (MCS). Abbreviations: Prom is promoter.

The vectors shown in Fig.8 are merely exemplary, each of them represents a family of vectors (e. g. with different selectable markers, different cloning sites, different origin of replications, different promoters, different modifications in the 5'UTR) that uses the principles described in the specification. DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the development of a new family of yeast expression vectors, which are based on the 5'UTR sequence of GCN4 as the regulatory element. This system affords efficient regulation of heterologous gene expression at the translational level. Expression from these vectors is tightly controlled and is induced under amino acid starvation conditions.

According to one preferred embodiment of the present invention, the post-transcriptional regulatory elements of the 5'UTR are used in conjunction with the GCN4 promoter for enhancement of heterologous gene expression. It will be appreciated by the artisan that any promoter could serve for this purpose.

The invention also discloses vectors in which the GCN4 5'UTR was modified. Modifications or fragments of the GCN4 5'UTR may advantageously be combined with other regulatory elements including an additional promoter. Expression from vectors comprising modifications of the GCN4 5'UTR may be either regulated or constitutive.

Biosynthesis of proteins (transcription and translation) in living organisms is similar. Therefore, in principle, any cell type may be used as a host for the expression of a foreign gene. Nevertheless, for practical reasons, to date only few cell-systems have been employed as "factories" for proteins. These practical reasons include, inter alia, the cost of growing the host cell, the efficiency in which the foreign protein is expressed, the cell's stabihty which may alter as a result of the expression of unrecognized proteins, the desire to obtain biologically functional proteins and the cost and efficacy of producing a purified protein.

The use of yeast for the expression of foreign proteins provides numerous advantages. While yeast are edible organisms and thus safe to use as a "factory" for pharmaceuticals. Prokaryotes, such as Escherichia coli, have toxic cell wall pyrogens, while mammalian cells may contain oncogenic or viral DNA, so that the products from these organisms must be tested more extensively. Further, yeast can be grown rapidly on simple media and to high cell density, their genetics are advanced, and they can be manipulated almost as readily as E. coli [Romanos M.A. et al. Yeast 8:423-488 (1992)]. Therefore, the use of yeast cells for the production of proteins reduces cost and is less time consuming. In addition, since yeast cells are eukaryotic cells, many of the post-translational processes necessary for the production of a biologically functional product are correctly performed.

As will be demonstrated by the following Examples, the system of the present invention differs from currently available expression vectors since it is controlled at the translational level, ensuring efficient translation and allowing rapid induction. All currently available expression systems ensure transcription, but cannot control or enforce translation. Under non-inducible conditions, expression from the vectors of the invention is quite well suppressed.

Further, the system of the invention allows fine control on the level of expression, since this level is directly proportional to the concentration of the inducer. Yet further, integrated versions of the expression vectors described in the invention are almost as efficient as multicopy versions. Therefore, the pGES expression system could be used under various growing conditions, including in rich, non-selective media, which is clearly an advantage.

The present invention thus relates to an expression cassette comprising: (a) a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor, or a functional derivative or fragment thereof; (b) a promoter operably linked upstream to said regulatory element; (c) a heterologous coding sequence downstream to said regulatory element; and (d) a termination signal operably linked downstream to said heterologous sequence;

Optionally, the expression cassette further comprises an operably linked selectable marker. It should be noted that as an alternative for the heterologous coding sequence, the expression cassette may comprise a genomic or cDNA library, which may be used for many different purposes, as known to the man skilled in the art, e.g. for screening.

By the term 'heterologous coding sequence' is meant any sequence which encodes a protein or peptide which one requires to be expressed. The coding sequence may be a naturally occurring sequence or a derivative thereof of any origin or an artificial sequence or a derivative thereof.

The term 'derivatives' used herein in connection with nucleic acids, either in a coding or in a non-coding sequence, means any modification thereof including, insertions, deletions, mutations and substitutions.

The term 'functional fragment' used herein in connection with nucleic acids, either in a coding or in a non-coding sequence, means any fragment which will provide substantially the same functional result as the original intact sequence in terms of enhanced expression as determined relative to an equivalent construct lacking the regulatory element of the invention.

The expression cassette of the invention may further comprise operably linked thereto a rephcation element, to provide an expression vehicle which will be substantially independent from the host cell rephcation elements.

When a rephcation element is required for the efficient expression of the coding sequence within the expression cassette of the invention, it may be any suitable replication element, for example, the 2μ element, which supports maintenance of many copies of the gene in the cell [Rose A.B. and Broach J.R. Meth. Enzymol. 185:234-279 (1990)] or a centromeric element (CEN) which rephcates together with the endogenous DNA and is maintained therefore as a single copy per cell. CEN based vectors are combined in many cases with auto-rephcating sequences I

(ARS). The CEN-ARS based vectors, which may also be employed, are maintained in few copies (usually 1-2).

However, as indicated hereinbefore, the expression cassettes of the invention may also be employed as an integrative single copy fragment, which does not require growing the cells on a selective medium.

Within the expression cassette of the invention, the said regulatory elements derived from the GCN4 transcription factor gene is preferably of the yeast Saccharomyces cereυisiae. In particular, the regulatory element employed in the expression cassette of the invention, comprises any sequence, or any fragment comprising at least part of the sequence from minus 600 to minus 1 of the 5'UTR of GCN4 and any functional derivative obtained by mutation thereof is a modular fragment that could be used in combination with any promoter of any origin.

Of particular interest is the regulatory element comprising the sequence substantially as denoted by SEQ ID NO: 15 or any sequence or any fragment thereof.

