WO2001048182A2

WO2001048182A2 - Amino acid inducible expression system

Info

Publication number: WO2001048182A2
Application number: PCT/EP2000/012887
Authority: WO
Inventors: Steen Holmberg; Camilla Ottendahl Samuelsen; Danny François Eve HUYLEBROECK; Jacques E. F. J. G. Remacle
Original assignee: Vlaams Interuniversitair Instituut Voor Biotechnologie
Priority date: 1999-12-28
Filing date: 2000-12-18
Publication date: 2001-07-05
Also published as: AU3013601A; WO2001048182A3

Abstract

The invention relates to an amino acid inducible expression system for eukaryotic cells, especially mammalian cells. In particular, a serine and/or threonine inducible system is described, by which a more than 100-fold induction can be obtained.

Description

Amino acid inducible expression system

The present invention relates generally to the fields of molecular and cellular biology. More particularly, the invention relates to products and methods for modulating the expression of recombinant genes in eukaryotic systems, preferentially mammalian systems.

The study of gene expression, especially of toxic genes, in complex genetic environments such as eukaryotic cells would highly profit from a system that would allow a stringent control of the expression of these genes. Ideally, such a system should possess the following features: in a first place, it should be specific and not respond to endogenous activators or interfere with cellular regulatory pathways. Secondly, it should exhibit low basal levels of expression in an uninduced state and rapidly achieve high levels of expression upon induction. In a third place, the system should be more than an 'on/off' switch, but it should allow modulation of expression levels. Finally, the inducer should be a cheap small molecule, that is not toxic for the cells neither for the environment.

Only a limited number of expression systems that meet more than one of above requirements. Several inducible systems have been developed that are based on endogeneous cellular elements that respond to exogeneous signals or stresses such as hormones (Klock et al., 1987; Israel & Kaufman, 1989), cytokines, metal ions (Searle et al., 1985) , heat shock (Voellmy, 1994) or hypoxia. The problem common to all of these inducers is that they have pleiotropic effects and are not specific at all. WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline responsive promoters. WO9601313 discloses tetracyciine-regulated transcriptional modulators. One of the advantages of this system is that the activity of the gene of interest can be modulated depending upon the concentration of the inhibitor. However, the system is dependent upon tetracycline or tetracycline analogs, which may be unwanted due to their toxic effect on the cells and/or on the environment, both in in vitro as well as in in vivo experiments. The same drawback is true for the vectors for tetracycline controlled gene expression, described in WO9837185.

WO9637609 and WO9738117 describe inducible systems based on the insect hormone ecdysone. However, steroids are difficult and expensive to make and the use of a steroid may be unwanted in agrochemical and pharmaceutical applications. It is the aim of the present invention to provide an inducible system for eukaryiotic cells, preferentially mammalian cells, whereby a simple, cheap and non-toxic inducer is used, preferentially an amino acid, more preferentially serine and/or threonine. Transcription in eukaryotes involves transcriptional activators, proteins that bind to specific sites distal from the TATA box termed enhancers or upstream activation sequences (UASs). When bound to a UAS, a transcriptional activator is able to stimulate the transcription initiation complex leading to synthesis of the mRNA.

