KR20190016383A - Method for analyzing interacting proteins in cells using peroxidase - Google Patents

Abstract

The present invention relates to a method for identifying a protein interacting with a target protein, which can be used to selectively crosslink two different proteins in a medical field. The method comprises the steps of: crosslinking interactive proteins to obtain a protein cross-linked body; and separating the protein cross-linked body including a target protein.

Description

Methods for analyzing interactions of proteins in cells using peroxidase {Methods for analyzing interacting proteins in cells using peroxidase}

The present invention relates to a method for obtaining a cross-linked product containing a target protein in a cell using a peroxidase and a method for identifying a protein interacting with a target protein by mass spectrometry of the cross-linked product, A fusion protein is produced by preparing a fusion polynucleotide in which a target gene and a gene encoding a peroxidase are linked to a cell organelle in which a target protein for interaction with the target protein exists and the fusion polynucleotide is introduced into the cell, To obtain a protein cross-linked product by cross-linking the proteins interacting in the organelle; and an antibody to the target protein is added to the cell lysate obtained by disrupting the cell to perform immunoprecipitation, The step of separating And a method for obtaining a protein crosslinked body containing the desired protein.

Most proteins interact with other proteins around them and function in vivo. One or more proteins in a cell exhibit biological functions through interaction (non-covalent, physical). Understanding the interactions between proteins is therefore of great importance in understanding biological functions. Efforts to identify protein interactions in diverse disciplines such as biochemistry, proteomics, molecular dynamics, and signal transduction have continued to date.

In order to confirm the interaction between these proteins, it has been known to use 'immunoprecipitation' using the affinity of an antibody and an antigen (in this case, a protein). Specifically, the immunoprecipitation method refers to a method of specifically separating an antigen from a solution using the affinity of the antibody and the antigen (in this case, a protein). In this case, when a protein factor interacting with an antigen in an aqueous solution exists in an aqueous solution, proteins that interact with the antigen may be included if the antigen bound to the antibody is separated. Therefore, And the proteins interacting with each other.

However, the conventional immunoprecipitation method has a limitation in revealing a protein factor having a transient covalent bond and binding to a specific protein. In addition, since proteins that do not interact with each other in a cell are likely to interact with each other in an extracellular environment, there is a problem in that proteins found to interact with conventional immunoprecipitation methods may not actually interact in cells There was.

In the conventional studies on protein crosslinking using a chemical crosslinker such as formaldehyde, secondary and tertiary crosslinking reactions with not only adjacent proteins but also nearby proteins occur, There was a problem that the information of the adjacent protein body could not be known accurately because the reaction could not be controlled.

Therefore, in order to solve the above-mentioned problems, it is required to develop a technique capable of specifically identifying proteins interacting with each other in a cell.

In order to solve the above problems and to develop a technique for identifying a target protein and a protein interacting with the target protein in a cell, the present inventors used a crosslinking reaction of a peroxidase to interact with a target protein and the target protein The present inventors have completed the present invention.

Since the peroxidase used in the present invention is linked to a signal sequence targeting various proteins or specific organelles present in the cell and is able to express in the cell and thereby cross-link the peripheral protein at the expressed position, Can be used as an important biologic research tool to solve the problems of immunoprecipitation method which could not find the nonspecific binding and to uncover new binding protein factors which have not been known until now.

An example of the present invention is to prepare a fusion polynucleotide in which a gene encoding a target gene or a target protein and a gene encoding a peroxidase are linked to a cell organelle in which a target protein to be identified interacts with a target protein in a cell, Introducing the fusion polynucleotide into a cell, treating the cell with hydrogen peroxide to obtain a protein cross-linked product by cross-linking proteins interacting with the cell organelles, and culturing the cell lysate obtained by disrupting the cell, And then separating the protein cross-linked product containing the desired protein. The present invention also provides a method for obtaining a protein cross-linked product containing the desired protein.

Another example of the present invention is a method for identifying a protein interacting with a target protein in a cell, comprising the steps of: (a) expressing a target protein to be identified with a target protein in the cell and confirming an electrophoretic pattern of the cell into which the gene encoding the peroxidase is introduced,

In this step, a fused polynucleotide in which a gene encoding a target gene or a target protein is linked to a gene encoding a peroxidase is introduced into a cell organelle in which a target protein exists, and the fused polynucleotide is introduced into cells,

Treating the cells with hydrogen peroxide to cross-link proteins interacting in the organelles to obtain a protein cross-linked product,

Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,

And electrophoresing the separated protein cross-linked product,

(b) confirming an electrophoretic pattern of a cell expressing a target protein to be identified for a protein interacting with a target protein in the cell but not a gene encoding the peroxide,

The step of treating the cells with hydrogen peroxide to cross-link proteins interacting with the cells to obtain a protein cross-linked product,

Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,

And electrophoresing the separated protein cross-linked product,

(c) comparing the protein pattern of the cell obtained in the step (a) with the protein pattern of the cell obtained in the step (b), and

(d) mass spectrometry of electrophoretic bands present in said step (a) but not present in said step (b).

And a method for identifying a protein that interacts with a target protein in a cell.

An example of the present invention relates to a method of crosslinking a target protein using a peroxidase and a protein interacting with the target protein.

Another embodiment of the present invention relates to a method for identifying a protein that interacts with a target protein through cross-linking between a target protein using a peroxidase and a protein interacting with the target protein.

Most proteins interact with various proteins in the cell and perform biochemical reactions. Signal transduction, mechanical movement, and metabolism are examples. However, it is not easy to identify the factors that interact with transient non-covalent bonds. This is because it is not easy to maintain temporary non-covalent bonding when using the conventional immunoprecipitation method.

Since peroxidase catalyzes the oxidation of one of the electrons of a phenolic compound, radicals are formed in the tyrosine group and are known to react with phenol present in adjacent proteins.

Recently, studies have been actively carried out to map various organelles using APEX, a peroxidase enzyme capable of expression and activity in most organelles of living cells. All the methods using APEX that have been established so far use phenol radicals such as biotin phenol as a phenoxy radical and then label proteins. However, when this method is used, the labeling radius becomes about 20 nm, There was a problem that it was difficult to apply it to confirm the action.

