Method for the preparation of a protein by yeasts using an inducible system, vectors and correspondence transformed strains

Construction of the PYERE/hER Vectors

Methods

The techniques of molecular biology used for the construction of the different plasmids are described in the manuals “Molecular cloning, a laboratory manual”, T. Maniatis, E. F. Fritsch and J. Sambrook, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1982) and “Current Protocols in Molecular Biology”, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Sruhl, Greene Publishing Associates and Wiley Intersciences (1987). They comprise, in particular, digestion of the DNA with restriction enzymes, separation of the DNA fragments by agarose or polyacrylamide gel electrophoresis, ligation of DNA fragments with the enzyme T4 DNA ligase, transformation of E. coli with the recombinant plasmids and determination of the nucleotide sequence by Sanger's technique.

The plasmids used carry the gene for resistance to ampicillin, thereby permitting selection of the bacteria transformed by a plasmid on agar dishes containing ampicillin (50 μg/ml).

The E. coli strain used is strain DH5, genotype: F-, enda1, HsdR17(rk-md+), supE44, thi-1, lambda-, reca&, gyrA96, reIAI (Hanahan: DNA cloning: a practical approach (Vol. 1=p. 109-135, ed. D. M. Glover, IRL Press, Oxford (1985)).

The techniques of transformation and growth of yeast strains are described in “Laboratory Course Manual for Methods in Yeast Genetics”, F. Sherman, G. R. Find and J. B. Hicks, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1986).

The yeast strain used is TGY14-1a, pep4.3, genotype Mat a, ura3-251-373-328, leu2, pep4.3.

The parent plasmid pTG848, described in European Patent No. 200,655, is an E. coli /yeast shuttle plasmid which permits the expression in yeast of an exogenous gene under the control of the phosphoglycerate kinase (PGK) gene promoter. This plasmid contains a pBR 322 origin of replication and the gene for resistance to ampicillin for selection in E. coli, and a 2 μm origin of replication and the ura3 and leu2 genes for selection in yeast, as well as the PGK gene promoter and its 3′ end containing the polyadenylation signal. This gene was modified so as to introduce a BglII site at the initiation ATG, thereby enabling the coding region for another protein to be inserted between this site and the BglII site situated upstream from the polyadenylation signal (FIG. 1 ).

Construction of Plasmids pUC18, ERE1, DUC18 EREm, pUC18 ERE3

Plasmid pUC18 (C. Yanisch-Perron, J. Vieira and J. Messing, Gene 33, 103-1190 (1986)) contains a pBR322 origin of replication, the gene for resistance to ampicillin and a polylinker comprising, in particular, XbaI and HindIII sites. This plasmid was digested with the restriction enzymes XmaI and HindIII; to the plasmid thus digested, the following are then added by complementary oligonucleotide techniques:

5′-CCGGGTCTAGAAGATCTA3′ (SEQ ID NO: 3)

3′-CAGATCTTCTAGATTCGA5′ (SEQ ID NO: 4)

The derived plasmid pUC18 BglII then has a polylinker which contains, in order, XmaI, XbaI, BglII and HindIII sites.

Into this plasmid, digested with BglII, the fragments consisting of the following complementary oligonucleotides are introduced respectively:

ERE1:

5′GATCCAATATTCCTGGTCAGGCGTGACCGGAGCTGA3′ (SEQ ID NO: 5)

3′GTTATAAGGACCAGTCGCACTGGCCTCGACTCTAG5′ (SEQ ID NO: 6)

EREm:

5′GATCCAATATTCCCCGTCAGCGTGACCGGAGCTGA3′ (SEQ ID NO: 7)

3′GTTATAAGGGGCAGTCGCACTGGCCTCGACTCTAG5′ (SEQ ID NO: 8)

ERE3 which consists of a direct repetition of three ERE1 fragments.

ERE1 contains the ERE (for “estrogen responsive element”) sequence -605 to -634 of the vitellogenin promoter.

EREm is a mutant in which a GC transversion in the ERE sequence considerably reduces its activity in MCF-7 cells.

Construction of Plasmids pLRERE1, PLREREm, pLRERE3

Plasmid pLR1Δ20 is described in the paper by R. W. West, R. R. Yocum and M. Ptashne, Molecular and Cellular Biology 4, 2467-2478 (1984). This plasmid contains an active and chimeric gene for beta-galactosidase under the control of the GAL1 promoter (FIG. 2 ).

The object of the construction is to replace the UASs (upstream activator sequences) of the Gal1 gene, situated between the XmaI and XhoI sites of the Gal1 promoter region, by the fragments ERE1, EREm and ERE3, respectively.

pLRERE1

Plasmid pUC18ERE1 is digested with the restriction enzyme BglII. The site is filled in with DNA polymerase I (Klenow fragment) in the presence of the 4 deoxynucleotide triphosphates. An XhoI linker (Biolabs ref. 1030) is then added to the ends: the plasmid is then digested with XmaI and XhoI and the XmaI-XhoI fragment containing the ERE1 sequence is isolated.

This fragment is then cloned into plasmid pLR1Δ20 between XmaI and XhoI sites to give plasmid pLRERE1.

pLREREm, pLRERE3

Plasmids pLREREm and pLRERE3 are constructed in the same manner by replacing pUC18ERE1 by pUC18EREm and pUC18ERE3, respectively, in the above protocol.