The ability of Saccharomyces cerevisiae to use serine or threonine as the sole nitrogen source depends on the CHA1 gene, which encodes the catabolic L-serine (L- threonine) dehydratase (Ramos and Wiame 1982; Bornaes et al. 1992). CHA1 is regulated by transcriptional induction by serine or threonine (Petersen et al. 1988). Recently, a deletion analysis of the CHA1 promoter identified two elements, UAS1 _CHA and UAS2CHA, each of which is sufficient to confer serine and threonine induction to Saccharomyces cerevisiae genes (Bornaes et al. 1993). It was also found that the multifunctional protein ABF1 binds to an element in the CHA1 promoter, irrespectively of CHA1 induction. Protein binding to either UASI CHA or UAS2_CHA was not detected using nuclear protein extracts prepared from cells grown in uninducing or inducing media (Bornaes et al. 1993). However, the regulated expression of CHA1 was expected to involve a transcriptional regulator(s) that, directly or indirectly, senses the presence or absence of serine/threonine in the cell. Holmberg and Schjerling (1996) identified Cha4p as frans-acting factor involved in transcriptional regulation the CHA1 gene. The transcriptional activator Cha4p has been shown to bind the serine/threonine response elements UAS1 _CHA and UAS2_CHA both in vitro (Holmberg and Schjerling, 1996) and in vivo, and to be necessary for inducible activation by the UASs in vivo (Bornaes et al., 1993). The activation is dependent on elevated levels of Ser or Thr. Cha4p belongs to a family of proteins characterized by an N-terminal DNA binding domain of the C₆ zinc cluster type and binds to the palindromic sequence CGGN-|₀CCG (Holmberg and Schjerling, 1996). The ORF encoding Cha4p indicates a monomer length of 648 amino acid residues (Holmberg and Schjerling, 1996). The C₆ zinc cluster extends from residue 43 to residue 72 and a coiled coil region, thought to be involved in dimehzation, are located between residues 87 and 105. A potential C-terminal located acidic activation domain is recognized between residue 618 and residue 643 (Scherling and Holmberg, 1996). Previously, many mutants expressing CHA1 independently of Ser and Thr induction have been isolated (Pedersen et al. , 1997). All of the isolated mutations were either alleles of HOM3 (increasing the pool of Thr 15- fold) or CHA4 suggesting that Cha4p is alone responsible for the Ser/Thr induction of CHA1.

This serine/threonine inducible system can be transferred to any other eukaryotic cell that does not contain a serine and/or threonine catabolic pathway, or where the system is not interfering with the endogenous serine and/or threonine catabolic pathway, by linking one or more UAS1 _CHA and/or UAS2_CHA elements to a suitable promoter, preferentially a minimal promoter, fusing the UAS-promoter unit to a gene of interest and transferring it to an eukaryotic host cell, together with the CHA4 gene placed after another suitable promoter that is functional in said host cell. Said suitable promoter in front of the CHA4 gene may be any promoter as long as it is functional in said host cell during induction with serine and/or threonine. Preferably, said suitable promoter is a constitutive promoter. Since no genes of the serine catabolic pathway are known in mammalian cells, these cells are suitable host cells. One aspect of the invention is a recombinant expression system, allowing amino acid induced expression in eukaryotic cells, preferentially eukaryotic cells that do not contain a catabolic pathway for said amino acid, or in which the recombinant expression system is not interfering with the catabolic pathway for said amino acid. A preferred embodiment of the invention is a recombinant expression system, allowing serine and/or threonine induced expression in eukaryotic cells, preferentially eukaryotic cells that do not contain a serine and/or threonine catabolic pathway, or in which the recombinant expression system is not interfering with the catabolic pathway for serine and/or threonine acid. A preferred embodiment of the invention is a recombinant expression system, allowing serine and/or threonine induced expression in mammalian cells.

Another aspect of the invention is a recombinant expression system allowing serine and/or threonine induction in eukaryotic cells, preferentially mammalian cells whereby said recombinant expression system comprises a serine and/or threonine response element. Another aspect of the invention is a recombinant expression system whereby said serine and/or threonine response element is linked to a minimal promoter. Still another aspect of the invention is a recombinant expression system allowing serine and/or threonine induction in eukaryotic cells, preferentially mammalian cells whereby said recombinant expression system comprises a transcriptional activator binding to said response element.

A preferred embodiment of the invention is said recombinant expression system whereby the said response element comprises UAS1_CHA with sequence identity n° 1 and/or UAS2_CHA with sequence identity 2 and/or the complement of said UAS sequences. Another preferred embodiment of the invention is said recombinant expression system whereby said transcriptional activator comprises a protein or a protein fragment with sequence identity n°4, and/or is encoded by a DNA sequence shown in sequence identity n° 3, or a biologically active fragment of said protein or protein fragment, or a biologically active variant. Said variant can be any mutation that still can bind the response element and cause e serine and/or threonine induced activation of transcritpion.

