WO2010137717A1 - タンパク質間相互作用の高感度検出方法 - Google Patents

タンパク質間相互作用の高感度検出方法 Download PDF

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WO2010137717A1
WO2010137717A1 PCT/JP2010/059160 JP2010059160W WO2010137717A1 WO 2010137717 A1 WO2010137717 A1 WO 2010137717A1 JP 2010059160 W JP2010059160 W JP 2010059160W WO 2010137717 A1 WO2010137717 A1 WO 2010137717A1
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amino acid
acid sequence
fusion protein
protein
complex
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French (fr)
Japanese (ja)
Inventor
小澤　岳昌
直美 三澤
研二 三浦
岡本　将
重明 西井
兼治 増田
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Probex
PROBEX Inc
Probex Inc
University of Tokyo NUC
Toyobo Co Ltd
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Probex
PROBEX Inc
Probex Inc
University of Tokyo NUC
Toyobo Co Ltd
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Priority to EP10780671.3A priority Critical patent/EP2436764B1/en
Priority to US13/375,142 priority patent/US8470974B2/en
Priority to CN201080023492.6A priority patent/CN102449147B/zh
Publication of WO2010137717A1 publication Critical patent/WO2010137717A1/ja
Anticipated expiration legal-status Critical
Priority to US13/906,475 priority patent/US20130323814A1/en
Priority to US14/169,750 priority patent/US9540678B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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 a method for detecting protein-protein interaction.
  • Such a complementarity method has been used for various reporter proteins including dihydrofolate reductase and ⁇ -lactamase green fluorescent protein.
  • luciferases have also been used, such as Renilla luciferase, firefly luciferase, red luminescent click luciferase, and green luminescent click luciferase.
  • An object of the present invention is to provide an assay system using split luciferase with extremely high detection sensitivity.
  • the present inventors have used a luciferase derived from a Brazilian red beetle (Pyrearinus termitilluminans) and used any one of C-terminal fragment having SEQ ID NO: 1 and SEQ ID NO: 2-6. It is found that when the N-terminal fragment having such is fused to two proteins that bind to each other and the two fusion proteins are bound, light having an intensity of about 30 times or more is emitted.
  • the present invention has been completed.
  • one embodiment of the present invention is a fusion protein having the amino acid sequence of amino acid sequence number 1.
  • Another embodiment is a fusion protein having an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6.
  • a protein has an amino acid sequence means that the protein includes the amino acid sequence, and may include an amino acid sequence other than the amino acid sequence.
  • the “fusion protein” refers to a peptide derived from Hikari-komushi luciferase (in the present invention, specifically, a peptide consisting of an amino acid sequence selected from the group consisting of amino acid sequence numbers 1 to 6), A protein fused with a peptide not derived from luciferase.
  • Another embodiment is a complex of a fusion protein having a peptide consisting of the amino acid sequence of amino acid sequence number 1 and a fusion protein having a peptide consisting of the amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6 .
  • DNA encoding a peptide consisting of an amino acid sequence selected from the group consisting of amino acid sequence numbers 1 to 6.
  • it may be an expression vector that can express a fusion protein containing this DNA and fused to a peptide consisting of an amino acid sequence selected from the group consisting of amino acid sequence numbers 1 to 6.
  • an expression vector for expressing a protein having a peptide consisting of the amino acid sequence of amino acid sequence number 1 and a protein having a peptide consisting of an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6 are used.
  • another embodiment is a method for detecting a fusion protein having the amino acid sequence of amino acid sequence number 1, wherein the fusion protein has an amino acid sequence selected from the group consisting of amino acid sequences number 2 to 6 And a step of interacting with a binding fusion protein that binds to the fusion protein to form a complex and a step of detecting light emitted from the complex.
  • Another embodiment is a method for detecting a fusion protein having an amino acid sequence selected from the group consisting of amino acid sequences Nos. 2 to 6, wherein the fusion protein has the amino acid sequence of amino acid sequence No. 1. And a step of interacting with a binding fusion protein that binds to the protein to form a complex, and a step of detecting light emitted from the complex.
  • Another embodiment has a first fusion protein having the amino acid sequence of amino acid sequence number 1 and an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6, and binds to the first protein
  • a method for detecting a complex with a binding fusion protein, the method comprising a step of detecting light emitted from the complex.
  • Another embodiment is a method for detecting the binding between a first fusion protein and a second fusion protein having binding ability to each other, wherein the first fusion protein has the amino acid sequence of amino acid SEQ ID NO: 1.
  • the second fusion protein has an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6, and the first fusion protein and the second fusion protein interact to form a complex.
