WO2018235787A1

WO2018235787A1 - Method using fluorescent proteins for determining interaction between proteins

Info

Publication number: WO2018235787A1
Application number: PCT/JP2018/023187
Authority: WO
Inventors: 拓渡部; 井上　健; ウリケシワシュル
Original assignee: 株式会社医学生物学研究所
Priority date: 2017-06-19
Filing date: 2018-06-19
Publication date: 2018-12-27
Also published as: JPWO2018235787A1

Abstract

This method determines the interaction between a first protein and a second protein by: introducing into cells, or inducing expression in the cells of, a first fusion protein including the first protein and a first tetramer fluorescent protein, and a second fusion protein including the second protein, and a tetramer fluorescent protein different to the first tetramer fluorescent protein; and detecting fluorescent bright spots generated by the association of the first fusion protein and the second fusion protein in the cells or detecting fluorescence resonance energy transfer (FRET) phenomenon signals via the formation of fluorescent bright spots.

Description

Method of determining protein-protein interaction using fluorescent protein

The present invention relates to a method of determining protein-protein interaction and its application, and a kit for use in the method.

The functions of many proteins are exerted by interactions with other proteins etc., and are deeply involved in the control of a wide variety of life-based systems such as signal transduction, transport, and metabolism. Therefore, analyzing protein-protein interactions is extremely important not only for elucidating protein functions and biological functions, but also for drug development through elucidation of disease mechanisms.

Many methods for analyzing protein-protein interactions have been developed, and various methods such as two-hybrid method, co-immunoprecipitation method, surface plasmon resonance method, and fluorescence resonance energy transfer (FRET) method are used.

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.

However, 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.

Therefore, 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.).

However, when the same tetrameric fluorescent protein (Azami-Green (Azamin green, AG) protein) is fused as both of the proteins capable of multimerization to both of the desired proteins, and expressed in cells, The present inventors have recently found that fluorescent luminescent spots are not formed and can not be used for determination of protein-protein interaction (D in Non-Patent Document 1, Supplemental Figure 5).

International Publication No. 2013/084950

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.

The present inventors examined using combinations of various fluorescent proteins to achieve the above purpose. As a result, as disclosed in 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.

However, when different tetrameric fluorescent proteins are fused to the target protein and expressed in cells, a fluorescent bright spot is formed, and the interaction between the target proteins is determined using the fluorescent bright spot as an index. It was possible to judge.

Further, even when one of the four-mer fluorescent proteins is a donor fluorescent protein and the other is an acceptor fluorescent protein, a 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.

In addition, when the combination of the aforementioned different tetrameric fluorescent proteins is used, 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.

Furthermore, in the case of using a combination of fluorescent proteins which can not form the above-mentioned fluorescent luminescent spots, when cells are immobilized, it becomes impossible to detect even the signal of the FRET phenomenon, while the combination of different tetrameric fluorescent proteins mentioned above It has been found that the signal of the FRET phenomenon can be detected even in the immobilized cells and the degree of protein-protein interaction to be detected can be analyzed with high sensitivity, and the present invention has been completed.

Therefore, 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.
<1> 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.
<2> 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 When 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. The method according to <1> or <2>, wherein an amino acid residue is determined to be involved in the interaction.
<5> 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 in the absence of the test compound, the test compound is selected as an inducer of the interaction, and the fluorescent spot is selected. A step of selecting the test compound as an inhibitor of the interaction, if the intensity of the light is attenuated more than the intensity of the fluorescent luminescent spot generated in the absence of the test compound.
<6> 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 A step of causing the first fusion protein containing the body fluorescent protein and the second fusion protein containing the second protein and the second tetrameric fluorescent protein to be expressed in cells or introduced into cells, 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 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;
(3) When the strength of the FRET phenomenon is higher than the strength of the FRET phenomenon generated in the absence of the test compound, the test compound is selected as an inducer of the interaction, and the strength of the FRET phenomenon is selected. And a step of selecting the test compound as an inhibitor of the interaction, when the intensity of the FRET phenomenon which occurs in the absence of the test compound is attenuated.
<7> The method according to any one of <1> to <6>, wherein the cell is a fixed cell.
<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 encoding a first fusion protein and a vector encoding a second fusion protein (i) First fusion protein (j) Second fusion protein (k 2.) A protein set comprising a first fusion protein and a second fusion protein.

According to the present invention, when the fluorescent protein is fused to both of the target proteins, and expressed or introduced in cells, 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.