The present invention utilizes the property of propagation of radicals to express APEX in a cell organelle in which a target protein exists, or to express APEX in association with a target protein to generate a radical in a tyrosine group present in a target protein, A method of cross-linking a target protein with an adjacent protein interacting with a target protein, and a method of identifying a protein interacting with a target protein cross-linked using the method.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing a fusion protein, which comprises preparing a fusion polynucleotide in which a gene encoding a target gene or a target protein and a gene encoding a peroxidase are linked to a cell organelle in which a target protein for identifying a protein interacting with a target protein in a cell exists, Introducing a polynucleotide into a cell, treating the cell with hydrogen peroxide to obtain a protein cross-linked by cross-linking proteins interacting in the cell organelle, and adding an antibody to the target protein to the cell lysate obtained by breaking the cell And performing immunoprecipitation and separating the protein cross-linked substance containing the target protein. The present invention also relates to a method for obtaining a protein cross-linked product containing a target protein.

In the present invention, the term " target protein " means a protein which intends to cross-link a protein interacting with the protein in the cell, or a protein which intends to identify a protein interacting with the protein in the cell. All my existing proteins can be targeted.

In the present invention, the term " crosslinking " means that the polymer chains are chemically connected to each other directly or via several bonds at arbitrary positions other than the terminals, and also referred to as crossing. Such a bond is called crosslinking (crosslinking or crosslinking) or crosslinking, and the structure formed by the bonding is referred to as a crosslinked structure or a crosslinked structure.

The crosslinking may refer to a chemical bond connecting a target protein with a protein interacting with the target protein, and the chemical bond may mean a covalent bond.

The peroxidase is an enzyme that catalyzes the oxidation reaction of an electron donor by hydrogen peroxide or organic peroxide, and can oxidize phenol, ascorbic acid, glutathione, cytochrome and the like. In the present invention, the peroxidase may be, for example, soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase, Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM (PCM), Phanerochaete chrysosporium PCM (PCM) , Coprinopsis cinerea peroxidase, Peanut peroxidase, Arabidopsis ATPA2, an enzyme consisting of the amino acid sequence of SEQ ID NO: 4, an amino acid sequence of SEQ ID NO: 7, An enzyme made of the amino acid sequence of SEQ ID NO: 8, and the like, It is not to be limited to.

The enzyme consisting of the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 7 is an enzyme that dehydrogenates hydrogen peroxide using ascorbate as a substrate. It is more effective than phenol oxidase in the presence of wild type ascorbate peroxidase Is an ascorbate peroxidase engineered to have an excellent ability to produce a phenoxy radical, and is preferable as a peroxidase enzyme to be applied to the present invention.

The gene sequence encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO:

Specifically, the amino acid sequence of wild-type ascorbate peroxidase may be partially mutated. The enzyme consisting of the amino acid sequence of SEQ ID NO: 4 has the amino acid sequence of the wild type ascorbate peroxidase and the lysine, which is the 14th amino acid, is mutated to aspartic acid (K14D) and the 41st amino acid tryptophan tryptophan is mutated into phenylalanine (W41F), and the 112th amino acid, glutamic acid, is mutated into lysine. The enzyme consisting of the amino acid sequence of SEQ ID NO: 7 has the amino acid sequence of the wild-type ascorbate peroxidase and the lysine, which is the 14th amino acid, is mutated to aspartic acid (K14D) and the 41st amino acid tryptophan tryptophan is converted to phenylalanine (W41F), the 112th amino acid glutamic acid is changed to lysine, and the 134th alanine is changed to proline.

In addition, wild-type ascorbate peroxidase is present as a dimer, whereas ascorbate peroxidase comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 of the present invention exists as a monomer have.

In the present invention, the enzyme consisting of the amino acid sequence of SEQ ID NO: 8 is Horseradish Peroxidase (HRP). The horseradish peroxidase exhibits activity in a secretory pathway with an oxidizing environment. In the present invention, in an endoplasmic reticulum or Golgi, which is an intracellular space on the secretory pathway naturally oxidized, The same effect as the ascorbate peroxidase consisting of SEQ ID NO: 7 can be obtained.

In an embodiment of the present invention, when a peroxidase is introduced into a cell and treated with hydrogen peroxide, the phenol molecule is converted into a phenoxyl radical by the action of the enzyme expressed in the cell, and the phenoxy formed in the phenol molecule The tyrosine group on the surface of the target protein is converted to a radical by a radical, so that the target protein and the protein interacting with the target protein can be crosslinked through a radical-radical coupling reaction.

In addition, since the radicals generated in the tyrosine group of the target protein exist only for a short period of time that is impossible to cross the phospholipid membrane, when radicals are generated in the tyrosine group of the target protein in each of the cell organelles surrounded by the membrane, It is possible to crosslink only the protein interacting with the target protein present in the target protein.

The introduction of the fusion polynucleotide into the cell is a cell organelle in which a target protein to be identified for a protein that interacts with a target protein in the cell is present, and a gene encoding a target gene and a gene encoding a peroxidase are linked, And a gene encoding a peroxidase are linked to each other to prepare a fusion polynucleotide, and the fusion polynucleotide is introduced into a cell, but the present invention is not limited thereto.

In the step of obtaining the protein cross-linked product, the fusion polynucleotide is introduced into a cell, the cell is treated with 0.1 to 2 mM of hydrogen peroxide and reacted for 1 to 10 minutes to crosslink proteins interacting in the cell organelle, May mean a process of obtaining a cross-linked product, and the protein cross-linked product may mean a complex that cross-links with a protein with which the target protein interacts, but is not limited thereto.

In the step of separating the cross-linked product, the cross-linked product is obtained, the cell is disrupted, the antibody against the target protein is added to the cell lysate obtained by immunocapsulation, and the protein cross-linked product containing the target protein is electrophoresed But it is not limited thereto.

Sequence genes targeted to the organelles in which the target protein is present include mitochondrial matrix expression genes, cytoplasmic expression genes, mitochondrial bilayer space expression genes, nuclear expression genes, extracellular membrane expression genes, endoplasmic reticulum expression genes, endoplasmic reticulum expression genes, Or an intracellular membrane expression gene, but are not limited thereto.

The organelles may be selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi apparatus, but the present invention is not limited thereto.

