Nanometer antibody-proximity marker enzyme fusion protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nano antibody-proximity marker enzyme fusion protein and application thereof. Specifically, the present disclosure provides a fusion protein for proximity labeling wherein the fusion protein is operably linked together by (a) a nanobody and (b) a proximity labeling enzyme; optionally, a peptide linker is also included between the nano antibody and the proximity marker enzyme. The nano antibody-proximity marker enzyme fusion protein disclosed by the invention is obtained through in-vitro expression and purification, target protein is targeted by using an antibody, and the nano antibody-proximity marker enzyme fusion protein does not depend on an overexpression system and has compatibility to various cell types, tissues and clinical fixed samples; proteins with post-translational modification and various organelles, such as membraneless organelles, can be targeted; and the positioning accuracy rate reaches 100%.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a nano antibody-proximity marker enzyme fusion protein and its application. Background Art

[0002] Protein interactions are the basis of various biological processes in cells, including cell signal transduction, metabolic regulation, gene transcription regulation, protein synthesis, cell structure assembly, etc. They are of great significance in biology and involve the interaction and mutual influence between proteins. Studying protein interactions helps to reveal the complex molecular networks and signal transduction pathways in cells and understand the regulatory mechanisms of cell functions and biological processes.

[0003] Proximity labeling technology provides a complementary option to traditional methods for studying protein interaction networks, organelle proteome and membrane contacts, protein-nucleic acid interactions and subcellular transcriptome analysis. This technology relies on a tool enzyme with proximity labeling function, which can biotinylate neighboring biological molecules at the living cell level. It has been widely used for the identification of macromolecular complex components, the analysis of protein species in organelles and the construction of protein interaction networks. The strategy of proximity labeling technology is usually to fuse the target protein with an enzyme with a biotin proximity label, so as to locate the target protein to a specific location in the cell. By adding small molecule substrates such as biotin and its derivatives, the proximity labeling enzyme can catalyze the biotinylation covalent linkage modification of proteins that are spatially adjacent to the target protein. Currently, the widely used tool enzymes include: biotin ligase A (BirA) and its mutants BioID / BioID2 and TurboID; peroxidase APEX (engineered ascorbate peroxidase, APEX) and APEX2, etc.

[0004] However, the current proximity labeling method relies on the expression of exogenous fusion proteins and cannot be used to identify post-translationally modified proteins. Although it has been reported that antibody-mediated protein A-APEX2 can be biotinylated in situ, thereby better preserving the spatial relationship of protein interactions; by determining the proteins that react with histone modifications through mass spectrometry analysis, we can further understand the functions and mechanisms of these modifications in cells. However, because protein A can interact with multiple proteins such as endogenous IgG, the orthogonality and reproducibility of protein A-APEX2 fusion protein with mammalian cells are poor. Although protein A-APEX2 can label post-translation modifications of histones in the cell nucleus, the labeling radius of protein A-APEX2 is large and cannot label fine structures in the cell, such as microtubules and mitochondria.

[0005] In traditional methods for studying protein interactions, such as immunoprecipitation, antibodies derived from mice and rabbits are indispensable tools in many basic research techniques and medical diagnostic tests. Typically, the method used to detect or fix these primary antibodies is to indirectly use polyclonal anti-IgG secondary antibodies. However, in order to meet the demand for a continuous supply of anti-IgG serum, a large number of goats, sheep, rabbits and donkeys need to be slaughtered, which is not only costly, but also involves important animal welfare and ethical issues. Researchers have currently developed a recombinantly expressed nanosecondary antibody, which is a single domain antibody from a camel heavy chain antibody, used as a substitute for polyclonal anti-IgG secondary antibodies. The nanosecondary antibody can bind to different regions of rabbit or mouse IgG, partially reducing the need for the use of animal-derived polyclonal secondary antibodies. Nanosecondary antibodies are considered powerful tools in fields such as cell biology and structural biology because of their small size, high specificity, the potential for regenerative recombinant fusion proteins, and excellent biophysical properties.

[0006] However, in the field of antibody-drug conjugate technology, nano-antibodies have not been widely used in the field of antibody conjugation due to their poor efficacy.

[0007] Furthermore, the identification of protein interactions in protein aggregates currently faces bottlenecks that are difficult to overcome. For example, (1) traditional biochemical methods cannot effectively separate the aggregates; (2) the formation of protein aggregates is highly dependent on the concentration of aggregated proteins, which results in the overexpression system of biotin ligase fused with the target protein not being able to represent the true endogenous phase transition protein concentration; (3) some overexpressed proteins will have abnormal localization, resulting in false positives. These directly or indirectly affect the detection and analysis of protein interactions.

