KR102072222B1 - Method for analyzing interacting proteins in cells using peroxidase - Google Patents

Abstract

The present invention relates to a method for identifying a protein that interacts with a target protein by crosslinking the protein of interest with the target protein in the cell.

Description

Method 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 cells using peroxidase and a method for identifying a protein interacting with the target protein by mass spectrometry of the cross-linked protein. To produce a fusion polynucleotide is linked to the target gene and the gene encoding the peroxidase as a cell organelle in which the target protein exists to identify the protein interacting with the fusion polynucleotide is introduced into the cell, and the cell is treated with hydrogen peroxide Cross-linking the interacting proteins in the cell organelles to obtain a protein crosslinked product, and adding the antibody to the protein of interest to the cell lysate obtained by crushing the cell to perform immunoprecipitation. Four stages to separate It relates to a method of obtaining a protein crosslinked product containing the desired protein.

Most proteins function in vivo by interacting with other proteins around them. In a cell, one or two or more proteins exhibit biological function through interaction (non-covalent, physical binding) with each other. Thus, understanding the interactions between proteins is of great importance in understanding biological function. Efforts to identify the interactions between proteins have continued to date in a variety of disciplines, including biochemistry, proteomics, molecular dynamics, and signal transduction.

It is known in the art to use 'immunoprecipitation' using the affinity of antibodies and antigens (in this case, proteins) to confirm the interaction between these proteins. Specifically, immunoprecipitation refers to a method of specifically separating an antigen from a solution by using an affinity between an antibody and an antigen (in this case, a protein). In this case, if there is a protein factor that interacts with the antigen in a non-covalent bond in an aqueous solution, is it possible to separate the antigen-binding antigen with the protein that interacts with the antigen? Proteins that interact with can be isolated.

However, conventional immunoprecipitation has a limitation in identifying protein factors that bind to specific proteins with transient covalent bonds. In addition, since proteins that do not interact with each other in the cell may interact in an extracellular environment, proteins identified as interacting by conventional immunoprecipitation may not actually interact in the cell. This existed.

In addition, in the study of protein crosslinking using a conventional chemical crosslinker such as formaldehyde, secondary and tertiary crosslinking reactions occur not only with adjacent proteins but also with non-contiguous proteins, and successive 2-3th-order crosslinking occurs. Since the reaction could not be controlled, there was a problem that the information of the proximal protein body was not known exactly.

Therefore, in order to solve the above problems, the development of a technique capable of specifically identifying proteins interacting in cells is required.

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 the cell, the present inventors interact with the target protein and the target protein using a cross-linking reaction of peroxidase. The present invention has been completed by developing a technology capable of crosslinking a protein.

The peroxidase used in the present invention can be expressed in a cell by being linked to a signal sequence targeting to various proteins or specific organelles present in the cell, thereby crosslinking the surrounding protein at the expressed position. It can be used as an important biological research tool to solve the problem of immunoprecipitation, which could not detect non-covalent binding, and to newly discover new binding protein factors that are not known.

An embodiment of the present invention is to produce a fusion polynucleotide in which a target gene or a gene encoding a target protein and a gene encoding a peroxidase are connected to a cell organelle in which a target protein exists to identify a protein that interacts with a target protein in a cell. Introducing the fusion polynucleotide into a cell and subjecting the cell to hydrogen peroxide to crosslink the interacting proteins in the cell organelle to obtain a protein crosslink, and to disrupt the cell with an antibody against a target protein. It is to provide a method for obtaining a protein cross-linked protein containing the target protein comprising the step of performing immunoprecipitation and separating the protein cross-linked protein containing the target protein.

Another embodiment of the present invention is a step of identifying the electrophoresis pattern of a cell expressing a target protein and a gene encoding a peroxidase to be introduced (a) to identify a protein that interacts with the target protein in the cell,

In the step, the fusion polynucleotide is introduced into a cell 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 the target protein is present.

