A proximity labeling method with spatiotemporal specificity

Abstract

The present invention provides a proximity labeling method with spatiotemporal specificity. Specifically, the method of the present invention uses SOPP3 and APEX2 systems to mediate and catalyze the reaction of biotin phenol in the presence of blue light, so that biotin is covalently linked to the target protein to achieve the effect of proximity labeling. The method of the present invention has the characteristics of high labeling efficiency, substantially non-toxicity, and accurate positioning.

Description

Technical Field

[0001] The present invention relates to the field of protein detection, and in particular to a proximity labeling method with spatiotemporal specificity. Background Art

[0002] Most proteins in cells exert their biological functions through interaction networks with other molecules. When these interaction networks become abnormal, they often lead to various human diseases, such as cancer and neurodegenerative diseases. The identification of protein interaction networks is crucial to understanding life processes at the system level. The traditional method of identifying protein-protein interactions is affinity purification coupled to mass spectrometry. The limitation of this method is that it destroys the original subcellular microenvironment and it is difficult to capture weaker protein-protein interactions. The emergence of proximity labeling (PL) can help us understand the interaction map between proteins and other molecules more comprehensively while maintaining the subcellular microenvironment.

[0003] The principle of proximity labeling is to fuse an enzyme with proximity labeling function with the target protein, and label the adjacent protein with biotin through the catalysis of the enzyme. There are currently two types of enzymes used for proximity labeling. Biotin ligases represented by BioID1 and TurboID2 can connect biotin to adjacent molecules within 10nm of the target protein, but the labeling efficiency is low and often takes an hour; while peroxidases represented by ascorbate peroxidase (APEX) can complete the labeling reaction within one minute under the activation of hydrogen peroxide.

[0004] TurboID is a highly efficient biotin ligase that can label biotin to neighboring proteins within 10nm of the target protein in cultured cells. For different types of organelle interfaces, the labeling reaction can be completed within 10 minutes to 1 hour. Kelvin et al. also developed the split-TurboID system based on TurboID, and identified GRP75, IP3R1, PDI, and VDAC, which are known to exist in mitochondrial-endoplasmic reticulum membrane contact sites, in 293T cells. Because this system is free from the dependence on hydrogen peroxide, it can be used in living animal models. Tetsuya et al. used split-TurboID in mice to identify the protein group enriched in the astrocyte-neuron membrane contact site in vivo.

[0005] The principle of APEX-induced proximity labeling is that it can catalyze the oxidation of biotin phenol to form free radical phenol. The half-life of this free radical phenol is short, only 1ms, so the radius of the system label is less than 20nm, and it can covalently react with electron-rich amino acids (Tyr, Trp, His and Cys). Therefore, the APEX protein can be located on the protein molecule of interest, and then the cells are incubated with hydrogen peroxide and biotin phenol. The molecules adjacent to the target protein will be modified by biotin, and then the biotinylated target protein is enriched by the streptavidin system, and finally identified by mass spectrometry. The advantages of the APEX labeling system are firstly that the spatial specificity of the labeled protein is good; secondly, the labeling efficiency of the system is very high, and the required reaction time is only 1min, which is very beneficial for highly dynamic subcellular structures; thirdly, unlike traditional co-immunoprecipitation, the system does not destroy the subcellular microenvironment, so that the protein interaction network can be more comprehensively restored.

[0006] In addition to the above-mentioned combination of APEX enzymes with other methods to achieve proteome identification within membrane contact sites, there is also a direct improved version of APEX, namely the split-APEX system. This system truncates the APEX enzyme into two parts, and then anchors the two parts at the two ends of the membrane contact sites of the two organelles to be studied. When the two organelles are close to each other, the recombinant APEX enzyme can mark the specific protein molecules in the membrane contact site at this time. Nazmiye et al. successfully used the split-APEX system to verify that after knocking down VAPA and VAPB in neurons, the number of endoplasmic reticulum-lysosome membrane contact sites formed was significantly downregulated, indicating that VAPA and VAPB play a very important role in endoplasmic reticulum-lysosome interactions.

[0007] However, the APEX-based proximity labeling method also has certain limitations. First, the introduction of hydrogen peroxide may affect certain biological processes in cells; second, the dependence on exogenous hydrogen peroxide also limits its application in living organisms. Therefore, if a method that can replace hydrogen peroxide is found, the efficient proximity labeling effect of APEX can be better utilized.

[0008] Among the current proximity labeling methods, BioID / TurboID has low labeling activity, while APEX relies on hydrogen peroxide, which is toxic to cells. Therefore, there is a need in the art to develop a sensitive and non-cytotoxic proximity labeling method to study biomolecular interactions. Summary of the invention

[0009] The purpose of the present invention is to provide a proximity labeling method which has high labeling efficiency, is substantially non-toxic and is easy to use.

[0010] In a first aspect of the present invention, a method for labeling interacting proteins and / or neighboring proteins is provided, the method comprising the steps of:

[0011] S1) connecting a peroxidase to a protein A involved in the interaction, or an element A expressing the protein A, thereby obtaining a peroxidase element A';

[0012] S2) connecting the inducible hydrogen peroxide (H2O2) producing protein to another protein B involved in the interaction, or to an element B expressing the protein B, thereby obtaining an H2O2 producing element B';

[0013] S3) contacting the peroxidase element A', the H2O2 generating element B' and biotin phenol, thereby catalyzing the attachment of biotin to adjacent proteins A and B and / or other adjacent proteins adjacent to proteins A and B.

