JPS63502397A - Expression of steroid receptor proteins in eukaryotic cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 7. @mammalian cells. The host cell according to claim 6.

8. A method for producing recombinant SR in a promoter animal, comprising: culturing the cells and collecting the 'SR protein.

Method.

96 by disrupting the cells, removing cell debris, and collecting the clear lysate. The SR is recovered. The method according to claim 8.

10, produced by the method set forth in claim 8.

The animal's recombinant steroid receptor.

11. Estrogen receptor. SR according to claim 10.

12. ER0 specification according to claim 11 leek" The present invention relates to the production of desired proteins using recombinant technology. In particular, two inventions are , steroid levels using eukaryotic hosts and expression systems compatible with these hosts. Concerning the production of Scepter protein.

Yijing and distortion Steroid hormone receptors control gene expression in eukaryotic cells is well known. The actions of steroid hormones in vertebrates are specific Involves interaction between intracellular steroid receptor proteins (SR) and the genome, and further In many cases, steroid hormones and SR proteins have been implicated in tumor growth. (Lippmann, M. Il', ).

Cancer: 7 and Shio 7, Noguchi (Reipan, NY 1975): Lippm ann.

M.E. et al., Nature (1975) 256: 592). this mutual Although the nature of the action and the location of the bond are unknown, the bond of steroids is It is thought to convert Pak into a form that binds strongly to nuclear DNA.

Steroid receptor proteins, especially estrogen receptors (ER), are divided into two tissues. The expression of this protein is regulated at one developmental stage.

ER has been purified from several sources and can have a molecular weight of 65-70 kd. (Redeuilh, G., et al., Eur. Biochem. (1980) 106: 481; Lubahn+ p, B, et al., J B iol Chem (1985) 260: 2215; Katzene llen-bogon+ B, S, et al., J Biol Chem (1983 ) 258: 3487; 5akai.

D. et al. Endocrinol (1984) 115: 2379; va’n,,0osberee,T,R.

et al., Anal Biochem (1984) 136: 321). Sara In addition, cDNA of the related human geltocortoid receptor (GR) was also prepared. (Hollenberg, S. M. et al., Nature (1985) 318:635).

Recently, cDNA encoding human estrogen receptor (hER) has been discovered in human milk. Prepared from cancer cell line MCF-7 and randomly primed λgtlO and isolated from the λgtll cDNA library.

Monoclonal anti-ER antibody and two peptide sequences of MCF-7 human ER Screened using matching synthetic oligonucleotides. This job is Waiter, P, et al., Proc Natl Acad 1 - A) (1985) Dish: 7889-7893, and references include: Adopted here. Oligonucleotide hybridization from cDNA clones A 2.1 kb cDNA clone was isolated by PCR. The cDN A was named OR8 and cross-hybridized with all other cDNAs, 2 It contained the expected sequence for two ER peptides. Furthermore, this cDNA teeth.

It selectively hybridizes with 6.2kb polyA RNA, and the polyA RNA is In vitro translation in the presence of 35S-methionine results in a small amount of immunoreactive 46 Together with the kd protein, it encodes the synthesis of the immunoreactive 65kd hER. was shown. The OR8 cDNA insert fragment contains the entire coding sequence of the 65kd protein. It was long enough to contain the body.

In the present invention, the OR8 cDNA clone was developed into an expression system compatible with mammalian host cells. 2. Recombinant hER using successfully integrated and transformed mammalian cell cultures was produced. Utility of hER and other steroid receptor protein expression systems by providing the ability to produce large amounts of purified steroid receptor protein. Its production in eukaryotic hosts depends on post-translational processing and folding. regenerates material that closely resembles naturally produced intracellular proteins It is possible.

summer dish The present invention provides a complete transcript encoding the human estrogen receptor protein (hER). The NA sequence is given along with the deduced amino acid sequence. Usefulness of this sequence information glucocortocoid receptor and v-erb-A (AEV) tumor gene. This is to allow comparisons with related receptor proteins such as gene products.

Additionally, suitable coding sequences can be functionally expressed in eukaryotic host cells, particularly mammalian host cells. Incorporated into the system. This makes it appropriate. and generally agonist and antagonist compounds Useful for compound design and research on the mechanism of action of steroid binding proteins, It is used in diagnostic assays for the park itself or for antibodies raised against it. many Provides protein.

