WO2000071743A1 - Two-hybrid method in mammalian cell - Google Patents

Abstract

A method for detecting an interaction between a first protein and a second protein in a mammalian cell, which comprises, in a mammalian cell having a DNA carrying a reporter gene ligated thereto in the downstream of a base sequence binding to a DNA-binding region, expressing a fused protein of the first protein with two or more transcriptional activation regions which are the same or different, and another fused protein of the second protein with the above-described DNA-binding region, and then detecting the expression of the reporter gene.

Description

 Specification

 Two-hybrid method in mammalian cells

 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

 The two-hybrid method is a method for detecting the interaction between two proteins using their respective fusion proteins (two hybrids). In the two-hydride method, 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. And a fusion protein of the second protein and the DNA binding region is expressed. Here, if the first protein and the second protein have the property of interacting with each other, the binding between the DNA binding region and the base sequence binding to the DNA binding region, and the binding between the second protein and the second protein, The interaction with the protein (1) draws the transcriptional activation region near the promoter region. As a result, the transcription efficiency of the repo overnight gene linked downstream increases, and the expression of the repo overnight gene product increases (Fig. 1). In other words, 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).

Conventionally, 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). However, there is no guarantee that the environment in yeast cells is the same as that in mammalian cells. In order to detect the interaction between white matter, it is appropriate to use the one-hybrid method using mammalian cells as a host.

 Attempts have also been made to screen for drugs that suppress the interaction between proteins using the two-hybrid method using yeast as host cells.However, drug permeability of the yeast cell wall is low, and For screening purposes, the two-hybrid method using mammalian cells that do not have a cell wall as a barrier to drug permeation was considered to be suitable.

 To date, the two-hybrid method using mammalian cells as a host has

 1. Relatively strong interaction between proteins (Fearon, ER, et al. (1992) Pro Nat 1. Acad. Sci. USA 89: 7958-7962, Rickles, JR, et al. (1998) Chem. Biol. 5: 529-538),

 2. Interactions between transcription factors (Fagan, R., et al. (1994) Cell 78: 799-811, Fagan, R., et al. (1994) Cell 78: 799-811, Dang, CV, et al. (1991) Mol. Ce 11 Biol. 11: 954-962, Kim, HJ, et al. (1998) J. Biol. Chem. 273: 28564-28567, Leahy, P., et al. (1999 ) J. Biol. Chem. 274: 8813-8822), has been shown to be detectable. However, we often experience cases where the interaction cannot be detected due to weak interaction or low sensitivity.When using mammalian cells as a host, the conventional two-hybrid method is not a versatile method. Not yet. Disclosure of the invention

 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.

 In the following, the first protein to be fused with the transcription activation region is referred to as protein X, and the second protein to be fused to the DNA binding region is referred to as protein Y.

In order to increase the sensitivity of detecting the interaction between protein X and protein Y by the two-hybrid method, 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.

 Then, by fusing multiple transcription activation regions to protein X, 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. When 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.

 That is, 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.

 Here, protein X and protein Y can be freely selected according to the purpose. For example, a peptide containing the SH3 region (src-SH3) in the src gene involved in cell proliferation signaling and a proline-rich motif (Rickles RJ, et. Al. (1995) Pro Natl. Acad. Sci. USA 92: 10909-10913) 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. Preferably, a combination of two or three transcription activation regions is used, and more preferably, a combination of two is used. Preferably, 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. For example, the GAL4 DNA binding domain (hereinafter abbreviated as GAL4DBD) involved in yeast galactose metabolism, the SRF DNA binding domain involved in mammalian growth factor response, and the LexA DNA binding involved in bacterial DNA damage response. Although the present invention is not limited thereto, the present invention is not limited thereto.

 A reporter gene refers to a gene whose expression can be measured by any means. For example, HIS3 gene (induction of expression in histidine synthase deficient cells allows growth in histidine-free medium), CAT gene, 一 -galactosidase gene, secreted alkaline phosphatase

The present invention is not limited to this. Preferably, 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.