Additionally and preferably, a fragment of said sequence, comprising at least part of the sequence from minus 600 to minus 1 of the 5'UTR of GCN4 and any derivative thereof further comprising elimi ation of at least one of the 4 upstream ORFs is a modular fragment that may be used in combination with any promoter of any origin.

A preferred embodiment relates to the expression cassette of the invention wherein the promoter may be the GCN4 transcription factor promoter of the yeast Saccharomyces cereυisiae.

In a specifically preferred embodiment this promoter can be taken from nucleotides minus 2000 to minus 600 upstream to the GCN4 coding sequence, substantially as denoted by SEQ ID NO: 11. Preferably, the expression cassette of the invention comprises promoter comprising nucleotides minus 1067 to minus 600 upstream to the GCN4 coding sequence and more preferably, it comprises the nucleotides from minus 904 to minus 600 of said sequence upstream to the GCN4 coding sequence, substantially as denoted by SEQ ID NO: 14 and 13 respectively, or any fragment or derivative thereof.

Alternativelly and preferably the expression cassette of the invention may comprise the GALl promoter or any functional fragments thereof.

In a preferred embodiment, the expression cassette according to the invention comprises a coding sequence, which encodes a naturally occurring amino acid sequence or a derivative thereof. In particular, the amino acid sequence corresponds to a natural protein, polypepti.de or peptide or to a functional derivative of said natural protein or peptide, to an essential fragment of said proteins or peptides or to a chimera of said proteins, peptides or fragments.

The term 'functional derivative' used herein means any modification of the naturally occurring sequence, including insertions, deletions or substitutions of nucleic or amino acids residues therein, or any other suitable modification which does not damage the biological function of the nucleic or amino acid sequence. The functional derivatives according to the invention may also be any dimeric or multimeric form of said amino acid according to the invention.

Functional proteins or peptides within the scope of the invention encompass any protein or peptide that one requires to have expressed, advantageously those proteins or peptides having a therapeutic value. Such proteins or peptides may be selected from the group consisting of enzymes, antibodies, cytokmes, hormones, receptors, transcription factors or any chimera of said therapeutic products with a different protein or peptide". The term "therapeutic" as used herein means any protein, polypeptide or peptide having an industrial application. Thus it may refer to any pharmaceutical, agricultural, veterinary or diagnostic product as well as for enzymes, toxins and inhibitors used in chemical industry, plastic industry, food industry and in chemical and biological research .

In one particular embodiment according to the invention, the expression cassette comprises a heterologous coding sequence, which encodes the human serum albumin (HSA) protein.

The termination signal according to the invention may be any suitable terminator which is capable of terminating the transcription of coding sequence and of adding polyadenylated ribonucleotides to the 3 end of the primary transcript obtained therefrom. The termination signal thus may be selected from the group consisting of the yeast alcohol dehydronease gene (ADHl), TRPl, glyceralkehyde-3-phosphate dehydrogenase (GAP, GAPDH), MF1, phosphate regulated terminator (e.g. of PH05), phosphoglyce ate kinase (PGK , CYCl or the GCN4 terminator. The termination signal can be selected from genes of any organism, not necessarily yeast.

In a second aspect, the invention relates to an expression vector comprising the expression cassette of the invention.

The expression vector of the invention may include, in addition to the expression cassette, any other element required for the efficient and regulated expression of the product encoded by the coding sequence, such as an ORI sequence. Further, the vector may include an appropriate polyA signal, and or an intron splicing signal, e.g. of the SV40 virus.

Evidently, the vector may include other elements such as sequences which encode peptides facilitating the secretion of the protein expressed by the heterologous coding sequence incorporated in the expression cassette into the growing media (secretion elements). Further, it may contain sequences which encode peptides capable of eliciting production of antibodies thereagainst, or even those encoding tagging proteins, which bind to specific columns during affinity purification (e.g. polyHis that binds to agarose-nickel beads, gluthatione S-transferase (GST) that binds to gluthatione beads, cellulose binding domain (CBD) which binds to cellulose).

According to further embodiments within the scope of the present invention, the vector may include combinations of several of the above elements. By way of a non-jumitative example the vectors may comprise a sequence encoding a leader peptide that facilitates secretion and a sequence encoding a tag such as CBD, GST, or polyhistidine. In one preferred embodiment, the expression vectors of the invention contain the leader peptide of the mating factor alpha, the CBD sequence, or the polyhistidine sequence (as will be exemplified hereinafter and in Fig. 8).

In one preferred embodiment, the expression vector according to the invention is the integrative plasmid substantially as shown in Figure 3A. Alternatively, according to a second preferred embodiment of the invention, the expression vector of the invention is the centromeric plasmid substantially as shown in Figure 3B. Yet, the expression vector of the invention may be the multicopy plasmid substantially as shown in Figure 3C.

In a third aspect, the invention relates to a method for the regulated production of a therapeutic protein or peptide in eukaryotic cells, which method comprises the steps of: (a) providing eukaryotic cells; (b) transforming said eukaryotic cells with an expression vector comprising:- (i) a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor, preferably that of the yeast S. cereυisiae, or a functional derivative or fragment thereof; (ϋ) a promoter operably linked to said regulatory element; (iii) a heterologous coding sequence downstream to said regulatory element; (iv) a termination signal operably linked to said heterologous sequence; and (v) optionally, an operably linked selectable marker; (c) selecting from the cell population obtained in (b) those cells which harbor said expression vector and growing the same; (d) inducing expression of said heterologous coding sequence in said cells under amino acid starvation conditions or under, conditions which mimic said starvation to obtain a functionally active protein or peptide encoded by said heterologous coding sequence; and (e) optionally, isolating said functionally active protein or peptide obtained in step (c) from said cells or from the cell's medium. As will be exemplified herein in some derivatives in which expression is constitutive induction is not required. Alternatively, in some derivatives, in which the 5'UTR of GCN4 is linked to other promoters induction may also require other treatments to induce transcription as are well known in the art, such as adding galactose or copper.