Accordingly, in certain aspects the invention provides methods for use in eukaryotic host cells.

These sequences were operably linked to control sequences capable of expression. receptor vertebrate steroid receptor protein, which contains DNA encoding the protein Regarding the expression system. In yet another aspect, 1 the present invention provides 9 recombinant expression systems comprising the expression system of the present invention. host eukaryotic cells, and these cells are used to produce steroid receptor proteins. and how it was produced.

Regarding these steroid receptor proteins.

Rulenη surprised skin Figure 1 shows the cDNA sequence encoding the human estrogen receptor protein. and the deduced amino acid sequence.

Figure 2 shows the relationship between hER, GR, and putative AEV oncogene proteins. A comparison of the amino acid sequences is shown.

Figure 3 shows the human protein expressed by OR8 cDNA in CHOK-1 cells. Figure 3 shows sedimentation analysis of estrogen receptors.

“Expression system” means a collection of components shown in sequence, particularly regulatory sequences, Furthermore, these sequences bound to an enhancer, or these sequences, and A code operably linked to a vector containing other DNA sequences involved in expression. may contain only code arrays.

“Human metallothionein-■” promoter (hMT-II) is a human MT - refers to regulatory sequences derived from the n gene or its functional equivalent. this gene The control sequences of Karin, M. et al.

Described in detail by Nature (1982) 299: 797-802. It is being

°'Chinese hamster ovary" (CHO") cells are derived from the standard cell line ATCC. CCL-61, and its derivatives as well as its close relatives isolated from the same tissue source. Includes limbs. A derivative is a variant of a system that is genetically or expressively different from the original system. However, it can be obtained by intentional or accidental mutation.

``derived from'' refers to ``derived from'' when it relates to one e.g. DNA or protein sequence. 9 means structurally similar ≧.

This does not necessarily mean physical induction.

“Purified” or “pure” means that it is found in its natural state. This means that the material is separated from its normal coexisting substances. Therefore, “pure” What is SR?5For example, in a human cell, it contains substances normally associated with its own environment. SR not included. Of course, “°pure SR coexists uniquely with Substances 9 containing, for example, sugar residues.

“Operably coupled” means that the components are arranged to perform their normal function. means the proximal state in which Therefore.

A control sequence or promoter operably linked to a coding sequence is a Expression of the sequence can be performed.

"Control sequences" are DNA sequences or sequences that when properly linked to a desired coding sequence. In this case, it means a sequence that can be expressed in a host that is compatible with such a sequence. Ru. Such control sequences include promoters and stop codons. en Additional factors that are necessary or useful for carrying out expression such as Hancer Children may also be identified. As used herein, “control sequence” simply means and any DNA sequences required to effect expression in the particular host used. means.

“cell” or “cell culture” or “recombinant host cell” or “host cell” ' is clear from the context.

Often used as synonyms. These terms refer to the direct target cell, and also Of course, it also includes its descendant cells.

Due to chance mutations or environmental differences, not all progeny cells are exactly like the parent cell. It is understood that they are not necessarily the same. However, first The progeny of the transformed cell retain properties related to those imparted to the transformed cell. To the extent such altered progeny cells are included in these terms.

(Margin below) B-21 ■ J O week The present invention provides steroid receptors under environmental conditions conducive to mimicking natural products. The first expression of a protein protein is disclosed herein. In order to perform such expression, A cDNA or genome encoding the desired steroid receptor protein of the animal Mu DNA.

or other DNA under the control of control sequences operable in eukaryotic cells for expression of placed. The invention is described below with respect to human estrogen receptors; However, using the expression systems of the present invention, including the expression systems described in detail, It is recognized that other steroid receptors can be successfully produced.

In order to obtain the effective structure of the natural protein, production is carried out in eukaryotic hosts. is desirable. Common eukaryotic hosts include yeast and mammalian cells. for mammalian cells Particular expression systems are described below; however, other expression systems are also available.

Make appropriate modifications to obtain expression of the appropriate steroid receptor-encoding sequence. I can give.