[0014] In another preferred embodiment, the peroxidase is selected from the following group: ascorbate peroxidase (APEX) or its derivatives, horseradish peroxidase (HRP) or its derivatives, or a combination thereof.

[0015] In another preferred embodiment, the APEX derivative includes a mutant protein of APEX.

[0016] In another preferred embodiment, the APEX derivative has the same or substantially the same function as APEX in catalyzing biotin phenol to form free radical phenol.

[0017] In another preferred embodiment, the HRP derivative includes a mutant protein of HRP.

[0018] In another preferred embodiment, the HRP derivative has the same or substantially the same function as HRP in catalyzing biotin phenol to form free radical phenol.

[0019] In another preferred embodiment, the inducible H2O2-producing protein is a protein that produces H2O2 under light induction.

[0020] In another preferred embodiment, the H2O2 production of the H2O2 producing protein increases with the increase of light induction time.

[0021] In another preferred embodiment, the H2O2 producing protein produces H2O2 locally.

[0022] In another preferred embodiment, the inducible H2O2-producing protein is selected from the following group: SOPP3 protein, SOPP2 protein, SOPP1 protein, or a combination thereof.

[0023] In another preferred embodiment, the illumination wavelength is selected according to the type of the inducible H2O2-producing protein.

[0024] In another preferred embodiment, the illumination is blue light.

[0025] In another preferred embodiment, the element A and element B are independently selected from: organelles, cells, or a combination thereof.

[0026] In another preferred embodiment, the cell organelles include: endoplasmic reticulum, mitochondria, Golgi apparatus, cell nucleus, or a combination thereof.

[0027] In another preferred embodiment, the step S1 comprises the steps of:

[0028] S1a) expressing a peroxidase on the element A, thereby linking the peroxidase to the element A; or

[0029] S1b) fusing peroxidase to the protein A, thereby linking the peroxidase to the protein A; or

[0030] S1c) contacting the protein A or element A with the antibody cross-linked with the peroxidase, wherein the antibody specifically binds to the protein A, thereby linking the peroxidase to the protein A or element A; or

[0031] S1d) contacting the protein A or element A, a first antibody that specifically binds to protein A, and a second antibody cross-linked with the peroxidase, thereby linking the peroxidase to protein A or element A, wherein the second antibody specifically recognizes the first antibody.

[0032] In another preferred embodiment, the step S2 includes the steps of:

[0033] S2a) expressing an inducible H2O2-producing protein on the element B, thereby linking the inducible H2O2-producing protein to the element B; or

[0034] S2b) fusing the inducible H2O2-producing protein to the protein B, thereby linking the inducible H2O2-producing protein to the protein B; or

[0035] S2c) contacting the protein B or element B with an antibody cross-linked with the inducible H2O2-producing protein, wherein the antibody specifically binds to protein B, thereby linking the inducible H2O2-producing protein to protein B or element B; or

[0036] S2d) contacting protein B or element B, a first antibody that specifically binds to protein B, and a second antibody cross-linked with the peroxidase, thereby linking the inducible H2O2-producing protein to protein B or element B, wherein the second antibody specifically recognizes the first antibody.

[0037] In another preferred embodiment, the step S3 includes the step of applying light to the peroxidase element A', the H2O2 producing element B' and the biotin-phenol, thereby catalyzing the connection of biotin to the interacting proteins A and B and / or their neighboring proteins.

[0038] In another preferred embodiment, the illumination is blue light.

[0039] In another preferred embodiment, the light wavelength is 430-480 nm, preferably 440-460 nm, and more preferably 450-452 nm.

[0040] In another preferred embodiment, the illumination time is ≤30s, preferably ≤5s, more preferably ≤1s.

[0041] In another preferred embodiment, the method further comprises a detection step S4): detecting the biotin to detect interacting neighboring proteins.

[0042] In another preferred embodiment, the detection of biotin is performed using streptavidin.

[0043] In a second aspect of the present invention, a reagent combination is provided, wherein the reagent combination is used to label interacting proteins and / or neighboring proteins, and the reagent combination comprises:

[0044] A) peroxidase, or its localization fusion protein, or its antibody cross-linked product, or a nucleic acid molecule encoding the same, or an expression vector containing the nucleic acid molecule; and

[0045] B) Inducible hydrogen peroxide-producing protein, or its localization fusion protein, or its antibody cross-linked product, or a nucleic acid molecule encoding the same, or an expression vector containing the nucleic acid molecule.

[0046] In another preferred embodiment, the interacting proteins include protein A and protein B.

[0047] In another preferred embodiment, the peroxidase localization fusion protein comprises the peroxidase and localization peptide A, and the localization peptide A localizes the fusion protein to element A where protein A is expressed.

[0048] In another preferred embodiment, the inducible hydrogen peroxide-producing protein localization fusion protein comprises the inducible hydrogen peroxide-producing protein and localization peptide B, and the localization peptide B localizes the fusion protein to element B where the protein B is expressed.

[0049] In another preferred embodiment, the peroxidase-antibody cross-linked product comprises the peroxidase and the antibody A cross-linked therewith, and the antibody A specifically binds to the protein A.