Treating the cells with hydrogen peroxide to crosslink the interacting proteins in the organelles to obtain protein crosslinks;

Performing immunoprecipitation by adding an antibody against the protein of interest to the cell lysate obtained by crushing the cells, and separating the protein crosslinked product containing the protein of interest,

Electrophoresing the separated protein crosslinked product,

(b) identifying the electrophoretic pattern of cells expressing the target protein to be identified with the target protein in the cell but not introducing the gene encoding the peroxidase,

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

Performing immunoprecipitation by adding an antibody against the protein of interest to the cell lysate obtained by crushing the cells, and separating the protein crosslinked product containing the protein of interest,

Electrophoresing the separated protein crosslinked product,

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

(d) mass spectrometry of the electrophoretic band present in step (a) but not present in step (b),

It is to provide a method for identifying a protein that interacts with the target protein in the cell.

One embodiment of the present invention relates to a method of crosslinking a target protein using 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 by crosslinking a target protein using peroxidase and a protein that interacts with the target protein.

Most proteins interact with various proteins in cells and carry out biochemical reactions. Examples include signal transmission, mechanical movements, and metabolism. But it is not easy to identify the factors that interact with the temporary noncovalent bond. This is because the conventional method, immunoprecipitation, is not easy to maintain temporary non-covalent bonds.

Peroxidases are known to produce radicals in the tyrosine group and to react with phenols present in other adjacent proteins because they oxidize one electron in the phenolic compound.

Recently, studies have been actively conducted to map various cell organelles using APEX, which is an expressive and active peroxidase in most organelles of living cells. All methods using APEX established up to now use the method of labeling proteins after making phenolic compounds such as biotin phenols into phenoxy radicals, but when using this method, the labeling radius is about 20 nm. There was a problem that it was difficult to apply to confirm the action.

The present invention utilizes the properties of propagation of radicals to express APEX in cell organelles in which a target protein is present, or to associate with the target protein to express APEX to generate radicals in tyrosine groups present in the target protein. It is an object of the present invention to provide a method for crosslinking a target protein with a target protein that interacts with the target protein and a method for identifying a protein that interacts with the target protein crosslinked using the method.

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

The present invention provides 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 exists to identify a protein that interacts with the target protein in a cell. Introducing a polynucleotide into a cell and subjecting the cell to hydrogen peroxide to crosslink the interacting proteins in the cell organelle to obtain a protein crosslink, and to add the antibody to the protein of interest to the cell lysate obtained by crushing the cell. By performing immunoprecipitation and separating the protein crosslinked product containing the target protein, the present invention relates to a method for obtaining a protein crosslinked product containing the target protein.

In the present invention, the term "target protein" refers to a protein that is intended to crosslink a protein that interacts with the protein in a cell, or a protein that is to identify a protein that interacts with the protein in a cell. Any protein in me can be the target.

In the present invention, the term "crosslinking" means that the polymer chains are chemically linked to each other directly or through several bonds at any position other than the terminal, and also referred to as cross. Such bonds are referred to as crosslinking or crosslinkage or crosslinking, and the structure formed by the bonds is called crosslinked or crosslinked structure.

The crosslinking may mean a chemical bond connecting a target protein with a protein that interacts with the target protein, and the chemical bond may mean a covalent bond.

The peroxidase is an enzyme catalyzing the oxidation reaction of the 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 is, for example, Soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, Spinach ascorbate peroxidase, Saccharin peroxidase Saccharomyces cerevisiae cytochrome c peroxidase, Panerocaete chrysosporium peroxidase PCL1, Panerocaete chrysosporium peroxidase PCM (Phanerochaete chrysosporium PCM) , Coprinopsis cinerea peroxidase, peanut peroxidase, Arabidopsis ATPA2, an enzyme consisting of the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 7 It may be an enzyme consisting of an enzyme consisting 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 enzyme consisting of the amino acid sequence of SEQ ID NO: 7 are enzymes for dehydrating hydrogen peroxide using ascorbate as a substrate, and are phenoxy compared to wild type ascorbate peroxidase. Ascorbate peroxidase engineered to have an excellent ability to generate phenoxyl radicals, and is preferable as the peroxidase 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 described in SEQ ID NO: 3.

