Fusion protein, recombinant vector, host cell and protein-protein interaction identification method

Abstract

The present invention relates to a fusion protein, a recombinant vector, a host cell and a protein-protein interaction identification method. The identification method comprises the steps of: fusing and expressing a photosensitive protein, a target protein and an Avi tag to obtain a first system containing a fusion protein; illuminating the first system with visible light so that the interacting protein of the target protein is cross-linked with the fusion protein to form a protein complex; performing biotinylation treatment using the Avi tag, separating and purifying the protein complex with a biotin label, and performing proteomics analysis to obtain information on the interacting protein. The labeling radius of the identification method is zero, and the confidence of the result far exceeds the prior art; the method of biotin enrichment reduces nonspecific adsorption; there is no need to perform any reagent treatment on living cells, and the protein interaction group in the real state of the cell is restored to the greatest extent; using light excitation, it can be applied in a time-resolved manner.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a fusion protein, a recombinant vector, a host cell and a protein-protein interaction identification method. Background Art

[0002] In order to study protein-protein interactions (PPI), many biochemical techniques have been used and developed to characterize the subcellular localization of proteins and the interaction targets of proteins. The three common research methods are: 1. Affinity purification-mass spectrometry technology; 2. Unnatural amino acid method; 3. Proximity labeling method. There is a light-activated dependent proximity labeling method in the prior art, which proposes a scheme of using a target protein and a photosensitive protein for fusion expression. This scheme uses photosensitive protein to generate singlet oxygen through photocatalysis, and modifies the histidine residues of neighboring proteins within a range of about 70 nm in a proximity-dependent manner, and then uses a probe to biotin-label the neighboring proteins to achieve pull-down of neighboring proteins. However, in actual use, it can be found that there are still a large number of non-interacting proteins in the pulled-down neighboring proteins. This may be because singlet oxygen indiscriminately modifies the histidine residues of neighboring proteins within the range, so that non-interacting proteins are also labeled by the probe. Therefore, the light-activated dependent proximity labeling method still has the problem of insufficient confidence in the detection results when applied to PPI research. Summary of the invention

[0003] The purpose of the present invention is to disclose a fusion protein, a recombinant vector, a host cell and a method for identifying protein-protein interactions, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0004] To achieve the above object, the present invention is implemented through the following technical solutions:

[0005] The first aspect of the present invention is to provide a method for identifying protein-protein interactions.

[0006] The second aspect of the present invention is to provide a fusion protein used in a method for identifying protein-protein interactions.

[0007] The third aspect of the present invention is to provide a recombinant vector containing a nucleic acid molecule encoding the fusion protein described in the second aspect of the present invention.

[0008] The fourth aspect of the present invention is to provide a recombinant cell containing the recombinant vector described in the third aspect of the present invention.

[0009] The protein-protein interaction identification method according to the first aspect of the present invention comprises the following steps:

[0010] The photosensitive protein (PS), the target protein (POI) and the biotinylated receptor tag are fused and expressed to obtain the first system containing the fusion protein;

[0011] The first system is subjected to illumination treatment using visible light, so that the interacting protein of the target protein is cross-linked with the fusion protein to form a protein complex;

[0012] The biotinylation receptor tag is used for biotinylation treatment, the protein complex with the biotin label is separated and purified, and proteomic analysis is performed to obtain the information of the interacting proteins.

[0013] Photosensitive proteins are a class of proteins that can respond to light of a specific wavelength, and in the present invention, specifically refer to proteins that can generate reactive oxygen free radicals in situ in cells.

[0014] Reactive oxygen species (ROS) refers to a series of chemical substances with unpaired electrons produced by aerobic cells during metabolism. These substances have strong oxidizing properties and the ability to compete for electrons. Reactive oxygen free radicals include various forms, such as superoxide anion (O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (HO·), singlet oxygen ( 1 O2) and peroxyl radicals (ROO·), etc.