Construction of Plasmids pYERE1, pYEREm, pYERE3

Plasmids pLRERE1, pLREREm and pLRERE3 possess a Tth111I site at the 3′ end of the lacZ (beta-galactosidase) chimeric gene (FIG. 2 ).

pYERE1

Plasmid pLRERE1 is digested with the enzyme Tth111I; the site is then filled in with DNA polymerase I (Klenow fragment). An XmaI linker (Biolabs ref. 1048) is then added to the ends. The DNA is digested with XmaI, and the XmaI-XmaI fragment containing the lacZ chimeric gene under the control of the hybrid promoter (EREA-Gal1) is inserted at the single XmaI site of plasmid pTG848, to give plasmid pYERE1.

pYEREm, pYERE3

Plasmids pYEREm and pYERE3 are constructed in the same manner by replacing pLRERE1 by pLREREm and pLRERE3, respectively, in the above protocol.

Construction of Plasmid pYGAL

Plasmid pLR1Δ20 is digested with the restriction enzyme Tth111I, and the site is then filled in with DNA polymerase I (Klenow fragment). An XmaI linker (Biolabs ref.1048) is added to the ends and the DNA is then digested with the enzyme XmaI. The XmaI fragment containing the lacZ chimeric gene under the control of the Gal1 promoter is inserted at the XmaI site of plasmid pTG848, giving plasmid pYGAL.

Construction of Plasmids pYERE1/hER, pYERE1/hERR, pYEREm/hER, pYGAL/hER

The object of these constructions is to place the cDNA coding for the human estrogen receptor (hER) under the control of the PGK promoter, at the BglII site of plasmids pYERE1, pYEREm, pYERE3 and pYGAL (FIG. 3 ).

Plasmid pKCR2-ER containing the cDNA of the human estrogen receptor (S. Green et al., Nature 320, 134-139 (1986)) is digested with the restriction enzyme EcoRI and the site is then filled in with DNA polymerase I (Klenow fragment). A BamHI linker (Biolabs ref. 102) is then added to the ends. After digestion with BamHI, the fragment coding for the human estrogen receptor is isolated.

It is inserted into the vector pYERE1 digested with BglII to create plasmids pYERE1/hER and pYERE1/hERR, which differ by the orientation of the hER cDNA relative to the PGK promoter.

In pYERE1/hER, the PGK promoter is situated on the 5′ side of the hER cDNA, and hence induces transcription of the mRNA coding for the hER protein.

In pYERE1/hERR, the orientation is reversed, and hence induces the transcription of an antisense RNA of the hER messenger RNA.

pYEREm/hER, pYERE3/hER and pYGAL/hER are constructed on the same model as pYERE1/hER, by replacing pYERE1 by pYEREm, pYERE3 and pYGAL, respectively, in the above construction.

These plasmids (FIG. 4 a ) constructed in E. coli DH5 are transferred into yeast strain TGY14.1a which carries the ura3 mutation. The presence of the ura3 gene on these plasmids permits complementation for the yeasts transformed by a plasmid, which are hence selected on agar dishes containing a medium without uracil (preparation: 0.67 g of Difco medium No. 0919-15, 0.5 g casamino acids, 1 g glucose, and 2 g agar per 100 ml).

Induction by Estradiol of Transcription for the ERE Gal1 Hybrid Promoters Carried by the pYERE/hER Vectors

The different yeasts containing plasmids pYERE1/hER, pYEREm/hER, pYERE3/hER, pYERE1/hERR and pYGAL/hER, respectively, are cultured in base medium for yeast (Difco No. 0919-15) on the basis of 0.67 g/100 ml and 0.5% casamino acids containing 1% glucose or 2% galactose or 100 nM estradiol (added to the culture 2 hours before the cells are recovered) as shown in FIG. 4 b.

The RNA is prepared from 10 9 cells according to the protocol described in Methods in Yeast Genetics, p. 143-144.

To measure the transcriptional activity of the Gal1 or ERE-Gal1 promoters, these RNAs are hybridized with a radiolabeled RNA probe derived from the 5′ end of the Gal1 gene (strand complementary to the messenger RNA). The hybridized fragments will be protected from digestion by RNases A and T1. The probe is prepared from the EcoRI fragment (containing the Gal1 promoter) of plasmid pLR1Δ20 (R. W. West, R. R. Yocum and M. Ptashne, Molecular and Cellular Biology 4, 2467-2478 (1984)) cloned into plasmid Bluescribe M13+ (Stratagene ref. 211201). The resulting plasmid is then linearized with HindIII and the radiolabeled probe is then synthesized from the phage T7 promoter with Stratagene Kit No. 211604. The probe size is of the order of 1000 nucleotides.

There are several transcription initiation sites in the Gal1 promoter; the probe is complementary to the Gal1 RNA over a length of 154 to 165 nucleotides.

The probe is incubated with 20 μg of RNA, and the mixture containing possible hybrids is digested with ribonucleases A and T1 according to the protocol described in Current Protocols in Molecular Biology.

The undigested fragments are then identified by urea/polyacrylamide gel electrophoresis and autoradiography (FIG. 4 b ).

For the constructions expressing the hER receptor, the induction of a messenger RNA specific to the ERE-Gal1 hybrid promoter is observed in the presence of estradiol; the transcription initiation sites are identical to those of the Gal1 (pYGAL) promoter induced by galactose: protected fragments of the same size.

The induction of transcription using the ERE-Gal1 hybrid promoter is strictly dependent on the presence of estradiol. The induction by estradiol requires the presence of the hER receptor (the construction pYERE/hERR is inactive). It also requires the presence of an ERE element (for the construction pYGAL/hER, there is no induction by estradiol).

The quantity of Gal1 RNA detected for cells containing pYERE3/hER is approximately 10 times as high as for cells containing pYERE1/hER: the repetition in tandem of the ERE elements (pYERE3 contains an ERE trimer, pYERE1 a monomer) hence appears to have a synergistic effect on the activation of transcription.

