WO2018235787A1 - Procédé de jugement de l'interaction de protéines mettant en œuvre des protéines fluorescentes - Google Patents

Procédé de jugement de l'interaction de protéines mettant en œuvre des protéines fluorescentes Download PDF

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WO2018235787A1
WO2018235787A1 PCT/JP2018/023187 JP2018023187W WO2018235787A1 WO 2018235787 A1 WO2018235787 A1 WO 2018235787A1 JP 2018023187 W JP2018023187 W JP 2018023187W WO 2018235787 A1 WO2018235787 A1 WO 2018235787A1
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protein
fluorescent
cells
interaction
tetrameric
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拓 渡部
井上　健
ウリケシ ワシュル
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株式会社医学生物学研究所
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    • C—CHEMISTRY; METALLURGY
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to a method of determining protein-protein interaction and its application, and a kit for use in the method.
  • FRET fluorescence resonance energy transfer
  • the present inventors have also previously developed a method for determining protein-protein interaction using a protein having multimerization ability (Patent Document 1). Specifically, a desired protein (a first protein and a second protein) is fused to a protein having multimerization ability (association inducing protein and a fluorescent protein having multimerization ability) and expressed in cells. In this case, if the first protein and the second protein interact with each other, the association between the proteins having multimerization ability is induced, whereby an aggregate (fluorescent spot) is formed autonomously. The present inventors have succeeded in developing a method for determining protein-protein interaction based on the finding of formation of the fluorescent luminescent spot.
  • PB1, SAM, etc. which are suitably used as the association-inducing protein, are functional domains possessed by the p62 protein for autophagy control, TEL protein, etc., which is a transcriptional repressor, and are deeply involved in the function of each protein in cells Do. Therefore, when the fusion protein containing such a functional domain is expressed in cells, the functions of endogenous p62 protein, TEL protein and the like of the cells will be disturbed, and thus determination of desired protein-protein interaction and There is a concern to affect their functional analysis.
  • the present inventors set a fluorescent protein to be a foreign protein for cells to be expressed together with a protein having both multimerizing ability to be fused to a desired protein, thereby providing a functional domain (PB1) in the endogenous protein It was conceived to determine protein-protein interactions without using domains, SAM domains, etc.).
  • the present invention has been made in view of the problems of the prior art described above, and a fluorescent luminescent spot formed when a fluorescent protein is fused to both of the target proteins and is expressed or introduced into cells
  • An object of the present invention is to provide a method for determining the interaction between target proteins as an indicator.
  • Non-Patent Document 1 Even if the same tetrameric fluorescent protein is fused to both of the target proteins and expressed in cells, the fluorescent bright spots are not formed, and the target The interaction between the proteins could not be determined. Furthermore, even when a monomeric fluorescent protein or a dimeric fluorescent protein is fused to any one of the proteins of interest and expressed in cells, a fluorescent bright spot indicating interaction between the proteins of interest is formed. It was not done.
  • fluorescent bright spot is formed depending on the interaction between proteins, and fluorescence resonance energy is further generated at the fluorescent bright spot. It has been found that movement or the like (FRET phenomenon) occurs.
  • the FRET obtained as compared with the case where a combination of fluorescent proteins which do not form a fluorescent bright spot when the subject interacts with each other is used. It was also revealed that the strength of the phenomenon (signal of FRET phenomenon, efficiency of FRET phenomenon) is increased.
  • the present invention relates to a method of determining protein-protein interaction and its application, and a kit for use in the method, and more specifically, provides the following invention.
  • a method for determining the interaction between a first protein and a second protein, wherein the first tetrameric fluorescent protein and the second tetrameric fluorescent protein are different proteins And a method (1) comprising the following steps (1) to (3): a first fusion protein comprising a first protein and a first tetrameric fluorescent protein, a second protein and a second tetrameric fluorescence Expressing the Cell into a Cell or Introducing a Cell into a Cell with a Second Fusion Protein Containing a Protein (2) Detecting a Fluorescent Spot Generated by Association of the First Fusion Protein with the Second Fusion Protein in the Cell Step (3) of determining the interaction between the first protein and the second protein by detecting the fluorescent luminescent spot.
  • One of the first four-mer fluorescent protein and the second four-mer fluorescent protein is a donor fluorescent protein, the other is an acceptor fluorescent protein, and in the step (3) The method according to ⁇ 1>, wherein the interaction between the first protein and the second protein is determined by detection of the FRET phenomenon through formation.
  • ⁇ 3> A method for screening a protein that interacts with a specific protein, wherein one of the first protein and the second protein is the specific protein, and the other is a test protein, The method as described in ⁇ 1> or ⁇ 2> which selects the protein which interacts with this specific protein by detection of the said fluorescent luminescent point or the said FRET phenomenon.
  • ⁇ 4> A method for identifying an amino acid residue in a first protein involved in the interaction or an amino acid residue in a second protein, which comprises the first protein and the second protein
  • a protein into which a mutation has been introduced is used and the intensity of the fluorescent spot or the FRET phenomenon is attenuated as compared to the case where a protein without a mutation is used, the mutation is introduced.
  • a method for screening a substance that regulates the interaction between the first protein and the second protein, wherein the first tetrameric fluorescent protein and the second tetrameric fluorescent protein are different A method (1) which is a protein and comprises the following steps (1) to (3): (1) a first fusion protein comprising a first protein and a first tetrameric fluorescent protein in the presence of a test compound, A step of expressing in a cell or introducing into a cell a second fusion protein comprising a second protein and a second tetrameric fluorescent protein, or A first fusion protein comprising a first protein and a first tetrameric fluorescent protein, and a second fusion protein comprising a second protein and a second tetrameric fluorescent protein in a cell Placing the cells in the presence of a test compound after introduction into cells or cells, (2) detecting a fluorescent bright spot generated by association of the first fusion protein and the second fusion protein in the cell; (3) When the intensity of the fluorescent spot is higher than the intensity of the fluorescent spot generated
  • a method for screening a substance that modulates the interaction between a first protein and a second protein, which is any one of a first tetrameric fluorescent protein and a second tetrameric fluorescent protein Method in which one is a donor fluorescent protein and the other is an acceptor fluorescent protein and includes the following steps (1) to (3) (1) the first protein and the first four amounts in the presence of the test compound
  • ⁇ 8> To be used in the method according to any one of ⁇ 1> to ⁇ 7>, including at least one substance selected from the group consisting of the following (a) to (k) and instructions for use: (A) A DNA encoding a first tetrameric fluorescent protein, and a DNA encoding any protein so as to be expressed fused to the first tetrameric fluorescent protein A vector containing a cloning site (b) insertion of DNA encoding any protein so as to be expressed fused to the second tetramer fluorescent protein and DNA encoding the second tetramer fluorescent protein (C) a vector encoding a first fusion protein (d) a vector encoding a second fusion protein (e) (a) or (c) And a vector set comprising the vector according to (b) or (d) (f) a transformed cell carrying a vector encoding a first fusion protein (g) a vector carrying a second fusion protein Transformed cell (h) Transformed cell carrying a vector
  • the interaction between the target proteins is determined using the formed fluorescent bright spots as an index. Is possible. Furthermore, the fluorescent protein to be fused is not inherently endogenous to the cell in which it is expressed or introduced. Therefore, there is little risk of disturbing the function of the cells, and protein-protein interaction can be determined.
  • the formation of a fluorescent bright spot is caused.
  • the FRET phenomenon can be generated efficiently, and the interaction between the target proteins can also be determined by detecting the FRET phenomenon.
  • the FRET phenomenon can be generated also in the immobilized cells, it is also possible to determine the protein-protein interaction by the signal of the FRET phenomenon.
  • the fluorescence microscope which shows the result of having observed the COS7 cell which made mAG1 or mEGFP which is monomer fluorescence protein fuse on one side of detection object (p53 and MDM2), and made it fuse and express it on the other is shown. It is a photograph. It is a fluorescence-microscope photograph which shows the result of having observed the COS7 cell which fused and expressed the same tetrameric fluorescent protein AG or Mmj to both detection object (p53 and MDM2). It is a fluorescence-microscope photograph which shows the result of having observed the COS7 cell which each fused different tetramer fluorescent protein (AG and Mmj) to detection object (p53 and MDM2), and was made to express it.
  • a protein (AG-MDM2) formed by fusing tetramer fluorescent protein AG with N terminal of MDM2 and a tetrameric fluorescent protein MR different from AG with C terminal of p53 It is each fluorescence image of AG and MR which show the result of having analyzed the HEK293 cell which made it express with the protein (p53-MR) which becomes, and a Ratio image which shows the efficiency of the FRET phenomenon of MR / AG. It is a graph which shows the result of having analyzed the HEK293 cell which made AG-MDM2 and p53-MR expressed before and after addition of Nutlin-3.
  • the graph shows the time course of the fluorescence intensity, and in the graph, the dotted line at the top shows the fluorescence intensity derived from AG (Em: 510), and the dotted line at the bottom shows fluorescence intensity from MR (Em: 610) Indicates Before and after the addition of Nutlin-3, a protein (AG-MDM2) formed by fusing tetramer fluorescent protein AG with N terminal of MDM2 and a tetrameric fluorescent protein MR different from AG with N terminal of p53 It is each fluorescence image of AG and MR which show the result of having analyzed the HEK293 cell which made it express with the protein (MR-p53) which becomes, and a ratio image which shows the efficiency of the FRET phenomenon of MR / AG.
  • AG-MDM2 a protein formed by fusing tetramer fluorescent protein AG with N terminal of MDM2 and a tetrameric fluorescent protein MR different from AG with N terminal of p53
  • HEK293 cells expressing p53-MR and AG-MDM2 are analyzed by images showing fluorescence intensities of AG and MR and ratio images showing the efficiency of FRET phenomenon. is there. It is a graph showing the result of having analyzed the efficiency of the FRET phenomenon before and behind addition of Nutlin-3 about HEK293 cells which made p53-MR and AG-MDM2 express.
  • the graph shows the time course of the fluorescence intensity, and in the graph, the dotted line at the top shows the fluorescence intensity derived from AG (Em: 510), and the dotted line at the bottom shows fluorescence intensity from MR (Em: 610) Indicates
  • AG and MR The results of analysis of HEK293 cells expressing p53-MR and a protein (mAG1-MDM2) fused with mAG1 at the N-terminus of MDM2 before and after the addition of Nutlin-3 are shown as AG and MR, respectively. It is an image which shows fluorescence intensity, and a Ratio image which shows the efficiency of a FRET phenomenon.
  • FIG. 6 is a graph showing the analysis results of the efficiency of the FRET phenomenon before and after the addition of Nutlin-3 for HEK293 cells expressing p53-MR and mAG1-MDM2.