Eukaryotic microorganisms, although less desirable than @mammalian cultures, can be used as hosts. It can be done. Although many other yeast strains are commonly used, Satucharomyces cerevisiae Experimental strains of baker's yeast are most commonly used. For example, Breach, J, Using the 2μ origin of replication of R., Math Enz (1983) 101:307 vector, or other yeast that is compatible with the origin of replication (e.g., Stinchc omb et al., Nature (1979) 282:39. Tschempe et al., Gene (1980), Published: 157 and C1arke, L. et al. (See Meth Enz (1983) 101:300) can be used. yeast The control sequences of the vector include a promoter for glycolytic enzyme synthesis (less et al., J Adv Enz me Re (1968) 7:149 HHo 1land et al., Biochemistr (197B) 17:4900) ’, Additional promoters known in the pig field today include 3-phosphog Promoter of lysate kinase (Hitzeman et al., J BiolCh ew (1980) 255:2073) and other glycolytic enzyme promoters. including tar. Other methods have the added advantage that transcription is controlled by growth conditions. As a promoter, alcohol dehydrogenase 2. isocytochrome C2 Acid phosphatase.

Degradative enzymes associated with nitrogen metabolism, and necessary for the utilization of maltose and galactose. There is an enzyme promoter region. A terminator sequence is placed at the 3' end of the coding sequence. It is also conceivable that something like this is desirable. Such a terminator is It is found in the <3' terminal untranslated region following the coding sequence in the maternally derived gene.

Yeast transformation is described by Van Solingen, P. et al., J. Bact. (1977) 130:946 or Hsiao, C.L., et al., Pro. c Natl Acad 5ci (USA) (1979) 76: 3829 The method can be carried out according to the method.

However, expression in @mammalian host cells is preferred, and for such expression The general technique of is known. For example, No. 4,399.21 of 1jxel et al. See No. 6. These systems are.

It has the additional advantage of being able to splice indolones. useful accommodation The main cell lines are VERO and eLa cells, and Chinese hamster eggs. Includes nest (CHO) cells. Expression vectors for such cells are usually 2 e.g. 1 Simian virus 40 (SV40) early and late promoters used (Fiers et al., Nature (1978) 273:113), or Polyoma, adenovirus2. bovine papillomavirus, or avian monkey Like promoters of other viruses, such as promoters from comaviruses Usually contains promoters and control sequences compatible with mammalian cells. controllable pro Motor 1111Tll [(Karin.

h, et al., Nature (1982) 299 Near 97-802) is also used. It can be done. It is also important that "enhancer" regions are important for optimal expression. now These are generally promoter biased in non-coding DNA regions; Sequences found upstream or downstream; these are also explained below. . If necessary, origins of replication can be obtained from viral material. However, to the chromosomes incorporation is a common mechanism for DNA replication in eukaryotes. mammalian For cells, Wigler as needed.

Gra modified by H. et al., Kabutan (1979) 16: 777-785. ham and van der Eb, Wandering Range ■ (1978) 52: 546 The calcium phosphate precipitation method can be used.

Linking the gene sequence to appropriate control sequences for desired expression in eukaryotic hosts and transformed into compatible host cells.

Cell cultures are then grown under appropriate conditions. Promoter whose regulatory sequence is inducible -, appropriate induction conditions are provided. steroid receptor Because the protein is intracellular, it lacks a signal sequence and typically contains a methionine initiation code. lead. Foreign proteins that lack this codon and can cause this protein to be secreted from the host Other constructs containing signal sequences may also be constructed. For example, renin, growth hormone 9 normally secreted mammalian proteins such as insulin. Gnar sequences are known in the art and can be used. For expression in yeast Yeast signal sequences, such as those associated with α-factor, are used for obtain.

C-Hanagi ratio What is the utility of purified steroid receptor protein in sufficient quantities?

Advantageous in designing diagnostic assays and means of controlling steroid-mediated metabolism That's true. Regarding the former, diagnosis of metabolic disturbances regulated by steroids as a means of estimating the levels of receptors present in various tissues, and estimating the susceptibility of various tumors to control by modulating steroid supply; I want it. Furthermore, it is possible to generate antibodies specific to steroid receptors. and the availability of large numbers of antigens facilitates their analysis.