According to the method of the invention, when necessary for efficient expression, the expression cassette further comprises a rephcation element. The cells provided for the expression of the coding sequence may be any eukaryotic cell capable of efficiently and sufficiently expressing said coding sequence, thus producing an active product. Particularly, the eukaryotic cell may be of a mammalian type.

Alternatively, the eukaryotic cells employed by the method of the invention may be yeast cells. .Evidently, the yeast, cells will be those capable of expressing said coding sequence and may include, inter alia, the yeast S. cereυisiae, Schizosaccharomycespom.be or Pichiapastoris.

In a preferred embodiment, the yeast cells are of the S. cereυisiae type, in particular, those of the strains SP1, W303, C13ABYS86, BJ2407, BJ2168, H625, H628, and H640 and is preferably of the BJ2168 strain. According to one currently preferred method of the invention, the regulatory element comprising sequences of the yeast S. cereυisiae GCN4 transcription factor 5'UTR. This regulatory element comprises nucleotides taken from the -600 to -1 sequence of the 5'UTR, or any derivative or fragments thereof, substantially as denoted by SEQ ID: 15. This modular fragment may be used in combination with any promoter. In a preferred embodiment the method of the invention utilizes the GCN4 promoter. The GCN4 prompter comprises nucleotides taken from the sequence of -2000 to -600 upstream to the GCN4 coding sequence, or any functional fragment thereof. Nevertheless, said promoter sequence may preferably comprise the nucleotides from -1067 to -600 upstream to the GCN4 transcription factor. Moreover, the sequence employed by one currently preferred method of the invention is that having the nucleotides from -1067 to -600, substantially as denoted by SEQ ID NO: -13, upstream to the GCN4 coding sequence as the promoter operablly linked to the regulatory elements of the invention.

It has now been found that advantageously a fragment comprising at least part of the sequence within the 5'UTR of the GCN4 comprising nucleotides -600 to -1 or any derivative thereof may be used modularly in combination with other promoters.

In a specifically preferred embodiment, the promoter employed by the method of the invention may be an inducible strong promoter such as the GALl, together with the 5'UTR of GCN4 (Fig. 8). This vector is highly inducible at the level of transcription (using galactose) and at the level of translation (using 3-aminotriazole). Thus a dramatic additive effect of very high transcription, rate and very high translation rate, gives rise to most efficient protein production.

By the method of the invention, any heterologous coding sequence, which encodes an amino acid sequence, may be utilized. The amino acid sequence may comprise naturally occurring amino acids and/or derivatives of the same, as defined hereinbefore.

The a ino acid sequences produced by utilizing the expression cassette of the invention will preferably correspond to a natural protein or peptide having an industrial purpose or to a functional derivative thereof, to a fragment of said proteins or peptides or to a chimera of said proteins, peptides or fragments thereof. Accordingly, the protein or peptide product may be an enzyme, an antibody, a cytokine, a hormone, a receptor, a transcription factor or a functional derivative thereof or any other protein or peptide having a biological and/or therapeutic function.

The product of the invention could also be a non-coding RNA, catalytic RNA or any functional or non-functional artificial (not occurring naturally) peptide.

The product could be also an array of many different peptides produced through the use of a cDNA or a genomic library. In a preferred embodiment, the human serum albumin protein is obtained by the method of the invention.

One substantial advantage of the method of the invention is the possibility to obtain the expressed protein in a controlled manner and with high yield.

As will be shown in the following Examples, under conditions, which mimic amino acid starvation, HSA is obtained in high quantities.

Starvation conditions may be mimicked by specific drugs which inhibit amino acid biosynthesis, such as 3-aminotriazole (3-AT), allowing stringent control and easy induction of expression of the foreign gene. Other examples for drugs capable of mimicl ng amino acid starvation may include 5-fluorotryptophan.

In the method according to the invention, the termination signal may be any suitable termination signal, taken from any organism. However, it is preferably selected from yeast, for example, the termination signal of ADHl, TRP1, GAP (GAPDH), MF1, PH05, PGK, CYC1 or of GCN4.

In a particular embodiment, the cells are transformed by the method of the invention with the expression vector which is the integrative plasmid substantially as shown in Figure 3A. As an alternative, the cells may be transformed by the method of the invention with the centromeric expression vector substantially as shown in Figure 3B. Yet, according to a further particular embodiment, the cells may be transformed by the method of the invention with the multicopy expression vector substantially as shown in Figure 3C.

As will be shown in the following Examples, the intracellular level of protein expression (per cell) obtained with the pGES vectors is somewhat higher than that obtained through the use of currently available vectors. Furthermore, as it could be used as an integrated vector and is still inducible in dense cultures (Fig. 6), these vectors may give rise to improved protein production (in quantity and quality). An improved transcription rate could also be obtained through a combination of a strong constitutive promoter (from another gene) with the regulatory element of GCN4 5'UTR. Such chimera may be more efficient than pGES and still be regulated at the translational level.

More preferred combinations will utilize an inducible strong promoter as is known in the art including but not hmited to that of the GALl gene for example, together with the 5'UTR of GCN4 (Fig. 8B and 8D). This vector is highly inducible at the level of transcription (using galactose) and at the level of translation (using 3-aminotriazole). Thus a dramatic additive effect of very high transcription rate and very high translation rate, gives rise to most efficient protein production.