 A fusion protein of two or more identical or different transcriptional activation regions and protein X in a mammalian cell having MA with a repo all-in-one gene downstream of a nucleotide sequence that binds to a DNA binding region; and Methods for expressing the fusion protein of the DNA binding region and protein Y include: (1) expressing, in a mammalian cell, a fusion protein of two or more transcription activation regions, the same or different, and protein X; (2) expressing a fusion protein of the DNA binding region and the protein Y; (3) downstream of the base sequence binding to the DNA binding region;

—Yuichi One method is to introduce DNA to which the gene is linked.

Usually, 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. When screening for a drug that inhibits 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. Rather than disrupting the transcription initiation complex by eliminating the involvement of the transcription activation region, rather than disrupting the transcription initiation complex, 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).

 Therefore, when the detection method of the present invention is 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. Is preferably 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. More preferably, the fusion protein in which the ligand binding site is fused is a fusion protein with a DNA binding region.

 Here, 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. For example, estrogen is used as the ligand, and the estrogen binding site of the estrogen receptor is used as the ligand binding site, but the present invention is not limited to these. For example, by fusing a DNA binding site with an estrogen binding site of an estrogen receptor, 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). With this configuration, 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 When the interaction between protein X and protein Y is not inhibited, transcriptional activation occurs, and the result of transcription of the reporter gene can be obtained.On the other hand, when 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."

 Examples of 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.

 In addition, 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. BEST MODE FOR CARRYING OUT THE INVENTION

 According to the detection method of the present invention, 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.

 In a fusion protein of two or more transcription activation regions and 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. When a ligand binding site is used, the same applies between the ligand binding site and the protein X or protein Y or the transcription activation region or the DNA binding region, as long as the change in the transcription activation activity by binding to the ligand is not prevented. It is.

 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.

 Hereinafter, an example of the construction of a vector used for expressing the fusion protein and for retaining the DNA containing the reporter gene in mammalian cells, and an example of the steps of the two-hybrid method will be described.

 1. Vector construction

One example is the combination of VP16AD and p53AD as transcriptional activation regions, GAL4DBD as DNA binding regions, src-SH3 as protein X and protein Y and peptides containing a proline-rich 'motif, and estrogen receptor as ligand binding site. The case where an estrogen binding site is used will be described. Further, in the following description, 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

 The operation described below can be performed according to a method described in an appropriate manual, for example, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

 1). 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.

 In some cases, DNAs encoding more transcription activation regions may be ligated.

 Similarly, immediately after the DNA encoding the linked transcription activation region, 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.

 2). 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.

 3). DNA encoding a fusion protein obtained by further fusing a ligand binding site to the fusion protein of 1) or 2): A fusion protein obtained by fusing a ligand binding site to a DNA binding region fusion protein of 2) will be described. 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.

 4). 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.

 Further upstream, a binding base sequence of the DNA binding region, for example, when the DNA binding region is GAL4DBD, an upstream activating sequence (UAS) 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.

2. Two-hybrid method

1). 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.

 2). Repo overnight event: The repo overnight event is performed by a method suitable for each repo overnight gene. Hereinafter, the case where the reporter gene is the secreted Alfa rifos phosphatase (PLAP) gene will be described, but the present invention is not limited to this.

After transfection of the DNA, 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

 Add 8 mM MgSO, pH 10) and Lumistin (Sumitomo Chemical Co., Ltd.), incubate at room temperature or at 37 C for 30 to 60 minutes, and measure the chemiluminescence in the reaction mixture to determine the alkaline phosphatase. Calculate the activity.

 3) Ligand induction: Ligand induction is performed in a manner appropriate for the respective ligand binding site. Hereinafter, a case where 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.

 24 hours after the DNA transfection, remove the culture supernatant, add a new culture medium containing 10 n / g of radioradiol, and incubate for another 24 hours. Then, go to 2) Repo one night at night. Example

Example 1 Detection of interaction between src-SH3 and Pro5 by two or more transcription activation regions

After 2.5 x 10 ⁴ monkey COS-1 cells are cultured on a 24-well plate for 1 day, 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). Was transfected with the combination shown in Fig. 3. Twenty-two hours later, 50 上清 1 of the culture supernatant was collected, and the amount of secreted lipophilic phosphatase contained therein was measured.