The cells to which the method of the present invention can be applied are not limited and may include all animal cells, plant cells, microorganisms, and the like. For example, it may be a mammalian cell capable of transfection. Specific examples thereof include kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, And may include muscle cells, blood cells, bone marrow cells, lymphocyte cells, mononuclear cells, lung cells, pancreatic cells, liver cells, stomach cells, intestinal cells, cardiac cells, bladder cells, urethral cells, embryonic germ cells or cumulus cells But is not limited thereto. More specifically, the animal cells can be cultured in an animal such as kidney cells (hek293T cells, hek293 cells, etc.), cancer cells (hela cells, etc.), monkey kidney cells (Cos- Cells, etc.), ovary cells (CHO cells, etc.), and the like.

The present invention relates to a method for identifying a protein that interacts with a target protein through cross-linking between a target protein and a protein interacting with the target protein using a peroxidase. More specifically, the present invention relates to: (a) Expressing a target protein to be identified, and confirming an electrophoresis pattern of a cell into which a gene encoding a peroxidase is introduced,

In this step, a fused polynucleotide in which a gene encoding a target gene or a target protein is linked to a gene encoding a peroxidase is introduced into a cell organelle in which a target protein exists, and the fused polynucleotide is introduced into cells,

Treating the cells with hydrogen peroxide to cross-link proteins interacting in the organelles to obtain a protein cross-linked product,

Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,

And electrophoresing the separated protein cross-linked product,

(b) confirming an electrophoretic pattern of a cell expressing a target protein to be identified for a protein interacting with a target protein in the cell but not a gene encoding the peroxide,

The step of treating the cells with hydrogen peroxide to cross-link proteins interacting with the cells to obtain a protein cross-linked product,

Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,

And electrophoresing the separated protein cross-linked product,

(c) comparing the protein pattern of the cell obtained in the step (a) with the protein pattern of the cell obtained in the step (b), and

(d) mass spectrometry of electrophoretic bands present in said step (a) but not present in said step (b).

To a method for identifying a protein that interacts with a desired protein in a cell.

In the step (a), the fusion polynucleotide is introduced into a cell, which is a cell organelle in which a target protein to be identified, which interacts with a target protein in a cell, is present, and a gene encoding the target gene and a peroxide- But is not limited to, preparing a fusion polynucleotide in which a gene encoding a target protein and a gene encoding a peroxidase are linked and introducing the fusion polynucleotide into a cell.

The step of disrupting the cells in the steps (a) and (b) to obtain a cell lysate and electrophoresis to separate the proteins further comprises the step of performing coomassie staining, silver staining, or western blotting of the electrophoresed gel can do.

The cell confirming the pattern of electrophoresis in the step (b) may be a cell expressing a target protein for identifying a protein interacting with a target protein in the cell, but not introducing a gene encoding the peroxidase, A cell into which a gene coding for a target protein and a peroxidase is introduced to identify a protein interacting with the protein, but the cell is different from the target protein in the peroxidase enzyme.

Immunoprecipitating the cell lysate in the step (a) or (b) may be performed by adding an antibody to a target protein or adding an antibody-bound bead to the target protein, It is not.

The step of comparing the protein patterns of the cells obtained in the step (a) or (b) in the step (c) may be performed by naked eye or by imaging. Any method of imaging a pattern can be used without limitation.

The mass spectrometry of the electrophoretic band in the step (d) may be performed using a mass spectrometer, for example, Orbitrap (Thermo), but is not limited thereto.

The present invention relates to a method of cross-linking a target protein in a cell with a protein interacting with the target protein using a peroxidase, wherein the target protein in the cell and the protein interacting with the target protein are cross- Can be used to identify a protein interacting with a target protein in a cell, and can be used for selectively crosslinking two different proteins in a medical field where a protein agent is to be used as a medicine.

1 shows a method of identifying a protein interacting with a target protein by cross-linking a protein interacting with a protein of interest (POI) with APEX, separating the cross-linked protein by immunoprecipitation, and analyzing with a mass spectrometer As shown in Fig.
FIG. 2 is a view showing specific crosslinking by APEX according to an embodiment of the present invention. FIG.
Fig. 3 shows a cleavage map of the MCU-APEX plasmid vector prepared according to the embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

In the following, the enzyme consisting of the amino acid sequence of SEQ ID NO: 4 was named APEX2 and APEX2 was purchased from USA addgene (www.addgene.org). APEX2 is formed by mutation in APEX, and is known to have a higher enzyme activity than APEX.

Example  1. Intracellular APEX protein expression and cross-linking

1-1. Mitochondrial inner membrane Targeting  Production of recombinant vector

To target the APEX protein to the mitochondrial inner membrane, a vector was constructed by inserting the polynucleotide with the Mitochondria Calcium Uniporter (MCU) gene and the APEX2 gene.

The MCU amino acid sequence and the gene sequence thereof, the amino acid sequence of APEX2, and the gene sequence thereof, the gene sequence of V5, and the gene sequence of MCU-V5-APEX2 are shown in Table 1 below.

SEQ ID NO: denomination One Gene sequence of MCU 2 Amino acid sequence of MCU 3 The gene sequence of APEX2 4 The amino acid sequence of APEX2 5 V5 gene sequence 6 The gene sequence of MCU-V5-APEX2

The vector was prepared according to the following method. Specifically, 1 μl of KpnI and BamHI restriction enzymes (purchased from New England biolab) were treated at 37 ° C. for 1 hour with polynucleotides to which the MCU gene, V5 gene, and APEX2 gene linked to Macrogen, A pCDNA5 vector (V601020, thermofisher) digested at 37 ° C for one hour was mixed with 1 μl of T4 DNA ligase (purchased from New England biolab) for ligation at room temperature for 1 hour. Subsequently, the cells were subjected to heat-shake transformation at 42 ° C. for 40 seconds in a Top10 E. coli cell (Invitrogen), followed by overnight incubation on an Ampicilin-Agar plate to isolate only the colonies containing the vector and extract plasmid DNA MCU-APEX plasmid DNA was obtained. The gene sequence of the polynucleotide to which the MCU gene, V5 gene and APEX2 gene are linked is shown in SEQ ID NO:

The structure and characteristics of the recombinant vector are summarized in Table 2 below, and the cleavage map thereof is shown in Fig.

