WO2000071743A1 - Methode bihybride dans une cellule de mammifere - Google Patents

Methode bihybride dans une cellule de mammifere Download PDF

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Publication number
WO2000071743A1
WO2000071743A1 PCT/JP2000/003353 JP0003353W WO0071743A1 WO 2000071743 A1 WO2000071743 A1 WO 2000071743A1 JP 0003353 W JP0003353 W JP 0003353W WO 0071743 A1 WO0071743 A1 WO 0071743A1
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protein
dna
transcription activation
region
gene
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PCT/JP2000/003353
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English (en)
Japanese (ja)
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Kappei Tsukahara
Takayuki Hida
Katsuji Nakamura
Hideki Yoshitomi
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Eisai Co., Ltd.
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Publication of WO2000071743A1 publication Critical patent/WO2000071743A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present invention relates to the use of a first protein fused to a transcriptional activation region and a second protein fused to a DNA binding region in an interaction between a first protein and a second protein in a mammalian cell. To a method for efficient detection by the two-hybrid method.
  • Conventional technology Conventional technology
  • the two-hybrid method is a method for detecting the interaction between two proteins using their respective fusion proteins (two hybrids).
  • the first protein and the transcriptional activation domain (domain) are combined in a cell having a base sequence that binds to the DNA binding region, a promoter sequence, and a DNA downstream of the reporter gene.
  • a fusion protein of the second protein and the DNA binding region is expressed.
  • the interaction with the protein (1) draws the transcriptional activation region near the promoter region.
  • the one-hybrid method uses the interaction between the first protein (X) and the second protein (Y) that occurs in cells to increase the transcription efficiency of the repo overnight gene, for example, the repo overnight gene product. (Warbrick, E (1997) Structure 15: 13-17).
  • the two-hybrid method is usually performed using yeast as a host cell, and has so far produced enormous results as a highly sensitive method for detecting proteins that interact with each other (Fields, S. and Song). , 0.K. (1989) Nature 340: 245-246, Dalton, S. and Trisman, R. (1992) Cel l 68: 597-612, Bartel, PL et al. (1993) In Cellular Interactions in Development: A Practical Approach).
  • yeast cells is the same as that in mammalian cells.
  • An object of the present invention is to establish a two-hybrid method that can detect even weaker protein interactions that cannot be detected by conventional methods and that can be applied to more types of protein interactions.
  • the purpose is to develop a drug screening method using the two-hybrid method.
  • the first protein to be fused with the transcription activation region is referred to as protein X
  • the second protein to be fused to the DNA binding region is referred to as protein Y.
  • the present inventors have proposed that the interaction between protein X and protein Y We thought that if the transcription activation region attracted to the vicinity of the sequence strongly binds to the transcription initiation complex, the transcription initiation complex could be formed efficiently.
  • the binding between the transcription activation region and the transcription initiation factor may be strengthened and the efficiency of the transcription initiation complex formation may be increased.
  • the two transcription activation regions were fused, it was found that an interaction between proteins that could not be detected only by fusing one transcription activation region could be detected. The present invention has been completed based on this finding.
  • the present invention provides a method for detecting an interaction between protein X and protein Y in a mammalian cell
  • a fusion protein comprising two or more identical or different transcription activation regions and protein X in a mammalian cell having a DNA to which a repo-all-one gene is bound downstream of a nucleotide sequence binding to a DNA-binding region; and A method comprising expressing a fusion protein of the DNA binding region and protein Y and detecting the expression of a reporter gene (hereinafter, also referred to as the “detection method of the present invention”).
  • a feature of the detection method of the present invention resides in that protein X is fused with two or more transcription activation regions.
  • protein X and protein Y can be freely selected according to the purpose.
  • a peptide containing the SH3 region (src-SH3) in the src gene involved in cell proliferation signaling and a proline-rich motif can be used as appropriate, and can be used for elucidation of intracellular signal transduction mechanism and drug screening, but protein X and protein Y are limited to this example. It is not something to be done.
  • the transcription activation region refers to a region capable of promoting transcription activity of a transcription factor protein.
  • Examples include the transcription activation region of the transcription activator VP-16 of the herpes simplex virus (hereinafter abbreviated as VP16A D) and the transcription activation region of the tumor suppressor gene p53 (hereinafter abbreviated as p53AD).