[0015] The reactive oxygen free radicals generated by the photosensitive protein after light treatment can modify the interacting proteins, so that the amino acids at the interaction interface between proteins are cross-linked together. Based on the above characteristics, the photosensitive protein, the target protein and the Avi tag are fused and expressed in the present invention, and the photosensitive protein is treated with light to generate reactive oxygen free radicals, and the reactive oxygen free radicals cross-link the target protein and its interacting proteins to form a protein complex, and then the biotinylated receptor tag is treated with biotin labeling, that is, the protein complex can be pulled down by strict washing steps for proteomics analysis. The labeling radius of the identification method is zero, and the confidence of the results far exceeds the existing technology; the biotin labeling enrichment method reduces nonspecific adsorption; there is no need to treat the living cells with any reagents, and the protein interaction group in the real state of the cell is restored to the greatest extent; the photosensitive protein is stimulated by light to produce reactive oxygen, so that the method can be applied in a time-resolved manner.

[0016] Furthermore, the inventors investigated the cross-linking phenomenon of reactive oxygen free radicals on the interaction interface between proteins. It was found that the addition of reagents that affect singlet oxygen, such as sodium azide or deuterated water, had no effect on the cross-linking phenomenon of the identification method, and it was inferred that the cross-linking of the interaction interface between proteins was actually mediated by peroxyl free radicals (mainly) and superoxide anions (secondarily).

[0017] In some embodiments of the present invention, the structure of the fusion protein can be arranged in the order of "light-sensitive protein-target protein-biotinylated receptor tag", but other arrangements and combinations are not excluded. For example, when SKP2 protein is used as the target protein, its C-terminus is the substrate binding region, so the light-sensitive protein and the biotinylated receptor tag need to be arranged at the N-terminus of the SKP2 protein fragment.

[0018] In some embodiments of the present invention, the photosensitive protein is selected from miniSOG, SOPP2 or SOPP3. MiniSOG, SOPP2 and SOPP3 are all genetically localizable photosensitive proteins that can be accurately expressed and localized in specific cells or tissues. Although the SOPP series of proteins are called "singlet oxygen photosensitive proteins", the reactive oxygen free radicals they actually produce still include superoxide anions, hydrogen peroxide, hydroxyl free radicals and other types.

[0019] Experiments have shown that the use of SOPP2 as the photosensitive protein in the identification method of the present invention can identify the most real and effective interacting proteins.

[0020] In some embodiments of the present invention, the biotinylated receptor tag is selected from an Avi tag or an Avi tag variant.

[0021] The Avi tag is a tag protein consisting of 15 amino acids, which can be linked to a biotin on a lysine residue by biotin ligase (BirA), thereby achieving biotinylation labeling of the fusion protein. The Avi tag variant refers to a truncated, extended or mutant Avi tag. The truncated body is formed by removing 1, 2, 3 or 4 amino acid residues from one or both ends of the Avi tag. The extended body is formed by adding 1, 2 or 3 amino acid residues from one or both ends of the Avi tag. The mutant is obtained by replacing some of the amino acid residues of the Avi tag. The Avi tag variant can still obtain biotin labeling in the presence of substrates such as BirA, ATP and biotin.

[0022] In some embodiments of the present invention, the first system is a stably transfected cell that stably expresses the fusion protein.

[0023] In some embodiments of the present invention, the method for constructing the stably transfected cells comprises the following steps:

[0024] 1) obtaining a nucleotide molecule encoding the fusion protein;

[0025] 2) transferring the nucleotide molecule into a plasmid vector, adding an envelope plasmid and a packaging plasmid to co-transfect packaging cells, and collecting lentiviral particles;

[0026] 3) Infecting the target cell line with the lentiviral particles to obtain stably transfected cells.

[0027] In some embodiments of the present invention, the plasmid vector is selected from pLX plasmid, pLVX plasmid or pCDH plasmid; the envelope plasmid is selected from pMD2.G plasmid; the packaging plasmid is selected from psPAX2 plasmid or pRSV-Rev plasmid; and the packaging cell is selected from HEK293T cells or cell lines derived therefrom.