In addition, when the donor tetrameric fluorescent protein is fused to one of the target proteins and the acceptor tetrameric fluorescent protein is fused to the other to be expressed or introduced into cells, 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. Furthermore, according to the present invention, since 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. Before and after addition of Nutlin-3, which is an inhibitor of the protein-protein interaction between p53 and MDM2, observe COS7 cells in which p53 and MDM2 were fused with different tetrameric fluorescent proteins (AG and Mmj) and expressed respectively Are fluorescence micrographs showing the results. It is a fluorescence-microscope photograph which shows the result of having observed the HEK293 cell which fused different tetramer fluorescent protein (AG, Mmj or MR) to p53 and MDM2 and expressed it before and behind addition of Nutlin-3. 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 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. It is a graph which shows the result of having analyzed the HEK293 cell which made AG-MDM2 and MR-p53 express 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 A pseudo-color image, each fluorescence showing the result of analyzing the efficiency of the FRET phenomenon in HEK293 cells in which different tetrameric fluorescent proteins (AG or MR) are fused and expressed to p53 and MDM2 by the acceptor photobleaching method It is the enlarged view of a luminescent point, and a graph. Before and after the addition of Nutlin-3, 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 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. It is a graph which shows the result of having analyzed the efficiency of the FRET phenomenon before and behind addition of Nutlin-3 about HEK293 cells which made MR-p53 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 Before and after the addition of Nutlin-3, HEK293 cells expressing MR-p53 and mAG1-MDM2 are analyzed by an image showing fluorescence intensity of mAG1 and MR and a Ratio image showing efficiency of 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. Analysis of signal and mAG1 / AG of FRET phenomenon in HEK293 cells expressing MR-p53 and AG-MDM2 and HEK293 cells expressing MR-p53 and mAG1-MDM2 before and after the addition of Nutlin-3 It is a graph which shows the result. In HEK293 cells expressing p53-MR and AG-MDM2, and in HEK293 cells expressing p53-MR and mAG1-MDM2, the signal of FRET phenomenon before the addition of Nutlin-3 is after the addition of Nutlin-3 It is a graph represented as a ratio to it. In HEK293 cells expressing MR-p53 and AG-MDM2, and in HEK293 cells expressing MR-p53 and mAG1-MDM2, the signal of FRET phenomenon before the addition of Nutlin-3 is after the addition of Nutlin-3 It is a graph represented as a ratio to it. HEK293 cells expressing p53-MR and AG-MDM2 (p53-MR / AG-MDM2) in the absence or presence of Nutlin-3, and 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 These are images showing fluorescence intensity of AG or mAG1 and MR after immobilizing MR-p53 / mAG1-MDM2) with paraformaldehyde and Ratio images showing the efficiency of the FRET phenomenon. Immobilized by paraformaldehyde in p53-MR / AG-MDM2, p53-MR / mAG1-MDM2, MR-p53 / AG-MDM2 and MR-p53 / mAG1-MDM2 in the absence or presence of Nutlin-3 It is a graph which shows the result of having analyzed the efficiency of the later FRET phenomenon. It is a fluorescence-microscope photograph which shows the result of having observed the cell which fused different tetrameric fluorescent protein (AG and MR) to detection object (MAX and Myc), respectively, and was made to express it. It is a fluorescence-microscope photograph which shows the result of having analyzed the cell which made each detection object (MAX and Myc) fuse | diffuse different tetrameric fluorescent protein (AG and MR), and was expressed by the acceptor photobleaching method. It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-BAK and AG-MCL1 express before and 5 minutes after addition of BAK and MCL1 interaction inhibitor (A-1210477) addition. "MR-BAK" represents a protein obtained by fusing tetramer fluorescent protein MR to N terminal of BAK, and "AG-MCL1" fuses tetrameric fluorescent protein AG to N terminal of MCL1. Represents a protein that Before and after addition of A-1210477, time course (upper in the figure) of each fluorescence intensity derived from MR and AG in cells expressing MR-BAK and AG-MCL1 and MR / AG ratio (Em: 610) It is a graph which shows time progress (in the figure, lower stage) of / Em: 510). In the upper graph, the dotted line located above indicates the fluorescence intensity (Em: 510) derived from AG, and the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR. In addition, the transition of the value which standardized the ratio of the fluorescence intensity or fluorescence intensity 60 seconds before addition of A-12 10477 as 1 respectively is shown. 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. 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-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). In the upper graph, the dotted line located above indicates the fluorescence intensity (Em: 510) derived from mAG1, and the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR. It is a graph which shows the result of having compared the Em: 610 / Em: 510 in the cell which made MR-BAK and mAG1-MCL1 express, and the cell in which MR-BAK and AG-MCL1 were made to express. 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. Before and after addition of A-1210477, the 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). In the upper graph, the dotted line located above indicates the fluorescence intensity (Em: 510) derived from AG, and 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). In the upper graph, the dotted line located above indicates the fluorescence intensity (Em: 510) derived from mAG1, and the dotted line located below indicates the fluorescence intensity (Em: 610) derived from MR. It is a graph which shows the result of having compared the Em: 610 / Em: 510 in the cell which made MR-BAX and mAG1-MCL1 express, and the cell in which MR-BAX and AG-MCL1 were made to express. 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, and "AG-FKBP12" fuses the tetrameric fluorescent protein AG to the N-terminal of FKBP12 Represents a protein that Time course (upper in the figure) of each fluorescence intensity derived from MR and AG in cells expressing MR-mTOR and AG-FKBP12 before and after addition of Rapamycin, and MR / AG ratio (Em: 610 / Em It is a graph which shows time progress (in the figure, lower stage) of: 510). In the upper graph, the dotted line at the top indicates the fluorescence intensity from MR (Em: 610), and the dotted line at the bottom indicates the fluorescence intensity from AG (Em: 510). It is a Ratio image which shows the efficiency of the FRET phenomenon in the cell which made MR-mTOR and mAG1-FKBP12 express before and 5 minutes after addition of Rapamycin. “MAG1-FKBP12” represents a protein obtained by fusing the monomeric fluorescent protein mAG1 to the N-terminus of FKBP12. Time course (upper in the figure) of each fluorescence intensity derived from MR and mAG1 in cells expressing MR-mTOR and mAG1-FKBP12 before and after addition of Rapamycin, and MR / mAG1 ratio (Em: 610 / Em It is a graph which shows time progress (in the figure, lower stage) of: 510). In the upper graph, the dotted line located at the top indicates the fluorescence intensity (Em: 610) derived from MR, and the dotted line located at the bottom indicates the fluorescence intensity derived from mAG1 (Em: 510). It is a graph which shows the result of having compared the Em: 610 / Em: 510 in the cell which made MR-mTOR and mAG1-FKBP12 express, and the cell in which MR-mTOR and AG-FKBP12 were made to express. Among living cells expressing MR-BAK and AG-MCL1 ((A) on the left side in the figure) and living cells expressing MR-BAK and mAG1-MCL1 ((B) on the right side in the figure) 3 is a graph showing the relationship between the signal of the FRET phenomenon (in each graph vertical axis) and the concentration of A-1210477 (in each graph horizontal axis). Cells expressing and immobilized MR-BAK and AG-MCL1 (in the figure (A) on the left) and cells expressing and immobilized MR-BAK and mAG1-MCL1 (in the figure (B) on the right) And a graph showing the relationship between the signal of the FRET phenomenon (in each graph vertical axis) and the concentration of A-1210477 (in each graph horizontal axis). Among living cells expressing MR-mTOR and AG-FKBP12 (in the figure, left (A)) and living cells expressing MR-mTOR and mAG1-FKBP12 (in the figure, right (B)) 3 is a graph showing the relationship between the signal of FRET phenomenon (in each graph vertical axis) and the concentration of Rapamycin (in each graph horizontal axis). Cells expressing and immobilized MR-mTOR and AG-FKBP12 (left (A) in the figure) and cells expressing and immobilized MR-mTOR and mAG1-FKBP12 (right (B) in the figure) And a graph showing the relationship between the signal of the FRET phenomenon (in each graph vertical axis) and the concentration of Rapamycin (in each graph horizontal axis). In the cells expressing MR-BAK and AG-MCL1, the concentration of A-1210477 and “sum of fluorescence intensities of AG at fluorescent spots in the field / total number of cells expressing AG in the field” In the figure, it is a graph showing the relationship with “the total of the fluorescence intensity of MR at the fluorescent light spot in the field of view / the total number of MR expressing cells in the field of view” (right side in the figure). In the cells expressing MR-BAX and AG-MCL1, the concentration of A-1210477 and “sum of fluorescence intensities of AG at fluorescent spots in the field / total number of cells expressing AG in the field” In the figure, it is a graph showing the relationship with “the total of the fluorescence intensity of MR at the fluorescent light spot in the field of view / the total number of MR expressing cells in the field of view” (right side in the figure). The ratio image (upper stage) which shows the efficiency of the FRET phenomenon in the cell which made mTOR-DsRed2 and AG-FKBP12 express before the rapamycin addition addition 10 minutes after addition is shown, and the graph which shows the signal of the FRET phenomenon (lower stage) . "MTOR-DsRed2" represents a protein obtained by fusing the tetrameric fluorescent protein DsRed2 to the C-terminus of mTOR. A ratio image (upper row) showing the efficiency of the FRET phenomenon and a graph (lower row) showing the signal of the FRET phenomenon in cells expressing FKBP12-AG and mTOR-COR5 before and 10 minutes after addition of rapamycin . "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, and "mTOR-KikGR1" fuses the tetrameric fluorescent protein KikGR1 to the C-terminal of mTOR 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 Indicates "Mmj-p53" represents a protein obtained by fusing the tetrameric fluorescent protein Momiji (Mmj) to the N-terminal of p53, and "MR-MDM2" represents the tetrameric fluorescent protein MR at the N-terminal of MDM2. Represents a protein formed by fusing It is a fluorescence microscope picture which shows the result of having observed the cell which made AG-FKBP12 and mTOR-mKO1 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 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. It is a fluorescence microscope picture which shows the result of having observed the cell which made AG-FKBP12 and mTOR-KO1 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 KO1. “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, and 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, and 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. It is a fluorescence-micrograph which shows the result of having observed the cell which made MR-FKBP12 and mTOR-mMiCy1 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 mMiCy1-derived fluorescence. “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. 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 MiCy1-derived fluorescence. "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.

Hereinafter, the present invention will be described in detail in line with its preferred embodiments. Further, with regard to the matters described in the following items, the same applies to other items etc. throughout the present invention, as a matter of course, unless otherwise specified.

<Method for Determining Protein-Protein Interaction>
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. Expressing or introducing the fusion protein of 2 into a cell (2): detecting a fluorescent luminescent spot produced by the association of the first fusion protein with the second fusion protein in the cell (3) It is a method including the step of determining the interaction between the first protein and the second protein by detecting the fluorescent spots.

In the present invention, "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.

As the "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” according to the present invention 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.

In Tables 1 to 4, the excitation wavelength and fluorescence wavelength of each fluorescent protein, and a typical sequence (sequence specified by SEQ ID NO) are also shown together.