[0008] So far, there has been no disclosure or development of a fusion protein or conjugate integrating a nanobody and a proximity-labeled tool enzyme in the art, and no breakthrough or improvement of the limitations or problems in traditional immunoprecipitation methods and proximity-labeling techniques in protein interaction network research using the spatiotemporal labeling properties of the tool enzyme and the biological properties of the nanobody. However, there has always been such a demand in the field of life science research, hoping to have the advantages of both tool enzymes and nanobodies to more efficiently and sensitively explore protein-protein interaction networks inside and outside cells or tissues. Summary of the invention

[0009] In order to solve the problems existing in the prior art, the present disclosure integrates the advantages of nanoantibodies and proximity labeling tool enzymes, and provides a fusion protein or conjugate of a nanoantibody and a proximity labeling enzyme, aiming to utilize the spatiotemporal labeling characteristics of the tool enzyme and the biophysical properties of the nanoantibody to break through or improve the application of traditional immunoprecipitation methods and proximity labeling technology in protein interaction network research.

[0010] In one aspect, the present disclosure provides a fusion protein for proximity labeling, wherein the fusion protein is operably linked together by (a) a nanobody and (b) a proximity labeling enzyme; and further comprises a peptide linker between the nanobody and the proximity labeling enzyme.

[0011] In another aspect, the present disclosure provides a nucleic acid molecule encoding the aforementioned fusion protein.

[0012] In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.

[0013] On the other hand, the present disclosure provides a kit, which comprises the aforementioned fusion protein: preferably, the kit further comprises a primary antibody targeting the target protein;

[0014] Preferably, the kit further comprises a biotinylation reaction solution;

[0015] Preferably, the kit further comprises fluorescent agent-conjugated streptavidin;

[0016] Preferably, the kit further comprises a secondary antibody coupled to a fluorescent agent;

[0017] Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP and biotin;

[0018] Preferably, the biotinylation reaction solution comprises biotin phenol and hydrogen peroxide.

[0019] On the other hand, the present disclosure provides a proximity labeling method, characterized in that the aforementioned fusion protein or the aforementioned kit is used to biotin-label the protein interacting with the target protein, which comprises the following steps:

[0020] Add a primary antibody targeting the target protein from rabbit or mouse species to the cells for incubation to allow the primary antibody to bind to the target protein;

[0021] According to the species of the primary antibody, a fusion protein of the corresponding species is added to the cells, and the cells are incubated to form a proximity marker enzyme-nano secondary antibody-primary antibody-target protein complex in the cells;

[0022] Adding biotinylation reaction solution to the cells, incubating, and biotin-labeling the protein molecules interacting with the target protein;

[0023] After washing, fluorescent agent-conjugated streptavidin and fluorescent agent-conjugated secondary antibody were added, incubated, washed, and detected.

[0024] In another aspect, the present disclosure provides a method for analyzing molecular interactions, the method comprising the following steps:

[0025] Using the aforementioned fusion protein or the aforementioned kit to biotin-label the protein interacting with the target protein;

[0026] Use magnetic beads or fluorescent agents coupled with streptavidin to enrich or fluorescently locate biotin-labeled protein molecules;

[0027] The enriched biotin-labeled protein molecules were analyzed and identified by LC-MS / MS.

[0028] In another aspect, the present disclosure provides the use of the aforementioned fusion protein, the aforementioned kit, and the aforementioned method, wherein the use comprises:

[0029] (a) Biotinylation proximity labeling and component analysis of the cytoskeleton and organelles;

[0030] (b) Biotinylation proximity labeling and component analysis of post-modified histones;

[0031] (c) Biotinylation proximity labeling and component analysis of different nucleolar substructures;

[0032] (d) Biotinylation proximity labeling of proteins in FFPE and OCT sections and analysis of interacting proteins;

[0033] (e) Dual biotinylation proximity labeling of the same sample; or

[0034] (f) Biotinylation proximity labeling of proteins and analysis of interacting proteins in model organisms.

[0035] The beneficial effects of the present disclosure are at least:

[0036] The above-mentioned technical scheme disclosed in the present invention does not rely on the overexpression system; by using antibodies that recognize histone modifications, the interacting proteins of the post-modified histones are biotin-labeled, thereby effectively analyzing the protein interaction network of the post-modified histones; it can also accurately analyze the protein components in the phase change particles; it does not rely on the construction of cloning vectors, and can use targeted antibodies to label proteins with larger molecular weights and identify their related interacting proteins; it can analyze the protein interactions of multiple localized proteins at specific locations with high resolution, such as distinguishing different protein interaction networks of the same protein in the nucleus and cytoplasm.

[0037] The Nanobody-Proximity Marker Enzyme Fusion Proteins provided by the present disclosure can be used for:

[0038] (a) Biotinylation proximity labeling and component analysis of the cytoskeleton and organelles;

[0039] (b) Biotinylation proximity labeling and component analysis of post-modified histones;

[0040] (c) Biotinylation proximity labeling and component analysis of different nucleolar substructures;

[0041] (d) Biotinylation proximity labeling of proteins in FFPE and OCT sections and analysis of interacting proteins;

[0042] (e) Double biotinylation proximity labeling of the same sample;

[0043] (f) Biotinylation proximity labeling of proteins and analysis of interacting proteins in model organisms.