In a further aspect, the invention concerns with eukaryotic cells transformed with the expression cassette or expression vector of the invention which cells are capable of expressing the heterologous coding sequence comprised therein. Eukaryotic cells transformed with the system of the invention may be mammalian cells, yeast cells, fungi, insect cells, avian cells or any other eukaryotic cell capable of efficiently producing the desired therapeutic protein or peptide.

Examples for insect cell lines which may be transformed with the system of the invention, but not limited thereto, are Spodeoptera frugiperda (e.g. sf9 cell line), Trichoplusia ni (Tn), Mamestra brassicae, Estigmene acrea, Anopheles gambiae, Aedes albopictus and Aedes aegypti Mos20. Examples for mammailan cells which may be transformed with the system of the present invention, but not limited thereto, are CV-1, COS, C127, NIH3T3, FR3T3 or the CHO cell lines.

Regulation of GCN4 translation is well studied and mutants are available in which GCN4 translation rate is constitutively high and not regulated (gcd mutants are described by Hinnebusch et al. |Hinnebusch A.G., Microbiol. Rev. 52:248-273 (1988); Hinnebusch A.G. In Broach J.R. Jones et al Ed.:

The Molecular and Cellular- Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY pp 319-414; and Harashima S. and Hinnebush A.G., Mol. Cell Biol. 6:3990-3998 (1986)]). Also GCN4 translation is induced by the yeast Ras/cAMP cascade and is therefore high in RASvall9 and bcyl strains [Engelberg D. et al, Cell 77:381-390 (1994)]. pGES plasmid could be used in gcd strains and in RASvall9 and bcyl strains as constitutive vectors to obtain large quantities of proteins for research and industry.

It' is clear therefore, that any protein or peptide, particularly therapeutic ones, produced by such cells, encompass part of the invention. One example for such a protein is the HSA protein.

Finally, the invention relates to the use of the expression cassette of the invention, wherein the , heterologous coding sequence encodes a therapeutic protein or peptide, in the production of a therapeutic protein or peptide.

Further, the invention relates to the use of the expression cassette of the invention, wherein the heterologous coding sequence encodes a therapeutic protein or peptide, in the preparation of a pharmaceutical composition containing said protein or peptide. Nevertheless, the expression cassette of the invention may be utilized for the expression of libraries, for example, for cloning via functional complementation of mutants, for screening genes which allow growth under certain conditions (e.g. toxic conditions, under extreme conditions or in the presence of drugs), for the detection of protein-protein interactions (through the expression of a sequence encoding a chimeric peptide), as with the two hybrid system. In the latter case, the nucleotide sequence of GAL4-DNA binding domain fused to a cDNA Hbrary, or alternatively, the sequence of GAL4 activation domain fused to a hbrary, will be expressed using the expression cassette of the invention. The expression cassette may also be utilized in the Sos recruitment system (SRS) or in the Ras recruitment system (RRS) system, in^" which the membrane localized cDNA Hbrary, the SOS -cDNA Hbrary or the RAS-cDNA Hbrary, may be expressed using the system of the invention.

The invention will now be described in an illustrative manner and it is to be understood that the terminology, which will be used is intended to be in the nature of the words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in Hght of the above teaching. It is therefore, to be understood that- within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

EXAMPLES

Example 1

Materials and methods

Yeast strains and growth conditions

The SP1 strain (MATa, his3, leu2, ura3, trpl, ade8 and can [Kataoka T. et al., Cell 37:437-445 (1984)] was obtained form Wigler M.) was used as a source of genomic DNA from which the GCN4 upstream sequences were loned by PCR. The BJ2168 strain (MATa, prcl-407, prb 1-1122, pep4-3, leu2, trpl, and ura3-52 [Zubenko, G.S. et al., Genetics 96:137-146 (1980)] was used as a host for. all expression plasmids and was obtained from the yeast genetic stock center at UC Berkley. Yeast ceHs were grown on either YPD (1% yeastextract, 2% bactopeptone, 2% glucose) or on SD media (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate (Difco), 0.5% NH4(S04)2, 2% glucose and the required amino acids and uracil (40 mg Hter)). Strains arboring plasmids were grown on SD (-uracil).

For expression experiments cultures were grown to logarithmic phase (unless otherwise indicated) on wither SD (-uracil), or YPD (for integrated vectors), coUected and re-suspended in SD (-uracil, -histidine) (or in SD (-his) for integrated vectors), supplemented with 3-AT (mostly at 40 mM, unless otherwise indicated).

PCR cloning of GCN4 fragments and plasmid construction

PCR reaction was performed using genomic DNA of yeast strain SP1 as a template and the foHowing primers:-

Primer 3' - 5'GCGCCGGATCCTTTATTTGTATTTAATTTATTTTCTTGAGC-3', substantially denoted by SEQ ID NO: 1. This primer contains the BamHI site and was used in all PCR reactions (Fig. 1).

Long promoter - 5'GCGCCGACTAGTTTGCCACGTACATGACATTA-3', substantially denoted by SEQ ID NO: 2. This primer was used in combination with primer 3' to clone the long promoter by amplifying the sequence from nucleotide number -1067 to nucleotide -1.

Mid promoter - 5'GCGCCGATAGTGCCGAACACCACCGGCATCTTG-3', substantiaUy denoted by SEQ ID NO: 3. This primer was used in combination with the primer 3' to clone the middle length promoter by amplifying the sequence from nucleotide number -904 to nucleotide -1. Short promoter - 5'GCGCCGACTAGTCGGAAGATAAATACTCCAAC-3*, substantially denoted by SEQ ID NO: 4. This primer was used in combination with primer 3' to clone the shorter promoter by amplifying the sequence from nucleotide number -706 to nucleotide -1.

