JP2009296973A - Method for analyzing protein interaction, polynucleotide and composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing protein interaction by which the dynamic state of the interaction between the proteins can be analyzed in good accuracy even when a target protein contains a localized nucleus signal sequence to improve the repeatability of experiments. <P>SOLUTION: The method comprises analyzing the dynamic state of the interaction between the proteins in a cell, including preparing an N-terminal side luciferase and a C-terminal side luciferase separated so that the luciferase activity may be recovered by bonding to each other, making a cell containing one protein fused with the N-terminal-side luciferase and the other protein fused with the C-terminal-side luciferase, adding a prescribed luminescent substrate to the prepared cell from exterior of the cell, picking up the luminescence image of the cell to which the prescribed luminescent substrate is added, and analyzing the dynamic state of the interaction of the protein in the cell, wherein the localized nucleus signal sequence of at least one protein is mutated in the preparation step. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a protein interaction analysis method, polynucleotide, and composition for non-invasively analyzing intracellular protein-protein interactions.

  Conventionally, in order to observe the interaction of proteins in cells, a luminescent enzyme such as luciferase is divided and fused to two target proteins, and the fusion protein expressed in the cells As a result of binding to each other by the interaction, the recovered luminescent enzyme activity was observed and measured.

  For example, in Non-Patent Documents 1 and 2, split split luciferase is bound to MyoD and Id, expressed as a fusion protein in the cell, and the luciferase activity recovered by the binding between split luciferases is observed. A method (split luciferase assay) has been developed to analyze the MyoD-Id interaction and localization thereof.

R. Paulmurugan, Y.M. Umezawa, S .; S. Gambir, "Nonvasive imaging of protein-protein interactions in living projects by using reporter compensation and propensity preparations." 99, no. 24, pp. 15608-15613, 2002 Kathlyn E.M. Luker, Matthew C.L. P. Smith, Gary D.M. Luker, Seth T .; Gammon, Helen Piwnica-Worms, David Piwnica-Worms, "Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals", Proceedings of the National Academy of Sciences, vol. 101, no. 33, pp. 12288-12293, 2004 Jody M.M. Lingbeck, Julie S. Trausch-Azar, Aaron Ciechanover, Alan L. Schwartz, “Determinants of Nuclear and Cytoplasmic Ubiquitin-Mediated Degradation of MyoD”, The Journal of Biological Chemistry, Vol. 278, no. 3, pp. 1817-1823, 2003 Julie S. Trausch-Azar, Jody Lingbeck, Aaron Ciechanover, Alan L. Schwartz, “Ubiquitin-Proteasome-mediated degradation of Id1 is modulated by MyD”, The Journal of Biological Chemistry, Vol. 279, no. 31, pp. 32614-32619, 2004

  However, for example, it has been reported in Non-Patent Documents 3 and 4 that the above-mentioned MyoD and Id have a nuclear localization signal (NLS) sequence. When a protein has a nuclear localization signal sequence, the target protein is localized in the nucleus, so that there is a problem that the divided luciferase is not efficiently reconstituted and the luminescence intensity is weak.

  In addition, in the case where a plurality of objects are detected by color at the same time using the same detection device, it is necessary to prevent the leakage by adjusting the emission intensity so that the difference in emission intensity does not become excessive, but in the conventional method, luciferase The gene had to be modified or adjusted with different types of luciferases. Further, even if the detection is not performed at the same time, if there is a difference in emission intensity, the sensitivity of the detection device or the like must be switched and set each time, and there is a problem in reproducibility.

  The present invention has been made in view of the above problems, and even when the target protein contains a nuclear localization signal sequence, the dynamics of protein-protein interaction can be analyzed with high accuracy. An object of the present invention is to provide a protein interaction analysis method, polynucleotide, and composition that can improve the reproducibility of experiments.