  • the present invention is not limited to this.
  • the number of transcription activation regions may be two or more, and any combination of the same type and different types is allowed.
  • a combination of two or three transcription activation regions is used, and more preferably, a combination of two is used.
  • one of the transcription activation regions is VP-16AD or p53AD.
  • VP16AD and p5 The combination of 3AD is more preferred.
  • the DNA binding region refers to a region of a transcription factor protein that can recognize and bind to a specific base sequence on DNA existing upstream of the promoter sequence.
  • GAL4 DNA binding domain (hereinafter abbreviated as GAL4DBD) involved in yeast galactose metabolism
  • SRF DNA binding domain involved in mammalian growth factor response
  • LexA DNA binding involved in bacterial DNA damage response.
  • a reporter gene refers to a gene whose expression can be measured by any means.
  • HIS3 gene induction of expression in histidine synthase deficient cells allows growth in histidine-free medium
  • CAT gene ⁇ -galactosidase gene
  • the present invention is not limited to this.
  • the cells when expressed in mammalian cells and X-gal is used as the substrate, the cells are stained blue and detectable.
  • the galactosidase gene is expressed in mammalian cells and secreted extracellularly and is detectable from the culture supernatant. A secreted allelic phosphatase is desirable.
  • the expression of the repo overnight gene is detected by detecting the promotion of repo overnight gene expression due to the interaction between protein X and protein Y.
  • the drug is added after the transcription initiation complex has been formed to inhibit the interaction between protein X and protein Y.
  • drugs are added before the formation of the transcription initiation complex to inhibit the interaction between protein X and protein Y, resulting in transcriptional activity. It has been reported that it is easier to inhibit the formation of a transcription initiation complex without involving the activation region (Chaudhuri B., et. al. (1995) FEBS letters 357: 221-226).
  • the detection method of the present invention when used for screening a drug that inhibits the interaction between protein X and protein Y, preferably, two or more transcriptional activities are added so that transcriptional activation occurs by adding a ligand.
  • the fusion protein between the ligated region and protein X, or the fusion protein between the DNA binding region and protein Y, is preferably selected so that the three-dimensional structure is changed by the binding of the ligand to change the transcription activation activity or DNA binding activity.
  • a fusion protein in which a ligand binding site is fused so that it has a transcriptional activation activity or a DNA binding activity only after its three-dimensional structure is changed by binding to a ligand.
  • the fusion protein in which the ligand binding site is fused is a fusion protein with a DNA binding region.
  • the ligand refers to a low-molecular compound that binds to the corresponding ligand receptor and causes a structural change of the receptor.
  • the ligand binding site is a site that binds to a ligand in a ligand receptor.
  • estrogen is used as the ligand
  • the estrogen binding site of the estrogen receptor is used as the ligand binding site, but the present invention is not limited to these.
  • the DNA binding activity of the fusion protein is promoted or nuclear translocation is promoted only when the estrogen binding site binds to estrogen. Then, the DNA binding site becomes able to bind to MA (Fig. 2).
  • transcriptional activation does not occur before the addition of the ligand due to the absence of the transcription initiation complex, and when the ligand is added in the presence of the drug, the protein X
  • transcriptional activation occurs, and the result of transcription of the reporter gene can be obtained.
  • the interaction between protein X and protein Y is inhibited by the drug, Can eliminate the involvement of the transcriptional activation region, inhibit transcriptional activation, and obtain the result of suppression of reporter gene transcription.
  • the present invention also provides a method for screening a drug using the detection method of the present invention, that is, a method for screening a first protein and a second protein using a first protein and a second protein having a property of interacting with each other.
  • a method of screening for drugs that affect an interaction A mammalian cell having a DNA to which a reporter gene is bound downstream of a base sequence that binds to a DNA binding region, a fusion protein comprising two or more identical or different transcription activation regions and a first protein, and the DNA
  • a method comprising expressing a fusion protein of a binding region and a second protein, culturing the mammalian cell in the presence of the drug, and screening for the drug based on the change in reporter gene expression (hereinafter, “ The present invention is also referred to as the "screening method of the present invention.”
  • protein X and protein Y include a combination of src-SH3 and a peptide containing a proline'ritichi motif, a peptide having the PDZ sequence in hDlg, a human homolog of the Drosophila tumor suppressor gene Dig (hereinafter abbreviated as hDlg-PDZ). And a peptide containing a C-terminal amino acid sequence of a shaker-type K channel Kv1.4 and the like, but the present invention is not limited thereto.