[0050] In another preferred embodiment, the inducible hydrogen peroxide-producing protein-antibody cross-linked product comprises the inducible hydrogen peroxide-producing protein and antibody B cross-linked therewith, and the antibody B specifically binds to the protein B.

[0051] In another preferred embodiment, the peroxidase-antibody cross-linked product comprises the peroxidase and a second antibody A' cross-linked therewith, wherein the second antibody A' specifically binds to the first antibody against the protein A.

[0052] In another preferred embodiment, the inducible hydrogen peroxide-producing protein antibody cross-linked product comprises the inducible hydrogen peroxide-producing protein and a second antibody B' cross-linked therewith, wherein the second antibody B' specifically binds to the first antibody against the protein B.

[0053] In a third aspect of the present invention, a fusion protein for labeling an interacting protein and / or a neighboring protein is provided, the fusion protein comprising:

[0054] Z1) peroxidase or an active fragment thereof;

[0055] Z2) Inducible hydrogen peroxide-producing protein or its active fragment.

[0056] In another preferred embodiment, the fusion protein has a structure shown in the following formula Ia or Ib from N-terminus to C-terminus:

[0057] Z1-L-Z2(Ia);

[0058] Z2-L-Z1(Ib);

[0059] in,

[0060] Z1 is the peroxidase or an active fragment thereof;

[0061] Z2 is the inducible hydrogen peroxide-producing protein or an active fragment thereof;

[0062] L is none or a linker peptide.

[0063] In another preferred embodiment, the inducible H2O2-producing protein is selected from the following group: SOPP3 protein, SOPP2 protein or SOPP1 protein.

[0064] In another preferred embodiment, the peroxidase is selected from the following group: ascorbate peroxidase (APEX) and its derivatives, horseradish peroxidase (HRP) and its derivatives, or a combination thereof.

[0065] In another preferred embodiment, the connecting peptide is a rigid connecting peptide or a flexible connecting peptide, preferably having a structure represented by (G4S)n, wherein n is an integer of 1-6.

[0066] In another preferred embodiment, the fusion protein is a localization fusion protein, which further comprises a localization peptide, and the localization peptide localizes the fusion protein to:

[0067] (a) Specific organelles;

[0068] (b) cell membrane;

[0069] (c) a specific DNA sequence or RNA sequence; or

[0070] (e) Any combination of (a) to (d) above.

[0071] In another preferred example, the localization peptide includes: an NLS sequence for localization to the cell nucleus (PKKKRKV, SEQ ID NO: 1), an MLS sequence for localization to the mitochondria (MGGSSSRIAAGLGAALFVGYCIYFDRKRRSDPN, SEQ ID NO: 2), and a TM sequence for localization to the cell membrane (ENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLF, SEQ ID NO: 3).

[0072] In another preferred embodiment, the localization peptide includes: a dCas9 protein for localizing to a specific DNA sequence, a dCas13a protein for localizing to a specific RNA sequence, or a combination thereof.

[0073] In another preferred embodiment, the fusion protein is further connected to a target protein portion, and the target protein portion is a protein to be studied that may be involved in the interaction.

[0074] In another preferred embodiment, the fusion protein is an antibody cross-linked protein, which further has an antibody portion, and the antibody portion is:

[0075] (a) a first antibody that specifically targets one of the interacting neighboring proteins; or

[0076] (b) a second antibody that specifically targets the first antibody.

[0077] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0078] In a fourth aspect of the present invention, a method for labeling interacting proteins and / or neighboring proteins is provided, the method comprising the steps of:

[0079] S1) connecting the fusion protein as described in the third aspect of the present invention to the protein A involved in the interaction, or the element A expressing the protein A, thereby obtaining a peroxidase-H2O2-producing element;

[0080] S2) contacting the peroxidase-H2O2 generating element with biotin-phenol, thereby catalyzing the attachment of biotin to the adjacent protein A and the protein B interacting with protein A, and / or to other adjacent proteins adjacent to proteins A and B.

[0081] In another preferred embodiment, the step S1 comprises the steps of:

[0082] S1a) expressing the fusion protein on the element A, thereby connecting the fusion protein to the element A; or

[0083] S1b) fusing the fusion protein with the protein A, thereby connecting the fusion protein to the protein A; or

[0084] S1c) contacting the protein A or element A with the antibody cross-linked with the fusion protein, wherein the antibody specifically binds to protein A, thereby linking the fusion protein to protein A or element A; or

[0085] S1d) contacting the protein A or element A, a first antibody that specifically binds to protein A, and a second antibody that is cross-linked with the fusion protein, thereby connecting the fusion protein to protein A or element A, wherein the second antibody specifically recognizes the first antibody.

[0086] In the fifth aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the fusion protein as described in the third aspect of the present invention.

[0087] In the sixth aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule as described in the fifth aspect of the present invention.

[0088] In another preferred embodiment, the vector is a plasmid.

[0089] In a seventh aspect of the present invention, a device for labeling interacting proteins and / or neighboring proteins is provided, the device comprising:

[0090] M1) a kit comprising the reagent combination as described in the second aspect of the present invention, or the fusion protein as described in the third aspect of the present invention, or the nucleic acid molecule as described in the fifth aspect of the present invention, or the vector as described in the sixth aspect of the present invention, or a combination thereof; and

[0091] M2) Lighting device.