  In order to solve the above-described problems and achieve the object, a protein interaction analysis method according to the present invention is a protein interaction analysis method for analyzing protein interactions in cells, and emits light by binding to each other. An N-terminal luminescent enzyme and a C-terminal luminescent enzyme that have been divided so as to recover the enzyme activity are prepared and fused with one of the proteins fused with the N-terminal luminescent enzyme and the C-terminal luminescent enzyme. A production step for producing the cell containing the other protein, an addition step for adding a predetermined luminescent substrate from outside the cell to the cell produced in the production step, and the predetermined luminescent substrate in the addition step. An imaging step of capturing a luminescent image of the given cell, and an interaction between the proteins in the cell based on the luminescent image captured in the imaging step Anda analysis step of the kinetic analysis, in the manufacturing process, to mutate nuclear localization signal sequence of at least one of said protein, characterized by.

  The protein interaction analysis method according to the present invention is the protein interaction analysis method described above, wherein the production step converts a lysine residue and / or an arginine residue of the nuclear localization signal sequence into an uncharged amino acid. It is characterized by replacing.

  The protein interaction analysis method according to the present invention is characterized in that, in the protein interaction analysis method described above, the uncharged amino acid is alanine or glycine.

  The protein interaction analysis method according to the present invention is the protein interaction analysis method described above, wherein one of the proteins is a wild-type Id protein and the other protein corresponds to the nuclear localization signal sequence. A mutated MyoD protein in which the arginine residues at amino acid numbers 102, 104, 112, and 103, 110, 111 are mutated to alanine residues. And

  The protein interaction analysis method according to the present invention is the protein interaction analysis method described above, wherein one of the proteins corresponds to the nuclear localization signal sequence at amino acid numbers 70, 81, and 84. It is a mutant Id protein obtained by mutating lysine residues and 68th and 80th arginine residues to alanine residues, and the other protein is a wild-type MyoD protein.

  The protein interaction analysis method according to the present invention is the above-described protein interaction analysis method, wherein the N-terminal luminescent enzyme comprises an N-terminal split comprising the amino acid sequence of amino acid numbers 1 to 416 of firefly luciferase. It is a luciferase, and the C-terminal luminescent enzyme is a C-terminal split luciferase comprising the amino acid sequence of amino acid numbers 399 to 550 of the firefly luciferase.

  The polynucleotide according to the present invention is a polynucleotide encoding a fusion protein of a C-terminal split luciferase and a MyoD protein containing a nuclear localization signal sequence, wherein a lysine in the nuclear localization signal sequence of the MyoD protein Residues and arginine residues have been mutated to encode alanine or glycine residues.

  The composition according to the present invention is a composition comprising at least the polynucleotide described above and a polynucleotide encoding a fusion protein of N-terminal split luciferase and wild-type Id protein. To do.

  The polynucleotide according to the present invention is a polynucleotide encoding a fusion protein of N-terminal split luciferase and an Id protein containing a nuclear localization signal sequence, wherein lysine in the nuclear localization signal sequence of the Id protein Residues and arginine residues have been mutated to encode alanine or glycine residues.

  The composition according to the present invention is a composition comprising at least the polynucleotide described above and a polynucleotide encoding a fusion protein of C-terminal split luciferase and wild-type MyoD protein. .

  According to the present invention, even if the target protein contains a nuclear localization signal sequence, the dynamics of protein-protein interaction can be analyzed with high accuracy, and as a result, the reproducibility of the experiment can be improved. .

  Embodiments of a protein interaction analysis method according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  First, the structure of the luminescence observation system 100 used in the protein interaction analysis method (specifically, the imaging step and the analysis step) according to the present invention will be described with reference to FIGS. FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100. 2 and 3 are block diagrams illustrating an example of the configuration of the light emission image capturing unit 106 of the light emission observation system 100. FIG. FIG. 4 is a block diagram illustrating an example of the configuration of the image analysis device 110 of the light emission observation system 100.

  As shown in FIG. 1, a luminescence observation system 100 includes a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) that contains cells 102, and a stage 104 on which the container 103 is arranged. And a light emission image pickup unit 106 and an image analysis device 110.