  • the present invention provides a compound found by the screening method of the present invention. Furthermore, the present invention relates to a fusion protein of two or more transcription activation regions, the same or different, used in the detection method of the present invention and protein X, a DNA encoding the fusion protein, a vector containing the DNA, and a vector containing the DNA. Also provided is a cell transformed by I. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a schematic diagram for explaining the one-hybrid method.
  • FIG. 2 is a schematic diagram for explaining a two-hybrid method in which two transcription activation regions are used and a ligand binding site is fused to a DNA binding region.
  • FIG. 3 shows the results of detecting the interaction of src-SH3 with a peptide containing a proline 'rich' motif by two or more transcription activation regions.
  • GAL4DBD GAL4 DNA binding region
  • SH3 src-SH3
  • VP16AD VP16 transcription activation region
  • Pro5 Proline 'rich motif
  • p53AD p53 transcription activation region
  • Pro6 mutant proline' rich 'motif.
  • FIG. 4 shows the results of induction of transcriptional activity using the ligand binding region of estrogen receptor.
  • GAL4DBD GAL4 DNA binding region
  • SH3 src-SH3
  • VP16 AD VP16 transcription activation region
  • Pro5 Proline-Ritichi motif
  • p53AD p53 transcription activation region
  • ERLBD Estrogen receptor binding site.
  • Figure 5 shows hDlg-PDZ and Kvl.4 C-terminal amino acid distribution with two or more transcription activation regions (Rule 26) The results of the detection of column interactions are shown.
  • GAL4DBD GAL4 DNA binding region
  • PDZ1 hDlg-PDZl
  • VP16 AD VP16 transcription activation region
  • PDZ2 hDlg-PDZ2
  • p53AD p53 transcription activation region
  • Kvl.4 Shaker type K channel.
  • FIG. 6 shows the results of detection of the interaction between src-SH3 and a peptide containing a proline 'rich' motif by three transcription activation regions.
  • GAL4DBD GAL4 DNA binding region
  • SH3 src-SH3
  • VP16AD VP16 transcription activation region
  • Prol5 Mutant proline 'rich' motif
  • p53AD p53 transcription activation region.
  • two or more fusion proteins of the transcription activation region and protein X are used.
  • Replacement forms (Rule 26 Other than using the fusion protein of the transcription activation region of No. 7 and protein X, the method may be the same as the conventional two-hybrid method.
  • the screening method of the present invention may be the same as a drug screening method using a conventional two-hybrid method except that the detection method of the present invention is used as a two-hybrid method.
  • a fusion protein of two or more transcription activation regions and protein X used in the detection method of the present invention, a DNA encoding the fusion protein, a vector containing the DNA, and two or more cells transformed by the vector can be obtained in the same manner as in the related art, except that the transcription activation region is fused to protein X.
  • a complex with the fusion protein of MA binding region and protein Y is formed between protein X and the transcription activation region and between the transcription activation regions.
  • a peptide that functions as a linker may be included as long as it does not interfere with the effect of transcriptional activation when performed.
  • the ligand binding site is preferably present between protein X or protein Y and the transcription activation region or DNA binding region, but may be located at other positions as long as the transcription activation activity is changed by binding to the ligand. There may be.
  • VP16AD and p53AD as transcriptional activation regions
  • GAL4DBD DNA binding regions
  • src-SH3 protein X and protein Y and peptides containing a proline-rich 'motif
  • estrogen receptor as ligand binding site.
  • an estrogen binding site will be described.
  • a peptide obtained by adding Arg-Tyr to the end of the sequence reported by Rickles et al. (Hereinafter referred to as Pro5) is used as a peptide containing a proline-rich motif. 8
  • DNA encoding the transcription activation region fusion protein encodes the transcription activation region! NA, eg, immediately after the DNA encoding VP16AD, DNA encoding another transcription activation region, eg, p53AD are ligated so that the codon reading frames are the same. If there is an appropriate restriction enzyme site, ligate using the restriction enzyme site. If not, add a restriction enzyme site to the PCR primers and incorporate the restriction enzyme site by PCR and ligate.
  • DNAs encoding more transcription activation regions may be ligated.