[0092] In another preferred embodiment, the illumination device is a blue light illumination device.

[0093] In another preferred embodiment, the light wavelength is 430-480 nm, preferably 440-460 nm, and more preferably 450-452 nm.

[0094] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0096] Figure 1 The ability of various light-sensitive proteins to activate APEX2 under light conditions was demonstrated.

[0097] Figure 2 It was shown that SOPP3 and APEX2 can mediate neighboring protein labeling between organelle interaction surfaces through blue light induction.

[0098] Figure 3 It was shown that the marked proteins detected by proteome enrichment were indeed interacting proteins between organelles.

[0099] Figure 4 It was shown that SOPP3- and APEX2-mediated protein labeling increased with the increase of blue light illumination time and was light-dependent.

[0100] Figure 5 It was shown that SOPP3 and APEX2, which are respectively localized on the cell membranes of neighboring cells, can mediate the labeling of interacting proteins between neighboring cells through light induction.

[0101] Figure 6 It was shown that the SOPP3-APEX2 protein fusion form can also mediate protein labeling through light induction, and the labeling effect is comparable to that of the non-fusion form.

[0102] Figure 7 Protein interaction markers mediated by SOPP3-APEX2 protein fusion forms are shown.

[0103] Figure 8 SOPP3 and APEX2 were shown to mediate neighbor protein labeling in living organisms. DETAILED DESCRIPTION

[0104] After extensive and in-depth research, the inventors have developed a proximity labeling method with spatiotemporal specificity for the first time. The inventors found that SOPP3 and APEX2 can mediate the catalytic reaction of biotin-phenol in the presence of blue light, so that biotin is covalently linked to the target protein, achieving the effect of proximity labeling. On this basis, the present invention was completed.

[0105] the term

[0106] As used herein, the term "peroxidase" refers to a class of enzymes that can catalyze the biotin-phenol reaction under the activation of hydrogen peroxide, thereby connecting biotin to the target protein. A representative peroxidase is ascorbate peroxidase (APEX). APEX is engineered from ascorbate peroxidase derived from plants. It can catalyze biotin-phenol to generate a free radical molecule with an extremely short half-life under the action of hydrogen peroxide. The free radical can react with electron-rich tyrosine, cysteine, tryptophan and histidine, so that biotin is covalently linked to the target protein to achieve the effect of proximity labeling. The peroxidase of the present invention can also be an APEX derivative, horseradish peroxidase (HRP) or a derivative thereof. The derivative can have one or more amino acid additions, deletions or changes relative to APEX (or HRP), and has substantially the same effect as APEX (or HRP) in catalyzing biotin-phenol to form free radical phenol.

[0107] As used herein, the term "inducible hydrogen peroxide-producing protein" or "inducible H2O2-producing protein" refers to a protein that can locally produce hydrogen peroxide under certain conditions. For example, the inducible hydrogen peroxide-producing protein used in the present invention can locally and accurately produce hydrogen peroxide under light conditions. The illumination wavelength used in the present invention can be selected according to the type of the inducible H2O2-producing protein. In some embodiments, the H2O2 production of the hydrogen peroxide-producing protein increases with the increase of the light induction time. Such proteins used in the present invention produce trace amounts of hydrogen peroxide. The "trace amount" refers to the amount of hydrogen peroxide generated that is sufficient to activate the peroxidase-catalyzed reaction, but does not cause a toxic reaction in cells or tissues. The inducible hydrogen peroxide-producing proteins used in the present invention include, but are not limited to, SOPP3 protein, SOPP2 protein, SOPP1 protein, or a combination thereof.

[0108] SOPP3 belongs to the light-oxygen-voltage (LOV) family. When it senses light signals, its domain changes to expose flavin mononucleotide (FMN). FMN is an important electron transporter in the respiratory chain in the body and is also a photosensitizer. After being stimulated by light, it produces singlet oxygen in a very short time.

[0109] As used herein, the term "protein of the present invention" refers to the above-mentioned peroxidase, or its active fragment, or its localization fusion protein, or its antibody cross-linked product, the above-mentioned inducible hydrogen peroxide-producing protein or its active fragment, or its localization fusion protein, or its antibody cross-linked product, or the fusion protein described in the third aspect of the present invention.

[0110] As used herein, the term "protein interaction" or "interaction" refers to the interaction between proteins that leads to changes in protein activity conformation and signal transduction, also known as protein-protein interaction (PPI). Common protein interactions in the art include interactions between polypeptide ligands and proteases, and polypeptide hormones and receptors.

[0111] In one embodiment of the present invention, when the proteins or elements (e.g., organelles, cells) involved in protein interactions are known, peroxidase (e.g., APEX) and inducible hydrogen peroxide-producing proteins can be separately connected to the two proteins or elements in the protein interaction, thereby detecting the known protein or element and other proteins adjacent to it.

[0112] In another embodiment of the present invention, when one or more of the proteins or elements involved in the protein interaction are unknown, a fusion protein containing peroxidase and an inducible hydrogen peroxide-producing protein can be linked to a known protein or element involved in the protein interaction, thereby marking an unknown protein or protein complex that may interact with the known protein or element, as well as other adjacent proteins.