Plasmid Characteristic Promoter / Vector Detail MCU-APEX2 KpnI-MCU-BamHI-V5-APEX2-Flag-Stop-XhoI CMV / pCDNA5 MCU NM_138357.2

1-2. Transformation, protein expression

To introduce the vector prepared in Example 1-1 into cells, the vector was introduced into HEK293T cells purchased from ATCC using a Lipofectamine transfection reagent.

The cells were then cultured in DMEM (Dulbecco Modified Eagle Medium) (Gibco) medium containing Fetal Bovine Serum at 36 ° C for 16-24 hours in a CO ₂ incubator (Esco).

1-3. Bridging Crosslinking ) reaction

Hydrogen peroxide (H ₂ O ₂ ) was treated to a concentration of 1 mM in a culture medium containing HEK293T cells transfected with the vector, and after 1 minute, 10 mM sodium ascorbate, 10 mM sodium azide, and 5 mM Trolox And washed three times with DPBS (Dulbecco's Phosphate Buffered Saline) (Thermofisher, 21300025) containing a small mineral solution (Sigma-Aldrich, 238813).

Example  2. Confirmation of protein expression

2-1. Cell lysis and sampling

In order to confirm the expression in the cell of the above prepared vector, the following experiment was conducted.

Specifically, the cells expressing the protein of Example 1 were pipetted into DPBS and transferred to a 1.5 ml microtube. The cells were centrifuged at 1500 rpm for 5 minutes to leave only the cell pellet. The supernatant was discarded and the 1X proteasome inhibitor cocktail (Elpis bio, EBA1149) supplemented with 1 mM PMSF (Biosesang, P1021) and a mineralsolution solution (Protease inhibitor cocktail (Sigma aldrich, P8340) lysis). The cells were centrifuged at 13000 rpm at 4 ° C for 10 minutes to remove cell debris. 10 μg of the supernatant was added to SDS-protein gel loading buffer containing 20 mM of DTT and heated at 95 ° C. for 10 minutes.

2-2. Western Blot  Confirmation of protein expression through

The sample prepared in Example 2-1 was subjected to SDS-PAGE gel electrophoresis at 150V for about 1 hour. Proteins were electrophoresed on a SDS-PAGE gel, transferred to a nitrocellulose membrane, washed with a TBST solution (mixture of Tris-Buffered Saline and Tween 20), washed with 2% dialyzed BSA solution for 1 hour at room temperature Lt; / RTI > The primary antibody (anti-V5, Invitrogen, R960-25) was diluted to 10000: 1 with 2% (w / v) dialyzed BSA solution and stirred at room temperature for 1 hour. Then, the membrane was rinsed with TBST 4 times for 5 minutes per one time. The secondary antibody (anti-mouse-HRP, Biorad, 1721011) was diluted to 3000: 1 with 2% (w / v) dialyzed BSA solution and stirred at room temperature for 20 minutes. Then, the membrane was rinsed with TBST 4 times for 5 minutes per one time. Developing solution (Clarity ECL product from Biorad) was developed and chemiluminescence imaging was performed using G box (Syngene).

Example  3. Identification of specific cross-linking by APEX

The following experiment was conducted to confirm whether the protein interacting with the target protein and the target protein was specifically crosslinked by APEX.

Specifically, the experiment was carried out in the same manner as in Example 1, The crosslinking reaction was carried out without adding H ₂ O ₂ in Example 1-3, and Western blotting was carried out in the same manner as in Example 2.

The results of the above experiment are shown in Fig.

As can be seen from FIG. 2, it was confirmed that the MCU cross-linked with the interacting protein only when the cross-linking reaction was performed.

Example  4. Cross-linking of intracellular interacting proteins using APEX

4-1. Cell lysis and Immune sedimentation

In order to identify intracellular interacting proteins using APEX, experiments were conducted to identify the interacting proteins of CHCHD3 protein known to be present in the inner membrane of mitochondria in the following manner.

Specifically, the cells expressing the protein of Example 1 were cross-linked, pipetted into DPBS, transferred to a 1.5 ml microtube, centrifuged at 1500 rpm for 5 minutes to leave only the cell pellet, discard the supernatant, Cells were treated with a protease inhibitor cocktail (Sigma aldrich, P8340), 1 mM PMSF (Biosesang, P1021), and RIPA lysis buffer (Elpis bio, EBA1149) supplemented with a minerals solution All were lysed.

Then, centrifugation was carried out at 4 ° C for 10 minutes using a centrifuge at 20,000 rpm to obtain only supernatant. 10 ul of the obtained supernatant was left as an input sample for the SDS-PAGE gel, and 40 μl of magnetic beads (Invitrogen, 10003D) coated with the anti-CHCHD3 antibody (Sigma Aldrich, SAB2106034) And incubated at room temperature (25 ° C) for 1 hour.

Plug in the magnetic stand and keep the same amount of input and flow through (FT) for testing purposes to see if the enrichment is good enough to leave the target protein. The magnetic beads were then washed three times with 2 M urea buffer and once with RIPA buffer. Then, 40 μl of SDS-protein gel loading buffer containing 20 mM of DTT was added and heated at 95 ° C. for 10 minutes, and then inserted into a magnetic stand and allowed to cool.

4-2. Western Blot  Check through

As in Example 3-1, the eluted samples in the beads were subjected to input, FT (flow through) and SDS-PAGE gel electrophoresis at 150V for about 1 hour.

Proteins were electrophoresed on a SDS-PAGE gel, transferred to a nitrocellulose membrane, washed with a TBST solution (mixture of Tris-Buffered Saline and Tween 20), washed with 2% dialyzed BSA solution for 1 hour at room temperature Lt; / RTI > The primary antibody (anti-CHCHD3 antibody, Sigma Aldrich, HPA042935) was diluted to 3000: 1 with 2% (w / v) dialyzed BSA solution and stirred at room temperature for 1 hour. Then, the membrane was rinsed with TBST 4 times for 5 minutes per one time. Developing solution (Clarity ECL product from Biorad) was developed and chemiluminescence imaging was performed using G box (Syngene).

Example  5 Identification of intracellular interacting proteins using APEX

5-1. Bridged  Sampling of protein bands

The same sample, which was identified by Western blotting in Example 4-2, was electrophoresed and coomassie staining was performed as follows.