[0044] In particular, by designing and providing nano-antibody-photosensitive proximity marker enzyme fusion proteins, efficient biotin labeling can be achieved in a flexible and controllable manner. Furthermore, in the future, it can be combined with a fully automatic AI recognition microscope imaging system. The AI ​​recognition algorithm can quickly and accurately identify structures of interest, thereby achieving the positioning and analysis of the target area. Furthermore, in combination with AI and microscopes, it is expected to achieve high-throughput, automated image acquisition and analysis and fixed-point biotinylation labeling, marking specific locations of hundreds of thousands of cells, thereby meeting the sample volume requirements of mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the working principle of the nanobody-proximity marker enzyme fusion protein disclosed in the present invention in actual sample detection.

[0046] Figure 2 This is a schematic diagram of the working principle of light-controlled nanoantibody-proximity marker enzyme fusion protein in actual sample detection.

[0047] Figure 3 It is an SDS-PAGE image of the purified anti-mouse or anti-rabbit nanobody-TurboID fusion protein, wherein lanes 1-8 represent the molecular map lanes of the constructed anti-mouse nanobody-TurboID fusion protein expressed in host cells and before and after purification; lane 9 is a protein standard (marker); lanes 10-16 represent the molecular weight of the constructed anti-rabbit nanobody-TurboID fusion protein expressed in host cells and before and after purification.

[0048] Figure 4 is the labeling radius of Nano-ID, Nano-APX, Pro-ID and Pro-APX in the mouse cytoskeleton. Figure 4 a is a proximity biotin ligase expressed by fusion of six different types of nanobodies, namely anti-rabbit nanobody-TurboID / APEX2, anti-mouse nanobody-TurboID / APEX2, and Protein A-TurboID / APEX2; Figure 4b is the experimental process of in vitro biotinylation. The cells are first fixed, blocked, and permeabilized. Then, the primary antibody targeting the region of interest is added. Protein A-TurboID / APEX2 or anti-rabbit nanoantibody-TurboID / APEX2 or anti-mouse nanoantibody-TurboID / APEX2 is added according to the species of the primary antibody. Figure 4 c-4n are the fluorescence localization results of different anti-mouse fusion proteins in U-2OS cells. The anti-β-actin primary antibody is used to mark actin filaments (4c, 4f, 4i, 4l), the anti-vimentin primary antibody is used to mark intermediate filaments (4d, 4g, 4j, 4m), and the anti-TubA4A primary antibody is used to mark tubulin (4e, 4h, 4k, 4n); Figure 4 c-4e added anti-mouse nanobody--TurboID, Figure 4 f-4h anti-mouse nanobody-APEX2, Figure 4 i-4k or 4l-4n add Protein A-TurboID / APEX2. The green marker is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa Fluor TM 560-conjugated secondary antibody shows the localization of the primary antibody. Scale bar, 5 μm; magnification, 2 μm.

[0049] Figure 5 for Figure 4 Representation of the fluorescence signal distribution in the area selected by the white line in (4c, 4f, 4i, 4l) (upper row) and the fluorescence signal index fitting (lower row).

[0050] Figure 6 To compare the labeling radius of Nano-ID, Nano-APX, Pro-ID and Pro-APX using mouse antibodies against different organelles. Anti-LAMP1 primary antibody was used for lysosome labeling (6a, 6d, 6g, 6j), anti-LaminB1 primary antibody was used for nuclear membrane labeling (6b, 6e, 6h, 6k), and anti-ATP5A1 primary antibody was used for mitochondrial labeling (6c, 6f, 6i, 6l); Figure 6 a-6c added anti-mouse nanobody-TurboID, Figure 6 d-6f Add anti-mouse nanobody-APEX2, Figure 6 g-6i or Figure 6 J-6l added Protein A-TurboID / APEX2, green labeled with Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa FluorTM 560-conjugated secondary antibody, showing the localization of the primary antibody. Scale bar, 5 μm; magnification, 2 μm.

[0051] Figure 7 To compare the labeling radius of Nano-ID, Nano-APX, Pro-ID and Pro-APX using rabbit antibodies against different organelles. Anti-Paxillin primary antibody was used for focal adhesion labeling (7a-7d), anti-PEX14 primary antibody was used for peroxisome labeling (7e-7h), and anti-EDC4 primary antibody was used for P body labeling (7i-7l). Figure 7 a, 7e, 7i added anti-rabbit nanobody-TurboID, Figure 7 b, 7f, 7j anti-rabbit nanobody-APEX2, Figure 7 c, 7g, 7k or Figure 7 Protein A-TurboID / APEX2 was added at d, 7h, and 7l. The green marker is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa Fluor TM 560-conjugated secondary antibody, showing the localization of the primary antibody. Scale bar, 5 μm; magnification, 2 μm.