Specifically, the amino acid sequence of the wild type ascorbate peroxidase may be a partial mutation. In the amino acid sequence of SEQ ID NO: 4, the lysine, the 14th amino acid, was transformed into aspartic acid (K14D) in the amino acid sequence of wild type ascorbate peroxidase (K14D), and the 41st amino acid tryptophan ( tryptophan is mutated to phenylalanine (W41F), and glutamic acid, the 112th amino acid, to lysine. The enzyme consisting of the amino acid sequence of SEQ ID NO: 7 is the lysine (14th amino acid) lysine (assic acid) mutated (K14D) in the amino acid sequence of the wild type ascorbate peroxidase (K14D), tryptophan (41st amino acid) tryptophan is mutated to phenylalanine (phenylalanine) (W41F), the 112th amino acid glutamic acid (glutamic acid) is transformed to lysine and 134th alanine (proline) is transformed to proline.

In addition, wild type ascorbate peroxidase is present as a dimer, ascorbate peroxidase consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 of the present invention is characterized by the presence of a monomer (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 the secretory pathway (Secretory Pathway) with an oxidizing environment. In the present invention, SEQ ID NO: 4 or the sequence in the endoplasmic reticulum or Golgi body, which is an intracellular space on the secretory pathway in which the oxidizing environment is naturally formed. It can produce the same effect as the ascorbate peroxidase which consists of number 7.

In one embodiment of the present invention, when the peroxidase is introduced into the 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 from the phenol molecule. By converting tyrosine groups on the surface of the target protein into radicals by radicals, the target protein and the protein interacting with the target protein may 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 time short enough to cross the phospholipid membrane, if the radicals are generated in the tyrosine group of the target protein in each of the cell organelles surrounded by the membrane, the target protein is formed in each cell organelle. Crosslinking is possible only with proteins that interact with the target protein present in it.

The introduction of the fusion polynucleotide into a cell is a cell organelle in which a target protein exists to identify a protein that interacts with a target protein in a cell, and a targeting gene and a gene encoding a peroxidase are linked or a gene encoding a target protein. It may mean that the fusion polynucleotides are introduced into a cell by preparing a fusion polynucleotide to which a gene encoding peroxidase is linked, but is not limited thereto.

The step of obtaining the protein cross-linked product may include introducing the fusion polynucleotide into a cell, and then treating the cell with 0.1 to 2 mM of hydrogen peroxide and reacting for 1 to 10 minutes to cross-link proteins that interact in the organelles. It may mean a process of obtaining a crosslinked body, and the protein crosslinked body may mean a complex that crosslinks with a protein to which a target protein interacts, but is not limited thereto.

Separation of the crosslinked product may be performed by immunoprecipitation by adding an antibody against the target protein to the cell lysate obtained by crushing the cells, and then electrophores the protein crosslinked product containing the target protein. It may mean a process of separating, but is not limited thereto.

The sequence genes targeted to the cell organelles in which the target protein exists include mitochondrial matrix expression genes, cytoplasmic expression genes, mitochondrial bilayer space expression genes, nuclear expression genes, extracellular membrane expression genes, endoplasmic reticulum expression genes, vesicle outer membrane expression genes, Or may be an endothelial expression gene, but is not limited thereto.