 The plasmid introduced into COS-1 cells was constructed as follows.

1). 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

(Made by INVITR0). 11

 2). 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. One was amplified by PCR. 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.

 Transfection was performed using FuGene 6 Transfection Reagent (Boehringer Mannheim) according to the instruction manual for the above reagent.

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.

As shown in Fig. 3, as a plasmid containing DNA encoding the DNA binding region, DNA encoding the fusion protein of GA L4DBD and src-SH3 (base sequence and amino acid sequence Nos. 1 and 2), and as a plasmid containing DNA encoding the transcription activation region, 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). On the other hand, as a plasmid containing DNA encoding the transcription activation region, 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

(Fig. 1) Strong transcriptional activation was observed when using the one containing (4). Also, instead of Pro5, use of Pro6, which has been reported to significantly reduce the interaction with src-SH3 by substituting Ala for the 3rd Leu of Pro5 with Ala and replacing 9th Pro with Ala When activated, transcriptional activation did not occur (5).

 When Pro5 was not fused to the transcriptional activation region VP16AD, which is a negative control (1, 2). And when src-SH3 was not fused to the DNA binding region GAL4DBD (1, 6), GAL4DBD was not added. In case (7), transcriptional activation did not occur.

These results indicate that the interaction between src-SH3 and Pro5, which could not be detected with a single transcription activation region, can be detected by connecting two transcription activation regions (3). (4). Furthermore, transcriptional activation did not occur in Pro6 with mutations in Pro5 (5), indicating that this transcriptional activation reflected the interaction between src-SH3 and Pro5. Example 2 Regulation of transcription activity using ligand binding region of estrogen receptor After culturing 2 × 10 ⁵ HEK293 cells for 1 day on a 24-well plate, the 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. After 5 hours, /?-Estradiol was added to a final concentration of 10 nM, and after 28 hours, 50/1 of the culture supernatant was recovered, and the amount of secreted alhi-riphosphatase contained therein was measured.

 The plasmid introduced into HEK293 cells was constructed as follows.

1). 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.

 The transfection and the measurement of the activity of the phospholipase were performed in accordance with Example 1.

 In each of the cases where src-SH3 is fused to the DNA binding region and Pro5 is fused to the transcription activation region, and where Pro5 is fused to the DNA binding region and src-SH3 is fused to the transcription activation region, Addition of 10 nM 5-estradiol increased the transcriptional activation of the repo overnight gene by about 3 to 4 times (Fig. 4).

 From these results, it was revealed that transcriptional activation can be controlled by? -Estradiol by fusing ERLBD.

 By this method, 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

 Interaction between a peptide having the PDZ sequence in the human homolog hDlg of the Drosophila tumor suppressor gene Dig and the shire-force type 1 K channel Kvl.4 by a two-hybrid method that fuses two or more transcription activation regions. Tried to detect.

After culturing 2 × 10 ⁴ monkey COS-1 cells on a 24- ゥ plate for 1 day, 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

 1). 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) and 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.

 2). 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.

 3). VP16AD-p53AD-Kv1.4 .: DNA encoding VP16AD and p53AD was prepared according to Example 1. As the 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. These 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 transfection and the measurement of the activity of the phospholipase were performed in accordance with Example 1.

As shown in Fig. 5, as a plasmid containing the DNA encoding the DNA binding region, the DNA encoding the fusion protein of GAL4DBD and the second PDZ counted from the Ν end of hDlg (base sequence and encoded amino acid sequence) Are shown in SEQ ID NOs: 12 and 13), or 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 consisting of one transcription activation region VP16AD and the C-terminal 15 amino acids of Kv1.4 (15 nucleotides; the nucleotide sequence and the encoded amino acid sequence are shown in SEQ ID NOs: 16 and 17). ), Only weak transcriptional activity could be detected (2, 4). On the other hand, 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).

 In the case of only the negative control, transcription activation region VP16AD and DNA binding region GAL4DBD (1), transcription activation did not occur.