  • a step of detecting light emitted from the complex also comprises a step of fusing the amino acid sequence of amino acid sequence number 1 to the first protein to produce the first fusion protein, and an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6 A step of fusing to a second protein to produce a second fusion protein.
  • another embodiment is a method of screening a binding fusion protein that binds to a first fusion protein from a fusion protein library, wherein the first fusion protein has the amino acid sequence of amino acid SEQ ID NO: 1,
  • the plurality of second fusion proteins contained in the protein library have an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6, and the step of causing the first fusion protein and the second fusion protein to interact with each other And identifying a binding fusion protein that forms a complex with the first fusion protein by detecting the emitted light.
  • Another embodiment is a method for screening a binding fusion protein that binds to a first fusion protein, wherein the first fusion protein has an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6.
  • FIG. 1A is a base sequence of cDNA of the click beetle luciferase.
  • FIG. 1B is a list of PCR primers used to make plucN and plucC in one example of the present invention.
  • FIG. 1C is the base sequence of DNA inserted into the multiple cloning site of pcDNA3.1 in pcDNA3.1 / myc-His (B).
  • FIG. 1D shows the nucleotide sequence of DNA inserted into the multiple cloning site of pcDNA4 in pcDNA4 / V5-His (B).
  • Example of this invention it is a graph showing the result of having investigated the luminescence intensity in the luciferase split assay using the combination of pFRB-lucC389-pFRB-lucC391 and plucN404-FKBP- plucN417-FKBP.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • it is a graph showing the result of having investigated the luminescence intensity in the luciferase split assay using the combination of pFRB-lucC392-pFRB-lucC394 and plucN404-FKBP- plucN417-FKBP.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • it is a graph showing the result of having investigated the luminescence intensity in the luciferase split assay using the combination of pFRB-lucC394-pFRB-lucC399 and plucN404-FKBP-plucN417-FKBP.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • Example of this invention it is a graph showing the result of having investigated the luminescence intensity in the luciferase split assay using the combination of pFRB-lucC400-pFRB-lucC403 and plucN404-FKBP-plucN417-FKBP.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • it is a graph showing the result of having investigated the luminescence intensity in the luciferase split assay using the combination of pFRB-lucC404-pFRB-lucC407 and plucN404-FKBP-plucN417-FKBP.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • Rap + is luminescence intensity when binding is induced
  • DMSO is luminescence intensity when binding is not induced (ie, background)
  • STDEV-R is standard deviation when binding is induced by Rap +
  • STDEV-D Represents the standard deviation when binding is not induced in DMSO.
  • the left bar of the bar graph is the result in the medium containing rapamycin
  • the right bar is the result in the medium containing DMSO.
  • it is a table
  • the symbols have the same meaning as in FIG.
  • HEK293 cells in which pSSTR2-lucC394 and plucN415-arrestin were introduced to transiently express SSTR2-lucC394 and lucN415-arrestin, respectively, when somatostatin was added or not It is a graph which shows the result of having compared luminescence intensity.
  • the x axis represents the presence (+) and absence ( ⁇ ) of somatostatin, and the y axis represents the number of photons (x10 4 ).
  • Example of this invention it is a graph which shows a dose-response curve when light emission is measured after stimulating HEK293-ARRB2-SSTR2 cell line with various concentrations of somatostatin or its analog (RIM23052 or BIM23056).
  • the x-axis is the concentration of each reagent (log [molar]), y-axis represents the number of photons (x10 4 pieces).
  • after stimulating HEK293-ARRB2-SSTR2 cell line with 1x10-6 M somatostatin it is a graph which shows a time-response curve when light emission is measured over time.
  • x axis represents time (minutes)
  • y-axis represents the number of photons (x10 4 pieces).
  • concentration EC50
  • T time after irritation
  • the present invention provides a luciferase split assay system with high detection sensitivity.
  • an amino acid sequence consisting of amino acid sequence No. 1 and an amino acid sequence selected from the group consisting of amino acid sequence Nos. 2 to 6 are used, which are derived from the click beetle luciferase (sequence shown in FIG. 1A).
  • the method for carrying out the present assay system will be described in detail, but the luciferase split assay is already a well-known technique, and those skilled in the art can perform parts not described in the present specification according to common general technical knowledge.
  • a second protein referred to as the second fusion protein having (this partial peptide is referred to as lucNmax) is synthesized.
  • the first protein and the second protein are proteins that can bind under specific conditions.
  • Each fusion protein may be chemically synthesized and introduced into the assay system, but as described later, an expression vector encoding the fusion protein may be constructed and the fusion protein expressed in the assay system. In that case, transient expression or constant expression may be used.