  • the graph shows the time course of the fluorescence intensity, and in the graph, the dotted line at the top shows the fluorescence intensity derived from AG (Em: 510), and the dotted line at the bottom shows fluorescence intensity from MR (Em: 610) Indicates
  • the analysis results of HEK293 cells expressing MR-p53 and AG-MDM2 before and after the addition of Nutlin-3 are images showing fluorescence intensities of AG and MR and Ratio images showing the efficiency of the FRET phenomenon. is there.
  • FIG. 8 is a graph showing the results of analysis of the efficiency of the FRET phenomenon before and after the addition of Nutlin-3 for HEK293 cells in which MR-p53 and mAG1-MDM2 were expressed.
  • the graph shows the time course of the fluorescence intensity, and in the graph, the dotted line at the top shows the fluorescence intensity derived from AG (Em: 510), and the dotted line at the bottom shows fluorescence intensity from MR (Em: 610) Indicates Before or after the addition of Nutlin-3, a signal of FRET phenomenon (MR / AG or mAG1 in HEK293 cells expressing p53-MR and AG-MDM2 and HEK293 cells expressing p53-MR and mAG1-MDM2 It is a graph which shows the result of having analyzed Em: 610 / Em: 510) and mAG1 / AG.
  • HEK293 cells expressing p53-MR and AG-MDM2 (p53-MR / AG-MDM2) in the absence or presence of Nutlin-3
  • HEK293 cells expressing p53-MR and mAG1-MDM2 (p53-MR and mAG1-MDM2) It is an image showing fluorescence intensity of AG or mAG1 and MR after immobilizing p53-MR / mAG1-MDM2) with paraformaldehyde and a Ratio image showing the efficiency of the FRET phenomenon.
  • HEK293 cells expressing MR-p53 and AG-MDM2 (MR-p53 / AG-MDM2) in the absence or presence of Nutlin-3 and HEK293 cells expressing MR-p53 and mAG1-MDM2
  • MR-p53 / AG-MDM2 Nutlin-3 and HEK293 cells expressing MR-p53 and mAG1-MDM2
  • A-1210477 It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-BAK and mAG1-MCL1 express before 5 minutes after addition.
  • “MAG1-MCL1” represents a protein obtained by fusing the monomeric fluorescent protein mAG1 to the N-terminus of MCL1.
  • time course (upper in the figure) of each fluorescence intensity derived from MR and mAG1 in cells expressing MR-BAK and mAG1-MCL1 and MR / mAG1 ratio (Em: 610) It is a graph which shows time progress (in the figure, lower stage) of / Em: 510).
  • the dotted line located above indicates the fluorescence intensity (Em: 510) derived from mAG1
  • the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR.
  • It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-BAX and AG-MCL1 express before and 5 minutes after addition of BAX and MCL1 interaction inhibitor (A-1210477) addition.
  • MR-BAX represents a protein obtained by fusing tetramer fluorescent protein MR to the N-terminus of BAX.
  • time course (upper in the figure) of each fluorescence intensity derived from MR and AG in cells expressing MR-BAX and AG-MCL1 and the MR / AG ratio (Em: 610) It is a graph which shows time progress (in the figure, lower stage) of / Em: 510).
  • the dotted line located above indicates the fluorescence intensity (Em: 510) derived from AG
  • the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR.
  • A-1210477 It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-BAX and mAG1-MCL1 express before 5 minutes after addition. Before and after addition of A-1210477, time course (upper in the figure) of each fluorescence intensity derived from MR and mAG1 in cells expressing MR-BAX and mAG1-MCL1 and MR / mAG1 ratio (Em: 610) It is a graph which shows time progress (in the figure, lower stage) of / Em: 510).
  • the dotted line located above indicates the fluorescence intensity (Em: 510) derived from mAG1
  • the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR.
  • It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-mTOR and AG-FKBP12 express before and 5 minutes after addition of mTOR and FKBP12 interaction inducer (Rapamycin) addition.
  • MR-mTOR represents a protein obtained by fusing the tetrameric fluorescent protein MR to the N-terminal of mTOR
  • AG-FKBP12 fuses the tetrameric fluorescent protein AG to the N-terminal of FKBP12
  • MAG1-FKBP12 represents a protein obtained by fusing the monomeric fluorescent protein mAG1 to the N-terminus of FKBP12.
  • the dotted line located at the top indicates the fluorescence intensity (Em: 610) derived from MR
  • the dotted line located at the bottom indicates the fluorescence intensity derived from mAG1 (Em: 510).
  • MTOR-DsRed2 represents a protein obtained by fusing the tetrameric fluorescent protein DsRed2 to the C-terminus of mTOR.
  • MTOR-COR5 represents a protein obtained by fusing the tetrameric fluorescent protein COR5 to the C-terminus of mTOR.
  • the ratio image (upper stage) which shows the efficiency of the FRET phenomenon in the cell which made MR-FKBP12 and mTOR-KikGR1 express before and 10 minutes after addition of rapamycin shows the graph which shows the signal of the FRET phenomenon (lower stage) .
  • MR-FKBP12 represents a protein obtained by fusing the tetrameric fluorescent protein MR to the N-terminus of FKBP12
  • mTOR-KikGR1 fuses the tetrameric fluorescent protein KikGR1 to the C-terminal of mTOR
  • KikGR1 represents a protein that A ratio image (upper part) showing the efficiency of FRET phenomenon and a graph (lower part) showing signals of FRET phenomenon in cells expressing FKBP12-DsRed2 and mTOR-KikGR1 before and 10 minutes after addition of rapamycin are shown.
  • the ratio image (upper part) which shows the efficiency of the FRET phenomenon in the cell which made FKBP12-COR5 and mTOR-KikGR1 express before the rapamycin addition 10 minutes after addition is shown, and the graph which shows the signal of the FRET phenomenon (lower) .
  • Ratio image (upper row) showing the efficiency of FRET phenomenon and the graph (lower row) showing the signal of FRET phenomenon in cells expressing Mmj-p53 and MR-MDM2 before and 10 minutes after addition of Nutlin-3
  • Mmj-p53 represents a protein obtained by fusing the tetrameric fluorescent protein Momiji (Mmj) to the N-terminal of p53
  • MR-MDM2 represents the tetrameric fluorescent protein MR at the N-terminal of MDM2.
  • the left side shows the result of detection and observation of fluorescence from AG, and the right side shows the result of detection and observation of fluorescence from mKO1.
  • MTOR-mKO1 represents a protein obtained by fusing the monomeric fluorescent protein mKO1 to the C-terminus of mTOR. It is a fluorescence-micrograph which shows the result of having observed the cell which made AG-FKBP12 and mTOR-mKO2 express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of fluorescence from AG, and the right side shows the results of detection and observation of fluorescence from mKO2.
  • MTOR-mKO2 represents a protein obtained by fusing the monomeric fluorescent protein mKO2 to the C-terminus of mTOR.
  • MTOR-KO1 represents a protein obtained by fusing the dimeric fluorescent protein KO1 to the C-terminus of mTOR. It is a fluorescence-micrograph which shows the result of having observed the cell which made AG-FKBP12 and mTOR-mKeima express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of fluorescence from AG, and the right side shows the results of detection and observation of fluorescence from mKeima.
  • MTOR-mKeima represents a protein obtained by fusing the monomeric fluorescent protein mKeima to the C-terminus of mTOR. It is a fluorescence microscope picture which shows the result of having observed the cell which made AG-FKBP12 and mTOR-dKeima express 30 minutes after Rapamycin addition.
  • the left side shows the result of detection and observation of fluorescence from AG, and the right side shows the result of detection and observation of fluorescence from dKeima.
  • MTOR-dKeima represents a protein obtained by fusing the dimeric fluorescent protein dKeima to the C-terminus of mTOR. It is a fluorescence-microscope photograph which shows the result of having observed the cell which made mAG1-FKBP12 and mTOR-mKO1 express 30 minutes after Rapamycin addition. The left side shows the results of detection and observation of fluorescence from mAG1 and the right side shows the results of detection and observation of fluorescence from mKO1. It is a fluorescence-microscope photograph which shows the result of having observed the cell which made mAG1-FKBP12 and mTOR-mKO2 express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of fluorescence from mAG1 and the right side shows the results of detection and observation of fluorescence from mKO2. It is a fluorescence-micrograph which shows the result of having observed the cell which made mAG1-FKBP12 and mTOR-KO1 express 30 minutes after Rapamycin addition. The left side shows the results of detection and observation of fluorescence from mAG1 and the right side shows the results of detection and observation of fluorescence from KO1. It is a fluorescence-microscope photograph which shows the result of having observed the cell which made mAG1-FKBP12 and mTOR-mKeima express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of fluorescence from mAG1 and the right side shows the results of detection and observation of fluorescence from mKeima. It is a fluorescence-microscope photograph which shows the result of having observed the cell which made mAG1-FKBP12 and mTOR-dKeima express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of fluorescence from mAG1 and the right side shows the results of detection and observation of fluorescence from dKeima. It is a fluorescence microscope picture which shows the result of having observed the cell which made MR-FKBP12 and mTOR-dAG (AB) express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of MR-derived fluorescence
  • the right side shows the results of detection and observation of dAG (AB) -derived fluorescence.
  • MTOR-dAG (AB) represents a protein obtained by fusing the dimeric fluorescent protein dAG (AB) to the C-terminus of mTOR. It is a fluorescence-microscope photograph which shows the result of having observed the cell which made MR-FKBP12 and mTOR-dAG (AC) express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of MR-derived fluorescence
  • the right side shows the results of detection and observation of dAG (AC) -derived fluorescence.
  • MTOR-dAG (AC) represents a protein obtained by fusing the dimeric fluorescent protein dAG (AC) to the C-terminus of mTOR. It is a fluorescence microscope picture which shows the result of having observed the cell which made MR-FKBP12 and mTOR-mUkG1 express 30 minutes after Rapamycin addition. The left side shows the results of detection and observation of MR-derived fluorescence, and the right side shows the results of detection and observation of mUkG1-derived fluorescence. “MTOR-mUkG1” represents a protein obtained by fusing the monomeric fluorescent protein mUkG1 to the C-terminus of mTOR.
  • MTOR-mMiCy1 represents a protein obtained by fusing the monomeric fluorescent protein mMiCy1 to the C-terminus of mTOR. It is a fluorescence microscope picture which shows the result of having observed the cell which made MR-FKBP12 and mTOR-MiCy1 express 30 minutes after Rapamycin addition.