[0113] The present invention provides a method for connecting the protein of the present invention to an element (such as a cell, an organelle, etc.). The connection method of the present invention comprises:

[0114] a) expressing the protein of the present invention on the element;

[0115] b) expressing the protein of the present invention by fusion with a protein involved in the interaction;

[0116] c) cross-linking the protein of the present invention with antibodies, connecting the protein of the present invention and proteins involved in the interaction through antibodies, for example, directly connecting antibodies or connecting through a double antibody sandwich method.

[0117] In order to express the protein of the present invention on an element, the protein of the present invention can be fused with a localization peptide for expression. The localization peptide can localize the protein of the present invention to the cell membrane surface, a specific organelle, a specific DNA or RNA sequence, but is not limited thereto.

[0118] Proximity Mark

[0119] Proximity labeling is an emerging technology for revealing interactions between biological molecules. It uses transgenic methods to fuse screened engineered enzymes, such as biotin ligase BioID, TurboID or peroxidase APEX, HRP, etc., with bait proteins in cells, and then uses biotin to label the target protein bound to the bait protein. Finally, mass spectrometry identification and bioinformatics analysis are used to further understand the molecules that interact with the bait protein.

[0120] The method of the present invention can be used to mark interacting proteins and / or neighboring proteins. As used in the present invention, the term "interacting protein" refers to a protein that participates in the interaction. The term "neighboring protein" refers to a protein near the interaction region, which may interact with a known protein. The two or more proteins involved in the interaction may be known or unknown, and the method of the present invention can be used to mark known interacting proteins, or to mark unknown proteins that may interact with known proteins.

[0121] The main advantages of the present invention include:

[0122] 1) Compared with the prior art of directly treating cells with hydrogen peroxide, SOPP3 locally produces hydrogen peroxide under blue light treatment conditions, which is very less toxic to living cells and can be applied to living organisms;

[0123] 2) The labeling reaction of the SOPP3-APEX2-mediated proximity protein labeling method of the present invention can be completed within seconds, so protein-protein interactions and cell-cell interactions in rapidly occurring biological processes can be captured;

[0124] 3) The SOPP3-APEX2-mediated neighboring protein labeling method of the present invention has good labeling specificity. By controlling the time of blue light irradiation and the area of ​​blue light irradiation, neighboring protein labeling in a specific time and a specific area can be achieved;

[0125] 4) The labeling method of the present invention is simple to operate and can be applied to the study of interacting proteins between known interacting subcellular organelles and between cells. By using the SOPP3-APEX2 fusion strategy, it is also possible to explore unknown subcellular organelles (or cells) that interact with the target subcellular organelles (or cells) and the interacting protein molecules.

[0126] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0127] 1) In vitro cell labeling and detection experimental methods:

[0128] After HeLa cells were cultured to cover 80% of the surface area of ​​a 3.5 cm culture dish (purchased from Corning), plasmid transfection was prepared. 1 μg of Tomm20mls-V5-APEX2 and 1 μg of SOPP3-HA-Sec61β plasmid were mixed with 100 μl of Opti-MEM (Gibco, Cat. No.: 31985070), and 1.5 μl of Lipofectamine TM 3000 (Thermo Fisher Scientific, Catalog No.: L3000015) and 100 μl Opti-MEM (Gibco, Catalog No.: 31985070) were mixed and allowed to stand at room temperature for 5 min. The plasmid solution was mixed with Lipofectamine TM 3000 solution was mixed and allowed to stand at room temperature for 15 min. 1 ml of 10% FBSDMEM (Gibco, Cat. No.: 11965118) medium was replaced for cells in 3.5 cm culture dish, and the mixed and allowed to stand plasmid solution was added to the cell culture dish and cultured at 37°C for 24 hours.

[0129] After 24 hours, the culture medium was removed and the cells were washed twice with 1 ml of DPBS buffer (Gibco, catalog number: C14190500CP). Complete culture medium containing 500 μM Biotin-phenol (Taoshu, catalog number: T19209) was added and incubated at 37°C for 1 h. Blue light (Shanshi Technology, 452 nm) was irradiated at a distance of 15 cm from the cell culture dish for the corresponding time. The culture medium was removed and the cells were washed five times with DPBS containing 5 mM Trolox (Taoshu, catalog number: T1710), 10 mM sodium azide, and 10 mM sodium ascorbate (Taoshu, catalog number: T6674).

[0130] For Western blot samples, add 100μl 1xSDS-PAGE protein loading buffer (Biyuntian, catalog number: P0015F, diluted with ddH2O) to the culture dish and denature at 98℃ for 10min. Separation gel concentration is 10%, stacking gel is 4%, and polyacrylamide gel thickness is 1.0mm (10% and 4% refer to the percentage of acrylamide in sodium dodecyl sulfate-PAGE gel) separation: 100V, 150 minutes. Transfer membrane (Roche, 0.2μM polyvinylidene fluoride membrane, PVDF, catalog number: 03010040001): constant current 250mA, 120 minutes. The transferred membrane was blocked in 5% BSA solution overnight, and then incubated with antibody horseradish peroxidase-labeled streptavidin (Invitrogen TM , Catalog No.: S911, 1:5000 dilution) and incubate at room temperature for 1 h. Add about 1 ml of chemiluminescent peroxidase substrate reagent (Millipore, Catalog No.: WBKLS0500) and detect the target protein level using the Tanon 5200s chemiluminescent imaging system.