Expression, Inducible by Estradiol, of Beta-galactosidase in Yeasts Containing the pYERE/hER Vectors

Beta-galactosidase activity is measured according to the protocol of West et al. (1984). Table 1 below shows the beta-galactosidase activity of yeast strains carrying plasmids pYERE1/hER, pYERE3/hER, pYEREm/hER, pYERE1/hERR and pYGAL/hER, respectively, in the presence or absence of glucose, galactose or estradiol, as seen in the table.

The level of constitutive activity of the promoters is very low and is not affected by the presence of the hER protein (pYGAL and pYGAL/hER or pYERE1/hER and pYERE1/hERR give the same degree of activity). In contrast, the addition of estradiol leads to a strong induction of the hybrid promoters of pYERE1/hER and pYERE3/hER.

The fact that the promoters of pYEREm/hER and pYGAL/hER are not sensitive to induction by estradiol shows the necessity of a functional ERE element in the promoter controlling the expression of lacZ, while the absence of induction for pYERE1/hERR shows that the presence of the hER receptor is also necessary for observing induction by estradiol.

The level of expression attained in comparable to that obtained using the Gal1 promoter (the case of pYGAL/hER) in the presence of galactose.

The presence of three ERE elements in tandem (the case of pYERE3/hER) gives an activity twice as high as the case where the promoter contains a single ERE element (pYERE1/hER).

The yeasts transformed by the plasmids indicated are cultured in the presence of 1% of glucose or 2% of galactose as stated in the table; for induction by estradiol, 100 nM hormone is added 2 hours before the cells are harvested.

The tests of beta-galactosidase activity are performed in a traditional manner (West R. W. jr., Yocum R. R. and Ptashne M., Mol. Cell. Biol., 4, 2467-2478 (1984)).

Integration of a Complementary DNA Coding for hER in the Genome of a Yeast Strain

Preparation of the Expression Vector Carrying a Cassette for the Expression of hER Inserted into the PRC1 Gene (pTG3809)

The cassette for the expression of hER is inserted into a fragment of the PRC1 gene in order to obtain, after transformation of a yeast strain with pTG3809, a yeast strain displaying elimination of the proteolytic function encoded by the PRC1 gene.

In the first place, a cassette for the expression of hER (M13TG2896) is prepared in the following manner:

the yeast PGK promoter (HindIII-BglII fragment) and its terminator (HindIII-BglII fragment) are introduced into phage M13TG130 (Kieny, M. P. et al. (1983), Gene 26, p. 91-99) linearized by HindIII digestion, to give phage M13 TG2890,

the BamHI fragment derived from pKCR2-hER carrying the human estrogen receptor is introduced into M13TG2890 linearized by BglII digestion, to give a cassette for the expression of hER referred to as M13TG2896.

This cassette for the expression of hER is then inserted into the PRC1 gene to give plasmid pTG3809 permitting its integration in the yeast genome.

The sequence of the PRC1 gene has been published by Valls, L. A. et al. (1987) (Cell 48, p. 887-897). Two oligonucleotides are constructed from this sequence. The first is complementary to the 5′ region of the gene, and its sequence is as follows:

5′ AAG AAA GAC TGG GAC TTT GTG 3′ (SEQ ID NO: 9)

The second is complementary to the 3′ region of the gene, and its sequence is as follows:

5′ GAT TGG ATG AAG CCT TAC CAC 3′ (SEQ ID NO: 10)

From a yeast genomic library (chromosomal DNA fragments partially digested with Sau3A, inserted into the BamHI site of pFL1 (Parent, S. A. et al. (1985), Yeast 1, p. 83-138)), and by hybridization with these two oligonucleotides, an E. coli clone containing the PRC1 gene inserted into pFL1, and referred to as pTG2863, is selected.

The 1.1-kb BamHI fragment of pTG2863 carrying a portion of the coding sequence of the PRC1 gene is introduced into the BamHI site of plasmid pUC8. This insertion takes place in tandem and plasmid pTG2893 (FIG. 5 a ) is obtained.

The coordinates of the first base of the restriction sites, relative to the adenine of the ATG of the PRC1 coding sequence, are as follows: BamHI-1: 482; EcoRV-1: 1102; EcoRV-2: 1153; EcoRV-3: 1234 and BamHI-2: 1574.

A deletion in the PRC1 coding sequence is accomplished by EcoRV digestion of pTG2893 to give pTG2894 (FIG. 5 b ).

The cassette for the expression of hER is introduced into the deleted inner fragment of the PRC1 gene of pTG2894 in the following manner: the expression cassette (M13TG2896) is digested with BamHI, the ends are filled in with Klenow polymerase and, after cleavage with EcoRV, fragment is isolated. This fragment carries the complete cassette; it is introduced into the EcoRV site of pTG2894 to give plasmid pTG3809 (FIG. 6 ), which carries, between two fragments of PRC1:

several recognition sites for the restriction enzymes SphI, XbaI and KpnI originating from M13TG130,

the PGK terminator yeast,

the complementary DNA of hER,

the PGK promoter of the yeast.

Transformation of the Yeast Strain with the Plasmid Carrying the hER Expression Cassette Inserted into a Fragment of the Deleted PRC1 Gene (pTG3809)

The yeast strain transformed is S. cerevisiae TGY2sp13b (MATa; ura3-251, -373, -328; leu2-3, -112).

Plasmid pTG3809 is cleaved with BamHI. The DNA sequences of the ends of the fragment thereby isolated are homologous with those of the PRC1 gene sequence. These sequences will enable the fragment to be integrated in the genomic portion of the PRC1 gene of the yeast strain. This insertion leads to destruction of the genomic PRC1 gene by deletion.