  Here, the light emitting image capturing unit 106 may be disposed below the stage 104. By disposing the light emitting image pickup unit 106 for measuring light emission weaker than fluorescence, disturbance light from above the sample due to opening and closing of the cover can be completely blocked, and the S / N ratio of the light emission image is increased. Can do. In the case of this configuration, the bottom of the container 103 may be made of a light-transmitting material, and the stage 104 may have a hole for transmitting light.

  The cell 102 includes, for example, a target protein (eg, Id protein) fused with a divided N-terminal luminescent enzyme (eg, N-terminal split luciferase) and a divided C-terminal luminescent enzyme (eg, C-terminal luminescent enzyme). A living cell containing the other target protein (for example, MyoD protein) fused to the side split luciferase), and at least one of the target protein nuclear localization signal sequences has a predetermined mutation to prevent nuclear localization Has been introduced. For example, the lysine residue and / or arginine residue of the nuclear localization signal sequence may be substituted with an uncharged amino acid (for example, alanine or glycine). Here, both fusion proteins are expressed in the cell 102 by introducing into the cell 102 an expression vector comprising a polynucleotide encoding the fusion protein and configured to express the fusion protein. You may comprise. The cell 102 is given a predetermined luminescent substrate (for example, luciferin) from the outside of the cell when imaging the cell.

The luminescence image capturing unit 106 is specifically an upright luminescence microscope and captures a luminescence image of the cell 102. As shown in the figure, the luminescent image capturing unit 106 includes an objective lens 106a, a dichroic mirror 106b, a CCD camera 106c, and an imaging lens 106f. Specifically, the objective lens 106a has a value of (numerical aperture / magnification) ² of 0.01 or more. The dichroic mirror 106b is used when light emitted from the cell 102 is separated by color and the amount of light emitted is measured by color using two colors of light. The CCD camera 106c takes a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the objective lens 106a, the dichroic mirror 106b, and the imaging lens 106f. The CCD camera 106c is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. The imaging lens 106f forms an image (specifically, an image including the cell 102) incident on the imaging lens 106f via the objective lens 106a and the dichroic mirror 106b. Note that FIG. 1 shows an example in which a light emission image corresponding to two light emissions separated by the dichroic mirror 106b is separately captured by the two CCD cameras 106c. The image capturing unit 106 may include an objective lens 106a, a single CCD camera 106c, and an imaging lens 106f.

  Here, when measuring the light emission amount and the light emission intensity for each color using the light emission of two colors, as shown in FIG. 2, the light emission image pickup unit 106 includes an objective lens 106a, a CCD camera 106c, and a split image unit 106d. And the imaging lens 106f. The CCD camera 106c captures a light emission image (split image) and a bright field image of a biological sample (such as the cell 102) projected onto the chip surface of the CCD camera 106c via the split image unit 106d and the imaging lens 106f. May be. The split image unit 106d separates light emitted from a biological sample (cell 102, etc.) by color, and is used when measuring the amount of light emitted and the intensity of light emitted by using two colors of light, similar to the dichroic mirror 106b. .

  Further, when measuring the emission amount and emission intensity for each color using light emission of a plurality of colors (that is, when using multicolor light emission), the light emission image pickup unit 106 includes an objective lens 106a and an objective lens 106a as shown in FIG. The CCD camera 106c, the filter wheel 106e, and the imaging lens 106f may be used. Then, the CCD camera 106c may capture a light emission image and a bright field image of a biological sample (such as the cell 102) projected on the chip surface of the CCD camera 106c via the filter wheel 106e and the imaging lens 106f. The filter wheel 106e is used when light emitted from a biological sample (such as the cell 102) is separated by color by exchanging filters, and the amount of emitted light and the intensity of light are measured by color using multiple colors of emitted light.

  Returning to FIG. 1 again, the image analysis apparatus 110 is, for example, a personal computer. As shown in FIG. 4, the image analysis device 110 is roughly divided into a control unit 112, a clock generation unit 114 that measures the system time, a storage unit 116, a communication interface unit 118, an input device 122, The input / output interface unit 120 is connected to the output device 124, and these units are connected via a bus.