Also, the 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). 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.

In the present invention, the "first tetrameric fluorescent protein" and the "second tetrameric fluorescent protein" are different proteins. Here, “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.

Further, in the present invention, as described later, the protein-protein interaction can be determined by detecting the FRET phenomenon through the formation of a fluorescent bright spot. From such a viewpoint, it is preferable that 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.

In the "first fusion protein" or the "second fusion protein" according to the present invention, 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. In addition, 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. There is no restriction | limiting in particular as another functional protein, According to the function to give to the fusion protein concerning this invention, it selects suitably. For example, 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. In addition, 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) And the DNA encoding the second fusion protein may be introduced into cells of a transgenic animal). Moreover, although the fusion protein concerning this invention will be normally introduce | transduced or expressed to the same cell, it may be a different cell. And, in such a case, it is possible to determine protein-protein interactions between different cells, such as interactions between cytokines and receptors, interactions between receptors, etc.

Further, as shown in the examples described later, according to the present invention, even between immobilized cells, protein-protein interactions can be detected. In the present invention, "immobilization" means to stop the dynamic change that usually occurs in all or part of the structure in cells. Further, 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. Furthermore, such 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. As 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. For insect cells, a method using baculovirus is mentioned. Further, for plant cells, the Agrobacterium method, the electroporation method, the particle gun method and the like can be mentioned. For yeast, the lithium acetate method, electroporation method and spheroplast method can be mentioned. Furthermore, for E. coli, a heat shock method (for example, calcium chloride method, rubidium chloride method), electroporation method and the like can be mentioned.

Further, in the present invention, 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 μm (usually 0.2 to 10 μm) 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 μm (usually 0.2 to 10 μm), 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. As such 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. A detection device of

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). In the detection of such fluorescent luminescent spots, 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.

For example, the 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) (the condition when all sensitivity is set to 100) "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.

Further, as shown in the examples described later, in the present invention, when 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 It becomes possible to efficiently detect the FRET phenomenon generated via the fluorescent luminescent spots formed by these fluorescent proteins. Therefore, in the present invention, the determination of protein-protein interaction can also be performed by “detection of FRET phenomenon through formation of a fluorescent bright spot”.

In the present invention, 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. (Re-absorption, pseudo-FRET, lecture and training live cell fluorescence imaging G Osaka University / Hokkaido University Microscope Course Book Practical Course: Fluorescence Microscopy of Living Cells (Page 127) October 25, 2007 First edition issued 1st publisher issued by Mitsujo Nanjo Publication office (refer to Kyoritsu Publishing Co., Ltd.).

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.

And, in the method of the present invention, if 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.

<Method for obtaining time information etc. of protein-protein interaction>
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.

Therefore, according to 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.

With regard to the detection of such “occurrence or loss of protein-protein interaction”, in the present invention, the intracellular region in which the protein-protein interaction takes place can also be identified.

Further, according to the present invention, through detection of such "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.

In addition, even if the interaction between the first protein and the second protein occurs or disappears in response to a particular stimulus, it can be detected in the present invention. Therefore, 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” according to the present invention 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) .

In addition, in such a method of the present invention, 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.

Furthermore, in 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). Thus, if the particular stimulus is a drug, 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.

Furthermore, in the method of the present invention, in the same cell, many types of protein-protein interactions, many types of protein-protein interactions depending on specific stimuli, and thus signal transduction involving these protein-protein interactions , Can be determined and detected.

<Method of screening for proteins that interact with specific proteins>
In the present invention, any protein-protein interaction can be detected. Therefore, 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.

The "test protein" according to the present invention 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>
In the present invention, 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” according to the present invention 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

In the present invention, when either 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 above determination can be performed using “the intensity of the FRET phenomenon through“ as an index. In this case, 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. Similarly, 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.

As “the efficiency of the FRET phenomenon”, a value usually calculated by E = 1− (F _D ′ / F _D ) can be used. Here, F _D indicates the fluorescence intensity of the donor fluorescent protein in the absence of the acceptor fluorescent protein, and F _D ′ indicates the fluorescence intensity of the donor fluorescent protein in the presence of the acceptor fluorescent protein.

In the case of using the acceptor photobleaching method as “the efficiency of the FRET phenomenon”, as shown in the examples below, E = ((Donorpost-Backpost)-(Donorpre-Backpre)) / (Donorpost-Backpost) The value calculated by can be used. Here, 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, and 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.

<Method of screening for substance that regulates protein-protein interaction>
In the method of the present invention, the strength of the protein-protein interaction can be grasped by using the intensity of the fluorescent spot as an index. Therefore, according to the present invention, (1) 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. 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.

Further, in the present invention, as described above, 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 is selected as an inducer of the interaction, and the intensity of the FRET phenomenon occurs in the absence of the test compound. And a step of selecting the test compound as an inhibitor of the interaction, in the case of attenuating due to the strength of the phenomenon.

The 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.

Further, 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.

<Kit for Use in the Method of the Present Invention>
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.

(A) a cloning site which allows insertion of DNA encoding any protein so as to be expressed fused to the first tetrameric fluorescent protein and the first tetrameric fluorescent protein And (b) a DNA encoding a second tetrameric fluorescent protein, and an insertion of a DNA encoding any protein so as to be expressed by fusing with a second tetrameric fluorescent protein (C) a vector encoding the first fusion protein (d) a vector encoding the second fusion protein (e) The vector according to (a) or (c) and (b) Or (d) a vector set comprising the vector according to (d) (f) a transformed cell carrying a vector encoding the first fusion protein (g) a second fusion protein (H) A transformed cell containing a vector encoding a first fusion protein and a vector encoding a second fusion protein (i) A first fusion protein (j) Second fusion protein (k) A set of proteins comprising a first fusion protein and a second fusion protein.

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. Such control sequences include promoters, enhancers, silencers, terminators, poly A tails, ribosome binding sequences (Shine-Dalgano (SD) sequences). Furthermore, the vector according to the present invention may contain a selection marker (drug resistance gene etc.) and a reporter gene (luciferase gene, β-galactosidase gene, chloramphenicol acetyltransferase (CAT) gene etc.). In addition, examples of such a vector according to the present invention include a plasmid vector, an episomal vector, and a virus vector.

As described above, 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 In order to further improve the efficiency of the DNA, 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. .

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. In addition, 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.

Hereinafter, the present invention will be more specifically described based on examples and comparative examples, but the present invention is not limited to the following examples.

(Example 1) Detection of protein-protein interaction 1
The present inventors previously fused desired proteins (a first protein and a second protein) with proteins having multimerization ability (association-inducing protein and fluorescent protein having multimerization ability), respectively, in cells. It has been found that, when expressed, the interaction between the desired proteins can be detected by detecting fluorescent luminescent spots produced by the association of these fusion proteins (Patent Document 1).

However, in such a method for detecting protein-protein interaction, 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).

Therefore, in the method for detecting protein-protein interaction using a protein having multimerization ability, conditions capable of detecting the interaction between these proteins, even when fluorescent proteins are fused and expressed on both of the desired proteins. The screening was attempted by the method described below.

The target of detection in this screening is the interaction between p53 protein and MDM2 protein. In addition, 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).

Further, as 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.

<Preparation of 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.

<Preparation of pAG-MDM2>
As a plasmid vector (pAG-MDM2) for expressing MDM2 (AG-MDM2) in which AG was fused to the N-terminal, the one contained in the kit was used.

<Preparation of pAG-p53>
The region encoding AG was excised by treating pAG-MDM2 with restriction enzymes NheI and AgeI. Then, 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.