These long, mid and short primers contain Spel sites and were used at the 5' side of the GCN4 promoter (see Fig. 1). PCR products were digested with Spel and BamHI and Hgated to pBluescript to obtain the plasmids pBSG-Short (pBSG-S), pBSG-Mid (pBSG-M) and p_.BSG-Long (pBSG-L). The cloned DNAs were sequenced and found identical to the sequence of this region as appears in the Saccharomyces genome database.

For the Hgation of the coding sequence of human serum albumin (HSA) downstream to the GCN4 sequences, the cDNA of HSA was cloned as a BamHI-EcoRI fragment to pBSG-S, pBSG-M and pBSG-L. These chimeric genes contained about 70 bp of the 5'UTR sequences of HSA which were found to reduce expression levels (data not shown). To remove this sequence from each of the three plasmids, a 885 bp BamHI-NcoI fragment, containing the entire 5'UTR and 815 bp of the HSA coding, was replaced with a 815 bp PCR product containing only the first 815 bp .of the HSA coding sequence. Primers used for this PCR reaction were as foHows:-

HSA-ATG - 5'GCGCGCGGATCCATGAAGTGGGTAACCTTTATTTCCC-3', substantially denoted by SEQ ID NO: 5.

HSA-900 - 5'GCAGATCTCCATGGCAGCATTCC-3\ substantially denoted by SEQ ID NO: 6.

The plasmids obtained were named pBSG-S-HSA, pBSG-M-HSA and pBSG-L-HSA. The GCS4-HSA moiety of each of these plasmids was removed as a Spel-EcoRI fragment and inserted into pRS316 (a centromeric yeast plasmid

[Sikorski R.S. and Hieter JP., Genetics 122:19-27 (1989)], pRS306 [Sikorski R.S. and Hieter P. (1989) ibid.] and pRS426 (a 2μ based vector [Chritianson T.W. et. al., Gene 110:119-122 (1992)]. The terminator of ADHI (0.6 Kb EcoRI fragment) was Hgated to the EcoRI site of wach of the 9 plasmids obtained.

The final constructs were named as follows:-

(a) pGES306-S-HSA, for the GCN4 based expression system, being derived from the pRS306 plasmid and containing the short GCN4 regulatory element and the HSA cDNA;

(b) pGES306-M-HSA, which is similar to pGES-S-HSA however containing the medium sized GCN4-derived regulatory element;

(c) pGES-L-HSA containing the long GCN4 regulatory element; (d) pGES316-S-HSA;

(e) pgES316-M-HSA:

(f pGES315-L-HSA pGES426-S-HSA;

(g) pGES426-M-HSA and (h) pGES426-L-HSA.

A schematic representation of these plasmids is provided in figure 3. To express the HSA under ADHl promoter, a BamHI EcoRI fragment containing the HSA cDNA was rendered blunt by DNA Pol I (Klenow fragment) and inserted into the Smal site of pAD4Δ [BaHester R. et al, Cell 59:681-686 (1989)].

For improving the vector for the user a convenient multicloning site was added to the various vectors of the pGES type (Fig. 8) using the foHowing ohgonucleotides (complementary to each other):

5'GATCCCTCGAGCCCGGGGCGGCCGCGAGCTCCCGCGGCGGCCGGTCGACG, substantiaUy denoted by SEQ ID NO: 9.

5'AATTCGTCGACCGGCCGCCGCGGGAGCTCGCGGCCGCCCCGGGCTCAGG, substantially denoted by SEQ ID NO: 10. The oHgonucleotides were hybridized. As a result cohesive ends of BamHI at one end and Ecorl at another end were created. The double stranded oHgonucleoti.de was Hgated to BamHI EcoRI digested versions of pGES plasmids.

The leader peptide of the mating factor alpha and the sequence encoding CBD were added using appropriate oHgonucleotides.

The GALl promoter-GCN4 5'UTR fusion was created by Hgation of the GALl promoter to the Seal site in the GCN4 element. Mutagenesis of the 4 upstream ORFs in the 5'UTR of the GCN4 gene was carried out using the quick-change ldt (Stratagene) according to manufacturer instructions.

Western Blot Analysis

Protein extracts were prepared as previously described [Gross E. et al., Mol. CeU. Biol. 12:2653-2661 (1992)]. AH SDS-PAGE gels contained 10% acrylamide. Protein blotting was performed -with a LKB semi-dry blotter for two hours under constant current (mA-0.8xgel area in cm2). Polyclonal anti HSA antibodies (RAHu/albumin) (from Nordic Immunology) were used in a dilution of 1:50,000. Peroxidase conjugated Goat anti rabbit antibodies (from Jackson Immunological) were used in a dilution of 1:25,000. Purified HSA (from SIGMA) was used as a standard positive control.

RNA preparation and primer extension analysis

The RNA preparation and primer extension analysis were performed as previously described [Engelberg D. et al., Mol. CeU. Biol. 14:4929-4937 (1994)] using the foUowing primers:-

HSA-extension: 5'ACCTCACTCTTGTGTGCATCTC 3', substantiaUy denoted by SEQ ID NO: 7.

GCN4-extension: 5'ATAATTCGCTAGTGAAACTGATGGGC 3', substantiaUy denoted by SEQ ID NO: 8. Primer extension products were separated on 6% acrylamide 7 M urea gel.