  • MTOR-MiCy1 represents a protein obtained by fusing the dimeric fluorescent protein MiCy1 to the C-terminus of mTOR. It is a fluorescence microscope picture which shows the result of having observed the cell which made MR-FKBP12 and mTOR-KCy1 express 30 minutes after Rapamycin addition.
  • the left side shows the results of detection and observation of MR-derived fluorescence, and the right side shows the results of detection and observation of KCy1-derived fluorescence.
  • MTOR-KCy1 represents a protein obtained by fusing the dimeric fluorescent protein KCy1 to the C-terminus of mTOR.
  • the method for determining protein-protein interaction of the present invention comprises A method for determining the interaction between a first protein and a second protein, wherein the first tetrameric fluorescent protein and the second tetrameric fluorescent protein are different proteins, and the following steps: (1) A first fusion protein containing a first protein and a first four-mer fluorescent protein, and a second fusion protein containing a second protein and a second four-mer fluorescent protein (1) to (3) 2.
  • protein means a molecule in which two or more amino acids are linked by peptide bond, and a modified form thereof. Therefore, the concept includes not only full-length proteins but also so-called oligopeptides and polypeptides. Protein modifications include, for example, phosphorylation, glycosylation, palmitoylation, prenylation (eg, geranylgeranylation), methylation, acetylation, ubiquitination, SUMOylation, hydroxylation, amidification.
  • first protein and the “second protein” according to the present invention, desired proteins for which an interaction is desired to be detected can be used. Also, the first protein and the second protein may be different or identical.
  • the “interaction between the first protein and the second protein” includes not only direct interaction but also another molecule (protein, etc.) between the first protein and the second protein. Also included are indirect interactions such as complex formation via nucleic acids, sugars, lipids, low molecular weight compounds etc.).
  • the “tetrameric fluorescent protein” according to the present invention may be a protein capable of emitting fluorescence and capable of forming a homotetramer in cells, for example, the fluorescence shown in Tables 1 to 4 below. Protein is mentioned.
  • amino acid sequences of these fluorescent proteins can be mutated in nature (ie, non-artificially). Also, mutations can be artificially introduced. Therefore, not only fluorescent proteins consisting of typical amino acid sequences shown in Tables 1 to 4 but also such mutants can emit fluorescence and form homotetramers in cells. As long as it can, it can be used in the present invention.
  • Such variants include, for example, the typical amino acid sequences shown in Tables 1 to 4 and 90% or more (eg, 91% or more, 92% or more, 93% or more, 94% or more, 94% or more, 95% or more, 96% or more And tetramer fluorescent proteins consisting of amino acid sequences having homology of 97% or more, 98% or more, 99% or more).
  • Sequence homology can be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990).
  • BLASTP amino acid level program
  • the specific procedure of the method of analyzing an amino acid sequence by BLASTP is known, and those skilled in the art can analyze, for example, using the default parameters of the program.
  • first tetrameric fluorescent protein and the "second tetrameric fluorescent protein” are different proteins.
  • “different” means a relationship in which the first tetrameric fluorescent protein and the second tetrameric fluorescent protein do not associate with each other and can not form a multimer.
  • the protein-protein interaction can be determined by detecting the FRET phenomenon through the formation of a fluorescent bright spot.
  • one of the first tetrameric fluorescent protein and the second tetrameric fluorescent protein is a donor fluorescent protein and the other is an acceptor fluorescent protein.
  • the “donor fluorescent protein” and the “acceptor fluorescent protein” may be in the overlapping relationship between the former fluorescence spectrum and the latter excitation spectrum in order to cause the FRET phenomenon.
  • a combination of these proteins can be selected based on the excitation wavelength and fluorescence wavelength of each fluorescent protein as shown in 4.
  • the first or second 4-mer fluorescent protein is the N-terminal side or the C-terminal side of the first or second protein. It may be fused to any of Furthermore, it may be fused directly to the first or second protein, or indirectly through the spacer protein.
  • another functional protein may be fused to the “first fusion protein” or the “second fusion protein” according to the present invention. In this case, the other functional protein is either the N-terminal side or C-terminal side or both sides of the fusion protein, or between the first or second tetrameric fluorescent protein and the first or second protein. Directly or indirectly.
  • functional proteins used to facilitate the purification of the fusion protein include Myc-tag (tag) protein, His-tag protein, hemagglutin (HA) -tag protein, FLAG-tag protein (registered trademark, Sigma-) Aldrich), glutathione-S-transferase (GST) protein.
  • the "cell” according to the present invention is not particularly limited, and may be a eukaryotic cell or a prokaryotic cell, for example, animal cells (HEK 293 cells, HeLa S3 cells, U2OS cells, etc.), insect cells (eg, insect cells). Sf9 cells, etc.), plant cells, yeast, and E. coli.
  • animal cells HEK 293 cells, HeLa S3 cells, U2OS cells, etc.
  • insect cells eg, insect cells.
  • plant cells yeast, and E. coli.
  • such cells may be in the state of being cultured ex vivo (for example, cells grown in or on the medium), and are in the state of being present in vivo (for example, encoding the first fusion protein)
  • the DNA encoding the second fusion protein may be introduced into cells of a transgenic animal).
  • the fusion protein concerning this invention will be normally introduce
  • immobilization means to stop the dynamic change that usually occurs in all or part of the structure in cells.
  • the method of immobilization is not particularly limited, and examples thereof include a method of treating cells with a protein coagulant or a protein crosslinker.
  • a drug is not particularly limited, but examples thereof include paraformaldehyde, formaldehyde (formalin), glutaraldehyde, alcohol (ethanol, methanol), formalin alcohol, picric acid, Bouin, Zenker, Hely, osmium, carnoir .
  • the expression of the fusion protein of the present invention in the cell may be transient expression or constant expression depending on the purpose.
  • Expression of the fusion protein in cells can be performed by introducing the vector according to the present invention described later into the cells.
  • a known method for introducing a vector into cells for animal cells, lipofection, electroporation, calcium phosphate method, DEAE-dextran method, viruses (adenovirus, lentivirus, adeno-associated virus, etc.) were used. The method is mentioned.
  • insect cells a method using baculovirus is mentioned.
  • the Agrobacterium method, the electroporation method, the particle gun method and the like can be mentioned.
  • yeast the lithium acetate method, electroporation method and spheroplast method can be mentioned.
  • a heat shock method for example, calcium chloride method, rubidium chloride method, electroporation method and the like can be mentioned.
  • the fusion protein can be introduced into the cells according to the type of cells by those skilled in the art.
  • Examples of known techniques for introducing proteins into cells include methods using protein transfer reagents, electroporation, and microinjection.
  • the “fluorescent spot” to be detected in the present invention is caused by the association of the first fusion protein and the second fusion protein, and typically, the first or second existing in the diffusion state
  • the region of 0.2 to 50 ⁇ m (usually 0.2 to 10 ⁇ m) having a fluorescence intensity higher than the fluorescence intensity of the fluorescent protein, preferably, the fluorescence intensities of the first and second fluorescent proteins are each in a diffusion state In the region of 0.2 to 50 ⁇ m (usually 0.2 to 10 ⁇ m), which both have higher fluorescence intensity than present.
  • the “detection of a fluorescent bright spot” can be performed using, for example, a commercially available fluorescence detection device as shown in the examples below.
  • a fluorescence device for example, a fluorescence microscope, a fluorescence scanner, a CCD camera type imager, an imaging cytometer such as IN Cell Analyzer (manufactured by GE Healthcare), a microtiter plate reader (a fluorescence plate reader), a flow cytometer, etc.
  • the fluorescent bright spot can also be detected by processing the obtained image with an image analysis program (for example, the icy spot detection program described later).
  • an image analysis program for example, the icy spot detection program described later.
  • filters, detectors, various parameters, etc. are adjusted according to the characteristics of the fluorescent luminescent spots to be detected (wavelength of fluorescence emitted from the fluorescent luminescent spots, intensity etc.) Selection and setting can be appropriately performed by those skilled in the art.
  • spot detection analysis program Icy J.-C. Olivo-Marin "Extraction of spots in biological images using multiscale products" Pattern recognition, vol. 35-9, pp. 1989-1996, 2002, http: // icy.
  • Set value Spot Detector plug-in scale1, scale2, scale3 all checked in bioimageanalysis. org
  • Detectable by analysis program “Fluorescence intensity value per unit” represents the background (eg, negative control Detection point at least 2.0 times or more, more preferably 2.5 times or more, still more preferably 5.0 times or more, and particularly preferably 10 times or more of the same fluorescence intensity value) It can be determined that it was possible.
  • the determination of protein-protein interaction can also be performed by “detection of FRET phenomenon through formation of a fluorescent bright spot”.
  • the “FRET phenomenon” detected in the present invention is that the excitation energy is transferred and transferred from the photoexcited donor fluorescent protein to the non-excited acceptor fluorescent protein without donor fluorescence emission, from the donor. It may be a phenomenon in which the fluorescence intensity decreases and the fluorescence intensity from the acceptor fluorescent protein increases (so-called ordinary “fluorescence resonance energy transfer (FRET)”), and fluorescence is once emitted from the donor fluorescent protein It may be a phenomenon (reabsorption, false FRET) in which the acceptor fluorescent protein is excited and the fluorescence intensity from the acceptor fluorescent protein is increased, but in the present invention, both are detected without discrimination.
  • FRET fluorescence resonance energy transfer
  • the detection of the FRET phenomenon can be performed by a conventional method, and can be performed, for example, using the above-mentioned fluorescence detection device. Furthermore, in order to remove the influence of background, it can also be performed using a device equipped with a time-resolved fluorescence detection function.
  • the fluorescent luminescent spot or the FRET phenomenon through the formation of the fluorescent luminescent spot is detected in the cell, it is considered that the first protein and the second protein interact with each other. It can be determined that if the fluorescent spot is not detected, it can be determined that the first protein and the second protein do not interact.
  • the method of the present invention can detect not only the occurrence of protein-protein interaction but also the disappearance of protein-protein interaction by using the presence or absence of the fluorescent luminescent spot according to the present invention as an indicator. In addition, the occurrence of such protein-protein interaction can also be traced over time. Furthermore, in the present invention, protein-protein interactions can also be detected in any region of the cell without being affected by the localization, signals and the like of association-inducing proteins such as PB1 and SAM.
  • the present invention by detecting the fluorescent luminescent spot according to the present invention or the FRET phenomenon through the formation of the fluorescent luminescent spot, generation or disappearance of protein-protein interaction, generation or disappearance of the interaction is achieved.
  • Methods can be provided to detect time, or duration of the interaction.
  • the intracellular region in which the protein-protein interaction takes place can also be identified.