Therefore, pure recombinant human estrogen receptors and other steroid hormone receptors Desiring to obtain a scepter.

It has several aspects. First, using this method, milligram quantities of the substance can be can get. The amount in milligrams is determined by three-dimensional structure research using X-ray diffraction and computer analysis. enables crystallization for research. This allows us to understand the shape of the molecule and therefore the hormone. Suitable forms of substances that can be used as agonists and antagonists are determined. Agents and antagonists are crucial in regulating steroid-mediated metabolism. These metabolisms include reproductive function, inflammatory responses, blood pressure, and secondary sexual characteristics. vinegar The dependence of some tumors on teroids is also known, but human ER, Lucocortocoid receptor and putative v-erb-A oncogene production The disclosure of the present invention makes it certain that there is partial homology between It will be done.

ER and other steroid receptors have DNA and steroid binding domains. do. and agonists and antagonists for steroid activity are identified in the SR. In order to increase the ability of a substance to actively and specifically interact with the It is a substance whose interaction with SR is stabilized by its three-dimensional structure.

This lock and key spatial arrangement is similar to that of the crystallized ER or other steroid receptors of the present invention. produced by molecules designed to be complementary to the surface topography of the target. "table ``face'' includes convolutions that may face inward; In particular, it should include both the normal sites of interaction and the internal structures that occur at these sites. It is understood that Furthermore, “complementary” refers to “compatible” spatial arrangements as well as We say that the interaction between a protein and a molecule that conforms to its surface shape is attractive and positive. It is understood to mean that These interactions are hydrogen bonding and ionic.

or hydrophobic affinity. antagonists and agonists.

It is a modified steroid with a complementary three-dimensional structure.

Second, even without the aid of three-dimensional structure determination, the purified receptor of the present invention can be evaluated. A special method for performing this is to screen agonists and antagonists in vitro. It is important as a reagent for testing. Impure receptors currently available Preparations cause data confounding due to the influence of impurities on test results. for example , contaminants that prove themselves to be agonists or antagonists to steroids, interfere with evaluation.

Therefore, current screening techniques for agonists and antagonists are Improvements are made by using purified human receptor proteins.

Furthermore, the availability of materials for the recombinant production of proteins is limited to 9 e.g. Sequences can be modified by position-directed mutagenesis to improve biological effects, such as ligand binding specificity. The properties of the product receptor can be modified. Therefore, the recombinant vector of the present invention is , provides a starting material for obtaining a series of modified receptors for processing transformation spectra. provide

Finally, the recombinant vector of the present invention targets human receptor proteins in biological samples. Provides specific and sensitive diagnostic analysis. Such an analysis9 For example, It is important in diagnosing tumor susceptibility to steroid metabolism. purified recombinant human The utility of the receptor is to provide material for standardization and calibration of direct immunoassays. Ru.

D, S”1 outside The following is the construction of an expression system effective for producing recombinant human estrogen receptor. be. This example is not presented to limit the invention.

Disaster relief Characterization of the cDNA encoding the human estrogen receptor Halter, p., et al. The OR8 cDNA obtained by the method described in (previously) was transformed into M13mp9 Eco Subcloned into the RI site. Then, create a clone containing both directions to the cDNA. Isolated, Sanger, F., et al., Proc NatlAcad Sci (USA) (1977) 74: 5463; 5chreirer, P, H, et al.

Sequence determined by the method of J Mol Biol (1979) 129:169. Established.

The resulting sequence is shown in FIG. Open reading containing 1785 nucleotides The frame corresponds to 595 amino acids and the molecular weight is calculated to be 66.200. calculated. The peptide sequence from purified hER is in this oven reading frame. and the position of the ATG start codon (terminal in frame) The first occurrence of nucleotides -54 to -52 downstream of Noether-TGA. ), was estimated. Deduced amino acid sequence and human glucocortocoid Known sequences of Scepter (GR) and v-erb-A (AEV) oncogene products When compared to , strong homology was observed in regions rich in lysine and arginine. Shown in Figure 2 As such, a high degree of homology is found in the various regions to which these proteins correspond.