Specifically, the gel electrophoresed with 0.1% (w / v) Coomassie Brilliant Blue R-250 (Biosesang, C1031) was stained for 60 minutes and discolored using 30% methanol and 10% acetic acid solution. After decolorization, the electrophoresis gel was washed with distilled water and a cross-linked Coomassie stained band that did not appear in the negative control was cut out.

5-2. Through mass analysis Bridged  Identification of proteins

The bands sampled in Example 5-1 were mass analyzed in the following manner to identify candidate proteins that interact with CHCHD3.

Specifically, the gel band cut in Example 5-1 was thoroughly washed with distilled water, the gel piece was completely dried with Speed vac (Eppendorf, 38-022820125), and then 100 mM ABC (Ammonium bicarbonate, Sigma-Aldrich, A6141) After reduction for 30 minutes with 10 mM DTT dissolved (Dithiothreitol, Sigm-Aldrich, 43817), alkylation was performed for 20 minutes with light blocked using 55 mM Iodoacetamide (Sigma-Aldrich, I1149) dissolved in 100 mM ABC. After washing with distilled water, the gel was completely dried, and 375 ng of trypsin was added to the solution. The protein was cut to the peptide level, and the peptide was dissolved in the above gel with 2: 1 mixture of acetonitrile and 5% (v / v) formic acid Extracted. The extracted peptides were completely dried and stored until analysis. The samples were stored in a dry state and dissolved in 2% (w / v) acetonitrile and 0.05% (v / v) TFA (Trifluoroacetic acid, Sigma-Aldrich) and then subjected to LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) Respectively.