  In the above configuration, the storage unit 116 is a storage unit, and specifically, a memory device such as a RAM or a ROM, a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like can be used. And the memory | storage part 116 memorize | stores the data etc. which were obtained by the process of each part of the control part 112. FIG. The communication interface unit 118 mediates communication between the image analysis device 110 and the CCD camera 106c. That is, the communication interface unit 118 has a function of communicating data with other terminals via a wired or wireless communication line. The input / output interface unit 120 is connected to the input device 122 and the output device 124. Here, in addition to a monitor (including a home television), a speaker or a printer can be used as the output device 124 (hereinafter, the output device 124 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input device 122 can be a monitor that realizes a pointing device function in cooperation with the mouse.

  The control unit 112 has an internal memory for storing a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data, and executes various processes based on these programs. . The control unit 112 is roughly composed of a light emission image capturing instruction unit 112a, a light emission image acquisition unit 112b, an image analysis unit 112c, and an analysis result output unit 112d.

  The luminescent image capturing instruction unit 112c instructs the CCD camera 106c to capture a luminescent image and a bright field image via the communication interface unit 118. The luminescent image acquisition unit 112b acquires the luminescent image and bright field image captured by the CCD camera 106c via the communication interface unit 118.

  The image analysis unit 112c analyzes fluctuations and dynamics of the interaction state of the target protein based on the plurality of luminescence images acquired by the luminescence image acquisition unit 112b. In other words, the image analysis unit 112c indicates the state of interaction between the target proteins, how the target protein that is interacting moves in the cell 102, based on the plurality of light emission images. To analyze. Further, the image analysis unit 112c quantifies and analyzes how the interaction state of the target protein changes based on the plurality of light emission images. The analysis result output unit 112d outputs the analysis result from the image analysis unit 112c to the output device 124. Specifically, the analysis result output unit 112d graphs and displays on the output device 124 time-series numerical data regarding the change in the interaction state between the target proteins obtained by the image analysis unit 112c.

  Examples of the present embodiment will be described below with reference to FIGS. Here, FIG. 5 is a diagram showing the detection principle of the split luciferase assay.

  The split luciferase assay is a luciferase such as firefly luciferase or Renilla luciferase, which is divided at a specific position to inactivate luminescent enzyme activity (bioluminescence ability), and then the N-terminal luciferase that is a fragment (fragment) thereof (NLuc) and C-terminal luciferase (CLuc) are reconstituted to recover the luminescent enzyme activity, and are used for detecting protein-protein interactions, detecting intracellular endoplasmic reticulum migration, and the like.

  FIG. 5 shows an example in which Id and MyoD are used as proteins that cause interaction. That is, when the Id gene site in the NLuc-Id fusion protein and the MyoD gene site in the MyoD-CLuc fusion protein that are expressed in a cell interact and bind to each other, the NLuc gene site and the CLuc gene site bind to each other. The luminescent enzyme activity is then restored and a detectable signal is emitted. In this example, an embodiment in which mutations are introduced into the nuclear localization signal sequence in the Id or MyoD gene in this split luciferase assay system will be described. Here, the flow (procedure) of the experiment in the present embodiment is as follows.

[Preparation of split luciferase gene and cloning of MyoD and Id genes]
[Procedure 1]
First, in order to produce a split luciferase gene (NLuc / CLuc), a synthetic oligo DNA used for PCR was prepared with the following sequence.
(Synthetic oligo DNA sequence for NLuc gene production)
NLuc_Forward (SEQ ID NO: 1): 5'-GCCACATGGGAAGATGCCAAAAAACTTT-3 '
NLuc_Reverse (SEQ ID NO: 2): 5′-CAGGGATCCGTCCTTGTCGATGAGAGCGTT-3 ′
(Synthetic oligo DNA sequence for CLuc gene production)
CLuc_Forward (SEQ ID NO: 3): 5′-ATCGGATCCGGCTACGTTAACAAACCCCGAG-3 ′
CLuc_Reverse (SEQ ID NO: 4): 5′-GACTCTAGAATTATTACACGGCGATCT-3 ′