  • generation or loss of protein-protein interaction generation and loss of signal transduction involving the protein-protein interaction
  • time until generation or loss of signal transduction and The duration of the signaling can be detected, as well as the region within the cell in which the signaling occurs can also be identified.
  • the present invention relates to the generation or disappearance of protein-protein interaction in response to a specific stimulus by detecting the fluorescent luminescent spot according to the present invention or the FRET phenomenon through the formation of the fluorescent luminescent spot. Methods can also be provided to detect the time to development or disappearance, or the duration of the interaction.
  • the “specific stimulation” may be a stimulation capable of inducing or inhibiting protein-protein interaction directly or indirectly, and stimulation by an endogenous factor generated in a cell (eg, intracellular calcium) It may be an increase or decrease of ion concentration, activation or inactivation of an enzyme, or an external stimulus given to cells (for example, administration of a ligand for a receptor (agonist or antagonist) to cells) .
  • generation or disappearance of a specific stimulus, generation or disappearance of the stimulus is detected by detecting the fluorescent luminescent spot according to the present invention or the FRET phenomenon through the formation of the fluorescent luminescent spot.
  • the time up to, or the duration of the stimulation can also be detected.
  • the method of the present invention it is also possible to detect an increase or decrease in protein-protein interaction depending on the degree of a particular stimulus (for example, if the particular stimulus is a drug, its concentration).
  • a 50% effective concentration (EC50) and a 50% inhibitory concentration (IC50) of the drug to protein-protein interactions can be determined according to the present invention.
  • the present invention provides a method for screening a protein that interacts with a specific protein, wherein one of the first protein and the second protein is the specific protein, and the other is the test protein. By doing this, it is possible to provide a method of selecting a protein that interacts with the specific protein by detecting the fluorescent luminescent spot according to the present invention or the FRET phenomenon through the formation of the fluorescent luminescent spot.
  • test protein is not particularly limited. From the viewpoint of being able to select proteins that interact with a specific protein efficiently in a comprehensive manner, a protein group encoded by a cDNA library can be suitably used.
  • ⁇ Method of identifying amino acid residues involved in protein-protein interaction the fluorescence intensity of the fluorescent spot and the strength of the protein-protein interaction are correlated. Therefore, according to the present invention, there is provided a method for identifying an amino acid residue in a first protein involved in protein-protein interaction or an amino acid residue in a second protein, said first protein and Using a protein in which a mutation has been introduced into any of the second proteins, the intensity of the fluorescent spot and / or the intensity of the FRET phenomenon through the formation of the fluorescent spot, the protein in which the mutation has not been introduced When attenuated as compared to the case of use, it can provide a method of determining that the amino acid residue into which the mutation has been introduced is involved in the interaction.
  • the “intensity of fluorescent luminescent spot” includes not only the fluorescent intensity of one fluorescent luminescent spot but also a certain area (eg, in one cell, in one field of view in fluorescence microscope observation, in one fluorescence image, plate reader Also included is the total fluorescence intensity of the fluorescent spot present in one well in the detection at
  • the above determination can be performed using “the intensity of the FRET phenomenon through“ as an index.
  • the result of the determination using the fluorescence intensity of the fluorescent light spot as an indicator can be confirmed by performing the determination using the intensity of the FRET phenomenon as an indicator.
  • the result of the determination using the intensity of the FRET phenomenon as an indicator can be confirmed by performing the determination using the fluorescence intensity of the fluorescent luminescent spot as an indicator.
  • the “intensity of FRET phenomenon” is not particularly limited, and the degree of decrease in fluorescence intensity of donor fluorescent protein, the degree of increase in fluorescence intensity of acceptor fluorescent protein, the fluorescence lifetime of donor fluorescent protein caused by occurrence of FRET phenomenon And more specifically, the efficiency of the FRET phenomenon and the signal of the FRET phenomenon.
  • F D indicates the fluorescence intensity of the donor fluorescent protein in the absence of the acceptor fluorescent protein
  • F D ′ indicates the fluorescence intensity of the donor fluorescent protein in the presence of the acceptor fluorescent protein.
  • E ((Donorpost-Backpost)-(Donorpre-Backpre)) / (Donorpost-Backpost)
  • Donorpost is the fluorescence intensity of the donor fluorescent protein after fading the acceptor fluorescent protein
  • Backpost is the fluorescence intensity of the background after the fading
  • Donorpre is the fluorescence intensity of the donor fluorescent protein before the fading
  • Backpre is the back of the fading before It shows the fluorescence intensity of the ground.
  • the “signal of the FRET phenomenon” can be expressed as the ratio of the fluorescence intensity of the acceptor fluorescent protein to the fluorescence intensity of the donor fluorescent protein, as shown in the examples below.
  • the “fluorescent intensity of the donor fluorescent protein” and the “fluorescent intensity of the acceptor fluorescent protein” according to the present invention are not only the fluorescent intensity of the donor fluorescent protein at one fluorescent bright spot and the fluorescent intensity of the acceptor fluorescent protein but also constant.
  • the total fluorescence intensity of the donor fluorescent protein and the total fluorescence intensity of the acceptor fluorescent protein per region (for example, in one cell, in one field in fluorescence microscopy, in a fluorescence image, in one well for detection with a plate reader) included.
  • the “protein in which a mutation is introduced into the first protein or the like” can be prepared by those skilled in the art by appropriately selecting known methods. Such known techniques include site-directed mutagenesis.
  • a first fusion protein comprising a first protein and a first tetrameric fluorescent protein in the presence of a test compound, a second protein and a second protein A step of expressing the second fusion protein containing the monomeric fluorescent protein intracellularly or introducing it into the cell, or a first fusion protein comprising the first protein and the first tetrameric fluorescent protein, (B) placing the cell in the presence of a test compound after intracellularly expressing the second fusion protein containing the two proteins and the second tetrameric fluorescent protein or introducing the same into the cell; 2) detecting a fluorescent luminescent spot generated by association of the first fusion protein and the second fusion protein in the cell, and (3) the intensity of the fluorescent luminescent spot is an odor in the absence of the test compound.
  • the test compound When the intensity of the fluorescent luminescent spot is increased, the test compound is selected as an inducer of the interaction, and the intensity of the fluorescent luminescent spot is higher than the intensity of the fluorescent luminescent spot produced in the absence of the test compound. In the case of attenuating, selecting the test compound as an inhibitor of the interaction can be provided.
  • protein-protein interaction can be determined not only with the intensity of the fluorescent bright spot but also with the FRET phenomenon through the formation of the fluorescent bright spot as an index. Therefore, according to the present invention, (1) in the presence of a test compound, a first fusion protein comprising a first protein and a first tetrameric fluorescent protein, a second protein and a second fourth amount A step of expressing the second fusion protein containing the somatic fluorescent protein intracellularly or introducing it into the cell, or a first fusion protein comprising the first protein and the first tetrameric fluorescent protein, and Placing the cell in the presence of a test compound after intracellularly expressing or introducing into the cell a second fusion protein comprising the B.) Detecting the FRET phenomenon through formation of a fluorescent bright spot caused by the association of the first fusion protein and the second fusion protein in the cell, and (3) the intensity of the FRET phenomenon When the intensity of the FRET phenomenon which occurs in the absence of the test compound is increased, the test compound
  • test compound used in the screening method of the present invention is not particularly limited, and examples thereof include expression products of gene libraries, synthetic low molecular weight compound libraries, peptide libraries, antibodies, bacterial substances, cells (microbes, plant cells , Extracts of animal cells) and culture supernatants, purified or partially purified polypeptides, extracts from marine organisms, plants or animals, soil, random phage peptide display libraries.
  • the state in the presence of the test compound means, for example, the state in which the test compound is in contact with the cell according to the present invention by addition of the test compound to the culture medium, etc. It includes the state of being introduced into cells.
  • the present invention can provide a kit to be used in the above method.
  • the kit of the present invention is a kit comprising at least one substance selected from the group consisting of the following (a) to (k) and instructions for use.
  • the vector of the present invention may be any vector as long as it contains the control sequences necessary for expressing (transcription and translation) the inserted DNA in the cell of the present invention.
  • control sequences include promoters, enhancers, silencers, terminators, poly A tails, ribosome binding sequences (Shine-Dalgano (SD) sequences).
  • the vector according to the present invention may contain a selection marker (drug resistance gene etc.) and a reporter gene (luciferase gene, ⁇ -galactosidase gene, chloramphenicol acetyltransferase (CAT) gene etc.).
  • examples of such a vector according to the present invention include a plasmid vector, an episomal vector, and a virus vector.
  • the proteins encoded in the vector according to the present invention are the first tetramer, the second tetramer fluorescent protein, and a fusion protein with these proteins, and the expression of the DNA encoding such a protein
  • a DNA for example, a DNA in which a codon is humanized
  • whose codon is optimized according to the species of the cell in which the protein is expressed is inserted into the vector according to the present invention. It may be
  • Examples of the "cloning site that allows insertion of DNA encoding any protein" include a multicloning site including one or more restriction enzyme recognition sites, a TA cloning site, and a GATEWAY (registered trademark) cloning site. .
  • a buffer In the preparation of the vector according to the present invention, other components such as a buffer, a stabilizer, a preservative, a preservative and the like may be added.
  • the transformed cell of the present invention can be prepared by introducing the vector of the present invention into cells as described above.
  • other components such as a medium necessary for storage and culture of the cells, stabilizers, preservatives, preservatives and the like may be added or attached to the preparation of transformed cells according to the present invention.
  • the "instructions" according to the present invention are instructions for using the vector or transformed cell in the method of the present invention. Instructions are, for example, the experimental method and conditions of the method of the present invention, and information on the preparation of the present invention (for example, information such as the vector map in which the nucleotide sequence of the vector, cloning site etc. are shown, transformed cells Information, such as the origin, nature, culture conditions of the cells, and the like.
  • Example 1 Detection of protein-protein interaction 1
  • desired proteins a first protein and a second protein
  • proteins having multimerization ability association-inducing protein and fluorescent protein having multimerization ability
  • Non-patent Document 1 when the same tetrameric fluorescent protein is fused to each of the desired proteins as the protein having the multimerization ability, the fluorescence is expressed in cells, The present inventors have also found that no bright spots are formed and the interaction between these proteins can not be detected (Non-patent Document 1).
  • the target of detection in this screening is the interaction between p53 protein and MDM2 protein.
  • Natrin-3 (Nutlin-3), which is known as an inhibitor of the interaction, was also used as described later in this screening (Vassilev LT et al., Science, Feb. 6, 2004, No. 303, No. 5659, 2004). , Pp. 844-848).