<Preparation of pMmj-MDM2>
The region encoding AG was cleaved off by treating pAG-MDM2 with restriction enzymes NheI and AgeI. Also, DNA (NheI-Mmj-AgeI, SEQ ID NO: 44) encoding Mmj 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 MDM2 (Mmj-MDM2) fused to Mmj at the N-terminus. A plasmid vector (pMmj-MDM2) was prepared.

<Preparation of pMmj-p53>
After cutting off the region encoding AG by treating pAG-p53 with restriction enzymes NheI and AgeI, 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.

<Preparation of pEGFP-p53>
The region encoding AG was cleaved off by treating pAG-p53 with restriction enzymes NheI and AgeI. In addition, DNA (NheI-EGFP-AgeI, SEQ ID NO: 45) 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. In addition, 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.

<Cell culture and gene transfer>
In order to introduce the plasmid vector prepared as described above, first, 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.). 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 μL per well.

Then, 100 ng of the above plasmid vector was mixed with 10 μL of Opti-MEM (manufactured by Thermo Fisher Scientific) in the combination of (A) to (I) shown below to prepare a DNA solution. 0.8 μL of a gene transfer reagent (Promega, product name: Fugene (registered trademark) HD) is added to the DNA solution and stirred, and then mixed with 90 μL of the culture solution, and then added to the COS 7 cells for 28 hours Cultured.
(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.

<Observation of cells>
As described above, COS7 cells that have been subjected to gene transfer treatment are IX-81 inverted microscope (manufactured by Olympus), U-MNIBA3 filter (manufactured by Olympus), UPlanApox20 (NA: = 0.7) (Olympus) And an ORCA-Flash 4.0 digital camera (manufactured by Hamamatsu Photonics) (excitation wavelength: 470 to 495 nm, fluorescence observation wavelength: 510 to 550 nm). The results obtained are shown in FIGS. 1A-1C. Moreover, the interaction inhibitor p53 and MDM2 inhibitor Nutlin-3 was added to the culture solution to a final concentration of 20 μM, 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.

As is clear from the results shown in FIG. 1A, in the COS7 cells in which the monomeric fluorescent proteins mAG1 or mEGFP are fused and expressed on one of the detection targets (p53 and MDM2), protein-protein interaction is exhibited. No fluorescent spots were identified. Further, although not shown in the figure, even in COS7 cells in which EGFP, which is a dimeric fluorescent protein, is fused and expressed to one of detection targets, a fluorescent bright spot indicating protein-protein interaction was not confirmed . Furthermore, in the case of COS7 cells in which the same tetrameric fluorescent protein AG or Mmj is fused and expressed to both of the detection targets, the results shown in d of Supplementary Figure 5 of Non-Patent Document 1 are also obtained. However, fluorescent spots showing protein-protein interaction were not confirmed (see FIG. 1B).

However, as is clear from the results shown in FIG. 1C, clear fluorescent spots showing protein-protein interaction were observed in COS7 cells in which different tetrameric fluorescent proteins were fused and expressed to be detected. . On the other hand, as shown in FIG. 1D, in the presence of Nutlin-3, which is an inhibitor of the protein-protein interaction between p53 and MDM2, the fluorescent luminescent spot showing these protein-protein interactions was resolved, so It was confirmed that the protein was formed depending on the protein-protein interaction.

As described above, when different tetrameric fluorescent proteins are fused to the detection target and expressed in cells, it is clear that the fluorescent bright spots are formed depending on the protein-protein interaction between the detection targets. became.

(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).

<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. Next, 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.

<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) A plasmid vector (pMR-MDM2) for expressing -MDM2 was constructed.

<Preparation of pMR-p53>
The region encoding p53 was excised by treating pAG-p53 with restriction enzymes BamHI and NotI. Subsequently, a plasmid vector (pMR-p53) for expressing p53 (MR-p53) fused to MR at the N-terminus is prepared by inserting into pMonti-Red-MCL treated with the same restriction enzyme combination. did.

<Cell culture and gene transfer>
In order to introduce the plasmid vector prepared as described above, first, 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 μL per well.

Then, 200 ng of the above plasmid vector was mixed with 10 μL of Opti-MEM (manufactured by Thermo Fisher Scientific) in the combination of (A) to (G) shown below to prepare a DNA solution. 0.8 μL of a gene transfer reagent (Fugene HD) was added to the DNA solution and stirred, and then mixed with 90 μL of a culture solution, and then added to the HEK293 cells and cultured for 48 hours.
(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.

<Observation of cells>
Observation of HEK 293 cells subjected to gene transfer treatment was performed in the same manner as in the case of the above-mentioned COS 7 cells. Images before and 15 minutes after addition of Nutlin-3 are shown in FIG.

Although not shown in the figure, 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 In HEK 293 cells fused and expressed with the fluorescent protein (mAG1), as in the case of the above-mentioned COS 7 cells, no clear fluorescent spot indicating protein-protein interaction was observed. Moreover, no change occurred when Nutlin-3 was added to these cells.

On the other hand, as is clear from the results shown in FIG. 2, as in the case of the above-mentioned COS7 cells, in HEK293 cells in which different tetrameric fluorescent proteins are fused and expressed to be detected, protein-protein interaction is exhibited. Clear fluorescent spots were observed. In addition, since the fluorescent luminescent spot showing the protein-protein interaction was resolved in the presence of Nutlin-3, which is an inhibitor of the protein-protein interaction between p53 and MDM2, the bright spot corresponds to the protein-protein interaction. It was confirmed that they were formed dependently (see the lower 3 images in FIG. 2).

From the above results, when different tetrameric fluorescent proteins are fused to the detection target and expressed in cells, regardless of the type of the cells, the interproteins are determined using the presence or absence of the fluorescent bright spot as an index. It became clear that the action could be determined.

Also, from the results shown in Examples 1 and 2, if the tetrameric fluorescent proteins to be fused to the detection target are different, the fluorescence wavelengths of these fluorescent proteins (wavelength of green region, wavelength of red region, converted fluorescence) It was also found that fluorescent luminescent spots were formed depending on the interaction between detection targets regardless of the wavelength).

Example 3 Application to a Method of Determining Protein-Protein Interaction Using FRET As described above, according to the method of determining protein-protein interaction of the present invention, 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.

<Preparation of pp53-MR>
First, 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). 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 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.

<Cell culture and gene transfer>
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.
(A) pp53-MR + pAG-MDM2
(B) pMR-p53 + pAG-MDM2.

<Observation of cells>
In detection of the FRET phenomenon of HEK293 cells subjected to gene transfer treatment, IX-81 inverted microscope (manufactured by Olympus), excitation filter 440 AF21 (manufactured by Omega, model number: XF1071), dichroic mirror 455 DRLP (manufactured by Omega, model number: XF2034) And fluorescent filters 510WB40 (manufactured by Omega, model number: XF3043) and 610ALP (manufactured by Omega, model number: XF3094). In addition, the HEK293 cells were observed using UPlan FLN 40 × Oil (NA = 1.3) (manufactured by Olympus), ORCA-Flash 4.0 digital camera (manufactured by Hamamatsu Photonics).

<Detection of FRET phenomenon>
Excitation was done with 440 AF21 and fluorescence on the donor side (AG) was detected by 510 WB 40 (Em: 510) and fluorescence on the acceptor side (MR) by 610 ALP (Em: 610). In addition, Nutlin-3 as an interaction inhibitor of p53 and MDM2 was added to the culture solution of HEK 293 cells to a final concentration of 20 μM, and image data was acquired at intervals of 10 seconds before and after the addition of the drug. Further, 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.

In addition, 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.

As apparent from the results shown in FIGS. 3A to 3D, in cells expressing p53-MR or MR-p53 and AG-MDM2, 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.