In addition, for the cloning of the MyoD gene and the Id gene, a synthetic oligo DNA used for PCR was prepared with the sequences shown below.
(Synthetic oligo DNA sequence for producing MyoD gene)
MyoD_Forward (SEQ ID NO: 5): 5′-GGGCCATGGAGCTTCTATCGCCGCCACTC-3 ′
MyoD_Reverse (SEQ ID NO: 6): 5′-GATGGATCCAAGCACCTGATAAAATCGCATT-3 ′
(Synthetic oligo DNA sequence for Id gene production)
Id_Forward (SEQ ID NO: 7): 5′-GAGGGATCCCTTGGTCTGTCGGAGCAAAGC-3 ′
Id_Reverse (SEQ ID NO: 8): 5′-GTGTTCTAGACTCAGCGCACAAGATGGCGAT-3 ′

[Procedure 2]
Then, using pGL4.10 (manufactured by Promega Corporation) as a template, the N-terminal luciferase gene (NLuc: including the first to 416th amino acids of the GL4.10 gene) and the C-terminal luciferase gene (CLuc) : 399th to 550th amino acids of GL4.10 gene) were amplified by PCR using the above synthetic oligo DNA as a primer.

[Procedure 3]
Then, using a mouse skeletal muscle cDNA library (manufactured by Takara Bio Inc.) as a template, a MyoD gene (including a region corresponding to the first to 318th amino acid sequences of the mouse MyoD gene), and an Id gene (mouse) The region corresponding to the amino acid sequence from the 29th to the 148th amino acid sequence of Id gene was amplified by PCR using the synthetic oligo DNA as a primer.

[Procedure 4]
Then, after sub-cloning the NLuc gene and MyoD gene amplified by PCR into the pBluescript II vector, the Id gene is located between the BamHI site and the XbaI site present downstream of the NLuc gene, and the BamHI site present downstream of the MyoD gene. CLuc genes were inserted between XbaI sites, respectively, to create fusion genes (NI: NLuc-Id and MC: MyoD-CLuc).

[Procedure 5]
Each fusion gene was incorporated into an expression vector pCI-neo (manufactured by Promega Corp.) for mammalian cells.

[Introduction of mutations into Myo gene and Id gene]
Subsequently, mutations were introduced into the nuclear localization signal sequences of MyoD and Id genes by the following procedure.

[Procedure 1]
First, in order to introduce a mutation into each gene, a synthetic oligo DNA was prepared with the following base sequence.
(Synthetic oligo DNA sequence for introducing mutation into MyoD gene)
Myo_mut_A_des (SEQ ID NO: 9): 5′-CTGCAAGGCGTCGCGGGCCGCGACCACCAACGC-3 ′
Myo_mut_A_opp (SEQ ID NO: 10): 5′-GCGTTGGGTGTCCGCGCCGCGCACGCCTTGCAG-3 ′
Myo_mut_B_des (SEQ ID NO: 11): 5′-CCAACGCTGATGCCGCCGCGGCCCCACCCATGC-3 ′
Myo_mut_B_opp (SEQ ID NO: 12): 5′-GCATGGTGGGCGCCGCGGCGCATCATGCGTTG-3 ′
(Synthetic oligo DNA sequence for introducing mutation into Id gene)
Id_mut_A_des (SEQ ID NO: 13): 5′-GGCTGCCTACTCAGCCCCTCGCGGAGCTGGTGCCC-3 ′
Id_mut_A_opp (SEQ ID NO: 14): 5′-GGGCACCAGCTGCCGGAGGGCTGAGTAGGCAGCC-3 ′
Id_mut_B_des (SEQ ID NO: 15): 5′-CCTGCCCCAGAACGCCGCAGTGGACAAGGTGG-3 ′
Id_mut_B_opp (SEQ ID NO: 16): 5′-CCACCCTGCTCACTGCGGCGGTCTGGGGCAGG-3 ′
Id_mut_C_des (SEQ ID NO: 17): 5′-GCCGCAGGTGCGCGGGTGGAGATCCTGC-3 ′
Id_mut_C_opp (SEQ ID NO: 18): 5′-GCAGGATCTCCCACCCGCTCCACTGCGGC-3 ′