Disaster ■Also Mitsui and ■Enzuki The host expression vector contains a metallothionein protein that is inducible in the presence of zinc ions. lomotor system (hMr-If), virus enhancer-2 and approximately 600 bp Hi 4. This expression vector contains the 3” untranslated region of growth hormone. From the vector named phGHg-SV (10) constructed as below , obtained by substituting the human growth hormone coding sequence. phGHg-3 Other vectors called V(9) (also described below) may also be used.

The construction of these vectors, which are used as sources for host vector sequences, is described below. Two dishes died Plasmid pH51 is +70 from the -765 red group ■ site of the hMT-II gene. Across the base BamHI cleavage site, p84H (Karin, M.

hM from Nature (1982) 299: 297-302) Contains 840 bp of T-II sequence. Completely digest plasmid p84H with BamHI treated with exonuclease Ba1-31 to remove terminal nucleotides, and Next, I disassembled it using the old nd111. The desired 840 bp HindI [[/blunt end truncation] The piece was pUc8 (Vieira, J. et al., Gene (1982) 19: 259-268) (Digested with HindI and old ncII to open the ring) connected to. Transform E. coli HBIOI into Amp” with this ligation mixture. p) One candidate plasmid, designated IsI, was isolated and p) The sequence was determined using the Shi method. pH51 is upstream of the polylinker containing convenient restriction sites. contains hMT-II regulatory sequences.

death of a star The genome sequence encoding hGH is p2.6-3 (DeNoto et al., Nuc leic Acids Res (1981) 19: 3719), B amHI (cuts at the 5′ end of the first exon) and EcoRI (cuts at the 5′ end of the first exon) and EcoRI (cuts at the 5′ end of the first exon) (cutting the 3' part of the gene) and purification using polyacrylamide gel. and isolated. The isolated fragment was digested with BamHI/EcoRI at pH51. and transform E. coli MC1061 into Amp'' with the ligation mixture. I changed it. Successfully obtained transformants were screened by restriction analysis and , the strain containing the desired plasmid (designated pMT-hG)Ig) was further propagated. plasmid DNA was prepared in large quantities.

, mutual 2ka ukeken gayo ku place SV40 enhancer and hG operably linked to MT-IIn promoter A pair of host expression vectors containing the 3° untranslated sequences of H. wife Then, the 1120bp 5V40 DNA fragment was inserted into the MT-Il protein in pMT-hGHg. It was constructed by inserting into the HindnI site in front of the promoter sequence. this The 5V40ONA fragment spans the SV40 origin of replication and contains nucleotides 5171 to nucleotide 5243 (starting point), nucleotides 107 to 250 and the 5' end of late viral mRNA. It continues up to nucleotide 1046 on the origin side including the end. This old nd m 1120b p fragment, SV40 DNA (Buchman+ A, R, et al. DNA Tumor Viruses, 2ded (J, Tooze+ed,), Co1d Spring Harbor Laboratory+ NewY ork (1981), pp, 799-841) obtained by the old ndl[I decomposition, Then, it was cloned into pBR322 and amplified. This cloning vector ndln, and the 1100bp SV40 DNA fragment was electromagnetized by gel electrolysis. pMT-h isolated by electrophoresis (digested with Htndll and treated with CIP) GHg (named (10)) is inserted in the opposite direction before the MT-n promoter. Contains fragments. In phGHg-SV (9), this enhancer is 5' Approximately 1600 bp from the mRNA start site; in the reverse direction, 5'...RNA start It is approximately 980 bp from the site. It can work in both directions.

Enhancer sequences oriented closer to the start site give higher expression levels. Ru. Deletions that position the enhancer 250 to 4 oobp upstream of the transcription start site are preferred. considered suitable.

Machimata j person kyodon no, phGHg-5V (10) prepared as above was treated with BamHI and SmaI. The hGH coding sequence was removed. (BaIII) I is a polylinker Smai is cleaved within the 3' untranslated region of hGH and vector Approximately 600 bp of the fragment remains. ) and the above-mentioned smooth containing estrogen receptors It was ligated with the terminated EcoRI 2.1 kb OR8 fragment. The obtained vector (ph ER-SV (named 10)) was cloned into E. coli and then Cl0-Kl cells were transformed and transformants were successfully isolated.