In addition, as shown in FIG. 3E, in cells expressing p53-MR and AG-MDM2, after fading (after irradiation with 530 to 550 nm excitation light), the MR fluorescence intensity at the fluorescent bright spot decreased to an average of 28% (N = 7). On the other hand, the fluorescence intensity of AG rose to 128% on average. In addition, the efficiency of the 22.1% FRET phenomenon was calculated from each fluorescence intensity.

Thus, it was revealed that the fluorescence intensity of AG was increased by fading the acceptor side (MR). Furthermore, since the efficiency of 22.1% of the FRET phenomenon was recognized, in the case where a fluorescent bright spot is formed by fusing and expressing different tetrameric fluorescent proteins (MR and AG) to the detection target, It has become clear that the FRET phenomenon can occur.

(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).

<Cell culture and gene transfer>
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.
(A) pp53-MR + pAG-MDM2
(B) pp53-MR + pmAG1-MDM2
(C) pMR-p53 + pAG-MDM2
(D) pMR-p53 + pmAG1-MDM2.

<Cell observation and detection of FRET phenomenon>
According to the method described in Example 3 above, observation of HEK 293 cells subjected to the gene transfer treatment and detection of the FRET phenomenon were performed. 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. 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. Were plotted, and in MR-p53 and AG-MDM2 and MR-p53 and mAG1-MDM2, the average values of the MR / AG ratio and the MR / mAG1 ratio in each of eight cells were plotted. Also, error bars represent standard errors.

Similar to the results shown in FIGS. 3A-3D, as shown in FIGS. 4A, 4B, 4E and 4F, in cells expressing p53-MR or MR-p53 and AG-MDM2, 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

Furthermore, as shown in FIGS. 4C, 4D, 4G and 4H, even in cells expressing p53-MR or MR-p53 and mAG1-MDM2, 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.

However, as apparent from the results shown in FIG. 4I, 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.

In addition, as shown in FIG. 4J, 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. Between 0 and 600 seconds after the addition of Nutlin-3, 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.

Furthermore, as shown in FIGS. 4K and 4L, assuming that each ratio at a point of 600 seconds after the addition of the inhibitor (when the FRET phenomenon is not occurring) is 1, the MR / before the addition of the inhibitor (when the FRET phenomenon is occurring) As a result of respectively calculating the AG ratio and the MR / mAG1 ratio, it became clear that the MR / AG ratio becomes 1.2 to 1.4 times higher than the MR / mAG1 ratio.

As described above, when the detection target interacts with proteins, 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.

Next, even if the cells were fixed, it was verified as shown below whether or not the fluorescent luminescent spots showing the interaction between the detection targets were maintained while being generated and furthermore the FRET phenomenon could also be detected. In addition to the combination of tetrameric fluorescent proteins (AG and MR) that form fluorescent spots when the detection targets interact, the combination of fluorescent proteins that do not form fluorescent spots (mAG1 and We also verified the case of using MR).

<Cell culture and gene transfer>
According to the method described in Example 2 above, HEK293 cells into which the plasmid vector was introduced by the combination of (A) to (D) shown below were prepared for each two wells.
(A) pp53-MR + pAG-MDM2
(B) pp53-MR + pmAG1-MDM2
(C) pMR-p53 + pAG-MDM2
(D) pMR-p53 + pmAG1-MDM2.

<Fixation of cells>
After culturing for 48 hours after gene transfer treatment, the whole culture solution was removed from one well of each of two wells (A) to (D), and 200 μL of 4% paraformaldehyde was added. After leaving still for 15 minutes at room temperature, 200 μL of HBSS containing 20 mM HEPES was added except for the total amount of paraformaldehyde. On the other hand, to each remaining 1 well, Nutlin-3 was added to a final concentration of 20 μM as an inhibitor of the interaction between p53 and MDM2. After 15 minutes, 200 μL of 4% paraformaldehyde was added except for the whole culture solution. After leaving still for 15 minutes at room temperature, 200 μL of HBSS containing 20 mM HEPES was added except for the total amount of paraformaldehyde.

<Observation of cells and detection of FRET phenomenon>
The HEK 293 cells subjected to gene transfer and fixation were observed by the method described in Example 3 and the FRET phenomenon was detected. 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.

As shown in (A) of FIG. 5A and (C) of FIG. 5B, in the cells expressing p53-MR or MR-p53 and AG-MDM2, the MR / AG ratio was increased centering on the fluorescent spot. On the other hand, it is decreased in the cells to which Nutlin-3 has been added, and the same results as in FIGS. 4A and C were obtained when the cells were immobilized.

On the other hand, in (B) of FIG. 5A and (D) of FIG. 5, unlike the results shown in FIG. 4C, 4D, 4G and 4H, cells expressing p53-MR or MR-p53 and mAG1-MDM2 On the other hand, by immobilizing the cells, a large difference in the MR / mAG1 ratio was not generated in addition / non-addition of Nutlin-3.

Further, as apparent from the results shown in FIG. 5C, in cells expressing p53-MR or MR-p53 and AG-MDM2, Nutlin-3 was added compared to the case where Nutlin-3 was added. The signal of the FRET phenomenon in the case of not being detected was detected at high value.

On the other hand, in cells expressing p53-MR or MR-p53 and mAG1-MDM2, no difference in the signal of the FRET phenomenon was observed depending on the presence or absence of Nutlin-3.

As described above, when a combination of fluorescent proteins that can not form a fluorescent luminescent spot when the detection target interacts with proteins is used, the FRET phenomenon can not be detected when the cells are immobilized. On the other hand, it becomes clear that 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. The

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.

<Preparation of pMR-Myc>
First, a nucleotide having a BamHI recognition sequence at the 5 'end of the DNA encoding the N-terminus of c-Myc (the region consisting of 383 to 454 amino acids of c-Myc protein) and a stop codon TAA and NotI recognition sequence at the 3' end. The sequence (SEQ ID NO: 47) was 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-Myc) for expressing Myc (MR-Myc) in which MR was fused to N-terminal was prepared.

<Preparation of pAG-Max>
First, 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.

Then, 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) A plasmid vector (pAG-Max) for expressing Max (AG-Max) in which AG was fused to N-terminal was constructed by inserting into.

<Cell culture and gene transfer>
In order to introduce the plasmid vector prepared as described above, 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 μL of Opti-MEM (manufactured by Thermo Fisher Scientific) to prepare a DNA solution. Separately, 0.2 μL of a gene transfer reagent Transficient (manufactured by MBL International) was added to 10 μL of OptiMEM, and the mixture was stirred to prepare a Transficient solution. Then, it was mixed with 10 μL of the DNA solution, reacted at room temperature for 20 minutes, added to the HEK293T cells, immediately centrifuged at 300 G, and the supernatant was replaced with the medium described in Example 2 and cultured for 48 hours.

<Observation of cells>
The observation of HEK293T cells subjected to the gene transfer treatment was carried out using Incell Analyzer 2200 (manufactured by GE) to detect green fluorescence from AG and red fluorescence from MR in the same field of view. The obtained results are shown in FIG. 6A. The cells were also observed using a fluorescence microscope ECLIPS TS100 (manufactured by NIKON). The obtained results are shown in FIG. 6B.

As is clear from the results shown in FIG. 6A, a protein (AG-MAX) in which AG is fused to the N-terminus of MAX and a protein (MR-Myc) in which MR is fused to the N-terminus of Myc are Even when they were expressed inside, fluorescent spots were detected. That is, when different tetrameric fluorescent proteins were fused and expressed in the detection target, it was confirmed that the presence or absence of protein-protein interaction can be determined using the fluorescent bright spot as an index regardless of the type of the detection target. .

In addition, in the microscopic image of cells in which AG-MAX and MR-Myc were expressed, as a result of fading the fluorescence of MR in a part of the region, as shown in FIG. 6B, the acceptor side (MR) was faded. The increase of the fluorescence intensity of AG was recognized by. That is, it was confirmed that the FRET phenomenon occurred.