Still another aspect of the invention is a vector system for transforming or transfecting an eukaryotic cell, said system comprising at least one vector, said system carrying at least one serine and/or threonine response element and at least one trancriptional activator. Said response element may and said activator may be situated on the same vector, or on different vectors. The vectors may be integrative vectors or self-replicating vectors, intended for either stable or transient expression. The vector can be specific for all kind of eukaryotic host cells, provided that the host cell does not contain a serine and/or threonine catabolic pathway, or that the cell does contain an endogenous serine and/or threonine catabolic pathway that is not interfering with the serine and/or threonine response element and the transcriptional activator on the vector. The vector is preferentially, but not limited to a mammalian cell vector, as known to the people skilled in the art, including viral type vectors such as adenoviral, retroviral and lentiviral vectors and including artificial chromosomes, knonw to the person skilled in the art. The vector may be a yeast vector, a fungal vector, an insect cell and plant cell vector, as known to the people skilled in the art. A special embodiment of the invention is a kit, comprising said vector system. Another aspect of the invention is an eukaryotic cell, preferentially a mamalian cell, comprising a recombinant expression system, allowing serine and/or threonine induced expression. Preferentially, said eukaryotic cell is transformed or transfected with a vector according to the invention. This eukaryotic cell may be any eukaryotic cell, such as a yeast cell, fungal cell, insect cell, mammalian cell or plant cell that does not contain a serine and/or threonine catabolic pathway, or does contain an endogenous serine and/or threonine catabolic pathway is not interfering with said recombinant expression pathway. The recombinant expression system may be totally or partially present on a vector according to the invention, or may be integrated in the genome of said eukaryotic cell. The eukaryotic cell, as mentioned here, is not limited to an individual cell or a cell culture, but can be part of an organism such as an animal or a plant. Another aspect of the invention is a transgenic animal or a transgenic plant comprising a recombinant expression system, allowing serine and/or threonine induced expression. Another aspect of the invention is a method of obtaining serine and/.or threonine induced expression of a gene of interest in eukaryotic cells, comprising the steps of ■ transforming or transfecting an eukaryotic cell with a vector system comprising at least one vector, carrying at least one serine and/or threonine response element and at least one trancriptional activator ■ growing the transformed or transfected cell in serine and/or threonine less or serine and or threonine poor medium ■ inducing the expression of a gene of interest by adding serine an/or threonine, whereby said eukaryotic cell does not contain a serine and/or threonine catabolic pathway, or whereby the cell does contain an endogenous serine and/or threonine catabolic pathway that is not interfering with the serine and/or threonine response element and the transcriptional activator on the vector. In a preferred embodiment, the vector systems contains two vectors, one carrying the response element linked to a minimal promoter which is fused to the gene of interest, and another vector carrying the transcriptional activator.

Definitions

The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein

Recombinant expression system as used her means any expression system, allowing the expression of one or more heterologous and/or homologous proteins in a host cell, whereby at least one functional element of said recombinant expression system is constructed by recombinant DNA techniques and not present as such in the wild type and/or untransformed or untransfected host cell. A functional element, as used here, is an element that is situated on or encoded by one contiguous piece of DNA. A promoter, with a response element modulating its activity is considered as one functional element. The recombinant expression system may comprise one or more functional elements.

Serine- and/or threonine-less medium: medium without measurable concentrations of serine and/or threonine, according to the measurement methods known to the people skilled in the art.

Serine and/or threonine poor medium: medium in which the expression of a gene of interest, operationally fused to a promoter which is linked to a serine and/or threonine response element as described in this invention still can be increased by increasing the concentration of serine and /or threonine in said medium. The serine and or threonine concentration in a serine and/or threonine poor medium is preferentially lower than 50 mg/l, more preferentially lower than 10 mg/l and even more preferentially lower than 1 mg/l.

Vector system: system comprising one or more vectors, suitable for transforming or transfecting the same host cell whereby the vector(s) can be maintained in the host cell for a period that is at least long enough to realize the induction of the expression. Transformation or transfection: is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including but not limited to electroporation, calcium phosphate coprecipitation, DEAE-dextran-mediated transfection, Agrobacterium mediated transformation and microinjection. Suitable methods can be found in Sambrook et al. (1989) and other laboratory textbooks.

Minimal promoter: promoter sequence or partial promoter sequence which defines the start of transcription for the fused sequence to be transcribed but which by itself is not capable of initiating transcription efficiently, if at all. Response element: DNA sequence on which a protein, proteineous molecule or protein complex can bind, either in the presence or in the absence of an inducer. Binding to the response element, or releasing of said binding, upon the addition of the inducer is resulting directly or indirectly in the activation of an otherwise inactive promoter

Transcriptional activator, protein, proteineous molecule or protein complex that can bind to a response element and directly or indirectly activates upon binding an otherwise inactive promoter. Proteineous molecule: chemically modified proteins or protein derivatives such as, but not limited to, glycoproteins and phosphorylated proteins.