<110> UNIST ACADEMY-INDUSTRY RESEARCH CORPORATION <120> Method for analyzing interacting proteins in cells using          peroxidase <130> DPP20146249 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 2962 <212> DNA <213> Homo sapiens <400> 1 ggcgccgttt ccagttgaga gatggcggcc gccgcaggta gatcgctcct gctgctcctc 60 tcctctcggg gcggcggcgg cgggggcgcc ggcggctgcg gggcgctgac tgccggctgc 120 ttccctgggc tgggcgtcag ccgccaccgg cagcagcagc accaccggac ggtacaccag 180 aggatcgctt cctggcagaa tttgggagct gtttattgca gcactgttgt gccctctgat 240 gatgttacaga tggtttatca aaatgggtta cctgtgatat ctgtgaggct accatcccgg 300 cgtgaacgct gtcagttcac actcaagcct atctctgact ctgttggtgt atttttacga 360 caactgcaag aagaggatcg gggaattgac agagttgcta tctattcacc agatggtgtt 420 cgcgttgctg cttcaacagg aatagacctc ctcctccttg atgactttaa gctggtcatt 480 aatgacttaa cataccacgt acgaccacca aaaagagacc tcttaagtca tgaaaatgca 540 gcaacgctga atgatgtaaa gacattggtc cagcaactat acaccacact gtgcattgag 600 cagcaccagt taaacaagga aagggagctt attgaaagac tagaggatct caaagagcag 660 ctggctcccc tggaaaaggt acgaattgag attagcagaa aagctgagaa gaggaccact 720 ttggtgctat ggggtggcct tgcctacatg gccacacagt ttggcatttt ggcccggctt 780 acctggtggg aatattcctg ggacatcatg gagccagtaa catacttcat cacttatgga 840 agtgccatgg caatgtatgc atattttgta atgacacgcc aggaatatgt ttatccagaa 900 gccagagaca gacaatactt actatttttc cataaaggag ccaaaaagtc acgttttgac 960 ctagagaaat acaatcaact caaggatgca attgctcagg cagaaatgga ccttaagaga 1020 ctgagagacc cattacaagt acatctgcct ctccgacaaa ttggtgaaaa agattgatct 1080 gcaaaaagcc tctgaatcct ggcagaagga acacctgttt gcctttttaa ttaaagcatt 1140 gcaggtggaa gctgggagcc atgtgggggg tagagcgttt ttacctttaa ttataaaaca 1200 aaaacagaaa ggatctgagg gaagaaggga atgttaaaac ctgaggatca ggcattgtgg 1260 aatataagct caaagggctt agtgaatatt gtcttaacca agtatctcag tttctggatg 1320 aaaatgatgc agttatatag ttgagagatt cataaagaga aaacaatgct gggggtgttc 1380 gtttcttgca tcttctttgc agagtcagca aaagagtaac acaccagcac cccactcgac 1440 tctatttgtt tttaatttaa ctgtccctat ttttgacata ggagtaaata aatatactag 1500 aaaagcaaat tctcatgata tgctaaaata tcattagcat ttattttaaa ttggacccag 1560 tctctgcaga gttaccagga atctttcctt ccagcatccc tttactgacc acctacctgt 1620 acctcttggt tacactcatt ttttccattt gataattgga accaacttat aactgtttaa 1680 taattgacac tttagattat ctcttaatac cttcttaaat gtctatatat cccagtgctc 1740 tggatcagtg tctaaaaatc actggcaaca ctgcatgagg ttgttggttt tgttttgttt 1800 tattaattag tctttcacag gaggaataat tgccctcctt tatatactta tctattgata 1860 atcccctctc cctccagaac acaaatcaga gggaaagggg gtgttcagct gtactaccaa 1920 atcaggaaga tgtaaggttt acaaattggc taagaatcat ggctctgtag ccatttcaac 1980 cagaataatt ttattgctaa tctgctttgt gtgacagcat tccaggccag ccagatggga 2040 ctgccttgtc tggaggcttt gttcatctcg aaggacacac acttccacac tgtttgtgag 2100 ccctcccacc tccacaactt cagttgtaaa tcaagtgtgt ggatctcaaa gggtgcaatt 2160 tatctttata taggaataca tttctagggc ttccttcaag cccactctct tcaccctatt 2220 ttttcttatc ttaaattgag agaaagagaa ttaatcttat actttgtcaa aacattttct 2280 accatatttc cagatgacat ctgcgcttga agagtcaaag gaatctgtgt ctaatatcct 2340 gtttttaact gctgtagggg caggatggaa aggatgatgg gggctgccac accactgatt 2400 ggccttttct ttcacgtgat tcatccttcc tcattgtggc aaggagtttc tttctctttt 2460 tcttcctcct ttgggatcat tgtgtatgaa aagaaaaact ttaaatgaca aacccagact 2520 ccaggtgcct tgcaaaggtt gaaggccagc caggattgct gctgctgctg ctactcctgc 2580 caacacccct ttcattggca tgacggaatg aaaggatgca tgtctccact tcctgaccct 2640 ccgcccactt ccttctccct ccaccacccc cagtcgtcag ctccttccct catttatttt 2700 tgttaagttg tgtgaattat ttttaaccca tttatcctgt ttgtgcatag ggtttttaag 2760 aagaaacagc acagtgcaac gagcaaatct ttttggggtg tgtgggaagc aagggaggga 2820 ggacatggag aaaagttctt taaacaaata gcaaactatt gaacatgtgt aaaatcctgt 2880 atcatttatg aaatatgtat aaaaagcaat gtaccttctg gaacaataaa tacttattca 2940 atttttgaaa aaaaaaaaaa aa 2962 <210> 2 <211> 351 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ala Ala Ala Gly Arg Ser Leu Leu Leu Leu Ser Ser Arg   1 5 10 15 Gly Gly Gly Gly Gly Gly Ala Gly Gly Cys Gly Ala Leu Thr Ala Gly              20 25 30 Cys Phe Pro Gly Leu Gly Val Ser Arg His His Gln Gln Gln His His          35 40 45 Arg Thr Val His Gln Arg Ile Ala Ser Trp Gln Asn Leu Gly Ala Val      50 55 60 Tyr Cys Ser Thr Val Val Ser Ser Asp Val Thr Val Val Tyr Gln  65 70 75 80 Asn Gly Leu Pro Val Ile Ser Val Arg Leu Pro Ser Arg Arg Glu Arg                  85 90 95 Cys Gln Phe Thr Leu Lys Pro Ile Ser Asp Ser Val Gly Val Phe Leu             100 105 110 Arg Gln Leu Gln Glu Glu Asp Arg Gly Ile Asp Arg Val Ala Ile Tyr         115 120 125 Ser Pro Asp Gly Val Val Ala Ala Ser Thr Gly Ile Asp Leu Leu     130 135 140 Leu Leu Asp Asp Phe Lys Leu Val Ile Asn Asp Leu Thr Tyr His Val 145 150 155 160 Arg Pro Pro Lys Arg Asp Leu Leu Ser His Glu Asn Ala Ala Thr Leu                 165 170 175 Asn Asp Val Lys Thr Leu Val Gln Gln Leu Tyr Thr Thr Leu Cys Ile             180 185 190 Glu Gln His Gln Leu Asn Lys Glu Arg Glu Leu Ile Glu Arg Leu Glu         195 200 205 Asp Leu Lys Glu Gln Leu Ala Pro Leu Glu Lys Val Arg Ile Glu Ile     210 215 220 Ser Arg Lys Ala Glu Lys Arg Thr Thr Leu Val Leu Trp Gly Gly Leu 225 230 235 240 Ala Tyr Met Ala Thr Gln Phe Gly Ile Leu Ala Arg Leu Thr Trp Trp                 245 250 255 Glu Tyr Ser Trp Asp Ile Met Glu Pro Val Thr Tyr Phe Ile Thr Tyr             260 265 270 Gly Ser Ala Met Ala Met Tyr Ala Tyr Phe Val Met Thr Arg Gln Glu         275 280 285 Tyr Val Tyr Pro Glu Ala Arg Asp Arg Gln Tyr Leu Leu Phe Phe His     290 295 300 Lys Gly Ala Lys Lys Ser Arg Phe Asp Leu Glu Lys Tyr Asn Gln Leu 305 310 315 320 Lys Asp Ala Ile Ala Gln Ala Glu Met Asp Leu Lys Arg Leu Arg Asp                 325 330 335 Pro Leu Gln Val His Leu Pro Leu Arg Gln Ile Gly Glu Lys Asp             340 345 350 <210> 3 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> gene of Engineered ascorbate peroxidase 2 (APEX2) <400> 3 atgggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag 60 cgctgagaag ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc 180 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt 240 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct 300 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag 360 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat 420 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct 480 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc 540 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa 600 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc 660 gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa 720 aagctttccg agcttgggtt tgctgatgcc 750 <210> 4 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Engineered ascorbate peroxidase 2 (APEX2) <400> 4 Met Gly Lys Ser Tyr Pro Thr Val Ser