  • one of the DNA encoding the protein X whose interaction is to be detected for example, the DNA encoding Pro5 and the DNA encoding src_SH3 is ligated.
  • DNA encoding DNA-binding domain fusion protein DNA encoding DNA-binding domain, for example, DNA encoding GAL4DBD, and DNA encoding protein Y whose interaction is to be detected immediately after encoding, eg, Pro5 And the other of the DNA encoding src-SH3 are ligated so that the reading frame of the codon is the same. If there is an appropriate restriction enzyme site, use the restriction enzyme site to ligate. If not, add the restriction enzyme site to the PCR primers and incorporate the restriction enzyme site by PCR for ligation.
  • the present invention is not limited to this.
  • a DNA encoding a DNA binding region for example, a DNA encoding a GAL4DBD, followed immediately by a DNA encoding a ligand binding site, for example, an estrogen binding site (ER-LBD) of an estrogen receptor, has a codon reading frame. Connect so that they are the same. If there is an appropriate restriction enzyme site, use the restriction enzyme site to ligate. If not, add the restriction enzyme site to the PCR primer and insert the restriction enzyme site by PCR and ligate. Similarly, the interaction is detected immediately after the DNA encoding the linked DNA binding region. 9 Connect the DNA encoding the desired protein Y.
  • ER-LBD estrogen binding site
  • Reporter DNA A reporter gene, for example, upstream of a PLAP gene encoding a secreted alkaline phosphatase gene (Goto, M. et. Al., Mol. Pharmacol. 49: 860-873, 1996).
  • a promoter sequence for example, the minimal promoter sequence of the simple virus thymidine kinase gene is ligated.
  • an upstream activating sequence is ligated.
  • the DNA of the above 1) to 4) is incorporated into a vector (plasmid) suitable for expression in mammalian cells, and a vector is constructed.
  • DNA transfection Culture lx lO 4 to 2 x 10 5 mammalian cells, such as COS-1, HEK293, etc. on a 24-well plate. The following day, a vector containing the DNA encoding the DNA-binding domain fusion protein, a vector containing the DNA encoding the transcriptional activation domain fusion protein, and a vector containing the reporter DNA are preferably used in appropriate amounts. Transfection with 400 ng.
  • Transflector Ekushi Yon method DEAE dextran method, calcium phosphate method, Ripofuekuchin method
  • the efficiency is frequently you wear if allowed any method of DNA in the introduction, preferably Ripofuekuchin method, more preferably FuGENE TM 6 Transfection reagent (base one ringer Mannheim).
  • Transfection of a vector containing a DNA encoding a DNA binding domain fusion protein and a vector containing a DNA encoding a transcriptional activation domain fusion protein in a mammalian cell having the repo overnight DNA May be.
  • Repo overnight event The repo overnight event is performed by a method suitable for each repo overnight gene.
  • the reporter gene is the secreted Alfa rifos phosphatase (PLAP) gene
  • PLAP Alfa rifos phosphatase
  • the cells are cultured for an appropriate time, preferably 6 to 24 hours, and the culture supernatant is collected.
  • the culture supernatant was heat-treated at 65 ° C for 10 to 20 minutes to inactivate the alkaline phosphatase contained in the serum, and then buffered (0.28 M carbonate buffer, Ten
  • Ligand induction is performed in a manner appropriate for the respective ligand binding site.
  • the ligand binding site is an estrogen binding site of an estrogen receptor will be described, but the present invention is not limited to this.
  • plasmid containing MA encoding the DNA binding region and plasmid containing DNA encoding the transcription activation region And a plasmid containing a promoter region containing the base sequence that binds to the DNA binding region (the base sequence is shown in SEQ ID NO: 11) and a reporter gene downstream of the promoter region (the secreted alkaline phosphatase (PLAP) gene).
  • PLAP secreted alkaline phosphatase
  • the plasmid introduced into COS-1 cells was constructed as follows.
  • GAL4DBD-SH3 GAL4DBD-encoding DNA was cut out from the vector pM contained in the Mammalian MATCHMAKER two-hybrid assay kit (manufactured by CL0NTECH).