Protein complex: complex comprising at least one protein or proteineous molecule, in combination with other proteins, proteineous molecules or other compounds such as, but not limited to amino acids or peptides. Functional fragment of a transcriptional activator: continuous or discontinuous (i.e. combination of more than one fragment) fragment of Cha4p, that still can bind the response element and cause a serine and/or threonine induced activation of transcription. As a non-limiting example, deletion mutants of Cha4p in which one or more amino acids between amino acid 120 and 165 are deleted are functional fragments. A preferred form of a functional fragment is a Cha4p deletion mutant in which amino acids 121 up to 164 are deleted.

Not interfering: means that the elements of the endogenous catabolic pathway, apart from the inducer itself, are not able to bind to the response element of the vector system and/or to promote the binding of the transcritptional activator of the vector system to the response element of the vector system and/or to activate the promoter linked to the response element of the vector system.

Brief description of the figures

Figure 1 : Fold induction of normalised luciferase activity in transfected 293T cells grown in the presence of different serine concentrations. The values are obtained by dividing the average luciferase activity under induced conditions by the average luciferase activity obtained in medium with normal, not dialysed serum, without addition of serine. Figure 2: Fold induction of normalised luciferase activity in transfected CHO cells grown in the presence of different serine concentrations. The values are obtained by dividing the average luciferase activity under induced conditions by the average luciferase activity obtained in medium with dialysed serum, without addition of serine.

Examples

Construction of plasmids for mammalian expression. Plasmids expressing Flag- tagged wt Cha4p (pCHA4) and Cha4p³⁵³ (pcha4³⁵³) from the CMV promoter were constructed as follows: pTK327 (Holmberg and Schjerling, 1996) was used as template to PCR amplify the CHA4 ORF with primer 1 (5'- CCGCGTGGATCCATGATGTTGGAGCCTTCACC-3') and primer 2 (5'- CGGGGCGGCCGCCTACCTCTTCACTCAAGATTACTTCGAC-3'), while pTK391 was used as template to PCR amplify the ORF of CHA4³⁵³ using the same primers. CHA4 contains a Trp to Cys mutation, previously described as SIL3 mutant (Pedersen et al., 1997) which results in a constitutive activation of the CHA1 promoter. The PCR-products were then cloned into the BamHI and Notl sites of pcDNA3. IflagC. The pcDNA3. IflagC was constructed from the pCDNA3.1 HisC vector (Invitrogen). The Hindlll-Asp718 fragment containing the Express-Tag and the HIS-tag was excised out of pCDNA3.1 HisC and replaced with the following oligonucleotide encoding the FLAG-tag peptide:

5'-AGCTTACCATGGATTACAAGGACGATGACGATAAG-3' (Hindlll-MDYKDDDDK- Asp⁷¹⁸).The reporter-plasmid (pGL3TKminUAS1_CHA) carrying three UAS1 _CHA sequences in front of the Tkmin promoter was constructed as follows: three copies of the following sequence 5'-ACCCAGCGGAAATGTAATTCCACTGAGTGTCA-3' was iigated into the pPCR-script Amp vector (Stratagene) into the Srfl site. The Sacl - Smal fragment was then isolated and inserted into pGL3min (Invitrogen) All constructed plasmids were sequenced to verify that the correct sequences had been cloned.

Cell culture and transfections. 293T cells were cultured in DM EM/4500 mg/l D- glucose (Gibco) containing either normal or dialysed 10% fetal calf serum, while CHO cells were cultured in F-12/L-glutamine and dialysed 10% fetal calf serum. Fetal calf serum was dialysed to remove possible traces of serine. Both cell line were grown in 5% CO₂ at 37°C. Cells were plated to about 40% confluency in 24 well-plates containing the appropriate media supplemented with L-se ine (Sigma) added to a finale concentration of 0 mg/l, 50 mg/l, 75 mg/l, 100 mg/l, 250 mg/l, or 500 mg/l.

After 24 hours, 50 ng of CMV-lacZ reporter, 100 ng of pGL3TKmin or pGL3TKminUAS1_CHA and 500 ng of pCHA4, pcha4³⁵³ or pcDNA3.1FlagC were transfected into the cells using Fugene 6 (Boehringer-Mannheim) according to manufacturer's instructions.