[Procedure 2]
Then, using the NI and MC gene expression vectors (NI and MC) prepared as described above as a template, using the above-mentioned synthetic oligo DNA, the MyoD gene and Id gene mutant gene expression vectors (NI_m, And MC_m). For the preparation of the mutant, QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) was used, and the preparation was performed according to the manual of the kit.

[Procedure 3]
Then, the sequences of the prepared wild type and mutant DNAs were confirmed by DNA sequencing. Here, FIG. 6 to FIG. 9 are diagrams showing the structures of the prepared fusion genes (NI, NI_m, MC, and MC_m), respectively. By performing the above operations, a fusion gene NI (SEQ ID NOS: 19 and 20) of N-terminal split luciferase NLuc and wild-type Id gene as shown in FIG. 6, and N-terminal split luciferase NLuc as shown in FIG. Gene NI_m (SEQ ID NO: 21 and 22), and a fusion gene MC (SEQ ID NO: 23, 24) of wild-type MyoD gene and C-terminal split luciferase CLuc as shown in FIG. 8, and As shown in FIG. 9, an expression vector having a fusion gene MC_m (SEQ ID NO: 25, 26) of a mutant MyoD gene and C-terminal split luciferase CLuc was obtained. The sequences of SEQ ID NOs: 19, 21, 23, and 25 are DNA sequences, and the sequences of SEQ ID NOs: 20, 22, 24, and 26 are amino acid sequences. Here, FIG. 10 and FIG. 11 are diagrams showing the wild-type and mutant amino acid sequences of nuclear localization signals of the MyoD gene and the Id gene.

  As shown in FIG. 10, in this example, the 102th, 104th, and 112th lysine residues, and the 103th, 110th, and 111th arginine residues of the amino acid sequence of wild-type MyoD were replaced with an alanine residue. Substituted for the group. Moreover, as shown in FIG. 11, the 70th, 81st, 84th lysine residues, and the 68th, 80th arginine residues of the amino acid sequence of wild-type Id were substituted with alanine residues. Thereby, since the MyoD protein and Id protein remain in the cytoplasm due to the mutation of the nuclear localization signal, which is a region involved in nuclear translocation, the split luciferase can remain in the cytoplasm.

[Transfection and luminescence measurement of split luciferase fusion gene]
The fusion gene expression vector (NI, NI_m, MC, and MC_m) prepared as described above was transfected into cells by the following procedure, and the luminescence was measured.

[Procedure 1]
HeLa cells were obtained from ATCC and cultured in a low glucose DMEM medium (GIBCO) in a 5% CO2 incubator.

[Procedure 2]
And it seed | inoculated at the cell density of 1 * 10 < ⁴ > / well to 96-well multipleplate (made by NUNC), and it culture | cultivated overnight.

[Procedure 3]
The combination of the NI fusion gene and the MC fusion gene, the NI fusion gene and the MC_m fusion gene, the NI_m fusion gene and the MC fusion gene, the NI_m fusion gene and the MC_m fusion gene, and the expression vector are mixed to produce Lipofectamine 2000 (Invitrogen Corporation). ) And was cultured overnight in a 5% CO2 incubator.

[Procedure 4]
Luciferin (manufactured by Promega Corp.) having a final concentration of 0.5 mM was added, and the amount of luminescence was measured with a luminometer (Lumisenser JNR-2000 (manufactured by Atto Corp.)). FIG. 12 is a graph showing the amount of luminescence measured with a luminometer in cells into which each expression vector has been introduced.