[0028] In some embodiments of the present invention, the light treatment is performed using blue light with a wavelength range of 400-500 nm. Further preferably, the wavelength of the blue light is preferably 420-480 nm, particularly selected from 430, 440, 450, 460 or 470 nm.

[0029] In some embodiments of the present invention, the duration of the light treatment is 1 to 15 minutes. More preferably, the duration of the light treatment is 5 to 12 minutes, particularly selected from 6, 7, 8, 9, 10 or 11 minutes.

[0030] In some embodiments of the present invention, the light treatment further comprises the step of: performing a lysis treatment on the stably transfected cells treated with the light. Since the light treatment has completed the cross-linking of the target protein and its interacting protein, the lysis treatment will not have a negative impact on the results of the identification method.

[0031] In some embodiments of the present invention, the lysis treatment is to resuspend the stably transfected cells using RIPA lysis buffer and a protease inhibitor. Further preferably, the protease inhibitor does not contain EDTA.

[0032] In some embodiments of the present invention, the lysis treatment is followed by ultrasonic disruption and then centrifugation to remove debris.

[0033] In some embodiments of the present invention, the biotinylation treatment refers to the addition of adenosine triphosphate, biotin and biotin ligase. Further preferably, the shaking incubation is carried out until the reaction is completed.

[0034] The fusion protein of the second aspect of the present invention contains a photosensitive protein, a target protein and a biotinylated receptor tag. The photosensitive protein can generate reactive oxygen free radicals under light conditions, and can fix the two cross-links when the interacting protein of the target protein and the fusion protein generate an interaction interface to form a protein complex; the biotinylated receptor tag can make the protein complex obtain a biotin label after treatment, and then undergo a strict washing step to pull it down for subsequent proteomics analysis.

[0035] Theoretically, the target protein can be selected from any protein. Even if the selected target protein has a large molecular weight or a complex spatial structure, the target protein can be added to the fusion protein so that the target protein does not directly interfere with the normal function of the photosensitive protein and the biotinylated receptor tag.

[0036] In some embodiments of the present invention, the target protein is selected from LaminA protein, H2BC11 protein or midnolin protein.

[0037] The recombinant vector described in the third aspect of the present invention is obtained by inserting a nucleotide molecule encoding the fusion protein into a plasmid vector.

[0038] In some embodiments of the present invention, the plasmid vector is selected from pLX plasmid, pLVX plasmid or pCDH plasmid.

[0039] The host cell in the fourth aspect of the present invention comprises the recombinant vector.

[0040] In some embodiments of the present invention, the host cell may be a stably transfected cell transfected with a lentiviral particle carrying the recombinant vector, or may be a transiently transfected cell transiently transfected.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The present invention has developed a novel identification method for studying protein interactomes. The zero-distance cross-linking between POI and its interacting proteins is used to covalently bind POI and interacting proteins together, thereby avoiding the destruction of the interactome when cells are lysed. At the same time, the enrichment strategy of biotinylated receptor tags is adopted, and strict washing steps can be performed, thereby enhancing the confidence of the identification results.

[0043] 2. By stimulating cross-linking through light, the identification method can be applied in a time-resolved manner without any reagent treatment of living cells, thereby restoring the protein interaction group in the real state of the cell to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the identification method constructed in Example 1;

[0045] Figure 2 This is the WB electrophoresis diagram of the fusion protein with H2BC11 as POI in Example 1;

[0046] Figure 3 This is the WB electrophoresis diagram of the fusion protein with LaminA as POI in Example 1;

[0047] Figure 4It is the volcano map of the fusion protein with H2BC11 as POI in Example 1;

[0048] Figure 5 It is the volcano map of the fusion protein with LaminA as POI in Example 1;

[0049] Figure 6 is the WB electrophoresis diagram of the fusion protein with midnolin as POI in Example 2;

[0050] Figure 7 It is the volcano map of the fusion protein with midnolin as POI in Example 2;

[0051] Figure 8 This is the electrophoresis diagram of the pull-down verification in Example 2;

[0052] Fig. 9 are the prediction result diagrams of the fusion protein in Example 2 and DNAJA1 and PPM1G respectively;

[0053] Fig.10 is the electrophoresis diagram of the substrate verification in Example 2;

[0054] Fig.11 This is the electrophoresis diagram of the ROS species that mediate the cross-linking phenomenon explored in Example 3. DETAILED DESCRIPTION

[0055] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0056] Example 1: Screening of photosensitive proteins.