Since the fragment does not carry a selection marker, plasmid pLRERE3 is used as a cotransformation vector. It permits the selection of Ura + prototrophs. Among Ura + prototrophs, the clones which have integrated the hER expression cassette carry a mutation in the PRC1 gene which leads to a loss of activity of carboxypeptidase yscY. These mutants are detected by a colorimetric test (Jones, E. W. (1977), Genetics 85, p. 23-33). The structure of the modified PRC1 locus is verified by Southern's method.

This S. cerevisiae strain which has integrated the cassette for the expression of hER in the deleted PRC1 gene is referred to as TGY2sp13b prc1-d :: hER.

Construction of a Plasmid Carrying the ERE3 Sequence and a Fragment of the PGK Gene Promoter (pGT3851)

An XhoI site is introduced into the vector M13TG2890 at the 5′, 3′ junction of the ends of the deletion accomplished by direct mutagenesis using the following oligonucleotide:

-506 . . . -401

5′ AATTACCGTCGCTCG AGCGACGGCTCACAG 3′ (SEQ ID NO:11)

XhoI

to give the vector M13TG3829 (FIG. 7 ). The positions indicated are specified relative to the ATG of the PGK gene. The deleted form of the PGK promoter will be designed hereinafter PGK-dp401. The conformity of the sequence is then verified by sequencing.

This vector carries the TATA sequence and the transcription initiation site, the second activator sequence downstream designated HSE from -256 to -377 (Piper, P. W. et al. (1988), Nucleic Acids Res. 16, p. 1333), but no longer carries the UAS from -402 to -479. The XhoI site permits ligation with the ERE3 sequence, and the BglII site the introduction of a gene which enables an activity to be detected by an enzymatic test.

The host strain for the M13 phages is E. coli strain JM103 (delta(lac, pro), thi, strA, supE, endA, sbcB, hsdR, F′traD36, proAB, lacIQ, lacZdeltaM15).

Plasmid pLRERE3 is digested with XmaI and EcoRI. The DNA fragment containing the ERE3 sequences is purified on agarose gel and then inserted into the XmaI-EcoRI sites of plasmid pTZ18R (Pharmacia, LKB Biotechnology) to give plasmid pTG3833. The host strain for the plasmids is E. coli strain 1106 (supE, hsdR, hsdM, met, supF).

Phase M13TG3829 (FIG. 7) is treated with XhoI and EcoRI and is then inserted at the XhoI-EcoRI site of plasmid pTG3833 to give pTG3834.

The lacZ gene is excised from pCH110 (Hall, C. V. et al. (1983), J. Mol. Appl. Gen. 2, p. 101-109) by HindIII-BamHI digestion and cloned into pPolyII (Lathe, R. et al. (1987), Gene 57 p. 193-201) to give pTG1174. Plasmid pTG2145 corresponds to plasmid pTG1174 in which the HindIII, PstI and SalI sites have been eliminated. It contains, in a BglII-BamHI fragment, the following E. coli gene fusion: gpt :: trDS :: lacZ.

The sequences in the vicinity of the ATG are modified so as to permit the correct translation in yeast in the following manner:

pTG2145 is digested with BglII and KpnI and the 200-base pair fragment containing the ATG is purified on acrylamide gel;

this fragment is inserted at the BamHI-KpnI sites of phage M13TG131 (Kieny, M. P. et al. (1983), Gene 26, p. 91-99) to give M13TG3838;

the “AGC” sequence corresponding to the second codon for a serine of M13TG3838 is exchanged for the sequence “TCT”, the cytosines at positions -3 and -1 relative to the ATG are replaced by adenines and a BglII site is generated on the 5′ side of the ATG to permit a fusion with genes to be inserted downstream from the hybrid promoter, by performing a directed mutagenesis using the following oligonucleotide:

5′ CGATGTATTTTTCAGACATTTTAAGATCTCCAGCCTGTTT 3′ (SEQ ID NO: 12) to give phage M13TG3842. The bases modified during this mutagenesis are underlined below:

GACACTTCACATGAGC- (M13TG3838) (SEQ ID NO: 14)

AGATCTTAAAATGTCT- (M13TG3842) (SEQ ID NO: 15)

The 130-base pair BglII-KpnI fragment of M13TG3842 is introduced into pTG2145 to replace the 200-base pair BglII-KpnI fragment therein. Plasmid pTG3843 is thereby obtained.

Plasmid pTG848 (FIG. 1) is digested with BglII and then religated to give pTG2886. The large HindIII-EcoRI fragment of pTG2886 is ligated in the presence of T4 ligaseth BwlII and thHindIII-EcoRI fragment of plasmid pFL1 (Parent S. A. et al. (1985), Yeast 1, p. 83-138) which carries a sequence of the S. cerevisiae plasmid, to give plasmid pTG2886 LEU2-d, URA3-d. The 0.9-kb HindIII fragment of plasmid pTG2800 described in the European publication of Patent EP-A-0,258,501 carrying the URA3-d gene is then inserted into the HindIII site of this plasmid to give pTG2886 URA3-d, delta LEU2-d. The SmaI-BglII fragment of M13TG131 (Kieny, M. P. et al. (1983), Gene 26, p. 91-99) which possesses several restriction sites is then introduced into this plasmid to give pTG3828.

Plasmid pTG3843 is digested with BglII and BamHI, and the fragment carrying the lacZ fusion gene is purified on agarose gel. This fragment is then inserted at the BglII site of pTG3846 (FIG. 8 ).

The plasmid pTG3834 is digested with XmaI and BglII, and the fragment is introduced into the XmaI and BglII site of pTG3846 to give plasmid pTG3851 (FIG. 8 ).