The cell organelle may be selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi apparatus, but 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 an animal cell capable of transfection. Specific examples include kidney cells, cancer cells, skin cells, ovarian cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, Muscle cells, blood cells, bone marrow cells, lymphocyte cells, monocytes, lung cells, pancreas cells, liver cells, gastric cells, intestinal cells, heart cells, bladder cells, urethral cells, embryonic germ cells, or oocytes and the like. However, it is not limited thereto. More specifically, the animal cells are kidney cells (hek293T cells, hek293 cells, etc.), cancer cells (hela cells, etc.), monkey kidney cells (Cos-7 cells, etc.), osteosarcoma cells (U2OS cells, etc.), skin cells (NIH3T3, etc.) Cells, etc.), ovarian cells (CHO cells, etc.) and the like, but are not limited thereto.

The present invention relates to a method for identifying a protein that interacts with the target protein through crosslinking of the target protein using peroxidase and the protein that interacts with the target protein, and specifically, (a) interacting with the target protein in the cell. As a step of confirming the electrophoretic pattern of a cell expressing a protein of interest and a gene encoding a peroxidase to be introduced,

In the step, the fusion polynucleotide is introduced into a cell 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 the target protein is present.

Treating the cells with hydrogen peroxide to crosslink the interacting proteins in the organelles to obtain protein crosslinks;

Performing immunoprecipitation by adding an antibody against the protein of interest to the cell lysate obtained by crushing the cells, and separating the protein crosslinked product containing the protein of interest,

Electrophoresing the separated protein crosslinked product,

(b) identifying the electrophoretic pattern of cells expressing the target protein to be identified with the target protein in the cell but not introducing the gene encoding the peroxidase,

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

Performing immunoprecipitation by adding an antibody against the protein of interest to the cell lysate obtained by crushing the cells, and separating the protein crosslinked product containing the protein of interest,

Electrophoresing the separated protein crosslinked product,

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

(d) mass spectrometry of the electrophoretic band present in step (a) but not present in step (b),

The present invention relates to a method for identifying a protein that interacts with a target 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 exists to identify a protein that interacts with a target protein in a cell. A fusion polynucleotide may be prepared by connecting a gene encoding a protein of interest and a gene encoding a peroxidase, and the fusion polynucleotide may be introduced into a cell, but is not limited thereto.

In step (a) and (b), the step of crushing the cells to obtain cell lysate and electrophoresis to separate the protein further comprises coomassie staining, silver staining, or western blotting. can do.

In the step (b), the cell confirming the pattern of electrophoresis is a cell expressing a target protein to identify a protein that interacts with the target protein in the cell, but not a gene encoding a peroxidase, or a target in the cell. Although a cell into which a gene encoding a target protein and a peroxidase to identify a protein that interacts with the protein has been introduced may be a cell having a different position than that of the target protein, the cell entrapment of the peroxidase may be, but is not limited thereto.

In the step (a) or (b), the immunoprecipitation of the cell lysate may be performed by adding an antibody to a target protein or adding a bead to which the antibody is bound to the target protein, but is not limited thereto. It is not.

Comparing the protein pattern of the cells obtained in the step (a) or (b) in the step (c) may be performed by the naked eye or by imaging, the method for imaging the protein pattern is a conventional protein Any method of imaging a pattern may be used without limitation.

Mass spectrometry of the electrophoretic band in 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 for crosslinking a target protein in a cell and a protein interacting with the target protein using a peroxidase, wherein the target protein in the cell and a protein interacting with the target protein are used using the peroxidase. It can be used to identify a protein that interacts with the target protein in the cell by using a method, and can be used to selectively crosslink two different proteins in the pharmaceutical field to use the protein preparation as a medicine.

1 is a method for identifying proteins interacting with a protein of interest by crosslinking a protein interacting with a protein of interest (POI) by APEX, immunoprecipitating the crosslinked protein, and analyzing it with a mass spectrometer. This is a picture of the model.
Figure 2 shows a picture confirming the specific cross-linking by APEX according to an embodiment of the present invention.
Figure 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.

Hereinafter, the enzyme consisting of the amino acid sequence of SEQ ID NO: 4 is named APEX2, APEX2 was purchased from the US addgene (www.addgene.org). APEX2 is formed by mutation of APEX and is known to have greater enzymatic activity than APEX.