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

In 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).

<Preparation of pAG-MCL1>
First, 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.

<Preparation of 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. By inserting, a plasmid vector (pmAG1-MCL1) for expressing MCL1 (mAG1-MCL1) in which mAG1 was fused to the N-terminal was prepared.

<Preparation of pMR-BAK>
First, 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.

<Preparation of pMR-BAX>
First, 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.

<Cell culture, gene transfer and observation of cells>
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.
(A) pMR-BAK + pAG-MCL1
(B) pMR-BAK + pmAG1-MCL1
(C) pMR-BAX + pAG-MCL1
(D) pMR-BAX + pmAG1-MCL1
Then, before observation, the whole culture solution is removed from each well, 200 μL of HBSS containing 20 mM HEPES is added, and HEK293 cells subjected to the gene transfer treatment are observed by the method described in <Observation of cells> in Example 1. did.

<Detection of FRET phenomenon>
In the detection of FRET phenomenon, A-1210477 was added to a culture solution of HEK 293 cells to a final concentration of 25 μM as an inhibitor of interaction between MCL1 and BAK or MCL1 and BAX instead of Nutlin-3. Except for the above, the method described in Example 3 was performed.

Regarding the obtained results, regarding the cells in which MR-BAK and AG-MCL1 were expressed, 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. In addition, when the FRET phenomenon is detected using a combination of tetrameric fluorescent proteins (MR-BAK and AG-MCL1) that forms a fluorescent luminescent spot, and a combination of fluorescent proteins that do not form a fluorescent luminescent spot (MR-BAK and The result of comparison with the case of detection using mAG1-MCL1) is shown in FIG. 7E. The results obtained for BAX and MCL1 as well as BAK and MCL1 are shown in FIGS. 7F to 7J.

As shown in FIGS. 7A, 7B, 7F and 7G, 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. As a change of each fluorescence intensity after A-1210477 addition, MR (Em: 610) decreased and AG (Em: 510) increased. This indicates that the addition of the protein-protein interaction inhibitor reduced the signal of the FRET phenomenon.

On the other hand, with regard to cells expressing MR-BAK or MR-BAX and mAG1-MCL1, as shown in FIGS. 7C, 7D, 7H and 7I, the same tendency was observed, but FRET with or without the addition of A-1210477. There was no noticeable difference in the signal of the phenomenon. In fact, as a result of comparing the MR / AG ratio with the R / m AG1 ratio, as shown in FIGS. 7E and 7J, 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

In the present Example 8, 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).

<Preparation of pAG-FKBP12>
Fluoppi: 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. In addition, phAG-MCL (manufactured by Medical Biology Laboratory Co., Ltd., code number: AM-VS0801M) was treated with the same restriction enzyme combination to cut out the region encoding AG protein. Then, by inserting the region into the plasmid vector, a plasmid vector (pAG-FKBP12) for expressing FKBP12 (AG-FKBP12) in which AG was fused to the N-terminus was prepared.

<Preparation of pmAG1-FKBP12>
pAG-FKBP12 was treated with NheI and AgeI to remove the region encoding AG from the plasmid vector. In addition, phmAG1-MCL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0039M) was treated with the same restriction enzyme combination to cut out the region encoding mAG1. Then, by inserting the relevant region into the plasmid vector, a plasmid vector (pmAG1-FKBP12) for expressing FKBP12 (mAG1-FKBP12) in which mAG1 is fused to the N-terminus was prepared.

<Preparation of pMR-mTOR>
First, 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.

<Cell culture, gene transfer, cell observation and detection of FRET phenomenon>
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.
(E) pMR-mTOR + pAG-FKBP12
(F) pMR-mTOR + pmAG1-FKBP12
Also, in place of A-1210477, observation of cells and FRET phenomenon are the same as in Example 7 except that Rapamycin necessary for interaction between mTOR and FKBP12 is added to the culture solution of HEK 293 cells to a final concentration of 1 μM. Detection of

Regarding the obtained results, 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. In addition, when the FRET phenomenon is detected using a combination of tetrameric fluorescent proteins (MR-mTOR and AG-FKBP12) that form fluorescent luminescent spots, and a combination of fluorescent proteins that do not form fluorescent luminescent spots (MR-mTOR and The result of comparison with the case of detection using mAG1-FKBP12) is shown in FIG. 8E.

As shown in FIGS. 8A and 8B, the MR / AG ratio of cells expressing MR-mTOR and AG-FKBP 12 increased, centered on the fluorescent spot formed after Rapamycin addition. As each change in fluorescence intensity, MR (Em: 610) increased and AG (Em: 510) decreased. This indicates that the addition of the protein-protein interaction inducer increased the signal of the FRET phenomenon.

On the other hand, for cells expressing MR-mTOR and mAG1-FKBP12, as shown in FIGS. 8C and 8D, the MR / mAG1 ratio was increased in the whole cell after Rapamycin addition. As the change in each fluorescence intensity, an increase in FRET event signal was observed as in the case of the MR-mTOR and AG-FKBP12. However, no significant difference in the signal was observed with and without the addition of a protein-protein interaction inducer. In fact, as a result of comparing the MR / AG ratio with the R / m AG1 ratio, as shown in FIG. 8E, 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.

As described above, according to the results shown in Examples 7 and 8, similar to the results shown in Example 6, according to the present invention, regardless of the subject, protein-protein interaction can be achieved through the detection of the FRET phenomenon through the formation of a fluorescent bright spot. It was confirmed that the effect could be detected. Further, similarly to the results shown in Example 4, when the detection target interacts with proteins, the combination of the fluorescent proteins that can not form a fluorescent bright spot is better for the combination of tetrameric fluorescent proteins that form a fluorescent bright spot. It has been confirmed that the efficiency of the FRET phenomenon is higher (improved 2.7 to 4 times) than that.

(Example 9) Detection of FRET Phenomenon Using Plate Reader It was confirmed by the method described below that in the present invention, the FRET phenomenon through formation of a fluorescent bright spot detected by a fluorescence microscope can also be detected by a plate reader. . In addition, the case of detection using a combination of tetrameric fluorescent proteins (AG and MR) forming a fluorescent bright spot, and the detection using a combination of fluorescent proteins not forming a fluorescent bright spot (mAG1 and MR) We also confirmed the effect of fluorescent spot formation on the efficiency increase of the FRET phenomenon by comparing. Furthermore, it was also confirmed in this example that the FRET phenomenon is maintained and detectable in immobilized cells.

<Cell culture and gene transfer>
(A) MR-BAK + AG-MCL1
(B) MR-BAK + mAG1-MCL1
(C) MR-mTOR + AG-FKBP12
(D) MR-mTOR + mAG1-FKBP12
In order to express the combinations of the proteins described in (A) to (D) in cells, the plasmids prepared above were introduced into HEK293 cells by the method shown below.

First, 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)). Next, the same cells were seeded on a 6-well plate (manufactured by Nunc, # 140675) the day before gene transfer, and cultured in 3 mL of culture solution per well. Then, a DNA solution prepared by mixing 3 μg of each plasmid in 300 μL of Opti-MEM (manufactured by Thermo Fisher Scientific) was diluted, 20 μL of Fugene HD (manufactured by Promega) was added, and the mixture was stirred. After standing at 25 ° C. for 10 minutes, the cells were added to cultured cells and cultured for 24 hours. In addition, cells not transfected were similarly cultured for background calculation. The cells were harvested by trypsinization and passaged to 24 wells of a 96 well plate (Nunc, # 137101). Then, the cells were cultured for 24 hours in 100 μL of culture solution per well.