Transcription assays. Reporter assays. 24-48 hours post-transfection cells were lysed on ice in the well-plates for 15 min using 100 μl lysis-buffer [50 mM Tris pH=7.2, 150 mM NaCI, 10% glycerol, 0.2% NP40] supplemented with 1 % Aprotinin. 7.5 μl of lysed cells was used to measure the luciferase activity using a luciferase assay (Promega). In order to normalise the luciferase activity 7.5 μl of lysed cells were used to measure the transfection efficiency using a LacZ reagent (Clontech).

Serine induction in 293T cells. Each induction experiment with the 293T cells was carried out three times. The individual measurements and the normalised average values are summarised in Table 1. There is a clear serine induction when Cha4p (produced by pCHA4) is present together with the UAS (UAS1 _CHA comprising sequence) linked to the TK minimal promoter, as well in medium with normal serum (Figure 1) as in medium with dialysed serum. The results clearly show that dialysis is not needed and that low concentrations of serine do not disturb the inducibility of the system. When the constitutive mutation Cha4p³⁵³ is used, high luciferase activity is found independent of the serine concentration: the minimal promoter is activated by the constitutive Cha4p³⁵³ transcriptional activator (Table 1). Elimination of either Cha4p or UAS out of the system results in a low luciferase activity, independent of the serine concentration (Table 1). Even if the constitutive Cha4p³⁵³ is used, no significant luciferase activity is obtained in absence of a UAS element linked to the TK minimal promoter. These results clearly indicate that both Cha4p and a UAS element are essential elements to obtain serine inducible expression. It is clear for the people skilled in the art that the UAS1_CHA can be replaced by a UAS2_CHA, or by a combination of both. Serine induction in CHO cells. Each induction experiment with the CHO cells was carried out two times, except the experiments without transcritional activator, with the use of a minimal promoter in front of the reporter gene, or without reporter construct, which were carried out only once. The individual measurements and the normalised average values are summarised in Table 2. There is a clear serine induction when Cha4p (produced by pCHA4) is present together with the UAS (UAS1 _CHA comprising sequence) linked to the TK minimal promoter in medium with dialysed serum (Figure 2). The results are completely in agreement with the results of the 293T cells, but the background value of luciferase activity as well as the induced values are higher. Although the background activity is higher, a more than 100-fold induction can be obtained. The results clearly indicate that the serine inducible system can be used independent of the cell type.

CELL MEDIA SERUM PROTEIN SERINE REPORTER GALACTOSIDASE LUCIFERASE AVERAGE STANDARD £-

LINE cone added PLASMID ACTIVITY (CPS) ACTIVITY LUCIFIRASE DEVIATION (%) (mg/l) (CPS) ACTIVITY (CPS)