[0131] For immunofluorescence samples, add PBS buffered saline containing 4% (w / v) paraformaldehyde to the cells, fix them at room temperature for 15 minutes, wash the cells three times with DPBS, add DPBS buffered saline containing 0.1% (v / v) Triton X-100, and permeabilize them at room temperature for 15 minutes. Then add 3% BSA, HA antibody (Cell signaling technology, catalog number: 3724S, 1:500 dilution), V5 antibody (ABclonal, catalog number: AE017, 1:500 dilution) and incubate at 4°C overnight. Change the secondary antibody (Invitrogen TM , goat anti mouse-Alexa fluor 647, Catalog No. A32728, 1:500 dilution; Invitrogen TM , goat anti rabbit-Alexa fluor 488, Catalog No. A11034, 1:500 dilution; Invitrogen TM Alexa Fluor 555 Streptavidin, Catalog No. S32355, 1:2000 dilution), incubate at room temperature for 1 hour in the dark. Wash the cells three times with 0.1% (v / v) Triton X-100 in DPBS buffered saline, add the nuclear fluorescent dye DAPI, and stain for five minutes at room temperature. Wash the cells five times with 0.1% (v / v) Triton X-100 in DPBS buffered saline, and finally image.

[0132] 2) Proteomic enrichment and detection experimental methods:

[0133] For Biotin-Streptavidin enrichment, take 10 plates of 100% density 3.5cm culture dishes, after being labeled and washed, add 100μl RIPA (Biyuntian, catalog number: P0013B) to each plate, mix the 10 plates of cell lysate, blow 20 times with a 1ml syringe, 12500rpm / min 4℃15min, take the supernatant for BCA quantification. Take 3mg protein solution and mix it with 100μl streptavidin-agarose (Millipore, catalog number: S1638), and incubate overnight at 4℃. 1000 rpm / min 4℃5min, aspirate the supernatant, wash streptavidin-agarose twice with RIPA, wash once with 1M KCL, wash once with 0.1M Na2CO3, wash once with 2M Urea (prepared with 10mM Tris, ph=8.0), wash twice with RIPA again, add 6xSDS-PAGE protein loading buffer after washing, and denature at 98℃ for 10min.

[0134] 3) Labeling and detection experimental methods in zebrafish:

[0135] Take 48hpf zebrafish embryos expressing Tomm20mls-V5-APEX2 and SOPP3-HA-Sec61β at the same time, and inject 500mM Biotin-phenol into the heart of the zebrafish embryo. The injection volume for each embryo is 10nl. After the injection, the embryos were immersed in PTU dissolved with 500mM Biotin-phenol for 2h. Irradiate with blue light for 3min. Fix with 4% PFA at 4℃ overnight, wash the embryos with PBS, digest with 40μg / ml proteinase K at room temperature for 10min, treat with 4% PFA at room temperature for 15min, and wash with DPBS buffered saline containing 0.1% (v / v) Tween20. Block the embryos with 5% BSA for 3 hours. HA antibody (Cell signalingtechnology, catalog number: 3724S, 1:500 dilution), V5 antibody (Invitrogen TM , Product No.: R960-25, 1:500 dilution) was added to the blocking solution and incubated at 4°C overnight. The embryos were washed with the blocking solution and the secondary antibody (Invitrogen TM , goat anti mouse-Alexa fluor 647, Catalog No. A32728, 1:500 dilution; Invitrogen TM , goat anti rabbit-Alexa fluor488, Catalog No. A11034, 1:500 dilution; Invitrogen TMAlexa Fluor 555 streptavidin, Catalog No. S32355, 1:2000 dilution), incubated overnight at 4°C. On the third day, embryos were washed with blocking solution and imaged with a confocal microscope.

[0136] Example 1 Testing the ability of various photosensitive proteins to activate APEX2

[0137] In this example, the ability of various light-sensitive proteins to activate APEX2 under light conditions was tested. Figure 1 As shown, the photosensitive proteins KillerRed, miniSOG and SOPP3 were co-transfected with APEX2 into HeLa cells, respectively. 24 hours after transfection, the cells were co-incubated with Biotin phenol (BP) required for APEX2 labeling at 37 degrees, and then the cells were treated with light. At the same time, a positive control group in which APEX2 was transfected alone and the labeling reaction was activated with hydrogen peroxide and a negative control group without transfection were set up. The experimental results showed that the experimental group co-transfected with SOPP3 and APEX2 produced a large amount of biotinylation signals after blue light irradiation, while the photosensitive proteins KillerRed and miniSOG did not detect APEX2 labeling activity after light induction, indicating that SOPP3's efficient hydrogen peroxide production ability enables it to activate the adjacent labeling activity of APEX2 after blue light irradiation.

[0138] The results confirmed that SOPP3 and APEX2-mediated neighboring protein labeling was best under light conditions compared with other light-sensitive proteins.

[0139] Example 2 Blue light-induced SOPP3 and APEX2 mediate neighboring protein labeling between organelle interaction surfaces

[0140] In vitro cell labeling and detection were performed in HeLa cells. Figure 2 As shown. When APEX2 protein localized on mitochondria and SOPP3 protein localized on the endoplasmic reticulum are expressed in living cells ( Figure 2 A), by using different powers of blue light sources for different periods of time (0, 1s, 5s), different levels of protein can be induced to be labeled with biotin ( Figure 2 B), immunofluorescence detection revealed that the proteins on the interaction surface between mitochondria and endoplasmic reticulum were labeled with biotin ( Figure 2 CD).