[0057] according to Figure 1 The idea shown here is to construct a protein-protein interaction identification method and screen different photosensitive proteins to obtain improvements with better cross-linking effects and less background signals. The candidate photosensitive proteins are miniSOG, SOPP2 and SOPP3.

[0058] Since the interacting proteins of LaminA and H2BC11 have been fully reported in many literatures, they can be directly selected as POIs.

[0059] S1) Construction of stable cells:

[0060] (1) Using miniSOG, SOPP2 or SOPP3 as light-sensitive proteins, six nucleotide molecules encoding fusion proteins such as "V5 tag-light-sensitive protein-H2BC11-Avi tag" and "V5 tag-light-sensitive protein-LaminA-Avi tag" were constructed. The six nucleotide molecules were used as plasmid vectors to construct recombinant lentiviral plasmids.

[0061] (2) HEK293T cells were plated at 2.0×10 per well. 5 The cells were seeded in 6-well plates and cultured for 24 hours until the cell confluence reached 80%. Then the recombinant lentiviral plasmid (2.4 μg), enveloped granules pMD2.G (1.2 μg) and packaging plasmid psPAX2 (1.5 μg) were co-transfected into HEK293T cells. After overnight transfection, the medium was replaced and incubated for another 24 hours. The lentiviral particles were collected after 48 hours and 72 hours, respectively.

[0062] (3) HEK293T cells were used as target cells. Before infecting the target cell line, the virus culture medium containing lentiviral particles was filtered with a 0.45 μm filter and polybrene was added to a concentration of 8 μg / mL. The target cell line was then placed in the virus culture medium for transfection for 24 hours. After transfection, the normal culture medium was replaced and stably transfected cells were selected with 5 μg / mL of blasticidin.

[0063] S2) Light treatment and lysis treatment:

[0064] (1) Ordinary HEK293T cells were selected as blank control cells. Each stable transfected cell and blank control cell were subjected to two conditions: illumination and non-illumination. The illumination group was used as the experimental group, and the non-illumination group was used as the control group. Each group had three biological replicates.

[0065] (2) When the cell confluence reached 80%, the cells were washed twice with fresh HBSS buffer. Then, the cells were irradiated with a blue LED for 10 min at room temperature in fresh HBSS buffer. After the irradiation, the HBSS buffer was discarded and the cells were resuspended in ice-cold RIPA lysis buffer containing EDTA-free protease inhibitors (MCE, #HY-K0011).

[0066] (3) Cells were lysed by ultrasonication for 30 seconds (1 second on and 2 seconds off, amplitude of 25%). The resulting mixture was centrifuged at 13,000 rpm for 10 minutes at 4°C to remove debris, and the concentration of the supernatant was adjusted to 4 mg / mL using a BCA protein assay kit (Beyotime, #P0009) to obtain 500 μL of lysate solution.

[0067] S3) In vitro biotinylation treatment: ATP (final concentration of 2 mmol / L), MgCl2 (final concentration of 5 mmol / L), biotin (final concentration of 10 μmol / L), and BirA (final concentration of 40 μg / mL) were added to 500 μL of the lysate solution in sequence, mixed thoroughly, and then incubated at 25°C, 700 rpm with shaking overnight (12-16 hours).

[0068] S4) Washing treatment:

[0069] (1) After the in vitro biotinylation reaction, the mixture was added to a mixed solution (MeOH:CHCl3:H2O volume ratio of 4:1:3), centrifuged at 4500 g for 10 min at room temperature, and the bottom and upper layers were discarded in turn, leaving the middle protein precipitate. Then, 1 mL of 8 mol / L urea (Aladdin, #U111902) solution formed by dissolving 25 mmol / L ammonium bicarbonate (ABC, Aladdin, #A110539) was added to dissolve the precipitate.