[Luminescence imaging using split luciferase]
[Procedure 1]
Cells were seeded in 35 mm diameter glass bottom dishes at a cell density of 2 × 10 ⁵ / dish and cultured overnight in a 5% CO 2 incubator.

[Procedure 2]
Then, an expression vector is mixed with a combination of NI fusion gene and MC fusion gene, NI fusion gene and MC_m fusion gene, NI_m fusion gene and MC fusion gene, NI_m fusion gene and MC_m fusion gene, and Lipofectamine 2000 (Invitrogen) ) And cultivated overnight in a 5% CO2 incubator.

[Procedure 3]
Then, 0.5 mM luciferin (manufactured by Promega Corp.) is added to the medium and set in a light emission microscope LV (LUMINOWEEW) -200 (manufactured by Olympus Corp.), CCD camera ORCA-ER (manufactured by Hamamatsu Photonics Corp.) ) Was taken into a personal computer configured as the image analysis device 110. The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c. Here, FIGS. 13 to 16 are diagrams showing luminescence images obtained in HEK293 cells into which the combinations of expression vectors (NI + MC, NI_m + MC, NI + MC_m, NI_m + MC_m) were introduced.

  As a result of the above experiment, as shown in FIG. 12, the case of introduction with a combination of NI_m and MC (FIG. 14) is compared to the case of introduction with a combination of NI and MC that are both wild type (see FIG. 13). The combination of NI and MC_m (see FIG. 15) shows a luminescence amount about four times stronger than the combination of both wild type NI and MC (see FIG. 13). It was. Note that the combination of NI_m and MC_m, both of which are mutant types (see FIG. 16), showed the same amount of luminescence as the combination of both wild-type NI and MC. As a result, if at least one of the nuclear localization signal sequences is mutated, it becomes possible to measure at about 1/10 times and 4 times the sensitivity of the wild type combination (NI + MC), respectively. It was found that the sensitivity can be adjusted according to the above.

  For example, if a combination of a mutant split luciferase that increases the amount of luminescence by about 4 times is used, the luciferase remaining in the cytoplasm is efficiently reconstituted, and a signal that is 4 times stronger than the conventional method can be obtained. Further, by using a combination of a mutant split luciferase whose luminescence amount is about 10 times lower, a signal that is 10 times weaker than the conventional one can be obtained, and the luminescence intensity can be adjusted so that the difference in luminescence intensity is not excessive.

  As described above, according to this example, even when the target protein includes a nuclear localization signal sequence, the dynamics of protein-protein interaction can be analyzed with high accuracy, and as a result, the reproducibility of the experiment is improved. be able to.

[Other embodiments]
In the embodiment described above, the split luminescent enzyme has been mainly described as split luciferase. However, the present invention is not limited to this, and split GFP may be used instead of split luciferase. In this case, excitation light can be irradiated instead of adding luciferin to the cells.

  As described above, the protein interaction analysis method, polynucleotide, and composition according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

1 is a diagram illustrating an example of an overall configuration of a light emission observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of an image analysis device 110 of the light emission observation system 100. FIG. It is a figure which shows the detection principle of a split luciferase assay. It is a figure which shows the structure of the produced fusion gene NI. It is a figure which shows the structure of the produced fusion gene NI_m. It is a figure which shows the structure of the produced fusion gene MC. It is a figure which shows the structure of the produced fusion gene MC_m. It is a figure which shows the wild-type and variant amino acid sequence of the nuclear localization signal of a MyoD gene. It is a figure which shows the wild-type and variant type | mold amino acid sequence of the nuclear localization signal of Id gene. It is a graph which shows the light-emission quantity measured with the luminometer in the cell which introduce | transduced each expression vector. It is a figure which shows the light emission image acquired in the HEK293 cell which introduce | transduced NI expression vector and MC expression vector. It is a figure which shows the light emission image acquired in the HEK293 cell which introduce | transduced the NI_m expression vector and MC expression vector. It is a figure which shows the light emission image acquired in the HEK293 cell which introduce | transduced NI expression vector and MC_m expression vector. It is a figure which shows the luminescence image acquired in the HEK293 cell which introduce | transduced the NI_m expression vector and MC_m expression vector.