Expression of a heterologous coding sequence requires a fragment of at least 904 bp upstream to the GCN4 coding sequence

To test whether elements controlling GCN4 expression could be used to regulate expression of a heterologous coding sequence, the inventors have cloned a sequence containing the regulatory elements of GCN4. Initially, a fragments containing nucleotides -706 to -1, prepared from a genomic DNA of the SP1 strain, was cloned. This fragment was reported to be sufficient for regulated expression of GCN4 [Hinnebusch A.G. et al, Mol. CeU Biol. 5:2349-2360 (1985)]. This DNA sequence was also sufficient for controUed expression of a GCN4/β-galactosidase chimeric coding sequence (containing 53 amino acids of Gcn4 fused to β-gal). The capability of these 706 bp alone (with no coding sequences) to control transcription and translation of an heterologous coding sequence has not been reported.

The cDNA encoding HSA was hgated downstream to the cloned 706 bp fragment. This GCN4-S-HSA cassette was inserted into a multicopy yeast plasmid as weU as into centromeric and integrated plasmids (Fig. 3). The terminator of the ADHl ' gene was inserted downstream to GCN4-S-HSA. The resulting plasmids were introduced into yeast and HSA expression was monitored by western blot analysis. Expression was tested under various growth conditions, including amino acid starvation and 3-AT treatment. In aU cases, using the short regulatory element, expression of HSA was not detectable (data not shown).

Therefore, the inventors have cloned two longer fragments of GCN4 upstream sequences, one containing the sequences from -904 to -1 and a longer one containing the sequence from -1067 to -1 (Materials & Methods, Figs. 1 and 3). When the HSA gene was hgated to these fragments to obtain plasmids pGES-M-HSA and pGES-L-HSA and introduced to yeast, high expression level of HSA was detected (Fig. 4). In aU constructs, HSA expression was barely detectable prior to induction with 3-AT, performed as described hereinafter. Expression level from constructs containing the Long regulatory element was usually higher than that obtained with the middle sized element (Mid.), as depicted from the levels of expression after 10 hours of induction (Fig. 4).

Expression of HSA under GCN4 regulatory elements is induced by 3-AT

To examine whether the cloned GCN4 elements could regulate HSA expression in a pattern similar to that of Gcn4 regulation, yeast ceUs (strain BJ2168) harboring pGES426-L-HSA were grown to logarithmic phase on SD(-URA). Then, ceUs were coUected and re-suspended in SD(-URA, -HIS) medium supplemented with 20 mM 3-AT. Samples were removed from the culture at indicated time points (Figure 5A) and expression of HSA was measured (Fig. 5A). Already 2 hrs after addition of 3-AT, expression of HSA could be easUy detected. As may be seen from the figures, expression reached its maximum level between 6 to 8 hours after induction.

These results presented in Figs. 4 and 5, show that a vector based on GCN4 upstream elements could be efficiently used for inducible expression of foreign genes. To examine whether the level of expression could be fine controUed by the level of amino acids starvation, various concentrations of 3-AT were provided in an experiment conducted in a similar manner as described above. Fig. 5B exhibit that the level of HSA expressed is prqportional to the level, of 3-AT introduced, up to 40mM.

Expression of HSA from GCN4-HSA constructs is regulated at the translational level

To verify that induction of HSA occurred at the translational level, HSA mRNA , and protein levels, before and after induction with 3-AT, were measured (Fig. 6).

Protein extracts were tested by Western blot analysis using anti HSA antibodies

(Fig. 6A), whereas RNA preparations were subjected to primer extension analysis

(Figs. 6B and 6C). It is evident from the results presented herein that the mRNA encoding HSA is expressed at a similar level before and after the 3-AT induction.

In addition, as may be seen from a compression between Fig. 6B and Fig. 6C most initiation sites used by RNA polymerase to transcribe the GCN4-HSA mRNA were identical to those found in GCN4 mRNA's [also in Hinnebusch A.G., PNAS USA 81:6442-6446 (1984)]. In contrast to the constitutive and uniform expression of HSA RNA, the HSA protein was barely detectable before induction with 3-AT and its expression increased in time following induction. It has thus been concluded that 3-AT dependent induction of HSA expression occurs at the translational level.

The GCN4 based vector induced efficient expression as an integrated copy Transcription rate of GCN4 seems to be high under most growth conditions [Hinnebusch et al. (1985) ibid.; Engelberg D. et. al. (1994) ibid.]. In addition, mRNA levels of HSA expressed from pGES plasmids are quite high (although lower than those of GCN4, see Fig. 6). It was therefore assumed by the inventors that the efficient expression from the pGES426-L-HSA stems mainly from efficient translation rate and less from the high copy number of the plasmid. To examine this assumption the GCN4-L-HSA fragments was inserted into an integrative vector obtaining the pGES306-L-HSA plasmid as described in the M&M. this construct was then integrated into the yeast genome and the resulting clones were grown on rich, non-selective media to high protein concentration (to about 5xl0⁸ ceUs/ml) prior to the addition of 3-AT. Under these conditions expression of HSA was stiU efficiently induced (Fig. 7). In fact, the level of HSA obtained per ceU was just sHghtly. less than that obtained using the multicopy pGES426-L-HSA vector. As the culture grown on YPD reached higher ceU concentration, the quantity of intraceUular HSA protein obtained was higher when the integrated vector was used as compared to HSA levels obtained with 2μ based plasmid (pGES-426-L-HSA; see below).