[0141] Example 3 Proteome enrichment detection verifies that the labeled proteins are interacting proteins between organelles

[0142] Figure 3A shows the basic flow chart of APEX2-SOPP3 proximity labeling combined with LC-MS for intracellular mitochondrial-endoplasmic reticulum interface proteomic analysis. APEX2 protein localized on the outer membrane of mitochondria and SOPP3 protein localized on the endoplasmic reticulum were expressed in cells, and blue light irradiation induced proximity labeling reaction, and then the biotin-phenol labeled protein was enriched by agarose coated with streptavidin. Coomassie brilliant blue staining showed that 5s blue light treatment could significantly enrich the proximity labeled protein compared with the non-irradiated group ( Figure 3 B). Western-blot further confirmed that the markers of the endoplasmic reticulum-mitochondria interface CANX, mitochondrial outer membrane proteins Tomm20 and VDAC1, and DRP1 and MFN2 that mediate mitochondrial fission all had significant enrichment effects. In contrast, the nuclear protein Histone3 was not enriched, proving that the APEX2-SOPP3-mediated proximity labeling method has good specificity ( Figure 3 C).

[0143] Example 4 Blue light dependence of protein labeling mediated by SOPP3 and APEX2.

[0144] Protein labeling was performed using different blue light irradiation times (0s, 1s, 5s, 20s, 30s), such as Figure 4 As shown in A, as the blue light illumination time increases, the protein labeling effect mediated by SOPP3 and APEX2 is also enhanced accordingly, and there is a clear and discernible protein labeling effect at 1s, and the protein labeling is very obvious at 5s, basically reaching saturation. Figure 4 As shown in B and 4C, SOPP3- and APEX2-mediated protein labeling occurs in the presence of blue light illumination, and when the blue light illumination is removed, the protein labeling reaction no longer continues.

[0145] Example 5 SOPP3 and APEX2 mediate interaction protein signatures between neighboring cells

[0146] like Figure 5 As shown in A, stable cell lines expressing SOPP3 and APEX2 elements located on the cell membrane were constructed respectively, and then the two cell lines were mixed together for culture. When the two cells were adjacent to each other, under the irradiation of blue light, the proteins on their interaction surfaces were labeled. Through protein immunodetection experiments, it was found that blue light treatment can induce protein labeling, and through immunofluorescence staining, it can be found that the proteins on the interaction surfaces of adjacent cells are labeled with biotin ( Figure 5 B) The results verified that SOPP3 and APEX2, which are respectively localized on the cell membranes of adjacent cells, can mediate the labeling of interacting proteins between adjacent cells through light induction.

[0147] Example 6 SOPP3-APEX2 protein fusion form can also mediate protein labeling through light induction, and the labeling effect is equivalent to that of the non-fusion form

[0148] like Figure 6 As shown, compared with expressing APEX2 and SOPP3 proteins separately, expressing APEX2-SOPP3 in the form of a protein fusion in living cells can also induce different degrees of protein labeling with biotin by irradiating with a blue light source for different periods of time, and the protein labeling efficiency is comparable to that of expressing the two proteins separately.

[0149] Example 7 Protein interaction labeling mediated by SOPP3-APEX2 protein fusion

[0150] like Figure 7 As shown in A, APEX2-SOPP3 expressed as a protein fusion localized on the cell membrane can be induced to be labeled with biotin to varying degrees by irradiating with blue light for different periods of time. Immunofluorescence staining revealed that in addition to the proteins on the cell membrane expressing the APEX2-SOPP3 fusion protein being labeled with biotin, the proteins on the cell membranes of the adjacent cells were also labeled with biotin ( Figure 7 B).

[0151] Sequence of SOPP3-APEX2-TM fusion protein (SEQ ID NO: 4):

[0152] METDTLLLWVLLLWVPGSTGDASYPYDVPDYAEFGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPD PTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADATGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSTS MEKSFVITDPRLPDNPIIFASDGFLELTEYSREEILGRNGRFLQGPETDQATVQKIRDAIRDQREITVQLINYTKSGKKFLNLLNLQPIRDQKGELQAFIGVVLDGLEFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEV SVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRR

[0153] Example 8 SOPP3 and APEX2 mediate neighboring protein labeling in living organisms

[0154] like Figure 8 As shown, SOPP3- and APEX2-mediated neighbor protein labeling can also occur between the mitochondria and sarcoplasmic reticulum interaction surfaces of muscle cells in living zebrafish muscle tissue.

[0155] discuss

[0156] In previous APEX-mediated biotin protein labeling methods, a relatively high concentration of hydrogen peroxide needs to be added exogenously. After the addition of high concentration of hydrogen peroxide, almost all cells die. Therefore, this method can only be limited to in vitro biotin labeling and cannot be applied in vivo.

[0157] After screening, the present invention unexpectedly discovered that SOPP3 can express a trace amount of H2O2 after illumination, and the expression amount can meet the requirements of catalyzing APEX reaction and will not be toxic to cells or organisms. At the same time, the labeling system of the present invention is induced by blue light illumination. The induction conditions are simple to apply in vivo, and have very little effect on the body, and basically no undesirable effects will be produced. Therefore, the labeling system of the present invention can be conveniently catalyzed in vivo, which is of great significance for in vivo experiments.