[0070] (2) 10 mmol / L dithiothreitol (Sangon, #A100281) was added for reduction at 55°C for 40 min, and then 15 mmol / L freshly prepared iodoacetamide (Sangon, #A600539) was added for alkylation at room temperature in the dark for 30 min. Subsequently, an additional 5 mmol / L dithiothreitol was added to stop the reaction to obtain a proteome solution.

[0071] (3) 70 μL of NeutrAvidin agarose resin beads (Thermo, #29202) washed three times with PBS were incubated with the above proteome solution at room temperature for 4 hours.

[0072] (4) Wash the beads three times with 5 mL PBS containing 0.2% SDS (Sangon, #A600485), three times with 5 mL PBS containing 1 mol / L urea, and three times with 5 mL PBS.

[0073] (5) Collect the beads by centrifugation and resuspend in 300 μL 25 mmol / L ABC containing 1 mol / L urea, 1 mmol / L CaCl2 (Macklin, #C805228), and 30 ng / μL trypsin (Promega, #V5280) and shake at 37°C overnight.

[0074] (6) Collect the supernatant by centrifugation and then add formic acid (Thermo, #A117-50) until the pH value of the solution reaches 2-3 and the reaction stops.

[0075] (7) The solution was desalted using a desalting column, and the desalted solution was dried using a vacuum concentration and stored at -20°C. Before LC-MS / MS analysis, the spin-dried sample was reconstituted with 0.1% formic acid water and injected after high-speed centrifugation.

[0076] S5) Analysis of mass spectrometry data: Proteome Discoverer data processing software and label free method were used to quantitatively analyze the proteome data. The volcano plot of mass spectrometry data was drawn using R language code. The abundance of endogenous biotinylated proteins Pyruvate carboxylase, 3-methylcrotonyl coA carboxylase, Propionyl coA carboxylase and Acetyl coA carboxylase in the experimental group and the control group was theoretically the same, which was used for data normalization. Proteins with significant differences were determined by abundance changes > 2 and p values ​​< 0.05. These proteins with significant differences were possible interacting proteins of POI.

[0077] A portion of the solution after BCA quantification was taken out for WB experiment to detect the V5 tag signal to confirm that the illumination produced cross-linked bands; the WB experiment of the sample after biotinylation treatment detected the biotin signal to ensure that the in vitro biotinylation reaction was normal. The results were as follows: Figure 2 and Figure 3 As shown in the figure, the size of the fusion protein with H2BC11 as POI is around 35 kDa, and the size of the fusion protein with LaminA as POI is around 100 kDa. After irradiation with blue light, a cross-linked upward band was generated, indicating that blue light irradiation can indeed induce zero-distance cross-linking of adjacent proteins.

[0078] Figure 4 and Figure 5 The volcano plots were drawn for each stable cell group after mass spectrometry data analysis. The optimal candidate photosensitive protein was determined by comparing the number of interacting proteins identified in the mass spectrometry experimental results of the three photosensitive proteins, miniSOG, SOPP2 and SOPP3, the authenticity of the identified interacting proteins, and the number of cross-linked bands in the WB experiment. Because the interacting proteins of LaminA and H2BC11 have been fully reported in many literatures, the mass spectrometry analysis results were directly compared with the literature reports, and the one with the highest degree of agreement was superior.

[0079] SOPP2 was finally determined to be the optimal light-sensitive protein.

[0080] Example 2: Protein-Protein Interaction Identification Method The interactome of midnolin protein was studied.

[0081] (1) Based on the screening results of Example 2, SOPP2 was used as a photosensitive protein to construct a nucleotide molecule encoding a fusion protein "V5 tag-SOPP2-midnolin-Avi tag", and HEK293T cells were used to make stably transfected cells.