  • the former is preferable when the assay system is an in-vitro system, and the latter is preferable when the assay system is an in-vivo system such as a cell.
  • lucCmax or lucNmax and each protein may be directly bonded or may be bonded via a linker.
  • the linker is preferably a partial peptide having an appropriate length.
  • luciferase activity when the assay system is a cell, luciferin may be administered to the cell culture medium, a cell extract may be prepared, and luciferase activity may be measured there, but commercially available Emerald Luc Luciferase Assay Reagent The activity can be easily measured by using / Lysis Solution (TOYOBO).
  • the amino acid sequence of lucC is amino acid sequence number 1 and the amino acid sequence of lucN is an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6, which is about 30 times or more than the conventional one.
  • the intensity of light emission can be obtained.
  • This vector may have the following configuration, for example.
  • a DNA encoding the target protein By inserting a DNA encoding the target protein into the multicloning site in a frame, a fusion protein of lucNmax and the protein can be easily expressed.
  • ATG which is the initiation codon, is downstream of the expression promoter functioning in the assay system, and DNA encoding the multicloning site and lucCmax is inserted in this order, followed by a transcription termination signal. It is a configuration that there is.
  • a fusion protein of lucCmax and the protein can be easily expressed by inserting a DNA encoding the target protein into the multicloning site in a frame.
  • the vector contains an amino acid sequence of amino acid sequence number 1, ie, a vector containing DNA encoding lucCmax, and an amino acid sequence selected from the group consisting of amino acid sequence numbers 2 to 6, ie, a vector containing DNA encoding lucNmax. If it is a kit, the expression vector of the fusion protein which has lucCmax or lucNmax can be constructed
  • a fusion protein having lucCmax can be detected.
  • the second fusion protein in which lucNmax is fused to the binding protein that binds to the protein to be detected is prepared as a probe. Introduce into the assay system. Then, the binding protein in the second fusion protein binds to the protein to be detected in the first fusion protein, and lucCmax and lucNmax interact to have luciferase activity. By detecting the luciferase activity, a fusion protein to be detected having lucCmax can be detected.
  • an expression vector that expresses the first fusion protein is prepared and introduced into a cell. After preparing an expression vector that expresses the second fusion protein and introducing it into the cell that expresses the first fusion protein, the fusion protein having lucCmax can be detected by measuring the luciferase activity of the cell as described above. It becomes like this.
  • a fusion protein having lucNmax can be detected.
  • the first fusion protein in which lucNmax is fused to the protein to be detected is in the assay system
  • the second fusion protein in which lucCmax is fused to the binding peptide that binds to the protein to be detected is prepared as a probe. And introduced into the assay system. Then, the binding peptide in the second fusion protein binds to the detection target peptide in the first fusion protein, and lucNmax and lucCmax interact to have luciferase activity. By detecting the luciferase activity, the fusion protein to be detected having lucNmax can be detected.
  • an expression vector that expresses the first fusion protein is prepared and introduced into a cell. After preparing an expression vector that expresses the second fusion protein and introducing it into a cell that expresses the first fusion protein, the fusion protein having lucNmax can be detected by measuring the luciferase activity as described above. Become.
  • a complex of a fusion protein having lucNmax and a fusion protein having lucCmax can be detected. This is because, when these fusion proteins form a complex, lucNmax and lucCmax interact to have luciferase activity, and the complex can be detected by detecting the luciferase activity.
  • the assay system in which the complex is present may be placed under conditions where luciferase activity can be detected. When the assay system is a cell, the luciferase activity may be measured as described above.
  • the first protein and the second protein having binding ability to each other. That is, if the lucNmax is fused to the first protein to synthesize the first fusion protein, and the lucCmax is fused to the second protein to synthesize the second fusion protein, the first fusion protein This is because the luciferase activity is detected when the first and second fusion proteins are bound.
  • this method for example, it can be examined whether or not the first protein and the second protein bind to each other.
  • the first protein and the second protein bind to each other, the luciferase activity is detected. If the first protein and the second protein do not bind, the luciferase activity is not detected.
  • expression vectors that express the first fusion protein and the second fusion protein are respectively prepared and introduced into the same cell. Thereafter, by measuring the luciferase activity as described above, if the reconstituted luciferase emits light, it can be determined that the first protein and the second protein are bound. It can be determined that the protein and the second protein do not bind.
  • a binding protein that binds to the first protein can be screened from the protein library. That is, lucNmax or lucCmax is fused to the first protein to produce a first fusion protein, and lucCmax or lucNmax is fused to the second protein in the protein library, respectively, to produce a second fusion protein.