Inducible Expression of lacZ

S. cerevisiae strain TGY2sp13b prc1-d :: hER is relieved of its cotransformation plasmid pLRERE3 and then transformed with plasmid pTG3851 by the lithium acetate method (Ito, H. et al. (1983), J. Bacteriol. 153, p. 163-168), and the Ura + prototrophs are selected. They are then cultured in an Erlenmeyer at 30° C. on a selective medium (0.7% of nitrogenous bases for yeasts without amino acids (Yeast Nitrogen Bases), 0.5% of casamino acids, 2% of glucose and, if necessary, 2% of agar). These cultures are set up with or without estradiol. For induction with estradiol, 100 nM hormone is added 2 hours before the cells are harvested. The cells are harvested, washed and ground in the presence of protease inhibitors, and the cell debris is then precipitated by centrifugation (10,000 rpm). The protein concentration is determined in the supernatant by the method described by Bradford (1976) (Anal. Biochem. 72, p. 248), and then the beta-galactosidase activity by the same assay method as that described in Example 3. The beta-galactosidase activity is also determined by the method used in Example 3; the assay is performed on permeabilized cells.

Table 2 shows the basal activity (in the absence of estradiol, [−(Est)]) and induced activity (in the presence of estradiol, [+(Est)]) of beta-galactosidase under the control of the ERE3 :: PGK-dp hybrid promoters. This activity is expressed as nmol of ONPG (O-nitro-phenyl-beta-D-glucopyranoside) hydrolyzed per minute and per mg of protein at 30° C.; the values in brackets are directly comparable with those shown in Table 1, expressed in “units”. The inducibility corresponds to the ratio specific activity without estradiol/specific activity with estradiol.

Preparation of a Vector for the Expression of Beta-galactosidase in Yeast which Also Carries a Sequence Coding for the hERG Receptor (pYERE1/hERG)

The starting material is plasmid pYERE1/hER. Using the following oligonucleotide:

5′GGAGCACCCAGGGAAGCTACTGT-3′ (SEQ ID NO: 13)

A mutation in the GTG codon coding for valine in the complementary DNA coding for the human estrogen receptor (hER) is produced by localized mutagenesis in the following manner: the complementary DNA coding for the hER receptor described by Green et al. (1986) (Nature 320, p. 134-139) is subcloned into the EcoRI site of the eukaryotic expression vector pSG 1 described by Green et al. (1988) (Nucl. Acids Res. 16, p. 369). The single-stranded DNA is then prepared and a point mutation is produced by localized mutagenesis in the GTG codon coding for valine at position 400 so as to obtain a GGG codon coding for glycine, using the oligonucleotide defined above (the change in nucleotide is underlined). The DNA sequences are verified. The vector pYERE1/hERG is then constructed by inserting the hERG complementary DNA into the vector pYERE1/hER.

Expression, Inducible by Estradiol, of Beta-galactosidase in Yeasts Containing the Vector pYER1/hERG

The conditions of transformation and culturing of the cells are identical to those described above. When the optical density reaches 600, 10 nM estradiol is added. FIG. 9 shows that 50% of the maximal beta-galactosidase activity is obtained for an estradiol concentration of 5 nM with pYERE1/hERG (triangles), whereas with pYERE1/hER (circles) a concentration of approximately 50 nM is required.

Moreover, it was possible to observe that the mutation which replaces val-400 by gly-400 in the estradiol-binding domain stabilizes the structure of the receptor and increases its affinity for estradiol to 25° C.

Saccharomyces strain TGY 14-1a pYERE3/hER was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM) (National Collection of Micro-organism Cultures) of the Institut Pasteur—25 rue du Docteur-Roux, 75724 Paris Cédex 15 (France) on Jun. 17, 1988 under No. I-770.

15

6450 base pairs

nucleic acid

single

linear

DNA (genomic)