[0052] Figure 8 Nano-ID is used to mark organelles or post-histone modifications. Among them, anti-GOLG2 primary antibody (mouse antibody, Golgi marker) (8a), anti-CD98 primary antibody (rabbit antibody, cell membrane marker) (8b), anti-SC35 primary antibody (mouse antibody, nuclear speckle marker) (8c), anti-PDI primary antibody (rabbit antibody, endoplasmic reticulum marker) (8d), anti-CEP250 primary antibody (rabbit antibody, centriole marker) (8e), anti-FUS primary antibody (mouse antibody) (8f), anti-H3K4me3 (mouse antibody) (8g), anti-H3K27Ac (rabbit antibody) (8h), anti-HAK119ub (rabbit antibody) (8i), anti-L-Lactyl Lysine (rabbit antibody) (8j); Figure 8 b, 8d, 8e, 8h, 8i, 8j, anti-rabbit nanobody-TurboID was added, Figure 8 a, 8c, 8f, 8g added anti-mouse nanobody-TurboID; green marker is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa Fluor TM488-conjugated secondary antibody, showing the localization of the primary antibody. Scale bar, 5 μm.

[0053] Fig. 9 To analyze the nucleolus using Nano-ID. Fig. 9 a is a schematic diagram of the nucleolus structure. Fibrillar Centers (FC), Dense Fibrillar Component (DFC), Granular Component (GC). Fig. 9 b-9d are the results of in situ biotin labeling of three different substructures of the nucleolus. Fig. 9 e is the immunoblot detection of biotin proteins in different layers of substructures. Fig. 9 f is the signal heat map of TCOF1, FBL and NPM1 after different nucleolar substructures were detected by mass spectrometry. Fig. 9 g is the heat map of mass spectrometry signals of nucleolar proteins in different layers. Fig. 9 h-9j Select representative protein interaction network diagrams enriched in FC structure (9h), DFC structure (9i) and GC structure (9j). The protein interaction network is displayed by StringDb.

[0054] Fig.10 The application of Nano-ID in FFPE and OCT sections. Fig.10 a is a representative immunofluorescence image of in vitro biotinylation of Nano-ID on FFPE sections of lung adenocarcinoma tissue. Fig.10 b is the detection results of biotinylated protein content of total protein in the experimental group (+, with PECAM1 primary antibody) and the control group (-, without PECAM1 primary antibody). Fig.10 c is the mass spectrometry signal intensity of PECAM1 in the FFPE samples from the above three patients after the total protein in the experimental group and the control group was enriched with streptavidin-coupled magnetic beads; ND, not detected. Fig.10 d Venn diagram showing the overlap in PECAM1 protein interactions among proteins in the three patients. Fig.10 e is the interaction network analysis result of the interacting proteins of PECAM1 detected in all three samples. The interaction network information comes from the Stringdb database, and different colors represent different signaling pathways in which the proteins participate.

[0055] Fig.11 The application of Nano-ID in mouse brain OCT slices. Fig.11 a is a representative immunofluorescence image of in vitro biotinylation of mouse brain OCT slices by Nano-ID. After fixation and permeabilization, the mouse brain OCT slices were added with GFAP antibody and Nano-ID for in vitro biotinylation reaction. After the reaction, the samples were incubated with Alexa FluorTM 560-conjugated secondary antibody labeled with GFAP, Alexa Fluor TM 488-coupled streptavidin to label biotinylated proteins. Fig.11 b is the detection results of total protein biotinylated protein content in the experimental group (+, with GFAP primary antibody) and the control group (-, without GFAP primary antibody). Fig.11 c is a Venn diagram of 689 endogenous wild-type biotin proteins and 795 D4-labeled proteins identified after enrichment. Fig.11 d shows the results of GO analysis of the proteins identified in 11c. Fig.11 e is a schematic diagram of the experimental process.

[0056] Fig.12 Application of Nano-ID in the adult mouse brain of the HTT disease model. Fig.12 a is a representative immunofluorescence image of HTT-adjacent proteins in HTT disease model mice. Fig.12 b is the detection results of biotinylated protein content of total protein in the experimental group (+, with HTT primary antibody) and the control group (-, without HTT primary antibody).

[0057] Fig.13 The application of Nano-ID in FFPE and OCT sections and fertilized egg samples. Fig.10 a and 10b are representative immunofluorescence images of in vitro biotinylation of Nano-ID on FFPE sections of lung adenocarcinoma tissues. Fig.13 a Add PDL1 antibody, Fig.13 b Add H3K27Ac antibody, Alexa Fluor TM 560-conjugated secondary antibody labeled with PDL1 (a) or H3K27Ac (b), AlexaFluor TM 488 coupled to streptavidin to label biotinylated proteins. Fig.13 c shows the biotin-labeled transcription factor PAX6 neighboring protein in the OCT section of the mouse brain at developmental day 12.5. Fig.13 d is a schematic diagram of the apical domain labeled using Nano-ID in a mouse 8-cell embryo.