Ala Asp Tyr Gln Asp Ala Val   1 5 10 15 Glu Lys Ala Lys Lys Lys Leu Arg Gly Phe Ile Ala Glu Lys Arg Cys              20 25 30 Ala Pro Leu Met Leu Arg Leu Ala Phe His Ser Ala Gly Thr Phe Asp          35 40 45 Lys Gly Thr Lys Thr Gly Gly Pro Phe Gly Thr Ile Lys His Pro Ala      50 55 60 Glu Leu Ala His Ser Ala Asn Asn Gly Leu Asp Ile Ala Val Arg Leu  65 70 75 80 Leu Glu Pro Leu Lys Ala Glu Phe Pro Ile Leu Ser Tyr Ala Asp Phe                  85 90 95 Tyr Gln Leu Ala Gly Val Val Ala Val Glu Val Thr Gly Gly Pro Lys             100 105 110 Val Pro Phe His Pro Gly Arg Glu Asp Lys Pro Glu Pro Pro Pro Glu         115 120 125 Gly Arg Leu Pro Asp Pro Thr Lys Gly Ser Asp His Leu Arg Asp Val     130 135 140 Phe Gly Lys Ala Met Gly Leu Thr Asp Gln Asp Ile Val Ala Leu Ser 145 150 155 160 Gly Gly His Thr Ile Gly Ala Ala His Lys Glu Arg Ser Gly Phe Glu                 165 170 175 Gly Pro Trp Thr Ser Asn Pro Leu Ile Phe Asp Asn Ser Tyr Phe Thr             180 185 190 Glu Leu Leu Ser Gly Glu Lys Glu Gly Leu Leu Gln Leu Pro Ser Asp         195 200 205 Lys Ala Leu Leu Ser Asp Pro Val Phe Arg Pro Leu Val Asp Lys Tyr     210 215 220 Ala Ala Asp Glu Asp Ala Phe Phe Ala Asp Tyr Ala Glu Ala His Gln 225 230 235 240 Lys Leu Ser Glu Leu Gly Phe Ala Asp Ala                 245 250 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> V5 <400> 5 ggcaagccca tccccaaccc cctgctgggc ctggacagca cc 42 <210> 6 <211> 1857 <212> DNA <213> Artificial Sequence <220> <223> MCU-V5-APEX2 <400> 6 atggcggccg ccgcaggtag atcgctcctg ctgctcctct cctctcgggg cggcggcggc 60 gggggcgccg gcggctgcgg ggcgctgact gccggctgct tccctgggct gggcgtcagc 120 cgccaccggc agcagcagca ccaccggacg gtacaccaga ggatcgcttc ctggcagaat 180 ttgggagctg tttattgcag cactgttgtg ccctctgatg atgttacagt ggtttatcaa 240 aatgggttac ctgtgatatc tgtgaggcta ccatcccggc gtgaacgctg tcagttcaca 300 ctcaagccta tctctgactc tgttggtgta tttttacgac aactgcaaga agaggatcgg 360 ggaattgaca gagttgctat ctattcacca gatggtgttc gcgttgctgc ttcaacagga 420 atagacctcc tcctccttga tgactttaag ctggtcatta atgacttaac ataccacgta 480 cgaccaccaa aaagagacct cttaagtcat gaaaatgcag caacgctgaa tgatgtaaag 540 acattggtcc agcaactata caccacactg tgcattgagc agcaccagtt aaacaaggaa 600 agggagctta ttgaaagact agaggatctc aaagagcagc tggctcccct ggaaaaggta 660 cgaattgaga ttagcagaaa agctgagaag aggaccactt tggtgctatg gggtggcctt 720 gcctacatgg ccacacagtt tggcattttg gcccggctta cctggtggga atattcctgg 780 gacatcatgg agccagtaac atacttcatc acttatggaa gtgccatggc aatgtatgca 840 tattttgtaa tgacacgcca ggaatatgtt tatccagaag ccagagacag acaatactta 900 ctatttttcc ataaaggagc caaaaagtca cgttttgacc tagagaaata caatcaactc 960 aaggatgcaa ttgctcaggc agaaatggac cttaagagac tgagagaccc attacaagta 1020 catctgcctc tccgacaaat tggtgaaaaa gataaggatc cgctagcagg caagcccatc 1080 cccaaccccc tgctgggcct ggacagcacc ggaaagtctt acccaactgt gagtgctgat 1140 taccaggacg ccgttgagaa ggcgaagaag aagctcagag gcttcatcgc tgagaagaga 1200 tgcgctcctc taatgctccg tttggcattc cactctgctg gaacctttga caagggcacg 1260 aagaccggtg gacccttcgg aaccatcaag caccctgccg aactggctcag cagcgctaac 1320 aacggtcttg acatcgctgt taggcttttg gagccactca aggcggagtt ccctattttg 1380 agctacgccg atttctacca gttggctggc gttgttgccg ttgaggtcac gggtggacct 1440 aaggttccat tccaccctgg aagagaggac aagcctgagc caccaccaga gggtcgcttg 1500 cccgatccca ctaagggttc tgaccatttg agagatgtgt ttggcaaagc tatggggctt 1560 actgaccaag atatcgttgc tctatctggg ggtcacacta ttggagctgc acacaaggag 1620 cgttctggat ttgagggtcc ctggacctct aatcctctta ttttcgacaa ctcatacttc 1680 acggagttgt tgagtggtga gaaggaaggt ctccttcagc taccttctga caaggctctt 1740 ttgtctgacc ctgtattccg ccctctcgtt gataaatatg cagcggacga agatgccttc 1800 tttgctgatt acgctgaggc tcaccaaaag ctttccgagc ttgggtttgc tgatgcc 1857 <210> 7 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Engineered ascorbate peroxidase (APEX) <400> 7 Met Gly Lys Ser Tyr Pro Thr Val Ser Ala Asp Tyr Gln Asp Ala Val   1 5 10 15 Glu Lys Ala Lys Lys Lys Leu Arg Gly Phe Ile Ala Glu Lys Arg Cys              20 25 30 Ala Pro Leu Met Leu Arg Leu Ala Phe His Ser Ala Gly Thr Phe Asp          35 40 45 Lys Gly Thr Lys Thr Gly Gly Pro Phe Gly Thr Ile Lys His Pro Ala      50 55 60 Glu Leu Ala His Ser Ala Asn Asn Gly Leu Asp Ile Ala Val Arg Leu  65 70 75 80 Leu Glu Pro Leu Lys Ala Glu Phe Pro Ile Leu Ser Tyr Ala Asp Phe                  85 90 95 Tyr Gln Leu Ala Gly Val Val Ala Val Glu Val Thr Gly Gly Pro Lys             100 105 110 Val Pro Phe His Pro Gly Arg Glu Asp Lys Pro Glu Pro Pro Pro Glu         115 120 125 Gly Arg Leu Pro Asp Ala Thr Lys Gly Ser Asp His Leu Arg Asp Val     130 135 140 Phe Gly Lys Ala Met Gly Leu Thr Asp Gln Asp Ile Val Ala Leu Ser 145 150 155 160 Gly Gly His Thr Ile Gly Ala Ala His Lys Glu Arg Ser Gly Phe Glu                 165 170 175 Gly Pro Trp Thr Ser Asn Pro Leu Ile Phe Asp Asn Ser Tyr Phe Thr             180 185 190 Glu Leu Leu Ser Gly Glu Lys Glu Gly Leu Leu Gln Leu Pro Ser Asp         195 200 205 Lys Ala Leu Leu Ser Asp Pro Val Phe Arg Pro Leu Val Asp Lys Tyr     210 215 220 Ala Ala Asp Glu Asp Ala Phe Phe Ala Asp Tyr Ala Glu Ala His Gln 225 230 235 240 Lys Leu Ser Glu Leu Gly Phe Ala Asp Ala                 245 250 <210> 8 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> Horseradish peroxidase <400> 8 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser   1 5 10 15 Asn Ile Val Arg Asp Thr Ile Val Asn Glu Leu Arg Ser Asp Pro Arg              20 25 30 Ile Ala Ser Ile Leu Arg Leu His Phe His Asp Cys Phe Val Asn          35 40 45 Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr      50 55 60 Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Pro Val  65 70 75 80 Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro Arg Thr Val                  85 90 95 Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln Ser Val Thr Leu             100 105 110 Ala Gly Gly Pro Ser Trp Arg Val Val Leu Gly Arg Arg Asp Ser Leu         115 120 125 Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro Phe Phe     130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn                 165 170 175 Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly             180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly         195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp Phe Asp Leu     210 215 220 Arg Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu Ile Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn                 245 250 255 Ala Thr Asp Thr Ile Pro Leu Val Arg Ser Phe Ala Asn Ser Thr Gln             260 265 270 Thr Phe Phe Asn Ala Phe Val Glu Ala Met Asp Arg Met Gly Asn Ile         275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu Asn Cys Arg Val     290 295 300 Val Asn Ser Asn Ser 305