1

GAGTTGTGCC TGGAGTGATG TTTAAGCCAA TGTCAGGGCA AGGCAACAGT CCCTGGCCGT 60

CCTCCAGCAC CTTTGTAATG CATATGAGCT CGGGAGACCA GTACTTAAAG TTGGAGGCCC 120

GGGAGCCCAG GAGCTGGCGG AGGGCGTTCG TCCTGGGAGC TGCACTTGCT CCGTCGGGTC 180

GCCGGCTTCA CCGGACCGCA GGCTCCCGGG GCAGGGCCGG GGCCAGAGCT CGCGTGTCGG 240

CGGGACATGC GCTGCGTCGC CTCTAACCTC GGGCTGTGCT CTTTTTCCAG GTGGCCCGCC 300

GGTTTCTGAG CCTTCTGCCC TGCGGGGACA CGGTCTGCAC CCTGCCCGCG GCCACGGACC 360

ATGACCATGA CCCTCCACAC CAAAGCATCT GGGATGGCCC TACTGCATCA GATCCAAGGG 420

AACGAGCTGG AGCCCCTGAA CCGTCCGCAG CTCAAGATCC CCCTGGAGCG GCCCCTGGGC 480

GAGGTGTACC TGGACAGCAG CAAGCCCGCC GTGTACAACT ACCCCGAGGG CGCCGCCTAC 540

GAGTTCAACG CCGCGGCCGC CGCCAACGCG CAGGTCTACG GTCAGACCGG CCTCCCCTAC 600

GGCCCCGGGT CTGAGGCTGC GGCGTTCGGC TCCAACGGCC TGGGGGGTTT CCCCCCACTC 660

AACAGCGTGT CTCCGAGCCC GCTGATGCTA CTGCACCCGC CGCCGCAGCT GTCGCCTTTC 720

CTGCAGCCCC ACGGCCAGCA GGTGCCCTAC TACCTGGAGA ACGAGCCCAG CGGCTACACG 780

GTGCGCGAGG CCGGCCCGCC GGCATTCTAC AGGCCAAATT CAGATAATCG ACGCCAGGGT 840

GGCAGAGAAA GATTGGCCAG TACCAATGAC AAGGGAAGTA TGGCTATGGA ATCTGCCAAG 900

GAGACTCGCT ACTGTGCAGT GTGCAATGAC TATGCTTCAG GCTACCATTA TGGAGTCTGG 960

TCCTGTGAGG GCTGCAAGGC CTTCTTCAAG AGAAGTATTC AAGGACATAA CGACTATATG 1020

TGTCCAGCCA CCAACCAGTG CACCATTGAT AAAAACAGGA GGAAGAGCTG CCAGGCCTGC 1080

CGGCTCCGCA AATGCTACGA AGTGGGAATG ATGAAAGGTG GGATACGAAA AGACCGAAGA 1140

GGAGGGAGAA TGTTGAAACA CAAGCGCCAG AGAGATGATG GGGAGGGCAG GGGTGAAGTG 1200

GGGTCTGCTG GAGACATGAG AGCTGCCAAC CTTTGGCCAA GCCCGCTCAT GATCAAACGC 1260

TCTAAGAAGA ACAGCCTGGC CTTGTCCCTG ACGGCCGACC AGATGGTCAG TGCCTTGTTG 1320

GATGCTGAGC CCCCCATACT CTATTCCGAG TATGATCCTA CCAGACCCTT CAGTGAAGCT 1380

TCGATGATGG GCTTACTGAC CAACCTGGCA GACAGGGAGC TGGTTCACAT GATCAACTGG 1440

GCGAAGAGGG TGCCAGGCTT TGTGGATTTG ACCCTCCATG ATCAGGTCCA CCTTCTAGAA 1500

TGTGCCTGGC TAGAGATCCT GATGATTGGT CTCGTCTGGC GCTCCATGGA GCACCCAGTG 1560

AAGCTACTGT TTGCTCCTAA CTTGCTCTTG GACAGGAACC AGGGAAAATG TGTAGAGGGC 1620

ATGGTGGAGA TCTTCGACAT GCTGCTGGCT ACATCATCTC GGTTCCGCAT GATGAATCTG 1680

CAGGGAGAGG AGTTTGTGTG CCTCAAATCT ATTATTTTGC TTAATTCTGG AGTGTACACA 1740

TTTCTGTCCA GCACCCTGAA GTCTCTGGAA GAGAAGGACC ATATCCACCG AGTCCTGGAC 1800

AAGATCACAG ACACTTTGAT CCACCTGATG GCCAAGGCAG GCCTGACCCT GCAGCAGCAG 1860

CACCAGCGGC TGGCCCAGCT CCTCCTCATC CTCTCCCACA TCAGGCACAT GAGTAACAAA 1920

GGCATGGAGC ATCTGTACAG CATGAAGTGC AAGAACGTGG TGCCCCTCTA TGACCTGCTG 1980

CTGGAGATGC TGGACGCCCA CCGCCTACAT GCGCCCACTA GCCGTGGAGG GGCATCCGTG 2040

GAGGAGACGG ACCAAAGCCA CTTGGCCACT GCGGGCTCTA CTTCATCGCA TTCCTTGCAA 2100

AAGTATTACA TCACGGGGGA GGCAGAGGGT TTCCCTGCCA CAGTCTGAGA GCTCCCTGGC 2160

TCCCACACGG TTCAGATAAT CCCTGCTGCA TTTTACCCTC ATCATGCACC ACTTTAGCCA 2220

AATTCTGTCT CCTGCATACA CTCCGGCATG CATCCAACAC CAATGGCTTT CTAGATGAGT 2280

GGCCATTCAT TTGCTTGCTC AGTTCTTAGT GGCACATCTT CTGTCTTCTG TTGGGAACAG 2340

CCAAAGGGAT TCCAAGGCTA AATCTTTGTA ACAGCTCTCT TTCCCCCTTG CTATGTTACT 2400

AAGCGTGAGG ATTCCCGTAG CTCTTCACAG CTGAACTCAG TCTATGGGTT GGGGCTCAGA 2460

TAACTCTGTG CATTTAAGCT ACTTGTAGAG ACCCAGGCCT GGAGAGTAGA CATTTTGCCT 2520

CTGATAAGCA CTTTTTAAAT GGCTCTAAGA ATAAGCCACA GCAAAGAATT TAAAGTGGCT 2580

CCTTTAATTG GTGACTTGGA GAAAGCTAGG TCAAGGGTTT ATTATAGCAC CCTCTTGTAT 2640

TCCTATGGCA ATGCATCCTT TTATGAAAGT GGTACACCTT AAAGCTTTTA TATGACTGTA 2700

GCAGAGTATC TGGTGATTGT CAATTCACTT CCCCCTATAG GAATACAAGG GGCCACACAG 2760

GGAAGGCAGA TCCCCTAGTT GGCCAAGACT TATTTTAACT TGATACACTG CAGATTCAGA 2820

GTGTCCTGAA GCTCTGCCTC TGGCTTTCCG GTCATGGGTT CCAGTTAATT CATGCCTCCC 2880

ATGGACCTAT GGAGAGCAAC AAGTTGATCT TAGTTAAGTC TCCCTATATG AGGGATAAGT 2940

TCCTGATTTT TGTTTTTATT TTTGTGTTAC AAAAGAAAGC CCTCCCTCCC TGAACTTGCA 3000

GTAAGGTCAG CTTCAGGACC TGTTCCAGTG GGCACTGTAC TTGGATCTTC CCGGCGTGTG 3060

TGTGCCTTAC ACAGGGGTGA ACTGTTCACT GTGGTGATGC ATGATGAGGG TAAATGGTAG 3120

TTGAAAGGAG CAGGGGCCCT GGTGTTGCAT TTAGCCCTGG GGCATGGAGC TGAACAGTAC 3180

TTGTGCAGGA TTGTTGTGGC TACTAGAGAA CAAGAGGGAA AGTAGGGCAG AAACTGGATA 3240