293T DMEM dialysed Cha4p 0 UAS 20456,8 316786 17,50 10,5 _

293T DMEM dialysed Cha4p 0 UAS 20536,8 367834

293T DMEM dialysed Cha4p 0 UAS 17740,7 338875

293T DMEM normal Cha4p 0 UAS 33248,5 358148 12,40 11 ,4

293T DMEM normal Cha4p 0 UAS 32147,3 421404

293T DMEM normal Cha4p 0 UAS 30092,2 400794

293T DMEM normal Cha4p 50 UAS 19449,7 2151228 107,64 5,5

293T DMEM normal Cha4p 50 UAS 17547,2 1769395

293T DMEM normal Cha4p 50 UAS 18945,3 21 12116

293T DMEM normal Cha4p 75 UAS 13303,8 2903879 235,96 6,8

293T DMEM normal Cha4p 75 UAS 12994,5 3117699

293T DMEM normal Cha4p 75 UAS 13766,8 3437201

293T DMEM normal Cha4p 100 UAS 10728,3 3684241 359,02 4,3

293T DMEM normal Cha4p 100 UAS 10310,8 3860227

293T DMEM normal Cha4p 100 UAS 8599,67 3089470

293T DMEM normal Cha4p 250 UAS 6415,17 4791760 766,19 2,3

293T DMEM normal Cha4p 250 UAS 9255,83 7234142

293T DMEM normal Cha4p 250 UAS 8303,67 6394223

293T DMEM normal Cha4p 500 UAS 8554,33 8108313 980,76 5,6

293T DMEM normal Cha4p 500 UAS 8040,83 8398060

293T DMEM normal Cha4p 500 UAS 8147,5 7740007

293T DMEM dialysed Cha4p353 0 UAS 10924 11180000 1063,81 3,9

293T DMEM dialysed Cha4p353 0 UAS 9709 10300000

293T DMEM dialysed Cha4p353 0 UAS 9077 10050000

293T DMEM normal Cha4p353 0 UAS 13333 9445360 727,15 3,3

293T DMEM normal Cha4p353 0 UAS 14482 10410000

293T DMEM normal Cha4p353 0 UAS 13763 10380000

293T DMEM normal Cha4p353 50 UAS 11187 9422711 867,05 6,4

293T DMEM normal Cha4p353 50 UAS 10647 8817811

293T DMEM normal Cha4p353 50 UAS 10351 9633267

293T DMEM normal Cha4p353 75 UAS 8545 7072729 828,37 5,5

I

00 CD

LO LO

ro E E E E E

U LU LU LU LU LU LU Έ

00 00 ro ro co OO 00 CO

CD CD CD CD CD CD

CM CM CM CM CM CM CELL MEDIA SERUM PROTEIN SERINE REPORTER GALACTOLUCIFE-RASE AVERAGE STANDARD 5¹ LINE (mg/l) SIDASE ACTIVITY LUCIFERASE DEVIATION (%) Φ ACTIVITY (CPS) (CPS) ACTIVITY ro

(CPS)

CHO F-12 Dialysed Cha4p 0 UAS 385,33 59184,3 189,22 26,6 CHO F-12 Dialysed Cha4p 0 UAS 280 62957

CHO F-12 Dialysed C a4p 50 UAS 615 1341203 2290,70 6,7 CHO F-12 Dialysed C a4p 50 UAS 657,33 1577210

CHO F-12 Dialysed Cha4p 100 UAS 771 7594847 11182,43 16,8 CHO F-12 Dialysed C a4p 100 UAS 637,33 7972997

CHO F-12 Dialysed C a4p 250 UAS 139 3222472 23285,74 0,3 CHO F-12 Dialysed Cha4p 250 UAS 128 2986631

CHO F-12 Dialysed Cha4p 500 UAS 297 6282916 19006,68 15,8 CHO F-12 Dialysed Cha4p 500 UAS 351 5920097

CHO F-12 Dialysed Cha4p353 0 UAS 111 1512488 14550,67 8,6 CHO F-12 Dialysed Cha4p353 0 UAS 97 1502259

CHO F-12 Dialysed Cha4p353 50 UAS 347

(J) 8852298 24176,11 8,0 CHO F-12 Dialysed Cha4p353 50 UAS 397 9065877

CHO F-12 Dialysed Cha4p353 100 UAS 554,67 16000000 30669,80 8,4 CHO F-12 Dialysed Cha4p353 100 UAS 505,33 16420000

CHO F-12 Dialysed C a4p353 250 UAS 264 6005384 22427,71 2,0 CHO F-12 Dialysed Cha4p353 250 UAS 294,67 6514493

CHO F-12 Dialysed Cha4p353 500 UAS 346,67 8934193 23994,97 10,5 CHO F-12 Dialysed Cha4p353 500 UAS 296 6576672

CHO F-12 Dialysed Cha4p 0 MIN 525,33 39013 84,93 17,8 CHO F-12 Dialysed Cha4p 0 MIN 374,67 35818

CHO F-12 Dialysed Cha4p 50 MIN 1005 131468 133,47 2,9 CHO F-12 Dialysed Cha4p 50 MIN 881 120018

CHO F-12 Dialysed Cha4p 100 MIN 824 188222 245,54 9,9 CHO F-12 Dialysed Cha4p 100 MIN 1074,67 282277

CHO F-12 Dialysed Cha4p 250 MIN 342,67 66837,5 202,19 5,0 CHO F-12 Dialysed Cha4p 250 MIN 267 55823,8

CHO F-12 Dialysed Cha4p 500 MIN 307 57971 ,5 229,23 24,8

References

■ Bornaes et al , 1992 Serine and threonine catabolism in Sacharomyces cerevisiae the CHA1 polypeptide is homologous with other serine and threonine dehydratases Genetics, 131 , 531-539 ■ Bornaes et al , 1993 A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes Mol Cell Biol 13, 7604-7611