[0058] Fig.14 Enables dual labeling of one sample for Nano-ID. Fig.14a is a schematic diagram of the double labeling experimental process. Intracellular ROI1 and ROI2 are labeled with rabbit and mouse antibodies, respectively. For example, in the first round of reaction, Nano-ID (Rb) recognizes the rabbit primary antibody and labels the rabbit primary antibody labeled ROI1 with wild-type biotin; after the first round of reaction, TEV enzyme is added for cleavage, Nano-ID-(Rb) is cleaved, and the free Turbo-ID will be washed; in the second round of reaction, Nano-ID (Ms) is added to recognize the mouse primary antibody, and then the biotin in the reaction solution is labeled with D4, and the second round of reaction is carried out to label the mouse-labeled ROI2 with D4. The cells are lysed, and the obtained protein precipitate is digested into peptides, which are then biotinylated and enriched at the peptide level, and finally the wild-type and D4-labeled peptides are obtained, thereby distinguishing the proteins labeled in the first and second rounds. Fig.14 b is according to Fig.14 As described in a, the first round of rabbit primary antibody anti-TFAM wild-type biotin was used to label mitochondria, and the second round of mouse primary antibody anti-NPM1 isotope D4 biotin was used to label nucleoli. The biotin proteins labeled in the first and second rounds were respectively labeled with Alexa Fluor TM 488 and Alexa Fluor TM 560 conjugated streptavidin for observation. Fig.14 The immunoblot results of single TFAM, NPM1 labeling, and double TFAM and NPM1 labeling in b are shown in Fig.14 c.

[0059] Fig.15 The results of Nb2-ID labeling in cell lines and model organisms stably expressing GFP fusion protein. Fig.15 a is a cell line overexpressing mEmerald-SRSF3, Fig.15 b is a cell line in which GFP was knocked into the SC35 gene locus using CRISPR. Fig.15 c is a cell line overexpressing mEmerald-ensconsin. Fig.15 d is a nematode stably expressing ajm-1-GFP with a developmental stage of 1.8 fold. After incubation with Nb2-ID, the above samples were subjected to in vitro biotinylation reaction, and Alexa Fluor TM 560-coupled streptavidin localizes biotinylated proteins.

[0060] Fig.16 This is the labeling result of nanobody-light-controlled proximity labeling enzyme in U-2OS cells. Fig.16 a Incubate with rabbit anti-PEX14 antibody, Fig.16b Incubate the mouse anti-Tubulin antibody, and add the corresponding species of nanoantibodies-photosensitive proximity labeling enzymes for biotinylation reaction. The purple label is Alexa Fluor TM 647-conjugated secondary antibody shows the localization of the primary antibody; the green marker is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins. Scale bar, 5 μm. DETAILED DESCRIPTION

[0061] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only the embodiments of the present application. For ordinary technicians in this field, other implementation methods can also be obtained based on these drawings.

[0062] definition

[0063] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or nouns used herein are intended to have the meanings commonly understood by those skilled in the art of chemistry and medicine. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and such definitions contained herein should not be interpreted as representing substantial differences from definitions of terms commonly understood in the art. In the event of ambiguity, chemical, pharmaceutical, and medical dictionaries may be used as further sources of supplementary information, to the extent consistent with the present invention.

[0064] Unless the context indicates otherwise, the term "a" or "an" characterizes a substance or component but does not limit its number / quantity and can refer to the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. For example, reference to "a pharmaceutical combination" should be understood as a combination of two or more components.

[0065] Herein, unless otherwise specified, the terms "comprises," "includes," and "contains" or equivalents are open-ended expressions, meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0066] Unless the context clearly indicates otherwise, singular terms include plural referents and vice versa. Similarly, the word "or" is intended to include "and" and vice versa unless the context clearly indicates otherwise.

[0067] As used herein, the terms "about", "substantially" refer to deviations within the range of -10% to +10% from the stated value. When the term "about" is used herein to refer to a number, it should be understood that another embodiment of the present invention includes the number that is not modified by the presence of the term "about".