To compare the new expression vectors of the present invention with commonly used vectors the cDNA encoding HSA was Hgated downstream to the ADHl promoter. In the previously described vector pAD4Δ [BaUester R. et al (1989) ibid.], cultures harboring either pADH-HSA, or pGES426-L-HSA were grown in paraUel. After induction of pGES426-L-HSA with 3-AT, the expression of HSA was compared. The levels of HSA expression obtained with pGES426-L-HSA was significantly higher than that obtained with pADH-HSA and reached the level of 6 mgtliter. The inventors current estimation is that the amount obtain with the integrated vector (pGES306-L-HSA) when ceUs rich high density on rich medium is at least twice the level obtained with pGES426-L-HSA. It was therefore concluded that the pGES system could be more efficient than currently available yeast expression vectors.

Example 2

Expression of a heterologous coding sequence in cells other than yeast cells pGES expression systems of the invention, which wiU include, inter alia, a heterologous coding sequence, for example the HSA coding sequence, and as a selectable marker neomycin or hygromycin resistance gene (suitable for selection either in mammalian ceUs, insect ceUs, plant ceUs or avian ceUs) wiU be prepared as described herein.

The plasmid obtained wiU be transferred into mammalian ceUs using electr op oration or calcium phosphate precipitation.

The ceUs wiU then be grown on a suitable medium to a concentration of about 2xl0⁷ceUs/ml and then transformed with the pGES based vectors described hereinbefore. When the expression vectors lack a selectable marker, the ceUs may be co-transfected with a second plasmid carrying the selectable marker.

FoUowing transfection, the transfected ceUs will be selected by culturing the population of ceUs obtained after transfection, in the presence of the relevant agent, such as an antibiotics (e.g. G418, hygromycin). The selected ceUs wiU then be exposed to amino acid starvation conditions mimicked by the addition of 3-AT, to induce expression of the HSA coding sequence.

Alternatively, induction of expression wiU be obtained through treatments known to induce PKR activity which is the homolog of the yeast Gcn2 kinase that activates GCN4 translation in response to starvation. Treatments known to induce PKR activity include but are not limited to exposure to double stranded RNA, TNF and UV radiation. According to yet further alternative embodiments it is now disclosed that some derivatives of the GCN4 5'UTR including but not Hmited to those mutated in the 4 upstream ORFs may give rise to constitutive translation and wiU not require induction.

Claims

CLAIMS:

1) An expression cassette comprising: -

a) a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor or a functional derivative or fragment thereof;

b) a promoter operably linked to said regulatory element;

c) a heterologous coding sequence downstream to said regulatory element;

d) a termination signal operably linked downstream to said heterologous sequence.

2) The expression cassette as claimed in claim 1 further comprising an operably Hnked selectable marker.

3) The expression cassette as claimed in any one of claims l and 2, further comprising at least one operably Hnked rephcation element.

4) The expression cassette as claimed in any one of claim 1, 2 and 3, wherein said GCN4 transcription factor is of the yeast Saccharomyces cereυisiae.

5) The expression cassette as claimed in claim 4, wherein said regulatory element comprises any functional fragment of the nucleotides -600 to -1 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO 15.

6) The expression cassette as claimed in claim 5, wherein said regulatory element comprises any functional derivative obtained by mutations thereof. 7) The expression cassette as claimed in claim 6, wherein said regulatory element or any functional derivative thereof further comprising ehmination of at least one of the 4 upstream ORFs.

8) The expression cassette as claimed in daim 4, wherein said promoter is the GCN4 transcription factor promoter of the yeast Saccharomyces cereυisiae.

9) The expression cassette as claimed in daim 8, wherein said promoter comprises any functional fragment taken from the nucleotides —2000 to -600 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 11.

10) The expression cassette as claimed in daim 9, wherein said promoter comprises any functional fragment of the nucleotides -1067 to -600 upstream to the GCN4 coding sequence.

11) The expression cassette as claimed in daim 10, wherein said promoter has the nucleotides -1067 to -600 upstream to said GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 12.

12) The expression cassette as claimed in claim 8, wherein said promoter comprises any functional fragment of the nucleotides -904 to -600 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 13.

13) The expression cassette as claimed in daim 4, wherein, said promoter is the GALl promoter.

14) The expression cassette as claimed in any one of claims 1 to 3, wherein said heterologous coding sequence encodes any of protein and peptide product.

15) The expression cassette as claimed in any one of claims 4 to 13, wherein said heterologous coding sequence encodes any of protein and peptide product. 16) The expression cassette as claimed in daim 14, wherein said coding sequence encodes a naturaUy occurring protein or peptide or . a derivative thereof, a fragment of said protein or peptide or a chimera of said protein, peptide or fragment thereof.

17) The expression cassette as claimed in daim 16, wherein said protein or peptide is a therapeutic product selected from the group consisting of enzymes, antibodies, cytokines, hormones, receptors, transcription factors, fragments thereof or any chimera of said therapeutic products.

18) The expression cassette as claimed in daim 17, wherein said heterologous coding sequence encodes a mammahan protein.

19) The expression cassette as claimed in claim 18, wherein said mammahan protein is the human serum albumin (HSA) protein.

20) The expression cassette as claimed in any one of claims 1 to 3 and 14, wherein said termination signal is selected from the group consisting of the termination signal of ADHl, TRP1, GAP (GAPDH), MF1, PH05, PGK, CYC1 or of GC 4.

21) The expression cassette as claimed in any one of claims 4 to 13 and 15 to 19, wherein said termination signal is selected from the group consisting of the termination signal of ADHl, TRP1, GAP (GAPDH), MFl, PH05, PGK, CYC1 or of GCN4.

22) The expression cassette as claimed in any one of claim 2 and 3, wherein said rephcation element is selected from 2μ, CEN or CEN-ARS.