  • the first fusion protein and the second fusion protein are allowed to interact, only the fusion protein having a binding protein that binds to the first protein in the second fusion protein is complexed with the first fusion protein. Form the body. Therefore, the second protein that forms a complex with the first protein can be identified by detecting the light emitted from the complex and identifying the fusion protein.
  • cells transformed with an expression vector that expresses the first fusion protein in which lucNmax is fused to the first protein are prepared.
  • a cDNA library constructed so that the protein is expressed in a form fused with lucCmax is introduced into the cells, and then luciferin is added to the medium to identify and clone the luminescent cells. From each clone, DNA derived from the library is collected to identify the expressed gene. In this way, cDNA of the second protein that forms a complex with the first protein can be obtained.
  • FKBP NM_054014
  • FRB NM_019906
  • LucNmax SEQ ID NOs: 2 to 6; amino acids 1 to 412 to 416
  • lucCmax SEQ ID NOs: 1; amino acid sequence at positions 394 to 542
  • PCR was carried out using the light beetle luciferase cDNA (sequence shown in FIG. 1A) as a template and the primers shown in FIG. 1B, and 14 types of amino acids 404 to 417 from the N-terminal amino acid were obtained.
  • 14 types of DNA fragments encoding peptides using N-PtGR-F001 and N-PtGR-R404 to R417 pairs
  • 389th to 413th amino acid sequences from the C-terminal amino acid
  • 25 types of DNA fragments C-PtGR-R542 and C-PtGR-F389 to F413 pairs were used
  • the DNA coding for the N-terminal region is cleaved with HindIII and BamHI, pFKBP with BamHI and XhoI, pcDNA3.1 / myc-His (B) with HindIII and XhoI, ligated in 3 molecules, and 14 types of plucN-FKBP Was made.
  • the DNA encoding the C-terminal region was cleaved with XhoI and IISacII, pFRB with BamHI and XhoI, pcDNA4 / V5-His (B) with BamHI and SacII, and ligation was carried out for 3 molecules. 25 types of pFRB-lucC Was made.
  • FIG. 1C shows the base sequence of DNA inserted into the multiple cloning site of pcDNA3.1
  • FIG. 1D shows pcDNA4 Are the plasmid vectors having the insertion sequences of SEQ ID NOs: 7 and 8, respectively.
  • each combination of expression vectors is transferred to COS7 cells using TtansIT Transfection Reagents (TAKARA) in a 96-well plastic culture dish. Transfected. About 16 hours after the transfection, the medium was replaced with a medium containing 1 ⁇ M rapamycin. After further incubation for 24 hours, ELA (TOYOBO) was added, and luminescence measurement was performed with TriStar LB941 (Berthold Technologies).
  • PTlucN-FKBP is a vector constructed by amplifying an N-terminal fragment by PCR using the following primer with the cDNA of red luminescent click luciferase and constructed similarly to plucN-FKBP.
  • PFRB-GlucC is a cDNA of green luminescent click luciferase Is a vector constructed by amplifying a C-terminal fragment by PCR using the following primers and constructing in the same manner as pFRB-lucC.
  • SSTR2 somatostatin receptor
  • NM_000794 somatostatin type 2 receptor
  • NM_004313 GPCR (G-protein coupled receptor)
  • GPCR G-protein coupled receptor
  • the C-terminus of SSTR2 is bound to the C-terminus of Eluc
  • the N-terminus of ⁇ -arrestin is bound to the N-terminus of Eluc
  • the fusion proteins are expressed in cultured cells
  • somatostatin is administered to the cultured cells. The luminescence from the cells was examined.
  • ARRB2-nestF2 AAAGGATCCATGGGGGAGAAACCCGGGACCAGGGTCT (SEQ ID NO: 54)
  • ARRB2-nestR-Eco AAGAATTCCAGCAGAGTTGATCATCATAGT (SEQ ID NO: 55)
  • SSTR2_start_Bam TTGGATCCATGGACATGGCGGATGAGCCAC (SEQ ID NO: 56)
  • SSTR2_R1107end_XhoI TTTCTCGAGCCGATACTGGTTTGGAGGTCTCCATTG (SEQ ID NO: 57)
  • the DNA encoding arrestin was cleaved with BamHI and EcoRI and ligated with plucN415 cleaved with BamHI and EcoRI to obtain plucN415-arrestin.
  • PlucN415 used here was obtained by excising DNA encoding lucN415 from plucN415-FKBP prepared in Example 1 with HindIII and BamHI and ligating with pcDNA3.1 / myc-His (B) cleaved with HindIII and BamHI. It was.