<Addition of interaction modifier (inhibitor or inducer)>
The whole culture solution was removed from each well and replaced with HBSS containing 20 mM HEPES. A-1210477, which is an inhibitor of MCL1 and BAK interaction, is adjusted to a final concentration of 16.67 μM as the maximum concentration and adjusted 8 points in 3-fold dilution series, and then 3 wells of each dilution of inhibitor are added. At 37 ° C., 5% CO ₂ for 30 minutes.

Prepare Rapamycin, an inducer of interaction between mTOR and FKBP, with a final concentration of 1 μM as the maximum concentration and adjust 8 points in 3-fold dilution series, then add 3 concentrations of each inducer dilution solution, 37 ° C, Let stand for 30 minutes in 5% CO ₂ .

<Detection of FRET phenomenon>
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).

<Fixation of cells>
From each well, 100 μL of 4% paraformaldehyde was added excluding the whole amount of the inhibitor dilution solution or the induction solution dilution solution. After leaving still for 15 minutes at room temperature, 100 μL of HBSS containing 20 mM HEPES was added except for the whole amount. After fixing the cells in this manner, the FRET phenomenon was detected in the same manner as described above.

<Data analysis>
The fluorescence value of the acceptor / the fluorescence value of the donor ([Ex Extracting each fluorescence value (Em. 620 and Em. 510) at 620 nm and 510 nm when the non-transfected cells are excited at 440 nm as the background value .440-Em.620] / [Ex.440-Em.510]) was calculated as a signal of the FRET phenomenon. In addition, the obtained signal values were normalized with an average value (n = 3) of the lowest concentrations of the respective interaction modifiers added as 1. Then, a dose response curve was drawn using a Kaleidagraph (manufactured by Synergy Software) to calculate IC ₅₀ . The results obtained are shown in FIGS. 9A and 9C for live cells and in FIGS. 9B and 9D for fixed cells. In each figure, error bars indicate standard errors.

As shown in FIGS. 9A and 9C, it was confirmed that 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.

Further, as shown in FIGS. 9B and 9D, it was confirmed that the FRET phenomenon can be detected by the plate reader even in the fixed cells when tetramer fluorescent protein is used. On the other hand, in the case of using the monomeric fluorescent protein, in the fixed cells, 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. In addition, as a result of comparing the case where the tetrameric fluorescent protein is used with the case where the monomeric fluorescent protein is used as described above, the former is 1.5 times the interaction between MCL1 and BAK, mTOR And FKBP12 interaction increased FRET ratio 1.9 times.

As described above, when the detection target interacts with proteins, 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.

(Example 10) Detection of interaction by image analysis using imaging system Determination of protein-protein interaction also by measuring fluorescence intensity of a fluorescent luminescent spot formed in the present invention using an imaging system We confirmed that we could do it.

<Cell culture and gene transfer>
(A) MR-BAK + AG-MCL1
(B) MR-BAX + AG-MCL1
In order to express the combination of the proteins described in (A) and (B) in cells, respectively, 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 μL per well. The cultures were cultured for 24 hours.

<Addition of interaction inhibitor and fixation of cells>
The whole culture solution was removed from each well and replaced with HBSS / 20 mM HEPES. After adjusting A-1210477, which is an inhibitor of MCL1 interaction with BAK or BAX, in 8-fold dilution series with a final concentration of 16.67 μM as the maximum concentration, adjust the inhibitor dilution solution of each concentration to 3 wells The mixture was added and allowed to stand at 37 ° C., 5% CO ₂ for 30 minutes. Thereafter, the medium was removed and 100 μL of 4% paraformaldehyde was added. After leaving still for 15 minutes at room temperature, 100 μL of HBSS containing 20 mM HEPES was added except for the whole amount.

<Imaging and Analysis>
Objective lens 20xAir, NA 0.4 is set in an imaging system (ParkinElmer, product name: operetta CLS), AG (time: 100 ms, power: 10%, excitation wavelength: 460 to 490 nm, fluorescence wavelength: 500 to 550 nm ), MR (time: 100 ms, power: 10%, excitation wavelength: 530 to 560 nm, fluorescence wavelength: 570 to 650 nm), Hoechst 33342 (time: 100 ms, power: 10%, excitation wavelength: 355 to 385 nm, fluorescence wavelength: 430) For each ̃500 nm), 49 fluorescence fields were acquired per well for each fluorescence image.

The obtained image was analyzed using software Harmony (manufactured by Parkin Elmer). The fluorescent spots were detected at a setting of Radius 2 μm, Contrast 0.3 or more, Uncorrected Spot to Region Intensity 0.5 or more, Distance 0.9 μm, and Spot Peak Radius 0 μm in Find Spots Method C, which is an analysis algorithm of Harmony. In addition, "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. These values were then normalized to the mean value (n = 3) at 0.01 μM addition of inhibitor A-1210477 and plotted on a graph. In addition, a dose response curve was drawn using a Kaleidagraph (manufactured by Synergy Software) to calculate IC ₅₀ . The analysis results for MR-BAK and AG-MCL1 are shown in FIG. 10A, and the analysis results for MR-BAX and AG-MCL1 are shown in FIG. 10B.

As shown in FIGS. 10A and 10B, 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.

<Preparation of pmTOR-DsRed2>
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. Moreover, pmTOR-AG (manufactured by Medical Biology Research Institute, code number: AM-8202M) is treated with the same restriction enzyme combination to remove the region encoding AG from the plasmid DNA, and then 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.

<Preparation of pmTOR-COR5>
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. Moreover, 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).

<Preparation of pmTOR-KikGR1>
The region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI. In addition, pmKikGR11-MNL (manufactured by Medical Biology Laboratory Co., Ltd., code number: AM-V0150M) was treated with the combination of the same restriction enzymes to cut out the DNA encoding KikGR1. Then, 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.

<Preparation of pFKBP12-AG>
First, 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. Then, 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) A plasmid vector (pFKBP12-AG) for expressing FKBP12 (FKBP12-AG) in which AG was fused to C-terminus was prepared.

<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.

<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.

<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.

<Cell culture and gene transfer>
(A) AG-FKBP12 + mTOR-DsRed2
(B) FKBP12-AG + mTOR-COR5
(C) MR-FKBP12 + mTOR-KikGR1
(D) FKBP12-DsRed2 + mTOR-KikGR1
(E) FKBP12-COR5 + mTOR-KikGR1
(F) Mmj-p53 + MR-MDM2
In order to express the combination of the proteins described in (A) to (F) in cells, HEK293 cells are cultured by the method described in Example 7, and the plasmid prepared above is used as the cells. Introduced to See Example 2 for the preparation of (F) and introduction into cells.

<Observation of cells and fixation of cells>
The observation and fixation of the transfected HEK 293 cells were performed using UPlanApo 20x (N.A. = 0.7) (Olympus) instead of UPlanFLN 40xOil (N.A. = 1.3) Except for the above, the method described in Example 3 was performed.

<Detection of FRET phenomenon and data analysis>
The donor side was AG, KikGR1 or Mmj, and the acceptor side was MR, DsRed2 or COR5. Furthermore, in mTOR and FKBP12, Rapamycin is added to the culture solution of HEK 293 cells to a final concentration of 1 μM 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 μM, 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.

With regard to the above (A) to (E), 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.

As shown in FIGS. 11A-11E, after addition of Rapamycin, the signal value of the FRET phenomenon (MR / AG ratio) rose around the formed fluorescent bright spot. That is, it was confirmed that the energy transfer efficiency between each tetrameric fluorescent protein was increased by interaction between mTOR and FKBP12, and the FRET phenomenon occurred.

Further, as shown in FIG. 11F, 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.

As described above, even if tetrameric proteins other than AG and MR are used, fluorescent luminescent spots are formed, and FRET phenomenon via the formation can also be detected, and these are used as indicators to judge protein-protein interactions It has been confirmed that it can.