Explanation of symbols

100 Luminescence observation system
103 container (petri dish)
104 stages
106 Luminous image pickup unit
106a Objective lens (for light emission observation)
106b Dichroic mirror
106c CCD camera
106d Split image unit
106e Filter wheel
106f Imaging lens
110 Image analyzer
112 Control unit
112a Luminous image capturing instruction unit
112b Luminescent image acquisition unit
112c Image analysis unit
112d Analysis result output part
114 Clock generator
116 storage unit
118 Communication interface
120 Input / output interface
122 Input device
124 Output device

Claims (10)

A protein interaction analysis method for analyzing protein interactions in cells,
One of the proteins fused with the N-terminal luminescent enzyme prepared by adjusting an N-terminal luminescent enzyme and a C-terminal luminescent enzyme that have been divided so as to recover the luminescent enzyme activity by binding to each other; and the C-terminal A production step of producing the cell containing the other protein fused with a side luminescent enzyme;
An addition step of adding a predetermined luminescent substrate from the outside of the cell produced in the production step;
An imaging step of capturing a luminescence image of the cell provided with the predetermined luminescent substrate in the adding step;
Based on the luminescence image captured in the imaging step, an analysis step of analyzing the dynamics of the interaction of the protein in the cell;
Including
In the manufacturing process,
Mutating the nuclear localization signal sequence of at least one of the proteins,
A protein interaction analysis method characterized by the above. The protein interaction analysis method according to claim 1,
The manufacturing process includes
Substituting lysine residues and / or arginine residues of the nuclear localization signal sequence with uncharged amino acids;
A protein interaction analysis method characterized by the above. The protein interaction analysis method according to claim 2,
The uncharged amino acid is alanine or glycine;
A protein interaction analysis method characterized by the above. The protein interaction analysis method according to claim 1,
One of the proteins is a wild type Id protein,
The other protein comprises amino acid number 102, 104, 112 lysine residues, and 103, 110, 111 arginine residues corresponding to the nuclear localization signal sequence, alanine residues. A mutant MyoD protein mutated to
A protein interaction analysis method characterized by the above. The protein interaction analysis method according to claim 1,
One of the proteins is obtained by mutating amino acid numbers 70th, 81st, 84th lysine residues and 68th, 80th arginine residues corresponding to the nuclear localization signal sequence to alanine residues. A mutant Id protein,
The other protein is a wild-type MyoD protein;
A protein interaction analysis method characterized by the above. In the protein interaction analysis method according to any one of claims 1 to 5,
The N-terminal luminescent enzyme is an N-terminal split luciferase comprising the amino acid sequence of amino acid numbers 1 to 416 of firefly luciferase,
The C-terminal luminescent enzyme is a C-terminal split luciferase comprising the amino acid sequence of amino acids 399 to 550 of the firefly luciferase;
A protein interaction analysis method characterized by the above. In a polynucleotide encoding a fusion protein of C-terminal split luciferase and MyoD protein containing a nuclear localization signal sequence,
Lysine and arginine residues in the nuclear localization signal sequence of the MyoD protein have been mutated to encode alanine or glycine residues;
A polynucleotide characterized by. A polynucleotide according to claim 7;
A polynucleotide encoding a fusion protein of N-terminal split luciferase and wild-type Id protein;
A composition comprising at least In a polynucleotide encoding a fusion protein of N-terminal split luciferase and Id protein containing a nuclear localization signal sequence,
The lysine and arginine residues in the nuclear localization signal sequence of the Id protein have been mutated to encode alanine or glycine residues;
A polynucleotide characterized by. The polynucleotide of claim 9;
A polynucleotide encoding a fusion protein of C-terminal split luciferase and wild-type MyoD protein;
A composition comprising at least

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