  • fluorescent proteins to be fused and expressed to be detected tetrameric fluorescent proteins (Azami-Green (AG), Momiji (Mmj)), dimeric fluorescent proteins (EGFP), monomeric fluorescent proteins (mAG1) , MEGFP) were used as evaluation targets, and were used in combination as appropriate as described later.
  • pmAG1-p53 A plasmid vector pAsh-p53 contained in a kit for protein-protein interaction analysis Fluoppi: Ash-hAG [p53-MDM2] (manufactured by Medical Biology Research Institute, code number: AM-8201M), with restriction enzymes BamHI and NotI By processing, the region encoding p53 was cut out. Next, p53 (mAG1-p53) in which mAG1 was fused to N-terminal by inserting into phmAG1-MCL (Medical Biology Research Institute, code number: AM-V0039M) treated with the same combination of restriction enzymes The plasmid vector (pmAG1-p53) for expressing was prepared.
  • pAG-p53 a plasmid vector for expressing p53 (AG-p53) in which AG is fused to N-terminal by inserting pAGM1-p53 excluding the region encoding p53 by treating with the same restriction enzyme combination and removing p53-encoding region (PAG-p53) was produced.
  • Mmj can be inserted by inserting the NheI-Mmj-AgeI treated with the same combination of restriction enzymes.
  • a plasmid vector (pMmj-p53) was constructed to express p53 (Mmj-p53) fused to the N-terminus.
  • pEGFP-p53 The region encoding AG was cleaved off by treating pAG-p53 with restriction enzymes NheI and AgeI.
  • DNA NheI-EGFP-AgeI, SEQ ID NO: 45
  • EGFP-p53 DNA encoding EGFP and having recognition sequences for NheI and AgeI at its ends was prepared by artificial synthesis. Then, the DNA is treated with the same restriction enzyme combination, and then inserted into a plasmid vector from which the region encoding AG is removed to express p53 (EGFP-p53) in which EGFP is fused to the N-terminus.
  • a plasmid vector (pEGFP-p53) was prepared.
  • ⁇ Preparation of pmEGFP-p53> The region encoding AG was cleaved off by treating pAG-p53 with restriction enzymes NheI and AgeI.
  • DNA (NheI-mEGFP-AgeI, SEQ ID NO: 46) encoding mEGFP and having recognition sequences for NheI and AgeI at its ends was prepared by artificial synthesis. Then, the DNA is treated with the same restriction enzyme combination, and then inserted into a plasmid vector from which the region encoding AG has been removed to express p53 (mEGFP-p53) fused to mEGFP at the N-terminus A plasmid vector (pmEGFP-p53) was prepared.
  • COS7 cells are cultured in DMEM [DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.).
  • DMEM DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.).
  • DMEM DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.).
  • the same cells were seeded on an 8-well chamber slide (manufactured by Nunc) the day before gene transfer, and cultured in a culture solution of 200 ⁇ L per well.
  • A pmAG1-p53 + pAG-MDM2
  • B pmEGFP-p53 + pAG-MDM2
  • C pmEGFP-p53 + pMmj-MDM2
  • D pEGFP-p53 + pAG-MDM2
  • E pEGFP-p53 + pMmj-MDM2
  • F pAG-p53 + pAG-MDM2
  • G pMmj-p53 + pMmj-MDM2
  • H pAG-p53 + pMmj-MDM2
  • I pMmj-p53 + pAG-MDM2.
  • the interaction inhibitor p53 and MDM2 inhibitor Nutlin-3 was added to the culture solution to a final concentration of 20 ⁇ M, and cells were also allowed to stand at room temperature for 15 minutes. Images before and 15 minutes after drug addition are shown in FIG. 1D.
  • Example 2 Detection of protein-protein interaction 2 As shown below, the same study as in Example 1 was performed using HEK293 cells instead of COS7 cells. Furthermore, it was examined whether the protein-protein interaction could be detected using the tetrameric protein Monti-Red (MR).
  • MR tetrameric protein Monti-Red
  • pmAG1-MDM2 ⁇ Preparation of pmAG1-MDM2>
  • the region encoding MDM2 was excised by treating the plasmid vector phAG-MDM2 contained in the above-described protein-protein interaction analysis kit with restriction enzymes BamHI and NotI.
  • a plasmid vector (pmAG1-MDM2) for expressing MDM2 (mAG1-MDM2) in which mAG1 was fused to N-terminal was constructed by inserting into phmAG1-MCL treated with the same combination of restriction enzymes.
  • pMR-MDM2 ⁇ Preparation of pMR-MDM2>
  • the region encoding MDM2 was excised by treating pAG-MDM2 with restriction enzymes BamHI and NotI. Then, by inserting the vector into pMonti-Red-MCL (Medical Biology Research Institute, code number: AM-VS0802M) treated with the same restriction enzyme combination, MDM is fused to N-terminal MDM2 (MR 2)
  • MR 2 N-terminal MDM2
  • pMR-MDM2 N-terminal MDM2
  • pMR-p53 plasmid vector for expressing p53 fused to MR at the N-terminus is prepared by inserting into pMonti-Red-MCL treated with the same restriction enzyme combination. did.
  • HEK293 cells were cultured in DMEM [DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.). Next, the same cells were seeded on an 8-well chamber slide (manufactured by Nunc) the day before gene transfer, and cultured in a culture solution of 200 ⁇ L per well.
  • DMEM DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.).
  • DMEM DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (Thermo Fisher) (Manufactured by Scientific Co.).
  • A pAG-p53 + pAG-MDM2
  • B pAG-p53 + pmAG1-MDM2
  • C pMmj-p53 + pMmj-MDM2
  • D pMmj-p53 + pmAG1-MDM2
  • E pMmj-p53 + pAG-MDM2
  • F pMmj-p53 + pMR-MDM2
  • G pMR-p53 + pAG-MDM2.
  • HEK 293 cells in which the same tetrameric fluorescent protein (AG or Mmj) is fused and expressed to both of the detection targets (p53 and MDM2), and a monomer to one of the detection targets
  • AG or Mmj tetrameric fluorescent protein
  • mAG1 fluorescent protein
  • Example 3 Application to a Method of Determining Protein-Protein Interaction Using FRET
  • a different tetrameric fluorescent protein is fused to a detection target to be intracellular It is characterized in that it is expressed in Therefore, it is shown below whether the FRET phenomenon (fluorescence resonance energy transfer, reabsorption) can be caused by using one tetrameric fluorescent protein as an acceptor and the other tetrameric fluorescent protein as a donor. It analyzed as follows.
  • pp53-MR> a nucleotide sequence was designed in which a BamHI recognition sequence and a NotI recognition sequence were added to the 5 'end and the 3' end of a DNA encoding a part of p53 (a region consisting of 1 to 70 amino acids of p53 protein).
  • a DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and NotI, and then treated with the same combination of restriction enzymes pMonti-Red-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM- By inserting into VS0802M), a plasmid vector (pp53-MR) for expressing p53 (p53-MR) in which MR was fused to C-terminal was prepared.
  • the plasmid vector was introduced into HEK293 cells in the combination of (A) to (B) shown below by the method described in Example 2 above.
  • the obtained image data was analyzed by MetaMorph ver 8.9.0 (manufactured by Molecular Device), and the fluorescence intensities of MR (Em: 610) and AG (Em: 510) of the whole cell area were calculated. Furthermore, the ratio of the fluorescence intensity on the acceptor side to the fluorescence intensity on the donor side (AG) (MR / AG ratio (Em: 610 / Em: 510)) was calculated as a signal of the FRET phenomenon to obtain a Ratio image.
  • the fluorescence images of AG and MR before and 15 minutes after the addition of Nutlin-3, and the ratio images of MR / AG are shown in FIGS. 3A and 3C. Furthermore, time-lapse graphs of each fluorescence intensity are shown in FIGS. 3B and 3D.
  • the efficiency of the FRET phenomenon in HEK 293 cells subjected to gene transfer treatment was measured by acceptor photo-bleaching method. More specifically, MR is bleached (bleach, bleach) by irradiating excitation light of 530 to 550 nm for 5 minutes using fluorescence mirror unit U-MWIG3 (manufactured by Olympus) to the cells before addition of the drug.
  • the efficiency of the FRET phenomenon was obtained by observing the change in the fluorescence intensity of AG using a fluorescent mirror unit U-MNIBA3 (excitation wavelength: 470 to 495 nm, fluorescence wavelength: 510 to 550 nm, manufactured by Olympus).
  • the AG fluorescence intensity image before (Post-bleach) and 530-550 nm excitation light irradiation (Pre-bleach) and after (Post-bleach), and the efficiency of the FRET phenomenon were determined using the following formula. ((Donorpost-Backpost)-(Donorpre-Backpre)) / (Donorpost-Backpost) Donorpost represents fluorescence intensity of AG after fading, Backpost represents fluorescence intensity of background after fading, Donorpre represents fluorescence intensity of AG before fading, and Backpre represents fluorescence intensity of background before fading.
  • the results obtained in this way are shown in FIG. 3E.
  • the MR / AG ratio is high centering on the fluorescent bright spot and after the addition of Nutlin-3. Decreased as it diffused throughout the cell. MR (Em: 610) decreased and AG (Em: 510) increased as a change in each fluorescence intensity after the addition of Nutlin-3. This indicates that the addition of the protein-protein interaction inhibitor (Nutlin-3) increases the fluorescence intensity on the donor side and decreases the fluorescence intensity on the acceptor side, that is, the signal of the FRET phenomenon decreases. There is.
  • Example 4 Comparison of the efficiency of FRET phenomenon When a fluorescent protein is fused to a detection target and expressed in cells, it is generated through the formation of interaction-dependent fluorescent luminescent spots between the detection targets. It verified about whether the efficiency of FRET phenomenon improves. More specifically, as described below, when a combination of tetrameric fluorescent proteins (AG and MR) that forms a fluorescent luminescent spot is used when the detection target interacts with a protein, the fluorescent luminescent spot is The efficiencies of the resulting FRET phenomena were compared to those with the combination of fluorescent proteins not formed (mAG1 and MR).
  • AG and MR tetrameric fluorescent proteins
  • the plasmid vector was introduced into HEK293 cells in the combination of (A) to (D) shown below by the method described in Example 2 above.
  • D pMR-p53 + pmAG1-MDM2.
  • FIGS. 4A, 4C, 4E and 4G The fluorescence images of AG and MR and the ratio images of MR / AG before and 15 minutes after Nutlin-3 addition are shown in FIGS. 4A, 4C, 4E and 4G. Furthermore, time-lapse graphs of each fluorescence intensity are shown in FIGS. 4B, 4D, 4F and 4H. Further, time-lapse graphs of the MR / AG ratio, the MR / mAG1 ratio, and the mAG1 / AG ratio by the addition of Nutlin-3 are shown in FIGS. 4I and 4J.