■ Holmberg and Schjerling, 1996 Cha4p of Saccharomyces cerevisiae activates transcntpion via serine/threonine response elements Genetics, 144, 467-478 ■ Israel and Kaufman, 1989 Highly inducible expression from vectorst containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor Nucl Acids Res, 12, 2589-2604

■ Klock et al , 1987 Oestrogen and glucocorticoid responsive elements are closely related but distinct Nature, 329, 734-736 ■ Pedersen et al , 1997 Locus-specific suppression of ιlv1 in Saccharomyces cerevisiae by deregulation of the CHA1 transcription Mol Gen Genet 255, 561- 569

■ Petersen et al , 1988 Molecular genetics of serine and threonine catabolism in Saccharomyces cerevisiae Genetics, 119, 527-534 ■ Ramos and Wiame, 1982 Occurrence of a catabolic L-seπne (L-threonine) deaminase in Saccharomyces cerevisiae Eur J Biochem , 123, 571-576

■ Sambrook et al Moleuclar Cloning A laboratory manual, 2^nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989

■ Schjerling and Holmberg, 1996 Comparative ammo acid sequence analysis of the C6 zinc cluster family of transcriptional regulators Nucleic Acids Res, 24, 4599-

4607

■ Searle et al , 1985 Building a metal-responsive promoter with synthetic regulatory elements Mol Cell Biol 5, 1480-1489

■ Voellmy, 1994 Transduction of the stress signal and mechanisms of transcriptional reagulation of heat shock / stress protein gene expression in higher eukaryotes

Crit Rev Eykaryot Gene Expr 4, 357-401

Claims

1. A recombinant expression system, allowing amino acid induced expression in eukaryotic cells

2. A recombinant expression system according to claim 1 , wherby said amino acid is serine and/or threonine.

3. A recombinant expression system, according to claim 1 or 2, whereby the eukaryotic cells are mammalian cells.

4. A recombinant expression system according to claim 1 , whereby the recombinant expression system comprises an amino acid response element

5. A recombinant expression system according to claims 1 - 4, whereby the recombinant expression system comprises a serine and/or threonine response element.

6. A recombinant expression system according to claim 4 or 5, whereby said response element is linked to a minimal promoter.

7. A recombinant expression system according to claim 5 or 6 whereby the response element comprises a DNA sequence shown in SEQ ID N° 1 and/or a DNA sequence shown in SEQ ID N°2 and/or the complement of said DNA sequences.

8. A recombinant expression system according to claim 4 - 7 whereby the recombinant expression system comprises a transcriptional activator binding to said response element.

9. A recombinant expression system according to claim 8 wherein the transcriptional activator comprises a protein or protein fragment with SEQ ID N° 4, or biologically active fragments thereof.

10. A recombinant expression system according to claim 8 whereby said protein or protein fragment is encoded by a DNA sequence shown in SEQ ID N° 3, or biologically active fragments of said protein or protein fragment.

11. An eukarytotic cell, comprising a recombinant expression system according to any of the previous claims.

12. A vector system for transforming or transfecting an eukaryotic cell, said system comprising of at least one vector, carrying at least a response element according to claim 4, 5, 6 or 7 and a transcriptional activator according to claim 8, 9 or 10, whereby the eukaryotic cell does not contain a serine and/or threonine catabolic pathway, or whereby the eukaryotic cell does contain an endogenous serine and/or threonine catabolic pathway that is not interfering with the serine and/or threonine response element and the transcriptional activator on the vector.

13. A kit, comprising a vector system according to claim 12.

14. A method of obtaining serine and/or threonine induced expression of a gene of interest in eukaryotic cells, comprising the steps a) transforming an eukaryotic cell with a vector system according to claim 12 b) growing the transformed cells in serine- and/or threonine-less or serine and/or threonine poor medium c) inducing the expression of a gene of interest by adding serine and/or threonine to the medium whereby said eukaryotic cell does not contain a serine and/or threonine catabolic pathway, or whereby the cell does contain an endogenous serine and/or threonine catabolic pathway that is not interfering with the serine and/or threonine response element and the transcriptional activator on the vector.