Actual + main ■Replacement” Needless■ Successfully obtained transformed colonies were transferred to IIMEM/Ham’s supplemented with zinc. F-12 medium (Ham, R, G, Proc Natl Aead Sci (USA) (1965) 53: 288) and harvested it. and released with EDTA and 10mM) Lys (p'H7,4) and 20mM Homogenize using a Polytron disruptor in a buffer containing M sodium molybutate. Naiz. The homogenate was centrifuged at 250,000X g for 30 minutes, and the The supernatant fraction was then treated with 0.5 nM estradiol (57 Ci/mmol) (200 a two-fold excess of non-radioactive control estradiol or diethylstilvertyrol. (with or without) for 60 minutes at 4°C.

In another experiment, one volume (200 a l) of the labeled extract was used for null ER antibody (10 μg D75P3. in final volume 22071.e.

at 4°C in the presence or absence of D547Sp, or H222SP, Incubate for 60 minutes.

・For sedimentation analysis, 200μ2 of the labeled extract or reaction solution was added to 10mM) Lis, 10mM sodium molybutate, 1.5mM EDTA.

pH 7,4 and 10mM KCI (low salt) or 400mM KCI (high salt) layer 1 on a 10-30% sucrose linear gradient (3,5d) prepared with either , SoLT O'C near 15 hours, 253,0OOX g T: Centrifuged .

Collect 100 Il of both sides, and use Triton χ-100) The radioactivity was measured in the solution mixture with a measurement efficiency of 35%. IC-labeled ovoa Parallel gradients of rubumin (3,6S) and c-labeled 1gG (7,05) It was used as a sedimentation marker.

Figure 3 shows the results of sedimentation analysis. Panel A shows 0.5 nM estradiol Extracts from hER-transformed cells labeled with (triangles) or non-transformed cells Figure 2 shows the profile of the extract (circled) in a low salt gradient. These results are , 8-9s Panel B was obtained with a high salt gradient. Indicates the corresponding profile. here Estradiol-labeled receptor protein from transformed cells as shown in Paku (triangle) now has a sedimentation constant of 4s. As a result, the production of ER It is demonstrated that this can be considered a diagnostic property of steroid receptors. This ER life Production of salt-sensitive hormone receptors from 8 to IOS in hypotonic extracts of responsive cells. Shown as formation of coalescence.

Furthermore, this is demonstrated by FIG. This homogenate was mixed with 0.5 nM Labeled estradiol/1100n is labeled with unlabeled estradiol ( (black circle) and the estrogen receptor complex disappeared. Figure 3 is also. the compound When the body was incubated with anti-receptor antibody 075P (white circle), the precipitation was 8. 8 complex. Furthermore, the concentration of the estrogen receptor complex is Adding IOT'H zinc ions to the culture medium 24 hours before collecting cells more than doubles the amount of , consistent with zinc induction of the metallothionein promoter (data (not shown). The aforementioned sedimentation characteristics are fully consistent with the expression of hER by transformed cells. do.

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Claims (12)

[Claims] 1. An expression system for vertebrate steroid receptor proteins, which encodes SR. and a DNA sequence operably linked to control sequences compatible with eukaryotic host cells. Including, expression system. 2. The expression system according to claim 1, wherein the host cell is a mammalian cell. 3. The expression system according to claim 2, wherein the host cell is a CHO cell. 4. Claim 1, wherein the SR is an estrogen receptor (ER). Expression system. 5. In claim 4, the ER is the human estrogen receptor (hER). Expression system described. 6. A recombinant host eukaryotic cell comprising an expression system according to claim 1. 7. 7. The host cell according to claim 6, which is a mammalian cell. 8. A method for producing a recombinant SR of a vertebrate, the method as set forth in claim 6. culturing the cells and collecting the SR protein; Method. 9. by disrupting cells, removing cell debris, and collecting clear lysate. 9. The method according to claim 8, wherein the SR is recovered. 10. A recombinant vertebrate strain produced by the method set forth in claim 8. terrorism receptor. 11. SR according to claim 10, which is an estrogen receptor. 12. ER according to claim 11, which is a human ER.