[0068] As used herein, the term "antibody" includes any immunoglobulin that binds to a specific antigen, including monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, or bispecific antibodies. A natural complete antibody includes two heavy (H) chains and two light (L) chains. Mammalian heavy chains are divided into α, δ, ε, γ, and μ, each heavy chain including a variable region (VH) and a first constant region, a second constant region, a third constant region, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are divided into λ or κ, and each light chain includes a variable region (VL) and a constant region. The antibody is "Y"-shaped, wherein the stem of the Y-shaped structure includes the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain usually contains three hypervariable regions, called complementarity determining regions (CDRs), of which the light chain CDRs include LCDR1, LCDR2, LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The three CDRs are separated by flanking segments called framework regions (FRs), of which the light chain FRs include LFR1, LFR2, LFR3, and LFR4, and the heavy chain FRs include HFR1, HFR2, HFR3, and HFR4; the framework regions are more highly conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0069] As used herein, the term "monoclonal antibody" refers to a highly uniform antibody produced by a single B cell (whose gene can only encode one antibody) clone and directed only to a specific antigen epitope. It is usually prepared using hybridoma technology. Hybridoma antibody technology is based on cell fusion technology, in which sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability are fused into B cell hybridomas. By culturing a single hybridoma cell with this characteristic into a cell population, a specific antibody directed to an antigen epitope, i.e., a monoclonal antibody, can be prepared.

[0070] As used herein, the term "polyclonal antibody" refers to a group of immunoglobulins secreted by plasma cells of an organism in response to an immune response stimulated by a heterologous antigen (macromolecule antigen, hapten conjugate). It usually contains at least 2 or more different antibodies, which can usually recognize multiple antigen epitopes and cause precipitation reactions. The preparation cost is low and the preparation speed is fast. The preparation process is simpler than that of monoclonal antibodies and is widely used in research and diagnosis.

[0071] As used herein, the term "primary antibody" or "first antibody" refers to a protein that can specifically bind to a non-antibody antigen (specific antigen). Types include monoclonal antibodies and polyclonal antibodies. That is, it is generally referred to as an antibody that can specifically bind to an antigen.

[0072] As used herein, the term "secondary antibody" or "secondary antibody" refers to an antibody that can bind to an antibody, i.e., an antibody of an antibody, and its main function is to detect the presence of the antibody and amplify the signal of the primary antibody. The secondary antibody utilizes the antigenic nature of the antibody as a macromolecular protein to immunize a xenogeneic animal, and the immunoglobulin produced by the immune system of the xenogeneic animal against this antibody. The secondary antibody is reactive against all antibodies (such as IgG, IgM or IgA, etc.) of a specific species (such as mouse).

[0073] As used herein, the term "antigen binding fragment" refers to an antibody fragment comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain a complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibody fragments, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen binding fragments are capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen binding fragment may include one or more CDRs from a specific antibody.

[0074] As used herein, the term "Fab" refers to a monovalent antigen-binding fragment of an antibody, which consists of a single light chain (variable region and constant region) connected to the variable region of a single heavy chain and the first constant region via a disulfide bond. Fab can be obtained by papain digestion of the N-terminal residues near the disulfide bond between the heavy chains in the hinge region of an antibody.

[0075] As used herein, the term "Fab'" refers to a Fab fragment comprising a portion of the hinge region, which can be obtained by pepsin digestion of residues near the C-terminal end of the disulfide bond between the heavy chains in the hinge region of an antibody, and therefore differs from Fab in a few residues in the hinge region (including one or more cysteines).

[0076] As used herein, the term "F(ab')2" refers to a dimer of Fab', which comprises two light chains and a portion of two heavy chains.

[0077] As used herein, the term "Fc" refers to an antibody portion that is composed of the second and third constant regions of the first heavy chain that are bound to the second and third constant regions of the second heavy chain via a disulfide bond. IgG and IgM Fc regions contain three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). It can be obtained by papain digestion of antibodies. The Fc portion of an antibody is responsible for various effector functions, such as ADCC, ADCP, and CDC, but does not play a role in antigen binding.

[0078] As used herein, the term "Fv" refers to the smallest antibody fragment with a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0079] As used herein, the term "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or through a peptide linker sequence. "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, a "scFv dimer" is a bivalent bifunctional antibody or a bivalent scFv (BsFv) comprising a VH-VL (connected by a peptide linker) dimerized with another VH-VL portion, so that the VH of one portion coordinates with the VL of the other portion and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific, bifunctional antibody comprising VH1-VL2 (linked by a peptide linker) combined with VL1-VH2 (also linked by a peptide linker) such that VH1 coordinates with VL1 and VH2 coordinates with VL2 and each coordination pair has a different antigenic specificity.

[0080] As used herein, the term "single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0081] As used herein, the term "nanobody" or "camelized single domain antibody", "heavy chain antibody (sdAb)" or "HCAb" has the same meaning and can be used interchangeably, all referring to cloning the variable region of the heavy chain of an antibody to construct a nanobody consisting of only one heavy chain variable region. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region. Heavy chain antibodies were originally obtained from Camelidae (camels, dromedaries and llamas), and although they do not contain light chains, camelized antibodies have a reliable antigen binding repertoire. The variable domain (VHH domain) of the heavy chain antibody represents the smallest known antigen binding unit produced by the acquired immune response.