  • the MA coding for SH3 is a human 5, -STRETCH PLUS cDNA library (lung) (manufactured by CL0NTECH), and the base sequence of AAAGAATTCCTGGCCGGTGGAGTGA (SEQ ID NO: 22) and TTTGGATCCCGGAG GGCGCCAC (SEQ ID NO: 23) was amplified by PCR using an oligodeoxyribonucleotide (oligo DNA) having the above as a primer. Using these restriction sites in the vector and the restriction sites designed in the primers, pcDNA3.1
  • VP16AD-P53AD-Pro5 DNA encoding VP16AD was excised from the vector pVP16 included in the Mammalian MATCHMAKER two-hybrid assay kit (manufactured by CLONTECH).
  • the DNA encoding p53AD was a p53 gene clone obtained from the Japan DNA Data Bank as type II, and an oligo DNA having the nucleotide sequence of AAACMTTGACCATGGAGGAGC (SEQ ID NO: 24) and AAAGMTTCGTCTTCAGTGA ACCATTGTTCAA (SEQ ID NO: 25) was used as a primer.
  • the DNA encoding Pro5 was synthesized by synthesizing an oligo DNA encoding an amino acid sequence containing the amino acid sequence shown in SEQ ID NO: 26 and an oligo DNA having a complementary sequence thereof, with restriction enzyme sites added to both ends, and annealing them. (Sometimes, a primer containing up to the Pro5 sequence was synthesized and the DNA encoding p53AD and Pro5 was amplified as a stretch of DNA.) These DNAs were incorporated into pcDNA3.1 (manufactured by INV ITR0) using restriction enzyme sites in the vector and restriction enzyme sites designed in the primer and in the synthetic oligo DNA.
  • Plasmids other than those described above were constructed by a method similar to the above.
  • Alkaline phosphatase activity was determined by recovering 15% of the culture supernatant, inactivating endogenous alkaline phosphatase by incubating at 65 ° C for 10 minutes, and then adding 60 ⁇ 1 of a carbonate buffer (0.28 M Na 2 C0 3 pH 10.0, 8 mM MgSCh) and 75 ⁇ 1 of Rumisutin (manufactured by Sumitomo Metal Industries) was added and after incubated for 30 minutes at 37 ° C for at the dark, and left for a further 30 minutes at room temperature, the chemiluminescence was measured in a microplate luminometer overnight.
  • a carbonate buffer (0.28 M Na 2 C0 3 pH 10.0, 8 mM MgSCh
  • Rumisutin manufactured by Sumitomo Metal Industries
  • DNA encoding the fusion protein of GA L4DBD and src-SH3 base sequence and amino acid sequence Nos. 1 and 2
  • a DNA encoding the fusion protein of one transcription activation region VP16AD and Pro5 base sequence And the encoded amino acid sequence shown in SEQ ID NOs: 3 and 4
  • no interaction between src-SH3 and Pro5 could be detected (3).
  • a DNA encoding the fusion protein of two transcription activation regions (VP16AD-p53AD) and Pro5 (base sequence and encoded amino acid sequence) SEQ ID NOS: 5 and 6 12
  • Example 2 Regulation of transcription activity using ligand binding region of estrogen receptor
  • DNA binding region and the ligand binding region of estrogen receptor Plasmid containing DNA base sequence and the encoded amino acid sequence are shown in SEQ ID NOS: 7 and 8) encoding DNA (base sequence and encoded amino acid) The sequence is shown in SEQ ID NOS: 9 and 10), and a plasmid containing a promoter and a plasmid containing the repo overnight gene (PLAP gene) in the region and downstream thereof in the combination shown in Fig. 4. Transfection.
  • the plasmid introduced into HEK293 cells was constructed as follows.
  • GAL4DBD-ERLBD-SH3 DNA encoding GAL4DBD and SH3 was prepared according to Example 1.
  • the DNA encoding ERLBD is a human 5'-STRETCH PLUS cDNA library (manufactured by CLON TECH), type III, and an oligo DNA having a base sequence of AAACAATTGTCTGCTGGAGACATGAGAGC (SEQ ID NO: 27) and A AAGAATTCGACTGTGGCAGGGAAACC (SEQ ID NO: 28). Amplified by PCR as primer. These DNAs are combined with restriction enzyme sites and vectors in the vector. 13 Using the restriction enzyme site designed in the primer, it was incorporated into pcDNA3.1 (manufactured by INVITR0).
  • Plasmids other than those described above were constructed by a method similar to the above.