(Comparative example) Detection of protein-protein interaction using dimeric and monomeric fluorescent proteins Monomeric fluorescent proteins (mKO1, mUkG1, mMiCy1, mKO2, mKeima) and dimeric fluorescence other than mAG1 and mEGFP Even if the proteins (KO1, dKeima, dAG (AB), dAG (AC), MiCy1, KCy1) are used, no fluorescent spots are formed, and it is confirmed by the following method that protein-protein interaction can not be determined. did.

<Preparation of pmTOR-mKO1>
The region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI. In addition, phmKO1-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0050M) was treated with the same restriction enzyme combination to cut out the DNA encoding mKO1. Then, 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.

<Preparation of pmTOR-mKO2>
The region encoding AG was removed from the pmTOR-AG by treatment with AgeI and XbaI. Moreover, phmKO2-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0140M) was treated with the same restriction enzyme combination to cut out the DNA encoding mKO2. Then, 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.

<Preparation of pmTOR-KO1>
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.

<Preparation of pmTOR-mKeima>
The region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI. In addition, 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.

<Preparation of pmTOR-dKeima>
The region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI. Furthermore, 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. Then, 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.

<Preparation of pmTOR-dAG (AC)>
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. Then, 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.

<Preparation of pmTOR-mUkG1>
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.

<Preparation of pmTOR-mMiCy1>
The region encoding AG was removed from pmTOR-AG by treatment with AgeI and XbaI. In addition, phmMiCy1-MNL (manufactured by Medical Biology Research Institute, Inc., code number: AM-V0110M) was treated with the same restriction enzyme combination to cut out the DNA encoding mMiCy1. Then, 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.

<Preparation of pmTOR-MiCy1>
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.

<Cell culture, gene transfer and addition of interaction inducer>
(A) AG-FKBP12 + mTOR-mKO1
(B) AG-FKBP12 + mTOR-mKO2
(C) AG-FKBP12 + mTOR-KO1
(D) AG-FKBP12 + mTOR-mKeima
(E) AG-FKBP12 + mTOR-dKeima
(F) mAG1-FKBP12 + mTOR-mKO1
(G) mAG1-FKBP12 + mTOR-mKO2
(H) mAG1-FKBP12 + mTOR-KO1
(I) mAG1-FKBP12 + mTOR-mKeima
(J) mAG1-FKBP12 + mTOR-dKeima
(K) MR-FKBP12 + mTOR-dAG (AB)
(L) MR-FKBP12 + mTOR-dAG (AC)
(M) MR-FKBP12 + mTOR-mUkG1
(N) MR-FKBP12 + mTOR-mMiCy1
(O) MR-FKBP12 + mTOR-MiCy1
(P) MR-FKBP12 + mTOR-KCy1
In order to express the combination of the proteins described in (A) to (P) in cells, HEK293 cells are cultured by the method described in Example 8, and the plasmid prepared above is Introduced to

After 48 hours of culture after addition of the plasmid DNA solution and Fugene HD, the whole culture solution is removed from each well, 200 μL each of HBSS containing 1 μM Rapamycin and 20 mM HEPES is added, 37 ° C., 5% CO ₂ incubator It stood for 30 minutes inside.

<Observation of cells>
The transfected HEK 293 cells were observed using an IX-81 inverted microscope (manufactured by Olympus). For filters and dichroic mirrors, for AG, mAG1, dAG (AB), dAG (AC), mUkG1, mM iCy1, MiCy1 and KCy1, use BP460-480HQ, BA495 and DM485HQ (U-MGFPHQ, manufactured by Olympus), mKO1 For 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 UPlanApo 20x (NA: = 0.7) (manufactured by Olympus), and the camera was an ORCA-Flash 4.0 digital camera (manufactured by Hamamatsu Photonics).

<Data analysis>
The obtained image data was analyzed by MetaMorph ver 8.9.0 (Molecular Devices), and each fluorescence image of the whole field of view was acquired. The results obtained are shown in FIGS. 12A-12P.

As shown in FIGS. 12A to 12P, when a monomeric fluorescent protein or a dimeric fluorescent protein was used, a fluorescent bright spot indicating an interaction between target proteins was not formed. Therefore, when different tetrameric fluorescent proteins are fused to the target protein and expressed in cells, a fluorescent bright spot is formed, and the interaction between the target proteins is determined using the fluorescent bright spot as an index. It has been confirmed that it can.

As described above, according to the present invention, when a fluorescent protein is fused to both of the target proteins and expressed or introduced in cells, 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.

In addition, when 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.

In particular, according to the present invention, even in immobilized cells, 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.

In addition, the process of image analysis, such as a fluorescent luminescent spot, can be reduced by detecting a FRET phenomenon in this way. That is, in the case of detecting a fluorescent bright spot instead of 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.

Therefore, 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

Claims

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 method comprising (1) to (3) (1) comprising a first fusion protein comprising a first protein and a first tetrameric fluorescent protein, and a second protein and a second tetrameric fluorescent protein A step of causing the second fusion protein to be expressed intracellularly or introduced into the cell (2) a step of forming a fluorescent luminescent spot caused by association of the first fusion protein and the second fusion protein in the cell (3) And b.) Determining the interaction between the first protein and the second protein by detecting the fluorescent spots.
Either 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, and in step (3), via the formation of the fluorescent luminescent spot The method according to claim 1, wherein the interaction between the first protein and the second protein is determined by the detection of the FRET phenomenon.
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 according to claim 1 or 2, wherein a protein that interacts with the specific protein is selected by point detection or detection of the FRET phenomenon.
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, comprising either the first protein or the second protein When a protein into which a mutation has been introduced is used and the intensity of the fluorescent bright spot or the FRET phenomenon is attenuated as compared to the case where a protein into which a mutation is not introduced is used, the amino acid residue into which the mutation is introduced The method according to claim 1 or 2, wherein it is determined to be involved in the interaction.
A method for screening a substance that regulates 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 comprising 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, and a second fusion protein A step of intracellularly expressing or introducing into the cell a second fusion protein comprising the protein and the second four-mer 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 in the absence of the test compound, the test compound is selected as an inducer of the interaction, and the fluorescent bright is selected. A step of selecting the test compound as an inhibitor of the interaction when the intensity of the point is attenuated more than the intensity of the fluorescent luminescent spot generated in the absence of the test compound.
A method for screening a substance that regulates 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 any 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, and a method comprising the following steps (1) to (3): (1) In the presence of a test compound, 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 Or expressing the fusion protein of SEQ ID NO.
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 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;
(3) When the strength of the FRET phenomenon is higher than the strength of the FRET phenomenon generated in the absence of the test compound, the test compound is selected as an inducer of the interaction, and the strength of the FRET phenomenon is selected. And a step of selecting the test compound as an inhibitor of the interaction, when the intensity of the FRET phenomenon which occurs in the absence of the test compound is attenuated.
The method according to any one of claims 1 to 6, wherein the cell is an immobilized cell.
A kit for use in the method according to any one of claims 1 to 7, comprising at least one substance selected from the group consisting of the following (a) to (k) and instructions for use: 2.) A DNA encoding a first tetrameric fluorescent protein and a cloning site enabling insertion of DNA encoding any protein so as to be expressed fused to the first tetrameric fluorescent protein (B) A DNA encoding a second tetrameric fluorescent protein, and an insertion of a DNA encoding any protein so as to be expressed fused to the second tetrameric fluorescent protein A vector containing the cloning site (c) a vector encoding the first fusion protein (d) a vector encoding the second fusion protein (e) The vector according to (a) or (c) And (b) 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 trait carrying a vector encoding a second fusion protein Transformed cell (h) Transformed cell carrying a vector encoding a first fusion protein and a vector encoding a second fusion protein (i) First fusion protein (j) Second fusion protein (k) A set of proteins comprising a first fusion protein and a second fusion protein.