CAGTTCTGAG CACAGCCAGA CTTGCTCAGG TGGCCCTGCA CAGGCTGCAG CTACCTAGGA 3300

ACATTCCTTG CAGACCCCGC ATTGCCTTTG GGGGTGCCCT GGGATCCCTG GGGTAGTCCA 3360

GCTCTTATTC ATTTCCCAGC GTGGCCCTGG TTGGAAGAAG CAGCTGTCAA GTTGTAGACA 3420

GCTGTGTTCC TACAATTGGC CCAGCACCCT GGGGCACGGG AGAAGGGTGG GGACCGTTGC 3480

TGTCACTACT CAGGCTGACT GGGGCCTGGT CAGATTACGT ATGCCCTTGG TGGTTTAGAG 3540

ATAATCCAAA ATCAGGGTTT GGTTTGGGGA AGAAAATCCT CCCCCTTCCT CCCCCGCCCC 3600

GTTCCCTACC GCCTCCACTC CTGCCAGCTC ATTTCCTTCA ATTTCCTTTG ACCTATAGGC 3660

TAAAAAAGAA AGGCTCATTC CAGCCACAGG GCAGCCTTCC CTGGGCCTTT GCTTCTCTAG 3720

CACAATTATG GGTTACTTCC TTTTTCTTAA CAAAAAAGAA TGTTTGATTT CCTCTGGGTG 3780

ACCTTATTGT CTGTAATTGA AACCCTATTG AGAGGTGATG TCTGTGTTAG CCAATGACCC 3840

AGGTAGCTGC TCGGGCTTCT CTTGGTATGT CTTGTTTGGA AAAGTGGATT TCATTCATTT 3900

CTGATTGTCC AGTTAAGTGA TCACCAAAGG ACTGAGAATC TGGGAGGGCA AAAAAAAAAA 3960

AAAAAGTTTT TATGTGCACT TAAATTTGGG GACAATTTTA TGTATCTGTG TTAAGGATAT 4020

GCTTAAGAAC ATAATTCTTT TGTTGCTGTT TGTTTAAGAA GCACCTTAGT TTGTTTAAGA 4080

AGCACCTTAT ATAGTATAAT ATATATTTTT TTGAAATTAC ATTGCTTGTT TATCAGACAA 4140

TTGAATGTAG TAATTCTGTT CTGGATTTAA TTTGACTGGG TTAACATGCA AAAACCAAGG 4200

AAAAATATTT AGTTTTTTTT TTTTTTTTTG TATACTTTTC AAGCTACCTT GTCATGTATA 4260

CAGTCATTTA TGCCTAAAGC CTGGTGATTA TTCATTTAAA TGAAGATCAC ATTTCATATC 4320

AACTTTTGTA TCCACAGTAG ACAAAATAGC ACTAATCCAG ATGCCTATTG TTGGATATTG 4380

AATGACAGAC AATCTTATGT AGCAAAGATT ATGCCTGAAA AGGAAAATTA TTCAGGGCAG 4440

CTAATTTTGC TTTTACCAAA ATATCAGTAG TAATATTTTT GGACAGTAGC TAATGGGTCA 4500

GTGGGTTCTT TTTAATGTTT ATACTTAGAT TTTCTTTTAA AAAAATTAAA ATAAAACAAA 4560

AAAAATTTCT AGGACTAGAC GATGTAATAC CAGCTAAAGC CAAACAATTA TACAGTGGAA 4620

GGTTTTACAT TATTCATCCA ATGTGTTTCT ATTCATGTTA AGATACTACT ACATTTGAAG 4680

TGGGCAGAGA ACATCAGATG ATTGAAATGT TCGCCCAGGG GTCTCCAGCA ACTTTGGAAA 4740

TCTCTTTGTA TTTTTACTTG AAGTGCCACT AATGGACAGC AGATATTTTC TGGCTGATGT 4800

TGGTATTGGG TGTAGGAACA TGATTTAAAA AAAAAACTCT TGCCTCTGCT TTCCCCCACT 4860

CTGAGGCAAG TTAAAATGTA AAAGATGTGA TTTATCTGGG GGGCTCAGGT ATGGTGGGGA 4920

AGTGGATTCA GGAATCTGGG GAATGGCAAA TATATTAAGA AGAGTATTGA AAGTATTTGG 4980

AGGAAAATGG TTAATTCTGG GTGTGCACCA AGGTTCAGTA GAGTCCACTT CTGCCCTGGA 5040

GACCACAAAT CAACTAGCTC CATTTACAGC CATTTCTAAA ATGGCAGCTT CAGTTCTAGA 5100

GAAGAAAGAA CAACATCAGC AGTAAAGTCC ATGGAATAGC TAGTGGTCTG TGTTTCTTTT 5160

CGCCATTGCC TAGCTTGCCG TAATGATTCT ATAATGCCAT CATGCAGCAA TTATGAGAGG 5220

CTAGGTCATC CAAAGAGAAG ACCCTATCAA TGTAGGTTGC AAAATCTAAC CCCTAAGGAA 5280

GTGCAGTCTT TGATTTGATT TCCCTAGTAA CCTTGCAGAT ATGTTTAACC AAGCCATAGC 5340

CCATGCCTTT TGAGGGCTGA ACAAATAAGG GACTTACTGA TAATTTACTT TTGATCACAT 5400

TAAGGTGTTC TCACCTTGAA ATCTTATACA CTGAAATGGC CATTGATTTA GGCCACTGGC 5460

TTAGAGTACT CCTTCCCCTG CATGACACTG ATTACAAATA CTTTCCTATT CATACTTTCC 5520

AATTATGAGA TGGACTGTGG GTACTGGGAG TGATCACTAA CACCATAGTA ATGTCTAATA 5580

TTCACAGGCA GATCTGCTTG GGGAAGCTAG TTATGTGAAA GGCAAATAAA GTCATACAGT 5640

AGCTCAAAAG GCAACCATAA TTCTCTTTGG TGCAAGTCTT GGGAGCGTGA TCTAGATTAC 5700

ACTGCACCAT TCCCAAGTTA ATCCCCTGAA AACTTACTCT CAACTGGAGC AAATGAACTT 5760

TGGTCCCAAA TATCCATCTT TTCAGTAGCG TTAATTATGC TCTGTTTCCA ACTGCATTTC 5820

CTTTCCAATT GAATTAAAGT GTGGCCTCGT TTTTAGTCAT TTAAAATTGT TTTCTAAGTA 5880

ATTGCTGCCT CTATTATGGC ACTTCAATTT TGCACTGTCT TTTGAGATTC AAGAAAAATT 5940

TCTATTCATT TTTTTGCATC CAATTGTGCC TGAACTTTTA AAATATGTAA ATGCTGCCAT 6000

GTTCCAAACC CATCGTCAGT GTGTGTGTTT AGAGCTGTGC ACCCTAGAAA CAACATACTT 6060

GTCCCATGAG CAGGTGCCTG AGACACAGAC CCCTTTGCAT TCACAGAGAG GTCATTGGTT 6120

ATAGAGACTT GAATTAATAA GTGACATTAT GCCAGTTTCT GTTCTCTCAC AGGTGATAAA 6180

CAATGCTTTT TGTGCACTAC ATACTCTTCA GTGTAGAGCT CTTGTTTTAT GGGAAAAGGC 6240

TCAAATGCCA AATTGTGTTT GATGGATTAA TATGCCCTTT TGCCGATGCA TACTATTACT 6300

GATGTGACTC GGTTTTGTCG CAGCTTTGCT TTGTTTAATG AAACACACTT GTAAACCTCT 6360

TTTGCACTTT GAAAAAGAAT CCAGCGGGAT GCTCGAGCAC CTGTAAACAA TTTTCTCAAC 6420

CTATTTGATG TTCAAATAAA GAATTAAACT 6450

595 amino acids

amino acid

single

linear

peptide