Claims (13)

A fusion protein is produced by preparing a fusion polynucleotide in which a target gene or a gene encoding a target protein and a gene encoding a peroxidase are linked to a cell organelle in which a target protein to be identified for interaction with a target protein in the cell is present, Lt; / RTI &gt;
Treating the cells with hydrogen peroxide to crosslink proteins interacting in the organelles to obtain a protein cross-linked product; and
Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation, and separating a protein cross-linked product containing the target protein, thereby obtaining a protein cross-linked product containing the target protein. The method according to claim 1, wherein the peroxidase is selected from the group consisting of Soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, Spinach ascorbate peroxidase, Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Phanerochaete chrysosporium PCM (PCM), Phanerochaete chrysosporium PCM (PCM) An enzyme comprising the amino acid sequence of SEQ ID NO: 4, an enzyme consisting of the amino acid sequence of SEQ ID NO: 7, an enzyme consisting of the amino acid sequence of SEQ ID NO: 7, an enzyme consisting of the amino acid sequence of SEQ ID NO: , And an enzyme consisting of the amino acid sequence of SEQ ID NO: 8 Lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt; 2. The method of claim 1, wherein the hydrogen peroxide is treated with 0.1 to 2 mM hydrogen peroxide for 1 to 10 minutes. The method according to claim 1, wherein the sequence gene targeted to the organelles in which the target protein is present includes a mitochondrial substrate expression gene, a cytoplasmic expression gene, a mitochondrial bilayer membrane spacer gene, a nuclear expression gene, an extracellular membrane expression gene, , An endoplasmic reticulum outer membrane expression gene, or an intracellular membrane expression gene. The method of claim 1, wherein the organelles are selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi. The method of claim 1, wherein the cells are selected from the group consisting of kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, Chondrocytes, macrophages, muscle cells, blood cells, bone marrow cells, lymphocyte cells, mononuclear cells, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, cardiac cells, bladder cells, urethral cells, Lt; / RTI &gt; (a) expressing a target protein for identifying a protein interacting with a target protein in a cell, and confirming an electrophoresis pattern of a cell into which a gene encoding a peroxidase is introduced,
In this step, a fused polynucleotide in which a gene encoding a target gene or a target protein is linked to a gene encoding a peroxidase is introduced into a cell organelle in which a target protein exists, and the fused polynucleotide is introduced into cells,
Treating the cells with hydrogen peroxide to cross-link proteins interacting in the organelles to obtain a protein cross-linked product,
Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,
And electrophoresing the separated protein cross-linked product,
(b) confirming an electrophoretic pattern of a cell expressing a target protein to be identified for a protein interacting with a target protein in the cell but not a gene encoding the peroxide,
The step of treating the cells with hydrogen peroxide to cross-link proteins interacting with the cells to obtain a protein cross-linked product,
Adding an antibody to a target protein to a cell lysate obtained by disrupting the cell, performing immunoprecipitation and separating a protein cross-linked product containing the target protein,
And electrophoresing the separated protein cross-linked product,
(c) comparing the protein pattern of the cell obtained in the step (a) with the protein pattern of the cell obtained in the step (b), and
(d) mass spectrometry of electrophoretic bands present in said step (a) but not present in said step (b).
A method for identifying a protein that interacts with a target protein in a cell. The method according to claim 7, wherein the peroxidase in the steps (a) and (b) is selected from the group consisting of soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase (Spinach ascorbate peroxidase), Saccharomyces cerevisiae cytochrome c peroxidase, Phanerochaete chrysosporium PCL1, Panerocaine tercrisporium peroxide An enzyme consisting of the amino acid sequence of SEQ ID NO: 4, an enzyme PCM (Phanerochaete chrysosporium PCM), Coprinopsis cinerea peroxidase, peanut peroxidase, Arabidopsis ATPA2, An enzyme consisting of the amino acid sequence of SEQ ID NO: 7 and an enzyme consisting of the amino acid sequence of SEQ ID NO: The method of one or more enzymes selected from the group consisting of. The method according to claim 7, wherein the step (a) or (b) is performed by disrupting the cells to obtain a cell lysate and separating the proteins by electrophoresis, the electrophoresis of the electrophoresed gel is carried out by coomassie dyeing, silver staining, The method further comprising performing the lot. 8. The method of claim 7, wherein treating the hydrogen peroxide in steps (a) and (b) comprises treating the hydrogen peroxide with 0.1 to 2 mM and performing for 1 to 10 minutes. [8] The method according to claim 7, wherein the step (a) comprises the steps of: (a) expressing a mitochondrial matrix expression gene, a cytoplasmic expression gene, a mitochondrial bilayer membrane spacer gene, a nuclear expression gene, An endoplasmic reticulum expression gene, an endoplasmic reticulum expression gene, or an endocardial expression gene. 8. The method of claim 7, wherein the organelles are selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi. 8. The method of claim 7, wherein the cells are selected from the group consisting of kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, Chondrocytes, macrophages, muscle cells, blood cells, bone marrow cells, lymphocyte cells, mononuclear cells, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, cardiac cells, bladder cells, urethral cells, Lt; / RTI &gt;

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