[0082] (2) The following groups were set up for comparative mass spectrometry experiments (three biological replicates were performed in each group):

[0083] Group A: HEK293T cells were treated with 10 μmol / L proteasome inhibitor (MG132) for 6 h and then irradiated with 450 nm light for 10 min;

[0084] Group B: the stably transfected cells were treated with 10 μmol / L MG132 for 6 hours and irradiated with 450 nm light for 10 minutes;

[0085] Group C is the stably transfected cells treated with 20 ng / mL phorbol ester (PMA) and 10 μmol / L MG132 for 6 hours and irradiated with 450 nm light for 10 minutes.

[0086] It is reported that midnolin protein mainly degrades proteins encoded by a class of immediate early genes, and PMA stimulation can activate the expression of these genes. Midnolin protein mediates the proteasome degradation of its substrates, but this process does not depend on ubiquitination. It binds to the proteasome through α-helix, uses its Catch domain to bind substrates, and uses ubiquitin-like domains to promote the degradation of substrates. Therefore, MG132 treatment was set up in the experiment to prevent its substrates from being degraded.

[0087] (3) Each group of cells was lysed with RIPA lysis buffer, and then the protein concentration of all samples was quantified by BCA protein assay kit to be unified at 4 mg / mL, with a volume of 500 μL, followed by in vitro biotinylation reaction. The protein was then precipitated with methanol and chloroform to remove the biotinylation reaction system, and then the protein was re-dissolved with urea, enriched with DTT, IAA, High Capacity Neutravidin agarose resin beads, and digested with trypsin, desalted, and entered into LC-MS / MS identification. The WB test results are shown in the figure. Figure 6 As shown, the size of the "V5 tag-SOPP2-midnolin-Avi tag" fusion protein is around 75 kDa. It can be clearly seen from the figure that after blue light irradiation, cross-linked bands were generated, indicating that blue light irradiation can indeed induce zero-distance cross-linking between interacting proteins.

[0088] Subsequently, the Proteome Discoverer data processing software and the Label Free method were used to quantitatively analyze the proteomic data, and the volcano map of the mass spectrometry data was drawn using the code written in R language (see Figure 7 ), the abundance of endogenous biotinylated proteins Pyruvate carboxylase, 3-methylcrotonyl coA carboxylase, Propionyl coA carboxylase and Acetyl coA carboxylase in the three groups is theoretically the same, which is used for data normalization. Proteins with significant differences are determined by abundance changes > 2 and p values ​​< 0.05. These proteins with significant differences are possible interacting proteins of midnolin protein.

[0089] After finding the interactome of midnolin protein through LC-MS / MS, five proteins including EGR1, DNAJA1, PPM1G, KAP1 and JUN were selected for pull-down verification, and antibodies for each of these proteins were purchased. First, the midnolin protein in living cells was covalently cross-linked with its interacting proteins by illumination, and then the Avi tag attached to the midnolin protein was biotinylated under lysate conditions, and then enriched with High Capacity Neutravidin agarose resin beads. Finally, Loading Buffer was added to the agarose beads bound to the midnolin protein and its interactome, heated at 95°C for 15 minutes, the bound proteins on the beads were boiled off, and then WB detection was run. By incubating the antibody of the candidate protein, it can be seen whether the POI pulls down the candidate protein, thereby proving the interaction between the midnolin protein and the candidate protein. WB results (see Figure 8 ) showed that these five proteins were cross-linked with midnolin protein under light, while no cross-linking was found in the group without light treatment. At the same time, the PLDDT score of the fusion protein and DNAJA1 protein predicted by AlphaFold Multimer was 54.9101, and the BSASA score was 2074.3672; the PLDDT score of the fusion protein and PPM1G protein was 50.6301, and the BSASA score was 2601.2656 (see Fig. 9 ). Based on the criteria of PLDDT>50 and BSASA>500, it was judged that DNAJA1 protein and PPM1G protein were likely to be the interacting proteins of midnolin protein.

[0090] Because midnolin proteins can mediate the degradation of their substrates in the proteasome, based on the identification results of the above-mentioned interacting proteins, it is expected to find new midnolin substrate proteins.