[0082] As used herein, the term "bifunctional antibody" includes small antibody fragments with two antigen binding sites, wherein the fragment comprises a VH domain connected to a VL domain in a single polypeptide chain (VH-VL or VL-VH). Because the linker is too short, the two domains on the same chain cannot pair, and therefore, the domains are forced to pair with the complementary domains of another chain, thereby generating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes).

[0083] As used herein, the term "enzyme" refers to a protein or RNA produced by living cells that has a high degree of specificity and catalytic efficiency for its substrate. The catalytic action of an enzyme depends on the integrity of the primary structure and spatial structure of the enzyme molecule. If the enzyme molecule is denatured or the subunits are depolymerized, the enzyme activity can be lost. Enzymes are biological macromolecules with a molecular mass of at least 10,000 or more, and the largest can reach one million. Enzymes are a very important class of biocatalysts. Due to the action of enzymes, chemical reactions in organisms can be carried out efficiently and specifically under extremely mild conditions. According to the chemical composition of the enzyme, enzymes can be divided into two categories: simple enzymes and conjugated enzymes. Simple enzyme molecules are enzymes that have only amino acid components after hydrolysis. Conjugated enzyme molecules are composed of protein parts and non-protein parts, such as metal ions, iron porphyrins or small organic molecules containing B vitamins.

[0084] As used herein, the term "protein interaction" or "protein interaction" refers to the process by which two or more protein molecules form a protein complex through non-covalent bonds, such as replication, transcription, translation, cell cycle regulation, material metabolism, etc.

[0085] As used herein, the term "Protein-Protein Interaction Networks (PPI)" is composed of proteins that participate in various aspects of life processes such as biological signal transmission, gene expression regulation, energy and material metabolism, and cell cycle regulation through interactions with each other. Systematic analysis of the interaction relationship between a large number of proteins in biological systems is of great significance for understanding the working principles of proteins in biological systems, understanding the reaction mechanisms of biological signals and energy material metabolism under special physiological conditions such as diseases, and understanding the functional connections between proteins. Currently, the commonly used research methods for finding protein interactions are mainly yeast two-hybrid (Y2H) screening and immunoprecipitation combined with mass spectrometry (IP-MS) analysis.

[0086] As used herein, the term "proximity labeling (PL)" refers to a technique in which certain enzymes are fused and expressed with target proteins by gene fusion, a specific reaction occurs under the catalytic action of the enzyme, and then the protein adjacent to the target protein space is labeled. Proximity labeling uses genetically modified enzymes, such as peroxidase or biotin ligase, which can catalyze the conversion of inert substrates into highly reactive and short-lived active substances. The active substance diffuses from the active site of the enzyme to the surroundings and covalently labels nearby biomolecules (proteins, nucleic acids), and the range of the labeling depends on the half-life of the active substance and the concentration of the quencher. The covalently labeled biomolecules are enriched with streptavidin magnetic beads and identified by mass spectrometry or nucleic acid sequencing. Proximity labeling is widely used to identify protein interaction networks, and is also used to study the interaction between proteins and RNA, proteins and DNA in living cells. It is also suitable for constructing interaction networks of insoluble proteins, and can detect proteins that interact transiently or dynamically. In addition, it is also suitable for analyzing protein components localized in subcellular organelles, or is used to study interaction networks in living organisms. In the past decade, proximity labeling technology has developed rapidly. At present, it has developed to nanometer-level spatial resolution and minute-level temporal resolution, and has been used to construct molecular interaction maps in living organisms.

[0087] As used herein, the term "proximity labeling enzyme" or "proximity labeling tool enzyme" refers to a tool enzyme with proximity labeling function, including peroxidase (e.g., APEX, HRP) and biotin ligase (e.g., BioID, TurboID), etc., which biotin-labels proximity biomolecules at the living cell level. Since the highly reactive small molecules catalyzed by these enzymes have a very short life span, the proximity labeling technology has a high spatial specificity. Combined with mass spectrometry-based proteomics technology and high-throughput sequencing technology, people can achieve large-scale analysis of proximity biomolecules.

[0088] The tool enzymes with ligation activity used can be mainly divided into two types from the structural point of view: intact type and split type. The intact proximity marker enzyme is mainly used to study the potential interacting proteins of a single target protein, while the split proximity marker enzyme is used to study proteins associated with known protein complexes or interacting proteins. The proximity marker assay based on the intact proximity marker enzyme is to fuse biotin ligase or ascorbate peroxidase (APEX enzyme) with the target protein and express it in living cells. After adding substrates such as biotin or biotin-phenol and hydrogen peroxide (H2O2) to the culture medium, the protein or RNA near the target protein can be labeled with biotin. By lysing the cells and incubating with streptavidin magnetic beads, the biotin-labeled protein or RNA can be enriched for subsequent LC-MS / MS or high-throughput sequencing analysis. The split proximity marker enzyme system used to identify the composition of protein complexes is a proximity marker enzyme in which the N-terminal and C-terminal parts are fused to a pair of known interacting proteins, respectively. When the pair of proteins interact in cells, the two halves of the proximity tag enzyme are pulled close together and reassembled into the complete proximity tag enzyme, which tags proteins near the protein complex. By lysing the cells and incubating with streptavidin magnetic beads, the biotinylated proteins can be enriched for subsequent LC-MS / MS sequencing analysis.