Example  1. Intracellular APEX Protein Expression and Crosslinking Reaction

1-1. Mitochondrial lining Targeting  Recombinant vector production

In order to target the APEX protein to the inner membrane of the mitochondria, a vector was prepared in which the polynucleotide linked to the Mitochondria Calcium Uniporter (MCU) gene and the APEX2 gene was inserted.

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

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

The group vector was produced according to the following method. Specifically, the polynucleotides linked to the MCU gene, the V5 gene, and the APEX2 gene prepared by Macrogen were treated with 1 ul of KpnI and BamHI restriction enzyme (purchased from New England biolab) for 1 hour at 37 ° C, respectively. The same enzyme was mixed with pCDNA5 vector digested at 37 ° C. for one hour (V601020, thermofisher), and then treated with 1 ul of T4 DNA ligase (purchased from New England biolab) for 1 hour at room temperature for ligation reaction. After 40 seconds of heat shot transformation in Top10 E. coli cells (Invitrogen) at 42 ° C, overnight incubation was carried out on an Ampicilin-Agar plate to isolate only the colonies containing the vector and extract the plasmid DNA. MCU-APEX plasmid DNA was obtained. The gene sequence of the polynucleotide to which the MCU gene, the V5 gene, and the APEX2 gene is linked is shown in SEQ ID NO: 6.

The configuration and characteristics of the recombinant vector are summarized in Table 2 below, and the cleavage map is shown in FIG. 3.

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

In order to introduce the vector prepared in Example 1-1 into the cells, the vector was introduced into HEK293T cells purchased from ATCC using a Lipofectamin transfection reagent.

Then, incubated in a CO ₂ incubator (Esco) at 36 ° C. for 16 to 24 hours in DMEM (Dulbecco Modified Eagle Medium) (Gibco) medium containing Fetal Bovine Serum.

1-3. Bridge Crosslinking ) reaction

The vector was introduced and treated with hydrogen peroxide (H ₂ O ₂ ) at a concentration of 1 mM in a medium for growing cultured HEK293T cells, and after 1 minute containing 10 mM Sodium ascorbate, 10 mM Sodium azide, and 5 mM Trolox. Washed three times with Dulbecco's Phosphate Buffered Saline (DPBS) containing the mineral solution (Sigma-Aldrich, 238813) (Thermofisher, 21300025).

Example  2. Confirmation of Protein Expression

2-1. Cell Lysis and Sampling

In order to confirm the expression in the cells of the prepared vector was carried out by the following method.

Specifically, the protein-expressing cells of Example 1 were pipetted into DPBS and transferred to 1.5 ml microtubes, followed by centrifugation at 1500 rpm for 5 minutes, leaving only the cell pellet and discarding the supernatant, 1X proteasome inhibitor cocktail. (Protease inhibitor cocktail) (Sigma aldrich, P8340), 1 mM PMSF (phenylmethylsulfonylfluoride) (Biosesang, P1021), and RIPA lysis buffer (Elpis bio, EBA1149) added with a quencher solution (Elpis bio, EBA1149) lysis). Cell residue was removed by centrifugation at 13000 rpm and 4 ° C. for 10 minutes, and 10 ug of the supernatant was sampled by adding 20 mM DTT-added SDS-protein gel loading buffer and heating at 95 ° C. for 10 minutes.