23) An expression vector comprising the expression cassette of claim 1. 24) An expression vector being the plasmid substantiaUy as shown in any one of Figures 2A, 2B, 2C, 8A, 8B, 8C, 8D, 8E and 8F.

25) A method for the regulated production of a therapeutic protein or peptide in eukaryotic ceUs, which method comprises the steps of:-

a) providing eukaryotic ceUs;

b) transforming said ceUs provided in (a) with an expression vector comprising: -

(i) a regulatory element comprising the nucleotide sequence of the 5'UTR of the gene encoding the GCN4 transcription factor or a functional derivative or fragment thereof;

(ύ) a promoter operably linked to said regulatory element;

(in) a heterologous coding sequence downstream to said regulatory element;

(iv) a termination signal operably linked to said heterologous sequence; and

(v) optionally, an operably Hnked selectable marker;

c) selecting from the ceU population obtained in (b) those ceUs which harbor said expression vector and growing the same;

d) inducing expression of said heterologous coding sequence in said ceUs under amino acid starvation conditions or under conditions which mimic said starvation to obtain a functionaUy active protein or peptide encoded by said heterologous coding sequence; and 26) The method as claimed in claim 25 further comprising the step of isolating said functionaUy active protein or peptide obtained in step (c) from said ceUs.

27) The method as claimed in any one of claims 25 and 26, wherein said eukaryotic ceUs are mammahan ceUs.

28) The method as claimed in any one of claims 25 and 26, wherein said eukaryotic ceUs are yeast ceUs.

29) The method as claimed in claim 28, wherein said yeast ceUs are selected from S. cereυisiae, Schizosaccharomyces pombe and Pichia pectoris.

30) The method as claimed in claim 29 wherein the yeast ceUs are S. cereυisiae.

31) The method as claimed in claim 30, wherein said yeast ceUs are of the strain BJ2168.

32) The method as claimed in any one of claims 25 and 26, wherein said expression cassette further comprises at least one rephcation element.

33) The method as claimed in claim 25 or 26, wherein said GCN4 transcription factor is of the yeast S. cerevisiae.

34) The method of claim 33 wherein said regulatory element comprises any functional fragment of the nucleotides -600 to -1 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO 15.

35) The method of claim 34, wherein said regulatory element is modified resulting in constitutive activation without induction. 36) The method of claim 35, wherein said regulatory element is modified by any one of mutations and elimination of at least one of the 4 upstream ORFs.

37) The method of claim 25 or 26 wherein said promoter is the GCN4 transcription factor promoter of the yeast Saccharomyces cereυisiae.

38) The method of claim 37 wherein said promoter comprises any functional fragment taken from the nudeotides -2000 to -600 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 11.

39) The method of claim 38 wherein said promoter comprises any functional fragment of the nucleotides -1067 to -600 upstream to the GCN4 coding sequence.

40) The method of claim 39, wherein said promoter has the nucleotides -1067 to -600 upstream to said GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 12.

41) The method of claim 40, wherein said promoter comprises any functional fragment of the nucleotides , -904 to -600 upstream to the GCN4 coding sequence, substantiaUy as denoted by SEQ ID NO: 13.

42) The method of claim 25, wherein said promoter is the GALl promoter.

43) The method as claimed in claim 25, wherein said heterologous coding sequence encodes a protein or peptide or a derivative thereof, a fragment of said proteins or peptides or to a chimera of said proteins, peptides or fragments thereof.

44) The method as claimed in claim 43, wherein said any of protein and peptide is a therapeutic product selected from the group consisting of enzymes, antibodies, cytokines, hormones, receptors, transcription factors or any chimera of said therapeutic products. 45) The method as claimed in claim 43, wherein said heterologous coding sequence encodes a mammaHan protein.

46) The method as claimed in claim 45, wherein said mammaHan protein is the human serum albumin (HSA) protein.

47) The method as claimed in claim 44, wherein said protein or peptide is produced in high quantities, relative to the level obtained without the regulatory element.

48) The method as claimed in claim 25, wherein said termination signal is the termination signal of ADHl, TRP1, GAP (GAPDH), MFl, PH05, PGK, CYC1 or of GCN4.

49) The method as claimed in claim 25 or 26, wherein said starvation conditions are mimicked by the addition of an effective amount of 3-AT.

50) The method as claimed in claim 25 or 26, wherein said ceUs are transformed with an_, expression being the plasmid substantiaUy as shown in any one of Figures 3A,3B, 3C, 8A, 8B, 8C, 8D, 8E, 8F.

51) A therapeutic protein or peptide produced by the method of any one of claims 25 and 26.

52) A therapeutic protein or peptide produced by the method of any one of claims 27 to 50.

53) The protein or peptide as claimed in claim 51 being the HSA protein.

54) The protein or peptide as claimed in claim 52 being the HSA protein. 55) Eukaryotic ceUs transformed with the expression cassette as claimed in claim 1 and capable of expressing said heterologous coding sequence.

56) CeUs according to claim 55 being mammaHan ceUs.

57) CeUs according to claim 55 being yeast ceUs.

58) Use of the expression cassette as claimed in claim 1, wherein said heterologous coding sequence encodes a therapeutic protein or peptide, in the production of a therapeutic protein or peptide.

59) Use of the expression cassette as claimed in claim 1, wherein said heterologous coding sequence encodes a therapeutic protein or peptide, in the preparation of a pharmaceutical composition containing said protein or peptide.

60) Use of the expression cassette as claimed in claim 1, wherein said coding sequence encodes a chimeric protein, in the detection of protein-protein interaction.