  • transcriptional activation does not occur only by transfection of DNA, but transcriptional activation occurs only when 5-estradiol is added. Therefore, if a test agent is added simultaneously with /?-Estradiol, It has become possible to verify whether or not the test agent inhibits transcriptional activation, that is, whether or not the test agent suppresses the interaction between the fusion proteins.
  • Example 3 Detection of interaction between hDlg-PDZ and Kv1.4 peptide having a C-terminal amino acid sequence by two or more transcription activation regions
  • the plasmid containing the DNA binding region, the plasmid containing the transcriptional activation region, the promoter region and the downstream Three types of plasmids containing the Yuichi gene PLAP (secreted lipophilic phosphatase gene) were transfected using the combinations shown in Fig. 5. Twenty-four hours later, 501 culture supernatants were collected, and the amount of secreted alkaline phosphatase contained therein was measured.
  • the plasmid introduced into COS-1 cells was constructed as follows. 14
  • GAL4DBD-PDZ2 DNA encoding GAL4DBD was prepared according to Example 1. The DNA encoding PDZ2 was prepared using QUICK-Screen Human cDNA library panel cat. 1ung (Agtll) of K1003-1 (manufactured by CLONTECH) as type III, and AAAGAATTCAGMGGAAACCAGTGTCAGAAA.
  • SEQ ID NO: 29 an oligo DNA having a base sequence of AAAGGATCCTCAAGGTTCCCTTGTAATTTCAT (SEQ ID NO: 30) were used as primers for amplification by PCR. These DNAs were incorporated into pcDNA3.1 (manufactured by INVITR0) using the restriction enzyme sites in the vector and the restriction enzyme sites designed in the primers.
  • GAL4DBD-PDZ1-PDZ2 MA encoding GAL4DBD was adjusted according to Example 1.
  • the DNA encoding PDZ2 PDZ2 was obtained from QUICK-Screen Human cDNA Library Panel cat. Number 3
  • the oligo DNA having the nucleotide sequence of 2) was used as a primer and amplified by PCR. These DNAs were incorporated into pcDNA3.1 (manufactured by INVITR0) using restriction enzyme sites in the vector and restriction enzyme sites designed in the Braima and the synthetic DNA.
  • DNA encoding VP16AD and p53AD was prepared according to Example 1.
  • DNA encoding Kvl 4 oligo DNA (base sequence: AMGMTTCGA TAAAAACAACTGTTCTAATGCAAAGGCTGTGGAGACTGATGTGTGAGGATCCAAA (SEQ ID NO: 33)) and an oligo DNA having a complementary sequence thereof were synthesized and anneal.
  • DNAs were incorporated into pcDNA3.1 (manufactured by INVITR0) using restriction enzyme sites in the vector and restriction enzyme sites designed in the primers and in the synthetic oligo DNA.
  • Plasmids other than those described above were constructed by a method similar to the above.
  • the DNA encoding the fusion protein of GAL4DBD and the second PDZ counted from the ⁇ end of hDlg are shown in SEQ ID NOs: 12 and 13
  • DNA coding for the fusion protein in the region containing the first and second PDZs counted from the N-terminus of GAL4DBD and hDlg base sequence and encoded amino acid.
  • Acid sequences shown in SEQ ID NOs: 14 and 15) and a plasmid containing DNA encoding the transcription activation region.
  • a DNA encoding a fusion protein of two transcription activation regions (VP16AD-p53AD) and the C-terminal 15 amino acids of Kvl.4 (the nucleotide sequence and the encoded amino acid sequence are shown in SEQ ID NOs: 18 and 19) ) Showed strong transcriptional activation when used (3, 5).
  • transcription activation region VP16AD and DNA binding region GAL4DBD (1) did not occur.
  • DNAs encoding GAL4DBD, VP16AD, p53AD, SH3, and Prol5 were prepared according to Example 1, and these DNAs were used as restriction enzyme sites in the vector and restriction enzyme sites designed in the primers and in the synthetic oligo DNA. It was incorporated into pcDNA3.1 (manufactured by INVITRO) using.
  • protein-protein interactions that could not be detected by the conventional two-hybrid method in mammalian cells can be detected.