 From these results, it became clear that the connection between hDlg_PDZ and the C-terminal amino acid sequence of Kv1.4 can be detected more reliably by connecting two transcription activation regions. Example 4 Detection of interaction between src-SH3 and Prol5 by two or more transcription activation regions

Interaction /}] was detected in the same manner as in Example 1 except that Prol5 was used, in which the interaction with src-SH3 was reduced by replacing the third Leu of Pro5 with Ala instead of Pro5. did.

 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.

 The transfection and the measurement of the alkaline phosphatase activity were carried out according to Example 1.

 As shown in Fig. 6, when a fusion protein with two transcriptional activation regions (VP16AD-p53AD) was used, even if Prol5 had a weak interaction with src-SH3, its interaction ffl Detected (2).

Furthermore, as a plasmid containing the DNA encoding the transcription activation region, a DNA encoding a fusion protein containing three transcription activation regions in which two p53ADs are connected to VP16AD (base sequence and encoded amino acid sequence SEQ ID NOs: 20 and 21) 16

When used (3), compared to the fusion protein containing two transcriptional activation regions (2), the transcriptional activity was further increased and the interaction with src-SH3 was reduced. It was possible to detect the interaction with higher sensitivity.

 When src-SH3 was not fused to the DNA binding region GAL4DBD, which is a negative control, transcriptional activation did not occur.

 These results revealed that even weaker interactions than the interaction between src-SH3 and Pro5 can be detected by connecting two transcription activation regions (2). Furthermore, it was shown that the sensitivity of detection was increased by connecting the transcription activation region (3). Industrial applicability

 According to the present invention, protein-protein interactions that could not be detected by the conventional two-hybrid method in mammalian cells can be detected.

Claims

17 Claims  1. A method for detecting an interaction between a first protein and a second protein in a mammalian cell,  A fusion protein comprising two or more identical or different transcription activation regions and a first protein in a mammalian cell having MA with a reporter gene downstream of a base sequence that binds to a DNA binding region; and A method comprising expressing a fusion protein of the DNA binding region and a second protein, and detecting expression of a reporter gene.  2. The method according to claim 1, wherein the number of transcription activation regions is two.  3. The method according to claim 1, wherein one of the transcription activation regions is a VP-16 transcription activation region.  4. The method according to claim 1, wherein one of the transcription activation regions is a p53 transcription activation region.  5. The method according to claim 1, wherein the transcription activation region comprises a VP-16 transcription activation region and a p53 transcription activation region.  6. The fusion protein of two or more transcription activation regions and the first protein, or the fusion protein of the DNA binding region and the second protein, changes the three-dimensional structure by binding to the ligand to activate transcription. 2. The method according to claim 1, which is a fusion protein further fused with a ligand binding site such that the activity or DNA binding activity is changed.  7. The method according to claim 6, wherein the ligand binding site is an estrogen binding site of an estrogen receptor.  8. The method according to claim 1, wherein the repo overnight gene is a secreted alkaline phosphatase gene.  9. The method according to claim 1, wherein the repo overnight gene is a 5-galactosidase gene.  10. A method for screening a drug that affects the interaction between a first protein and a second protein, using the first protein and the second protein that have a property of interacting with each other, A mammalian cell that has a reporter gene-linked DNA downstream of the nucleotide sequence that binds to the DNA-binding region, and is composed of two or more identical or different transcription activation regions and the first protein. 18 A fusion protein with the DNA-binding domain and a second protein, culturing the mammalian cells in the presence of the drug, and screening the drug based on the change in reporter gene expression. A method that includes performing  11. The method according to claim 10, wherein one of the first protein and the second protein interacting with each other is src-SH3, and the other is a peptide containing a proline-rich motif.  1 2. Either the first protein or the second protein interacting with each other is a peptide having a PDZ sequence in the human homolog hDlg of the Drosophila tumor suppressor gene Dig, and the other is a shaker-type K channel Kvl.4. The method according to claim 10, which is a peptide comprising the C-terminal amino acid sequence of:  13. A compound obtained by the method according to claim 10.  14. A protein comprising the same or different two or more transcription activation regions and a first protein fused to be used in the method according to claim 1.  15. A DNA encoding the protein of claim 14.  16. A vector comprising the DNA according to claim 15.  17. A mammalian cell transformed with the vector of claim 16.