2-2. Weston Blot  Confirmation of protein expression through

The sample prepared in Example 2-1 was subjected to electrophoresis on SDS-PAGE gel at 150V for about 1 hour. The proteins electrophoresed on the SDS-PAGE gel were transferred to a nitrocellulose membrane, washed with TBST solution (mixture of Tris-Buffered Saline and Tween 20), and then at room temperature for 1 hour with 2% of dialyzed BSA solution. Stirred Primary antibody (anti-V5, Invitrogen, R960-25) was diluted to 10000: 1 in 2% (w / v) dialysed BSA solution and stirred at room temperature for 1 hour. The membranes were then washed with TBST four times for five minutes each. Secondary antibody (anti-mouse-HRP, Biorad, 1721011) was diluted to 3000: 1 in 2% (w / v) dialysed BSA solution and stirred at room temperature for 20 minutes. The membranes were then washed with TBST four times for five minutes each. After developing with a developing solution (Biorad's Clarity ECL product), Chemiluminescence imaging was performed using a G box (Syngene).

Example  3. Confirmation of Specific Crosslinking by APEX

In order to confirm whether the target protein and the protein interacting with the target protein are specifically crosslinked by APEX, the following experiment was performed.

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

The experimental results are shown in FIG.

As can be seen in Figure 2, only when the cross-linking reaction was confirmed that the MCU cross-links the interacting protein,

Example  4. Confirmation of crosslinking of intracellular interacting protein using APEX

4-1. Cell lysis and Immunoprecipitation

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

Specifically, after the cross-linking reaction to the protein expressed cells of Example 1 pipette with DPBS and transfer to 1.5 ml microtubes, centrifuged at 1500 rpm for 5 minutes, leaving only the cell pellet, discard the supernatant, 1X protea Cells were extracted with a Protease inhibitor cocktail (Sigma aldrich, P8340), 1 mM PMSF (phenylmethylsulfonylfluoride) (Biosesang, P1021), and RIPA lysis buffer (Elpis bio, EBA1149) with the addition of a mineral solution. All were lysed.

Then, by centrifugation at 20,000 rpm for 10 minutes using a centrifuge at a temperature of 4 ℃ to obtain only the supernatant. 10 ul of the obtained supernatant was left as an input sample for SDS-PAGE gel, and the remaining samples were 40 ul of magnetic beads (Invitrogen, 10003D) coated with anti-CHCHD3 antibody (Sigma Aldrich, SAB2106034), which is a primary antibody against the protein of interest. Incubated at room temperature (25 ℃) for 1 hour.

Plugged into a magnetic stand, the input and the same amount of supernatant (Flow through, FT) are stored separately for testing purposes to ensure the enrichment of the desired protein is complete. Magnetic beads were then washed three times with 2 M urea buffer and once with RIPA buffer. Then, 40 ul of SDS-protein gel loading buffer to which 20 mM DTT was added, heated at 95 ° C. for 10 minutes, and cooled in a magnetic stand.

4-2. Weston Blot  Confirmation via

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

The proteins electrophoresed on the SDS-PAGE gel were transferred to a nitrocellulose membrane, washed with TBST solution (mixture of Tris-Buffered Saline and Tween 20), and then at room temperature for 1 hour with 2% of dialyzed BSA solution. Stirred The primary antibody (anti-CHCHD3 antibody; Sigma Aldrich, HPA042935) was diluted to 3000: 1 in 2% (w / v) dialysed BSA solution and stirred at room temperature for 1 hour. The membranes were then washed with TBST four times for five minutes each. After developing with a developing solution (Biorad's Clarity ECL product), Chemiluminescence imaging was performed using G box (Syngene).

Example  5 Identification of intracellular interacting proteins using APEX

5-1. Crosslinked  Sampling of Protein Bands

The same sample identified by Western blot in Example 4-2 was electrophoresed and Coomassie staining was performed in the following manner.

Specifically, gels electrophoresed with 0.1% (w / v) Coomassie Brilliant Blue R-250 (Biosesang, C1031) were dyed for 60 minutes and decolorized using 30% methanol and 10% acetic acid solution. After bleaching, the electrophoretic gels were washed with distilled water and cross-linked Coomassie stained bands which were not shown in the negative control were cut out.

5-2. Through mass spectrometry Crosslinked  Identification of Protein

The band sampled in Example 5-1 was subjected to mass spectrometry to identify candidate proteins interacting with CHCHD3.