Abstract

L'invention concerne une méthode de détection d'une interaction entre une première protéine et une seconde protéine dans une cellule de mammifère, laquelle consiste, dans une cellule de mammifère présentant un ADN portant un gène rapporteur lié à celui-ci en aval d'une séquence de base se fixant à une région de liaison d'ADN, à exprimer une protéine fusionnée de la première protéine avec deux régions d'activation de transcription ou davantage lesquelles sont identiques ou différentes, ou une autre protéine fusionnée de la seconde protéine avec la région de liaison d'ADN ci-dessus décrite, et ensuite à détecter l'expression du gène rapporteur.
PCT/JP2000/003353 1999-05-25 2000-05-25 Methode bihybride dans une cellule de mammifere WO2000071743A1 (fr)

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
JP2005531303A (ja) * 2002-05-30 2005-10-20 センター ナショナル ド ラ ルシェルシュ サイエンティフィーク（シーエヌアールエス） トランスジェニックツメガエル胚および環境中の内分泌攪乱物質の検出剤としてのその使用
US7531326B2 (en) * 2001-02-20 2009-05-12 Intrexon Corporation Chimeric retinoid X receptors and their use in a novel ecdysone receptor-based inducible gene expression system
US7563879B2 (en) 2001-09-26 2009-07-21 Intrexon Corporation Leafhopper ecdysone receptor nucleic acids, polypeptides, and uses thereof
US7807417B2 (en) 2000-03-22 2010-10-05 Intrexon Corporation Ecdysone receptor-based inducible gene expression system
US8105825B2 (en) 2000-10-03 2012-01-31 Intrexon Corporation Multiple inducible gene regulation system
US8691527B2 (en) 2001-02-20 2014-04-08 Intrexon Corporation Substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system
US9163256B2 (en) 2004-04-30 2015-10-20 Intrexon Corporation Mutant receptors and their use in a nuclear receptor-based inducible gene expression system
US9249207B2 (en) 2001-02-20 2016-02-02 Intrexon Corporation Substitution mutant receptors and their use in an ecdysone receptor-based inducible gene expression system
US9493540B2 (en) 2001-02-20 2016-11-15 Intrexon Corporation Ecdysone receptor/invertebrate retinoid X receptor-based inducible gene expression system

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010510A2 (fr) * 1997-08-26 1999-03-04 Ariad Gene Therapeutics, Inc. Proteines de fusion a domaine de dimerisation, de trimerisation ou de tetramerisation, et a domaine additionnel d'activation de transcription heterologue, d'inhibition de transcription, de liaison d'adn ou de liaison de ligand

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010510A2 (fr) * 1997-08-26 1999-03-04 Ariad Gene Therapeutics, Inc. Proteines de fusion a domaine de dimerisation, de trimerisation ou de tetramerisation, et a domaine additionnel d'activation de transcription heterologue, d'inhibition de transcription, de liaison d'adn ou de liaison de ligand

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHIEN C.T. ET AL.: "The two-hybrid system: a method of identify and clone genes for proteins that interact with a protein of interest", PROC. NATL. ACAD. SCI. USA, vol. 88, no. 21, 1991, pages 9578 - 9582, XP002930194 *
FEARON E.R. ET AL.: "Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interaction in mammalian cells", PROC. NATL. ACAD. SCI. USA, vol. 89, no. 17, 1992, pages 7958 - 7962, XP002930193 *
LUO Y. ET AL.: "Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system", BIOTECHNIQUES, vol. 22, no. 2, 1997, pages 350 - 352, XP002930192 *
NATESAN S. ET AL.: "A general strategy to enhance the potency of chimeric transcriptional activators", PROC. NATL. ACAD. SCI. USA, vol. 96, no. 24, November 1999 (1999-11-01), pages 13898 - 13903, XP002930191 *

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US8105825B2 (en) 2000-10-03 2012-01-31 Intrexon Corporation Multiple inducible gene regulation system
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US7531326B2 (en) * 2001-02-20 2009-05-12 Intrexon Corporation Chimeric retinoid X receptors and their use in a novel ecdysone receptor-based inducible gene expression system
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US9029152B2 (en) 2001-02-20 2015-05-12 Rheogene, Inc. Substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system
US8680249B2 (en) 2001-09-26 2014-03-25 Intrexon Corporation Leafthopper ecdysone receptor nucleic acids, polypeptides, and uses thereof
US8497093B2 (en) 2001-09-26 2013-07-30 Intrexon Corporation Leafhopper ecdysone receptor nucleic acids, polypeptides, and uses thereof
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