  • FIG. 4K The results of comparing the signals of the FRET phenomenon (MR / AG ratio and MR / mAG1 ratio) are shown in FIG. 4K.
  • 4B, 4D, 4F and 4H mean values of MR / AG ratio and MR / mAG1 ratio in each of 11 cells in p53-MR and AG-MDM2 and p53-MR and mAG1-MDM2 respectively.
  • the MR / AG ratio indicates the fluorescent spot. It was high in the center and decreased with diffusion throughout the cells after Nutlin-3 addition. MR (Em: 610) decreases and AG (Em: 510) increases as changes in fluorescence intensity after Nutlin-3 addition, and a decrease in the signal of FRET phenomenon due to the inhibition of protein-protein interaction is observed.
  • the MR / mAG1 ratio is higher after addition than that of Nutlin-3. It decreased overall. As each change in fluorescence intensity, MR (Em: 610) decreased, AG (Em: 510) increased, and a decrease in the signal of the FRET phenomenon due to the inhibition of protein-protein interaction was observed. Thus, it was confirmed that the protein-protein interaction can be detected by the detection of the FRET phenomenon even when using a combination of fluorescent proteins (mAG1 and MR) that do not form a fluorescent luminescent spot.
  • the MR / AG ratio in cells expressing p53-MR and AG-MDM2 and the ratio MR / mAG1 in cells expressing p53-MR and mAG1-MDM2 As a result, the difference at 600 seconds was the highest between 0 and 600 seconds after the addition of Nutlin-3, and the MR / AG ratio decreased 1.2 times after the addition of the inhibitor.
  • the MR / AG ratio in cells in which MR-p53 and AG-MDM2 were expressed was compared with the MR / mAG1 ratio in cells in which MR-p53 and mAG1-MDM2 were expressed.
  • the difference at 160 seconds was the highest, and the MR / AG ratio dropped 1.52 times. In addition, it decreased by 1.4 times at 600 seconds after the addition.
  • the combination of tetrameric fluorescent proteins that form fluorescent luminescent spots is more efficient in the FRET phenomenon than the combination of fluorescent proteins that can not form fluorescent luminescent spots. It became clear that it became high (it improved 1.2 to 1.52 times). The reason why the efficiency of the FRET phenomenon is thus improved is not necessarily clear, but the aggregation density of these fluorescent proteins is increased in the aggregates (fluorescent spots) formed by using different tetrameric fluorescent proteins. Is presumed to be due to
  • Example 5 Comparison of efficiency of FRET phenomenon in cells after fixation As shown in Examples 3 and 4 above, by combining different tetrameric fluorescent proteins with the object to be detected and expressing them in cells, the fluorescence intensity can be enhanced. It has become clear that FRET phenomena can occur when points are formed.
  • FIGS. 5A and 5B The fluorescence images of AG and MR obtained with or without Nutlin-3 and the ratio images of MR / AG are shown in FIGS. 5A and 5B.
  • a graph of the signal of each FRET phenomenon (Em: 610 / Em: 510, MR / AG ratio, MR / mAG1 ratio) is shown in FIG. 5C.
  • the data shown in FIG. 5C is an average value of three fields of fluorescence intensity ratio in the whole field of view, and error bars indicate standard errors.
  • the FRET phenomenon can not be detected when the cells are immobilized.
  • the FRET phenomenon can be maintained and detected even in the immobilized cells when using a combination of tetrameric fluorescent proteins that form fluorescent luminescent spots when the detection targets interact with each other.
  • Example 6 Detection of Protein-Protein Interaction 3
  • the target of detection in Example 3 above was changed from p53 and MDM2 to MAX and Myc, and it was verified whether the interaction between these proteins could also be detected.
  • DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and NotI, and then treated with the same combination of restriction enzymes pMonti-Red-MCL (Medical Biology Research Institute, Inc. Code No. AM- By inserting into VS0802M), a plasmid vector (pMR-Myc) for expressing Myc (MR-Myc) in which MR was fused to N-terminal was prepared.
  • pMR-Myc plasmid vector for expressing Myc
  • pAG-Max a 4-amino acid sequence (N-terminal) of the amino acid region from which the nuclear localization signal (amino acids 153 and 154 of Max protein) and the DNA binding region (region consisting of 1-35 amino acids of Max protein) are removed from the full length of Max Nucleotide sequence (SEQ ID NO: 49) obtained by inserting SEQ ID NO: 48) and adding BamHI recognition sequence to the 5 'end of the DNA encoding the obtained amino acid sequence and stop codon TAA and XhoI recognition sequence at the 3' end Designed.
  • a DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and XhoI, and treated with the same combination of restriction enzymes, phAG-MCL (Medical Biology Research Institute, Inc., code number: AM-VS0801M)
  • phAG-MCL Medical Biology Research Institute, Inc., code number: AM-VS0801M
  • a plasmid vector (pAG-Max) for expressing Max (AG-Max) in which AG was fused to N-terminal was constructed by inserting into.
  • HEK293T cells were first cultured by the method described in Example 2 above. Then, 200 ng of each of the above plasmid vectors (pMR-Myc and pAG-Max) was mixed with 10 ⁇ L of Opti-MEM (manufactured by Thermo Fisher Scientific) to prepare a DNA solution. Separately, 0.2 ⁇ L of a gene transfer reagent Transficient (manufactured by MBL International) was added to 10 ⁇ L of OptiMEM, and the mixture was stirred to prepare a Transficient solution.
  • Opti-MEM manufactured by Thermo Fisher Scientific
  • Example 7 Detection of Protein-Protein Interaction 4
  • the target of detection in Example 3 above was changed from p53 and MDM2 to another protein to detect the interaction between these proteins. More specifically, it is confirmed that fluorescent light spots are formed even by protein interactions other than p53 and MDM2 by the method described below, and that the formation of the fluorescent light spots causes an increase in the efficiency of the FRET phenomenon. did. Furthermore, when the protein-protein interaction is detected using a combination of tetrameric fluorescent proteins that form fluorescent spots (AG and MR) and a combination of fluorescent proteins that do not form fluorescent spots (mAG1 and MR) Were compared with those detected using
  • Example 7 the target protein-protein interaction is an interaction between MCL1 and BAX or BAK. Both BAX and BAK have been shown to interact with MCL1 via the BH3 domain (see Ku B et al., Cell Res., April 2009, Vol. 21, No. 4, pages 627-641). Also, A-1210477 (BH3 mimetic) is known as a compound that binds to the BH3 binding groove of MCL1 and inhibits the interaction with BH3 (Xiao Y et al., Mol Cancer Ther., August 2015, 14 Volume 8, pages 1837 to 1847).
  • pAG-MCL1 a nucleotide sequence in which a BamHI recognition sequence, a stop codon and a NotI recognition sequence are added to the 5 ′ end and the 3 ′ end of the nucleotide sequence encoding a part of MCL1 (region consisting of 173 to 327 amino acids of MCL1 protein) Number: 50) designed. Then, a DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and NotI, and then treated with the same combination of restriction enzymes, phAG-MCL (Medical Biology Research Institute Co., Ltd. code number: AM-VS0801M) A plasmid vector (pAG-MCL1) for expressing MCL1 (AG-MCL1) in which AG was fused to the N-terminus was constructed by inserting into.
  • phAG-MCL1 a plasmid vector for expressing MCL1 (AG-MCL1) in which AG was fused to the N-terminus was constructed
  • pmAG1-MCL1 A nucleotide sequence including a part of MCL1 to which a BamHI recognition sequence and a NotI recognition sequence are added is treated with phmAG1-MCL (Medical Biology Research Institute, code number: AM-V0039M) treated with the same restriction enzyme combination.
  • phmAG1-MCL Medical Biology Research Institute, code number: AM-V0039M
  • a plasmid vector (pmAG1-MCL1) for expressing MCL1 (mAG1-MCL1) in which mAG1 was fused to the N-terminal was prepared.
  • pMR-BAK a nucleotide sequence in which a BamHI recognition sequence, a stop codon and a NotI recognition sequence are added to the 5 ′ end and the 3 ′ end of the nucleotide sequence encoding a part of BAK (region consisting of 72-87 amino acids of BAK protein) Number: 51) designed. Then, DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and NotI, and then treated with the same combination of restriction enzymes pMonti-Red-MCL (Medical Biology Research Institute, Inc. Code No. AM- By inserting into VS0802M), a plasmid vector (pMR-BAK) for expressing BAK (MR-BAK) in which MR was fused to N-terminal was prepared.
  • pMonti-Red-MCL Medical Biology Research Institute, Inc. Code No. AM-
  • pMR-BAX a nucleotide sequence in which a BamHI recognition sequence, a stop codon and a NotI recognition sequence are added to the 5 'end and the 3' end of the nucleotide sequence encoding a part of BAX (region consisting of 35-55 amino acids of BAX protein) Number: 52) designed. Then, DNA consisting of the nucleotide sequence is artificially synthesized, digested with BamHI and NotI, and then treated with the same combination of restriction enzymes pMonti-Red-MCL (Medical Biology Research Institute, Inc. Code No. AM- By inserting into VS0802M), a plasmid vector (pMR-BAX) for expressing BAX (MR-BAX) in which MR was fused to N-terminal was prepared.
  • pMonti-Red-MCL Medical Biology Research Institute, Inc. Code No. AM-
  • HEK293 cells were cultured by the method described in Example 2 above, and 200 ng of each of the plasmid vectors prepared above was introduced into the cells in combination of (A) to (D) shown below.
  • FIG. 7A Ratio images before and 5 minutes after addition of the drug are shown in FIG. 7A, and the time course graph of each fluorescence intensity and the MR / AG ratio ( A time lapse graph of Em: 610 / Em: 510) is shown in FIG. 7B.
  • Figure 7C shows the ratio images of MR-BAK and mAG1-MCL1 expressing cells before and after addition of the drug in Figure 7C, and the time course graph of each fluorescence intensity and the time course graph of the MR / AG ratio are shown in Figure 7D. Shown in.
  • the MR / AG ratio of cells expressing MR-BAK or MR-BAX and AG-MCL1 is high, centering on the fluorescent spot, and after addition of A-1210477, cells are added. It decreased while spreading throughout.
  • MR Em: 610
  • AG Em: 510
  • the difference at 600 seconds is the highest between 0 and 600 seconds after the addition of A-1210477
  • the MR / AG ratio decreased significantly after the addition of the inhibitor (a 4-fold decrease for MR-BAK and AG-MCL1 and a 2.8-fold decrease for MR-BAX and AG-MCL1). That is, the combination of MR / AG indicates that the energy transfer efficiency by FRET is higher, suggesting that the efficiency of the FRET phenomenon is improved by increasing the aggregation density using the tetrameric fluorescent protein. doing.