  • the DNA fragment encoding SSTR2 was cleaved with BamHI and XhoI, and inserted into the multiple cloning site of pcDNA4 / V5-His (B) to obtain pSSTR2. Furthermore, the DNA encoding lucC394 having a linker length extended to 22 amino acids was cleaved with XhoI and SacII and inserted into the XhoI-SacII site of pSSTR2 to obtain pSSTR2-lucC394.
  • linker length of lucC394 was obtained by PCR amplification using pFRB-lucC394 as a template and using linkerC12-F-XhoI (SEQ ID NO: 58) and PtGR-R542-SacII (SEQ ID NO: 61) primers as XhoI and SacII.
  • linkerC12-F-XhoI AGGCTCGAGTGGCGGTGGAGGTAGTGGAGGCGGCGGAACAAA
  • linkerC17-F-XhoI AGGCTCGAGTGGTGGTGGGGGCAGTGGCGGTGGAGGTAGTGG
  • linkerC22-F-XhoI AGGCTCGAGTGGAGGTGGCGGTTCTGGTGGTGGGGGCAGTGGCGGT (SEQ ID NO: 60)
  • PtGR-R542-SacII TTTCCGCGGCAGCTTAGAAGCCTTCTC (SEQ ID NO: 61)
  • PSSTR2-lucC394 and plucN415-arrestin thus prepared were transfected into HEK293 cells cultured in a 96-well plastic culture dish using TtansIT Transfection Reagents (TAKARA). About 40 hours after transfection, the cells were cultured for 12 minutes in a medium containing 1 ⁇ M somatostatin, ELA (TOYOBO) was added, and luminescence measurement was performed with TriStar LB941 (Berthold Technologies). As a control, luminescence was measured in the same manner using cells to which no somatostatin was added, and the results were compared. As shown in FIG. 6, by adding somatostatin, the luminescence was significantly enhanced and its intensity was increased. Increased 8 times.
  • TtansIT Transfection Reagents TtansIT Transfection Reagents
  • HEK293 cells similarly transfected with plucN415-arrestin using a 6 cm plastic culture dish are cultured in a medium containing 0.8 mg / mL G418 for 20 days, thereby constitutively expressing lucN415-arrestin.
  • HEK293 cell line HEK293-ARRB2 was generated.
  • This cell line was further transfected with pSSTR2-lucC394 in the same manner, and cultured for 20 days in a medium containing 0.8 mg / mL G418 and 0.04 mg / mL Zeocin, so that lucN415-arrestin and SSTR2-lucC394 were
  • the HEK293 cell line HEK293-ARRB2-SSTR2 that expresses constitutively was produced.
  • the cells were cultured in 96-well plastic culture dishes and stimulated with various concentrations of somatostatin or analogs thereof (RIM23052 or BIM23056) for 12 minutes, and luminescence measurements were similarly performed. Based on the obtained results, a dose-response curve representing the relationship between the ligand concentration and the luminescence intensity was created.
  • the luciferase split assay of the present invention is a GPCR other than SSTR2, that is, ADRB2 (adrenergic beta2 receptor, surface) (NM_000024), AGTRL1 (apelin receptor) (NM_00516), EDNRB (endothelin receptor type B) (NM_000115), CCKBR This is applicable to (cholecystokinin B receptor) (NM_17685), and in the same experimental system, results as shown in FIG. 9 were obtained.
  • an assay system using split luciferase with extremely high detection sensitivity can be provided.

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PCT/JP2010/059160 2009-05-29 2010-05-28 タンパク質間相互作用の高感度検出方法 Ceased WO2010137717A1 (ja)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP10780671.3A EP2436764B1 (en) 2009-05-29 2010-05-28 Method for highly sensitive detection of protein-protein interaction
US13/375,142 US8470974B2 (en) 2009-05-29 2010-05-28 Method for highly sensitive detection of protein-protein interaction
CN201080023492.6A CN102449147B (zh) 2009-05-29 2010-05-28 蛋白质间相互作用的高灵敏度检测方法
US13/906,475 US20130323814A1 (en) 2009-05-29 2013-05-31 Method for highly sensitive detection of protein-protein interaction
US14/169,750 US9540678B2 (en) 2009-05-29 2014-01-31 Method for highly sensitive detection of protein-protein interaction

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JP2009-131481 2009-05-29
JP2009131481 2009-05-29
JP2010037921A JP4849698B2 (ja) 2009-05-29 2010-02-23 タンパク質間相互作用の高感度検出方法
JP2010-037921 2010-02-23

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