Specifically, the gel band cut in Example 5-1 was sufficiently washed with distilled water, and after drying the gel piece completely with Speed vac (Eppendorf, 38-022820125), 100 mM ABC (Ammonium bicarbonate, Sigma-Aldrich, A6141) After reducing for 30 minutes using dissolved 10 mM DTT (Dithiothreitol, Sigm-Aldrich, 43817), alkylation was performed for 20 minutes while blocking light using 55 mM Iodoacetamide (Sigma-Aldrich, I1149) dissolved in 100 mM ABC. After alkylation, wash with distilled water thoroughly, dry the gel completely, add 375 ng of Trypsin and cut the protein to peptide level. Peptide was removed from the gel with a solution of Acetonitrile and 5% (v / v) Formic acid 2: 1. Extracted. The extracted peptides were completely dried and stored until analysis. The sample stored in dry state was dissolved in 2% (w / v) acetonitrile, and 0.05% (v / v) Trifluoroacetic acid (Sigma-Aldrich), and then LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) Analyzed.

<110> UNIST ACADEMY-INDUSTRY RESEARCH CORPORATION <120> Method for analyzing interacting proteins in cells using          peroxidase <130> DPP20146249KR <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 gatgttacag 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 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 Arg 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 Pro Ser Asp 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 Arg 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 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca 120 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 aactggctca 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 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 Pro 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)