[0091] According to literature reports, after cells were serum starved overnight, 20% serum stimulation increased the expression of midnolin protein. Therefore, we used the common 293T cell line and the stable cells, and performed serum starvation and serum stimulation tests at the same time. The WB test results are as follows: Fig.10 As shown. It can be found that:

[0092] (1) The EGR1 protein in 293T cells gradually decreased with the extension of serum stimulation time, while the degradation rate of EGR1 protein in stable cells was significantly faster than that in 293T cells, indicating that overexpression of midnolin protein accelerated the degradation of EGR1 protein. This phenomenon has been reported in the literature and was used as a positive control.

[0093] (2) Although no changes in ATF4 protein were observed in 293T cells with prolonged serum stimulation, it was not expressed in stable cells, indicating that ATF4 protein may be a substrate protein of midnolin protein.

[0094] (3) The behavior of Jun protein is the same as that of EGR1 protein, indicating that it may also be a substrate protein of midnolin protein.

[0095] (4) The phenomenon of TNPO1 protein is similar to that of ATF4. The expression level of TNPO1 protein shows a downward trend in stable transfected cells, indicating that TNPO1 protein may also be a substrate protein of midnolin protein.

[0096] Previously, we had conducted experiments to verify the interaction between midnolin protein and ATF4 protein. However, due to the limitations of the antibody (because midnolin protein will cross-link with its interacting protein to form a large complex, once the site recognized by the antibody is wrapped inside the complex, it may result in the interaction being detected by mass spectrometry but not by the pull-down experiment), no signal of ATF4 protein being pulled down could be detected in the illumination group.

[0097] Example 3: Mechanism exploration of protein-protein interaction identification method.

[0098] Photosensitive proteins can generate a large number of reactive oxygen free radicals under blue light irradiation. In order to study which reactive oxygen free radical mediates the cross-linking reaction of the identification method, different reagents are used to quench or stabilize different reactive oxygen free radicals. The amounts of experimental reagents are shown in Table 1.

[0099]

[0100] After the stable transfected cells stably expressing "V5 tag-photosensitive protein-LaminA-Avi tag" reached 80% confluency, they were washed twice with fresh HBSS buffer. The cells were incubated with the experimental reagents at 37°C for 1 hour, and then irradiated with blue light at room temperature for 10 minutes. After the illumination, the buffer was discarded and the cells were washed twice with fresh HBSS buffer. The cells were then resuspended in ice-cold RIPA lysis buffer for lysis. After the protein concentration was quantitatively adjusted by BCA, Loading Buffer was added and denatured at 95°C for 10 minutes. The prepared protein samples were run with WB to detect the V5 tag to observe the effect of the experimental reagents on the cross-linking phenomenon.

[0101] WB results are as follows Fig.11 As shown, Trolox treatment of cells in advance will lead to the complete disappearance of cross-linked bands; vitamin C treatment of cells in advance will weaken the cross-linking phenomenon; hydrogen peroxide, D-mannitol, deuterated water and sodium azide treatment have no effect on the cross-linking phenomenon. Based on the effects of the above six reagents on active oxygen species, it is speculated that peroxyl radicals (mainly) and superoxide anions (secondarily) mediate the cross-linking reaction of the identification method.

[0102] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for identifying protein-protein interactions, characterized in that: Includes steps: The light-sensitive protein SOPP2, the target protein and the biotinylated receptor tag are fused and expressed to obtain the first system containing the fusion protein; The first system is subjected to light treatment so that the interacting protein of the target protein is cross-linked with the fusion protein to form a protein complex; The biotinylation receptor tag is used for biotinylation treatment, the protein complex with the biotin label is separated and purified, and proteomic analysis is performed to obtain the information of the interacting proteins.

2. The identification method according to claim 1, characterized in that: The biotinylated receptor tag is selected from an Avi tag or an Avi tag variant.

3. The identification method according to claim 1, characterized in that: The light treatment is performed by irradiating with blue light having a wavelength range of 400-500 nm.

4. The identification method according to claim 1, characterized in that: The biotinylation treatment refers to the addition of adenosine triphosphate, biotin and biotin ligase.

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