2

Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His

1 5 10 15

Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys

20 25 30

Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys

35 40 45

Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala

50 55 60

Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr

65 70 75 80

Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly

85 90 95

Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His

100 105 110

Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val

115 120 125

Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala

130 135 140

Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly

145 150 155 160

Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met

165 170 175

Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala

180 185 190

Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe

195 200 205

Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr

210 215 220

Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys

225 230 235 240

Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg

245 250 255

Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp

260 265 270

Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala

275 280 285

Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn

290 295 300

Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu

305 310 315 320

Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro

325 330 335

Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg

340 345 350

Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val

355 360 365

Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu

370 375 380

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Val

385 390 395 400

Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys

405 410 415

Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser

420 425 430

Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu

435 440 445

Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser

450 455 460

Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp

465 470 475 480

Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr

485 490 495

Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser

500 505 510

His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met

515 520 525

Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu

530 535 540

Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val

545 550 555 560

Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser

565 570 575

His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro

580 585 590

Ala Thr Val

595

18 base pairs

nucleic acid

single

linear

DNA (genomic)

3

CCGGGTCTAG AAGATCTA 18

18 base pairs

nucleic acid

single

linear

DNA (genomic)

4

CAGATCTTCT AGATTCGA 18

36 base pairs

nucleic acid

single

linear

DNA (genomic)

5

GATCCAATAT TCCTGGTCAG GCGTGACCGG AGCTGA 36

35 base pairs

nucleic acid

single

linear

DNA (genomic)

6

GTTATAAGGA CCAGTCGCAC TGGCCTCGAC TCTAG 35

35 base pairs

nucleic acid

single

linear

DNA (genomic)

7

GATCCAATAT TCCCCGTCAG CGTGACCGGA GCTGA 35

35 base pairs

nucleic acid

single

linear

DNA (genomic)

8

GTTATAAGGG GCAGTCGCAC TGGCCTCGAC TCTAG 35

21 base pairs

nucleic acid

single

linear

DNA (genomic)

9

AAGAAAGACT GGGACTTTGT G 21

21 base pairs

nucleic acid

single

linear

DNA (genomic)

10

GATTGGATGA AGCCTTACCA C 21

30 base pairs

nucleic acid

single

linear

DNA (genomic)

11

AATTACCGTC GCTCGAGCGA CGGCTCACAG 30

40 base pairs

nucleic acid

single

linear

DNA (genomic)

12

CGATGTATTT TTCAGACATT TTAAGATCTC CAGCCTGTTT 40

23 base pairs

nucleic acid

single

linear

DNA (genomic)

13

GGAGCACCCA GGGAAGCTAC TGT 23

16 base pairs

nucleic acid

single

linear

DNA (genomic)

14

GACACTTCAC ATGAGC 16

16 base pairs

nucleic acid

single

linear

DNA (genomic)

15

AGATCTTAAA ATGTCT 16