[0158] Another important significance of the present invention is that the SOPP3-APEX labeling system has a very high sensitivity. After 1 second of blue light illumination, a clearly discernible protein labeling effect has been produced, and at 5 seconds, the protein labeling is very obvious and basically reaches saturation. Therefore, compared with the protein labeling time of hours or minutes in the prior art, the labeling time of the labeling system of the present invention is greatly shortened, and can be used to detect protein interactions that occur instantaneously (seconds level) in organisms, and has extremely high application prospects in the field of life science research.

[0159] In addition, since SOPP3 only releases a small amount of H2O2 locally after blue light irradiation, the SOPP3-APEX labeling system of the present invention also has spatial specificity. By fusing SOPP3 protein and APEX protein to the target protein to be detected, or coupling them with corresponding antibodies, or localizing and expressing them on the cell membrane, proteins in specific parts, organelles or protein interactions between cells can be accurately labeled and detected.

[0160] At the same time, the SOPP3-APEX labeling system of the present invention can also be used multiple times at different time points. The labeling system of the present invention can be repeatedly activated by multiple illuminations at intervals, and the protein interactions at multiple time points in the same sample can be monitored, which is of great significance for observing the biological reaction process over a period of time.

[0161] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for labeling interacting proteins and / or neighboring proteins, characterized in that: The method comprises the steps of: S1) connecting a peroxidase to a protein A involved in the interaction, or an element A expressing the protein A, thereby obtaining a peroxidase element A'; S2) connecting the inducible hydrogen peroxide (H2O2) producing protein to another protein B involved in the interaction, or to an element B expressing the protein B, thereby obtaining an H2O2 producing element B'; S3) contacting the peroxidase element A', the H2O2 generating element B' and biotin phenol, thereby catalyzing the attachment of biotin to adjacent proteins A and B and / or other adjacent proteins adjacent to proteins A and B.

2. The method according to claim 1, characterized in that The peroxidase is selected from the group consisting of ascorbate peroxidase (APEX) or its derivatives, horseradish peroxidase (HRP) or its derivatives, or a combination thereof.

3. The method according to claim 1 or 2, characterized in that The inducible H2O2-producing protein is selected from the group consisting of SOPP3 protein, SOPP2 protein, SOPP1 protein, or a combination thereof.

4. The method according to claim 1, characterized in that The element A and element B are independently selected from: organelles, cells, or a combination thereof.

5. The method according to claim 1, characterized in that The step S1 includes the following steps: S1a) expressing a peroxidase on the element A, thereby linking the peroxidase to the element A; or S1b) fusing peroxidase to the protein A, thereby linking the peroxidase to the protein A; or S1c) contacting the protein A or element A with the antibody cross-linked with the peroxidase, wherein the antibody specifically binds to the protein A, thereby linking the peroxidase to the protein A or element A; or S1d) contacting the protein A or element A, a first antibody that specifically binds to protein A, and a second antibody cross-linked with the peroxidase, thereby linking the peroxidase to protein A or element A, wherein the second antibody specifically recognizes the first antibody.

6. The method according to claim 1, characterized in that The step S2 includes the following steps: S2a) expressing an inducible H2O2-producing protein on the element B, thereby linking the inducible H2O2-producing protein to the element B; or S2b) fusing the inducible H2O2-producing protein with the protein B, thereby linking the inducible H2O2-producing protein to the protein B; or S2c) contacting the protein B or element B with an antibody cross-linked with the inducible H2O2-producing protein, wherein the antibody specifically binds to protein B, thereby linking the inducible H2O2-producing protein to protein B or element B; or S2d) contacting protein B or element B, a first antibody that specifically binds to protein B, and a second antibody cross-linked with the peroxidase, thereby linking the inducible H2O2-producing protein to protein B or element B, wherein the second antibody specifically recognizes the first antibody.

7. The method according to claim 1, characterized in that The step S3 includes the step of applying light to the peroxidase element A', the H2O2 producing element B' and the biotin-phenol, thereby catalyzing the connection of biotin to the interacting proteins A and B and / or their neighboring proteins.

8. A reagent combination for labeling interacting proteins and / or their neighboring proteins, characterized in that: The reagent combination comprises: A) peroxidase, or its localization fusion protein, or its antibody cross-linked product, or a nucleic acid molecule encoding the same, or an expression vector containing the nucleic acid molecule; and B) Inducible hydrogen peroxide-producing protein, or its localization fusion protein, or its antibody cross-linked product, or a nucleic acid molecule encoding the same, or an expression vector containing the nucleic acid molecule.

9. A fusion protein for labeling an interacting protein and / or a neighboring protein, characterized in that: The fusion protein comprises: Z1) peroxidase or an active fragment thereof; Z2) Inducible hydrogen peroxide-producing protein or its active fragment.

10. A method for labeling interacting proteins and / or neighboring proteins, characterized in that: The method comprises the steps of: S1) connecting the fusion protein according to claim 9 to the protein A involved in the interaction, or the element A expressing the protein A, thereby obtaining a peroxidase-H2O2-producing element; S2) contacting the peroxidase-H2O2 generating element with biotin-phenol, thereby catalyzing the attachment of biotin to the adjacent protein A and the protein B interacting with protein A, and / or to other adjacent proteins adjacent to proteins A and B.

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