  • Example 8 Detection of protein-protein interaction 5 Similarly to Example 7 above, a fluorescent bright spot is formed also by a drug-induced protein interaction other than p53 and MDM2 by the method described below, and the efficiency of FRET phenomenon due to the fluorescent bright spot formation. Confirmed that a rise in Furthermore, when the protein-protein interaction is detected using a combination of tetrameric fluorescent proteins that form fluorescent spots (AG and MR) and a combination of fluorescent proteins that do not form fluorescent spots (mAG1 and MR) Were compared with those detected using
  • the targeted protein-protein interaction is an interaction between the FRB domain of mTOR protein and the FKBP12 protein, and it is known that these interact in the presence of rapamycin. (See Chen J et al., Proc Natl Acad Sci USA., May 23, 1995, Vol. 92, No. 11, pp. 4947-4951).
  • a plasmid vector pAsh-FKBP12 contained in Ash-hAG [mTOR-FKBP12] (Medical Biology Research Institute, code number: AM-8202M) is treated with NheI and AgeI, and the Ash peptide from the plasmid vector is processed. Removed the coding region.
  • phAG-MCL manufactured by Medical Biology Laboratory Co., Ltd., code number: AM-VS0801M
  • pAG-FKBP12 was treated with NheI and AgeI to remove the region encoding AG from the plasmid vector.
  • phmAG1-MCL manufactured by Medical Biology Research Institute, Inc., code number: AM-V0039M
  • a plasmid vector for expressing FKBP12 (mAG1-FKBP12) in which mAG1 is fused to the N-terminus was prepared.
  • pMR-mTOR a nucleotide sequence (SEQ ID NO: 53) in which an EcoRI recognition sequence and an XhoI recognition sequence are added to the 5 'end and the 3' end of the nucleotide sequence encoding a part of mTOR (region consisting of 2025-2114 amino acids of mTOR protein). ) Designed. Then, a DNA consisting of the nucleotide sequence is artificially synthesized, digested with EcoRI and XhoI, and then treated with the same combination of restriction enzymes pMonti-Red-MCL (Medical Biology Research Institute, Inc. Code No. AM- By inserting into VS0802M), a plasmid vector (pMR-mTOR) for expressing a part of mTOR (MR-mTOR) in which MR was fused to N-terminal was prepared.
  • pMR-mTOR plasmid vector for expressing a part of mTOR (MR-mTOR) in which MR was fused
  • HEK 293 cells were cultured in the same manner as in Example 7 described above, and 200 ng of each of the plasmid vectors prepared above was introduced into the cells in the combination of (E) and (F) shown below.
  • F pMR-mTOR + pmAG1-FKBP12
  • Rapamycin necessary for interaction between mTOR and FKBP12 is added to the culture solution of HEK 293 cells to a final concentration of 1 ⁇ M.
  • FIG. 8A the ratio images of the cells expressing MR-mTOR and AG-FKBP12 before and 5 minutes after addition of the drug are shown in FIG. 8A, and the time course graph of each fluorescence intensity and the MR / AG ratio ( The time lapse graph of Em: 610 / Em: 510) is shown in FIG. 8B.
  • Figure 8C shows the ratio images of MR-mTOR and mAG1-FKBP12-expressing cells before and 5 minutes after drug addition, and the time course graph of each fluorescence intensity and the time course graph of MR / AG ratio are shown in FIG. 8D. Shown in.
  • the difference at 320 seconds is the highest between 0 seconds and 430 seconds after the addition of rapamycin, and the MR / AG ratio Increased 2.7 times after the induction agent was added. That is, similar to the result of the above-mentioned Example 7, the MR / AG combination shows that the energy transfer efficiency by FRET is higher, and the fluorescence density is increased by using the tetrameric fluorescent protein to increase the aggregation density. A bright spot is formed, and it is suggested that the efficiency of the FRET phenomenon is improved by interposing the fluorescent bright spot.
  • HEK 293 cells were cultured in a culture solution (DMEM High glucose (manufactured by SIGMA ALDRICH), 10% FBS (manufactured by EQUITECH), 1% penicillin / streptomycin (manufactured by Thermo Fisher Scientific)).
  • DMEM High glucose manufactured by SIGMA ALDRICH
  • FBS manufactured by EQUITECH
  • penicillin / streptomycin manufactured by Thermo Fisher Scientific
  • the 96-well plate is set in a multi-label plate reader (Perkin Elmer, product name: EnVision 2102 Multilabel Reader), excitation 430/8 filter (2100-5250, Perkin Elmer), D480 (custom-made dichroic mirror) and Optical filter
  • the fluorescence value of the donor was measured using a Fura2 51/10 filter (2100-5320, manufactured by Perkin Elmer). Further, the fluorescence value of the acceptor was measured using an excitation 430/8 filter, D480, Cy5 620/40 filter (2100-5760, manufactured by Perkin Elmer).
  • the signal of the FRET phenomenon was changed depending on the concentration of the interaction modifier, and could be detected by the plate reader. Furthermore, when the maximum value of the dose-response curve is FRET + and the minimum value is FRET-, the FRET ratio (FRET + / FRET-) is calculated, and the case where a tetrameric fluorescent protein is used and the case where a monomeric fluorescent protein is used As a result, the FRET ratio in the former was increased 1.6 times in the interaction between MCL1 and BAK and 2.0 times in the interaction between mTOR and FKBP12.
  • the FRET phenomenon can be detected by the plate reader even in the fixed cells when tetramer fluorescent protein is used.
  • the IC 50 may not be calculated, or the IC 50 measured in the living cells may largely deviate from the value, and an accurate evaluation could not be performed.
  • the former is 1.5 times the interaction between MCL1 and BAK, mTOR And FKBP12 interaction increased FRET ratio 1.9 times.
  • the combination of tetrameric fluorescent proteins that form fluorescent luminescent spots is more efficient in the FRET phenomenon than the combination of fluorescent proteins that can not form fluorescent luminescent spots. Since it became high, it was confirmed that the said phenomenon can be detected by a plate reader, and the degree of protein-protein interaction to be detected can be analyzed with high sensitivity. Further, similarly to the results shown in Example 5, when a combination of tetrameric fluorescent proteins that form fluorescent bright spots when the detection targets interact with each other, the FRET phenomenon is maintained even in the immobilized cells. It was also confirmed that it could be detected.
  • HEK293 cells are cultured by the method described in Example 9, and the plasmid prepared above is used as the cells. Introduced to Then, add the plasmid DNA solution and Fugene HD, culture for 24 hours, collect the cells after trypsin treatment, pass to 24 wells of a 96 well plate (Corning # 356640), and add 100 ⁇ L per well. The cultures were cultured for 24 hours.
  • the obtained image was analyzed using software Harmony (manufactured by Parkin Elmer).
  • the fluorescent spots were detected at a setting of Radius 2 ⁇ m, Contrast 0.3 or more, Uncorrected Spot to Region Intensity 0.5 or more, Distance 0.9 ⁇ m, and Spot Peak Radius 0 ⁇ m in Find Spots Method C, which is an analysis algorithm of Harmony.
  • "sum of fluorescence intensities of AG at fluorescent spots in the field / total number of cells expressing AG in the field” and "sum of fluorescence intensities of MR at fluorescent spots in the field / MR in the field”"Total number of cells expressing" was calculated.
  • both the AG-derived and MR-derived fluorescence intensities at the fluorescent spots changed in an inhibitor concentration-dependent manner. Therefore, it was confirmed that protein-protein interaction can also be determined by analyzing a fluorescence microscope image obtained using an imaging system.
  • Example 11 Detection of fluorescent spot formation and FRET phenomenon using tetrameric fluorescent proteins other than AG and MR Even tetrameric fluorescent proteins (DsRed2, COR5, KikGR1) other than AG and MR can be used. It was confirmed by the method described below that the formation of fluorescent spots and the detection of the FRET phenomenon can be performed.
  • a nucleotide sequence was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence (SEQ ID NO: 27) encoding DsRed2, respectively. Then, a DNA consisting of the nucleotide sequence was artificially synthesized and cleaved with AgeI and XbaI.
  • pmTOR-AG manufactured by Medical Biology Research Institute, code number: AM-8202M
  • AM-8202M the same restriction enzyme combination to remove the region encoding AG from the plasmid DNA
  • DsRed2 the DsRed2 is The coding DNA was inserted, and a plasmid vector (pmTOR-DsRed2) for expressing mTOR (mTOR-DsRed2) in which DsRed2 was fused to C-terminal was prepared.
  • a nucleotide sequence was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence encoding COR5 (SEQ ID NO: 23), respectively. Then, a DNA consisting of the nucleotide sequence was artificially synthesized and cleaved with AgeI and XbaI.
  • pmTOR-AG is treated with a combination of the same restriction enzymes to remove the AG coding region from the plasmid DNA, and then the DNA coding for the COR5 is inserted and mTOR (COR5 is fused to the C-terminal)
  • a plasmid vector (pmTOR-COR5) was constructed to express mTOR-COR5).
  • pmTOR-KikGR1 ⁇ Preparation of pmTOR-KikGR1>
  • the region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI.
  • pmKikGR11-MNL manufactured by Medical Biology Laboratory Co., Ltd., code number: AM-V0150M
  • the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-KikGR1) for expressing mTOR (mTOR-KikGR1) in which KikGR1 is fused to C-terminal.
  • a nucleotide sequence (SEQ ID NO: 54) was designed in which an EcoRI recognition sequence and an XhoI recognition sequence were added to the 5 ′ end and the 3 ′ end of the nucleotide sequence encoding the full-length FKBP12, respectively.
  • a DNA consisting of the nucleotide sequence is artificially synthesized, digested with EcoRI and XhoI, and treated with the same combination of restriction enzymes, phAG-MNL (Medical Biology Research Institute Co., Ltd., code number: AM-VS0801M)
  • phAG-MNL Medical Biology Research Institute Co., Ltd., code number: AM-VS0801M
  • a plasmid vector (pFKBP12-AG) for expressing FKBP12 (FKBP12-AG) in which AG was fused to C-terminus was prepared.
  • pFKBP12-DsRed2 ⁇ Preparation of pFKBP12-DsRed2>
  • the region encoding AG was removed from the pFKBP12-AG by treatment with AgeI and XbaI. Further, the pmTOR-DsRed2 was treated with the same restriction enzyme combination to cut out the DNA encoding DsRed2. Then, the DNA was inserted into the restriction enzyme-treated pFKBP12-AG to prepare a plasmid vector (pFKBP12-DsRed2) for expressing FKBP12 (FKBP12-DsRed2) in which DsRed2 is fused to C-terminal.