Preparation of a fusion polynucleotide in which a gene encoding a signal sequence targeting a cell organelle in which a target protein exists to identify a protein interacting with a target protein in a cell or a gene encoding a target protein and a gene encoding a peroxidase Introducing the fusion polynucleotide into a cell,
Treating hydrogen peroxide to the cells into which the fusion polynucleotide is introduced, thereby crosslinking the target protein and proteins interacting with the target protein in the cell organelles to form protein crosslinks, and
A method of immunoprecipitation by adding an antibody that specifically binds a target protein to the cell lysate obtained by crushing the cell, and separating a protein crosslinked product containing the target protein, and the protein interacting with the target protein. As a method of obtaining a protein crosslinked product containing a target protein,
The forming of the protein crosslinked product includes generating a radical in a tyrosine group present in the target protein and crosslinking the target protein with a target protein interacting with the target protein by the radical. How to get a sieve. The method of claim 1, wherein the peroxidase is Soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, Spinach ascorbate peroxidase, Saccharomyces Saccharomyces cerevisiae cytochrome c peroxidase, Panerocaete chrysosporium peroxidase PCL1 (Phanerochaete chrysosporium PCL1), Panerocaete chrysosporium peroxidase PCM (Phanerochaete chrysosporium PCM), Prinopsis cinerea peroxidase, Peanut peroxidase, Arabidopsis ATPA2, an enzyme consisting of the amino acid sequence of SEQ ID NO: 4, an enzyme consisting of the amino acid sequence of SEQ ID NO: 7 And in the group consisting of the enzyme consisting of the amino acid sequence of SEQ ID NO: 8 The at least one enzyme selected. The method of claim 1, wherein treating the hydrogen peroxide is performed with 0.1 to 2 mM of hydrogen peroxide and performed for 1 to 10 minutes. According to claim 1, The sequence gene targeting the cell organelles in which the target protein is present, Mitochondrial matrix expression gene, cytoplasmic expression gene, mitochondrial bilayer interspace expression gene, nuclear expression gene, extracellular membrane expression gene, endoplasmic reticulum expression gene , Endoplasmic reticulum expression gene, or intracellular expression gene. The method of claim 1, wherein the cell organelle is selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi apparatus. According to claim 1, wherein the cells are kidney cells, cancer cells, skin cells, ovary cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, melanin cells, Chondrocytes, macrophages, muscle cells, blood cells, bone marrow cells, lymphocyte cells, monocytes, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, heart cells, bladder cells, urethral cells, embryonic germ cells or eggs The cell. (a) identifying the electrophoretic pattern of a cell expressing a protein of interest and identifying a protein that interacts with the protein of interest in the cell and into which a gene encoding a peroxidase is introduced,
In the step, the fusion polynucleotide is introduced into a cell by preparing a fusion polynucleotide in which a gene encoding a signal sequence targeting a cell organelle in which a target protein is present or a gene encoding a target protein and a gene encoding a peroxidase are connected. step,
Treating hydrogen peroxide to the cells into which the fusion polynucleotide is introduced to crosslink the target protein and proteins interacting with the target protein in the organelle to form a protein crosslinked product,
Performing immunoprecipitation by adding an antibody that specifically binds a target protein to the cell lysate obtained by crushing the cells, and separating a protein crosslinked product containing the target protein, and
Electrophoresing the separated protein crosslinked product,
(b) identifying the electrophoretic pattern of cells expressing the target protein to be identified with the target protein in the cell but not introducing the gene encoding the peroxidase,
Said step is the step of treating hydrogen peroxide on the cells that do not introduce the gene encoding the peroxidase,
Performing immunoprecipitation by adding an antibody that specifically binds a protein of interest to the cell lysate obtained by crushing the cell,
Electrophoresing the separated protein crosslinked product,
(c) comparing the protein pattern of the cells obtained in step (a) with the protein pattern of the cells obtained in step (b), and
(d) mass spectrometry of the electrophoretic band present in step (a) but not present in step (b),
A method of identifying a protein that interacts with a protein of interest in a cell. The method according to claim 7, wherein the peroxidase in step (a) and (b) is soybean ascorbate peroxidase, Arabidopsis ascorbate peroxidase, spinach ascorbate peroxidase (Spinach ascorbate peroxidase), Saccharomyces cerevisiae cytochrome c peroxidase, Panerocaete chrysosporium peroxidase PCL1 (Phanerochaete chrysosporium PCL1), Panerocaete chrysosporium peroxide Enzyme PCM (Phanerochaete chrysosporium PCM), Coprinopsis cinerea peroxidase, Peanut Peroxidase, Arabidopsis peroxidase ATPA2, enzyme consisting of amino acid sequence of SEQ ID NO: 4, 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: 8 The method of one or more enzymes selected from the group consisting of. 8. The method of claim 7, wherein in step (a) and (b), the step of crushing the cells to obtain cell lysate and electrophoresis to separate the protein comprises: coomassie staining, silver staining, or western blotting the electrophoretic gel. Further comprising doing a lot. 8. The method of claim 7, wherein the treatment of hydrogen peroxide in steps (a) and (b) is performed with 0.1 to 2 mM hydrogen peroxide and carried out for 1 to 10 minutes. According to claim 7, wherein the step (a) the sequence gene targeting the cell organelles in which the target protein is present, mitochondrial matrix expression gene, cytoplasmic expression gene, mitochondrial bilayer space expression gene, nuclear expression gene, extracellular membrane expression gene, A endoplasmic reticulum expression gene, a endoplasmic reticulum expression gene, or a cell endometrial expression gene. 8. The method of claim 7, wherein said cell organelle is selected from the group consisting of mitochondria, endoplasmic reticulum, nucleus, cytoplasm and Golgi apparatus. According to claim 7, wherein the cells are kidney cells, cancer cells, skin cells, ovary cells, synovial cells, peripheral blood mononuclear cells, fibroblasts, fibroblasts, neurons, epithelial cells, keratinocytes, hematopoietic cells, melanin cells, Chondrocytes, macrophages, muscle cells, blood cells, bone marrow cells, lymphocyte cells, monocytes, lung cells, pancreatic cells, liver cells, gastric cells, intestinal cells, heart cells, bladder cells, urethral cells, embryonic germ cells or eggs The cell.

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