  • pFKBP12-COR5 ⁇ Preparation of pFKBP12-COR5> The region encoding AG was removed from the pFKBP12-AG by treatment with AgeI and XbaI. In addition, the pmTOR-COR5 was treated with the same restriction enzyme combination to cut out the DNA encoding COR5. Then, the DNA was inserted into the restriction enzyme-treated pFKBP12-AG, and a plasmid vector (pFKBP12-COR5) was constructed to express FKBP12 (FKBP12-COR5) in which COR5 was fused to C-terminal.
  • pMR-FKBP12 ⁇ Preparation of pMR-FKBP12> From pAG-FKBP12 prepared in Example 8, the region encoding AG was removed by treatment with NheI and AgeI. Also, pMonti-Red-MCL (Medical Biology Research Institute, Inc., code number: AM-VS0802M) was treated with the same restriction enzyme combination to cut out the DNA encoding Monti-Red (MR). Then, the DNA was inserted into the restriction enzyme-treated pAG-FKBP12 to prepare a plasmid vector (pMR-FKBP12) for expressing FKBP12 (MR-FKBP12) in which MR was fused to N-terminal.
  • pMR-FKBP12 plasmid vector for expressing FKBP12 (MR-FKBP12) in which MR was fused to N-terminal.
  • the donor side was AG, KikGR1 or Mmj, and the acceptor side was MR, DsRed2 or COR5.
  • Rapamycin is added to the culture solution of HEK 293 cells to a final concentration of 1 ⁇ M as its interaction inducer, or in p53 and MDM2, Nutlin-3 is used as its interaction inhibitor.
  • the culture solution of HEK 293 cells was added to a final concentration of 20 ⁇ M, and the change in each fluorescence intensity for 10 minutes before and after the addition of the drug was measured. And it carried out by the method of Example 3 except the above-mentioned matter.
  • FIGS. 11A to 11E Ratio images before and after addition of Rapamycin and graphs relating to signal values of the FRET phenomenon are respectively shown in FIGS. 11A to 11E. Further, regarding (F), FIG. 11F shows a ratio image before and after the addition of Nutlin-3 and a graph regarding the signal value of the FRET phenomenon, respectively.
  • the signal value (MR / AG ratio) of the FRET phenomenon before the addition of Nutlin-3 was high centering on the fluorescent bright spot, and after the addition, it decreased while diffusing to the whole cell. That is, it was confirmed that the energy transfer efficiency between the tetrameric fluorescent proteins (Mmj and MR) was reduced and the FRET phenomenon was reduced by the inhibition of the interaction between p53 and MDM2.
  • pmTOR-mKO1 ⁇ Preparation of pmTOR-mKO1>
  • the region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI.
  • phmKO1-MNL manufactured by Medical Biology Research Institute, Inc., code number: AM-V0050M
  • the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-mKO1) for expressing mTOR (mTOR-mKO1) in which mKO1 was fused to C-terminal.
  • pmTOR-mKO2 ⁇ Preparation of pmTOR-mKO2>
  • the region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI.
  • phmKO2-MNL manufactured by Medical Biology Research Institute, Inc., code number: AM-V0140M
  • the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-mKO2) for expressing mTOR (mTOR-mKO2) in which mKO2 was fused to C-terminal.
  • a nucleotide sequence (SEQ ID NO: 55) was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 ′ end and the 3 ′ end of the nucleotide sequence encoding KO1, respectively. Then, DNA consisting of the nucleotide sequence is artificially synthesized, cleaved with AgeI and XbaI, treated with the same restriction enzyme combination, and inserted into pmTOR-AG from which the region encoding AG has been removed, thereby KO1 is inserted.
  • a plasmid vector (pmTOR-KO1) for expressing mTOR (mTOR-KO1) fused to the C end was prepared.
  • pmTOR-mKeima ⁇ Preparation of pmTOR-mKeima>
  • the region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI.
  • phmKeima-Red-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0090M) was treated with the same restriction enzyme combination to cut out the DNA encoding mKeima. Then, the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-mKeima) for expressing mTOR (mTOR-mKeima) in which mKeima was fused to C-terminal.
  • pmTOR-dKeima ⁇ Preparation of pmTOR-dKeima>
  • the region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI.
  • phdKeima-Red-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0100M) was treated with the same restriction enzyme combination to cut out the DNA encoding dKeima. Then, the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-dKeima) for expressing mTOR (mTOR-dKeima) in which dKeima was fused to C-terminal.
  • ⁇ Preparation of pmTOR-dAG (AB)> A nucleotide sequence was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence (SEQ ID NO: 56) encoding dAG (AB), respectively.
  • a DNA consisting of the nucleotide sequence is artificially synthesized, cleaved with AgeI and XbaI, treated with the same restriction enzyme combination, and inserted into pmTOR-AG from which the region encoding AG has been removed, to obtain dAG (
  • a plasmid vector (pmTOR-dAG (AB)) was constructed to express mTOR (mTOR-dAG (AB)) in which AB) was fused to C-terminus.
  • a nucleotide sequence was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence (SEQ ID NO: 57) encoding dAG (AC), respectively.
  • a DNA consisting of the nucleotide sequence is artificially synthesized, cleaved with AgeI and XbaI, treated with the same restriction enzyme combination, and inserted into pmTOR-AG from which the region encoding AG has been removed, to obtain dAG (
  • a plasmid vector (pmTOR-dAG (AC)) was constructed to express mTOR (mTOR-dAG (AC)) in which AC) was fused to C-terminus.
  • a nucleotide sequence (SEQ ID NO: 58) was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence encoding mUkG1, respectively. Then, DNA consisting of the nucleotide sequence is artificially synthesized, cleaved with AgeI and XbaI, treated with the same restriction enzyme combination, and inserted into pmTOR-AG from which the region encoding AG has been removed, mUkG1 can be obtained.
  • a plasmid vector (pmTOR-mUkG1) for expressing mTOR (mTOR-mUkG1) fused to the C end was constructed.
  • pmTOR-mMiCy1 The region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI.
  • phmMiCy1-MNL manufactured by Medical Biology Research Institute, Inc., code number: AM-V0110M
  • the DNA was inserted into the restriction enzyme-treated pmTOR-AG to prepare a plasmid vector (pmTOR-mMiCy1) for expressing mTOR (mTOR-mMiCy1) in which mMiCy1 was fused to C-terminal.
  • a nucleotide sequence (SEQ ID NO: 59) was designed in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence encoding MiCy1, respectively. Then, DNA consisting of the nucleotide sequence is artificially synthesized, cleaved with AgeI and XbaI, treated with the same combination of restriction enzymes, and inserted into pmTOR-AG from which the region encoding AG has been removed, whereby MiCy1 is inserted.
  • a plasmid vector (pmTOR-MiCy1) for expressing mTOR (mTOR-MiCy1) fused to the C end was prepared.
  • ⁇ Preparation of pmTOR-KCy1> A nucleotide sequence (SEQ ID NO: 60) in which an AgeI recognition sequence, a stop codon and an XbaI recognition sequence were added to the 5 'end and the 3' end of the nucleotide sequence encoding KCy1, respectively, was artificially synthesized and cleaved with AgeI and XbaI
  • a plasmid vector for expressing mTOR (mTOR-KCy1) in which KCy1 is fused to C-terminal by treating with the same restriction enzyme combination and inserting into pmTOR-AG from which the AG coding region has been removed pmTOR-KCy1) was produced.
  • the transfected HEK 293 cells were observed using an IX-81 inverted microscope (manufactured by Olympus).
  • mAG1, dAG (AB), dAG (AC), mUkG1, mM iCy1, MiCy1 and KCy1 use BP460-480HQ, BA495 and DM485HQ (U-MGFPHQ, manufactured by Olympus), mKO1
  • mKO2 and KO1 use BP520-540HQ, BA555-600HQ, DM545HQ (FSET-KOHQ, manufactured by Olympus), and for mKeima and dKeima, use 440AF21, 610ALP, 590DRLP (manufactured by Olympus), and for MR , BP 530-550, BA 575IF, and DM 570 (U-MWIG3, manufactured by Olympus Corporation) were used.
  • the objective lens was
  • the fluorescent bright spots formed are used as an index between the target proteins. It is possible to determine the interaction. Furthermore, since the fluorescent protein to be fused is not intrinsically inherent to the cell to be expressed or introduced, there is little risk of disturbing the function of the cell, and protein-protein interaction can be determined.
  • the donor tetrameric fluorescent protein is fused to one of the target proteins and the acceptor tetrameric fluorescent protein is fused to the other and expressed or introduced into cells, a fluorescent luminescent spot is formed,
  • the interaction between the target proteins can also be determined by efficiently detecting the signal of the increased FRET phenomenon via that.
  • the FRET phenomenon can be caused to occur, and the protein-protein interaction can be determined by the signal detection of the FRET phenomenon or the FRET phenomenon. Therefore, the present invention can be suitably used in high throughput screening or the like which requires immobilization of a sample (cell) in order to make detection conditions uniform.
  • the process of image analysis such as a fluorescent luminescent spot
  • detecting a FRET phenomenon the region of the fluorescent bright spot is designated manually or by a program for designating the region on an image such as a computer, and the fluorescence intensity in that region is measured and calculated (although it is necessary to perform image analysis, on the other hand, in the case of detection of the FRET phenomenon, the presence or absence of interaction can be determined without passing through the stage of image analysis.
  • the method of the present invention for determining protein-protein interactions and the like, and the vector or kit to be used in these methods are elucidating various signal transductions in the living body, control of various biological reactions, etc. It is useful in the development of medicines etc. through the elucidation of

Abstract

L'invention concerne un procédé selon lequel l'interaction entre une première protéine et une seconde protéine est jugée par expression à l'intérieur d'une cellule ou par induction dans une cellule d'une première protéine hybride contenant une première protéine et une première protéine fluorescente tétramère, et une seconde protéine hybride contenant une seconde protéine et une protéine fluorescente tétramère distincte de la première protéine fluorescente tétramère, et par détection de points brillants fluorescents dus à l'association de la première et de la seconde protéine hybride dans ladite cellule, ou d'un signal de phénomène de FRET à travers la formation de points brillants fluorescents.
PCT/JP2018/023187 2017-06-19 2018-06-19 Procédé de jugement de l'interaction de protéines mettant en œuvre des protéines fluorescentes WO2018235787A1 (fr)

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CN114380904A (zh) * 2021-12-03 2022-04-22 河南省华隆生物技术有限公司 检测car-t细胞car阳性表达率的荧光融合蛋白及其应用

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