[0089] There are many proximity labeling enzymes used for protein interaction identification, among which the commonly used proximity labeling tool enzymes are the mutant of Escherichia coli biotin ligase BirA (BioID) and ascorbate peroxidase (APEX). Based on this, many other proximity labeling enzymes have been optimized and developed, such as the optimized enzyme APEX2 of the APEX series; the optimized enzymes BioID2, AirID, BASU, etc. of the BioID series; and some small-scale tool enzymes such as HRP, EXCELL, PUP-IT, NEDDylation. The development of these tool enzymes has continuously expanded the application scope of proximity labeling technology.

[0090] As used herein, the term "fusion protein" has the common and customary meaning understood by those of ordinary skill in the art based on the reference books in the art and the specification of this application. In the present application, "fusion protein" is the expression product of two genes recombined by DNA recombination technology, and two different proteins can be connected into a large molecule by gene fusion. In addition to the nano antibodies listed in this application, the fusion protein in this application also includes optional tag sequences that assist in expression and / or purification (e.g., 6xHis tags, GGGS sequences, FLAG tags); or includes optional polypeptide molecules or fragments with therapeutic functions; or optional protein functional domains that assist in physicochemical or pharmaceutical (e.g., molecules that can prolong the half-life of nano antibodies in vivo, such as Fc fragments, HLE, ABD).

[0091] In the present application, the nanobody-proximity marker enzyme fusion protein is formed by coupling a nanobody (Nanobody, referred to as Nano) and a proximity marker enzyme. When the proximity marker enzyme is TurboID, it can be referred to as "Nano-ID (Ms / Rb)", "Nanobody-ID (Ms / Rb)" or "anti-rabbit / anti-mouse nanobody-TurboID". When the proximity marker enzyme is APEX2, it can be referred to as "Nano-APX (Ms / Rb)", "Nanobody-APX (Ms / Rb)" or "anti-rabbit / anti-mouse nanobody-APEX2". The fusion protein formed by coupling protein A and the proximity marker enzyme can be referred to as "ProteinA-TurboID", "Pro-ID", "ProteinA-APEX2", "Pro-APX". In some embodiments, "Nb2-ID", "Nanobody-light-controlled proximity marker enzyme" is also a type of nanobody-proximity marker enzyme fusion protein.

[0092] As used herein, the term "post-translational modifications" refers to the chemical modification process of proteins after protein translation. These modifications include acetylation, methylation, phosphorylation, ubiquitination, ADP ribosylation, etc., which can occur on the amino acid residues of histones, thereby changing the structure and function of chromatin, thereby regulating gene expression. "Post-modified histones" refer to histones that are modified after translation.

[0093] Detailed description of embodiments

[0094] In one aspect, the present disclosure provides a fusion protein for proximity labeling, wherein the fusion protein is operably linked together by (a) a nanobody and (b) a proximity labeling enzyme; and further comprises a peptide linker between the nanobody and the proximity labeling enzyme.

[0095] In some embodiments, a peptide linker is further included between the Nanobody and the proximity labeling enzyme.

[0096] In some embodiments, the proximity labeling enzyme comprises a peroxidase and / or a biotin ligase. In some embodiments, the peroxidase is horseradish peroxidase HRP or ascorbate peroxidase. In some embodiments, the ascorbate peroxidase is APEX or APEX2. In some embodiments, the biotin ligase is selected from any one of Mini TurboID, TurboID, AirID, BioID, BASU or BirA. In some embodiments, the biotin ligase is TurboID. In some embodiments, the bio-ligase is an engineered light-controlled TurboID. In some embodiments, the amino acid sequence of the light-controlled TurboID is shown in any one of SEQ ID NO.25-29.

[0097] In some embodiments, the nanobody is a primary antibody targeting a target protein or a secondary antibody targeting an immunoglobulin. In some embodiments, the immunoglobulin is a rabbit immunoglobulin, a human immunoglobulin, or a mouse immunoglobulin. In some embodiments, the immunoglobulin is selected from IgG, IgM, IgD, IgE, IgA, or IgY. In some embodiments, the nanobody is a secondary antibody targeting IgG, and the secondary antibody specifically binds to a primary antibody targeting a target protein. In some embodiments, the nanobody is a secondary antibody, and its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.11, or SEQ ID NO.18.

[0098] In some embodiments, the nanobody is a nanobody that recognizes GFP. In some embodiments, the amino acid sequence of the nanobody that recognizes GFP is shown in SEQ ID NO.46.

[0099] In some embodiments, the peptide linker is a flexible linker or a rigid linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the amino acid sequence of the flexible linker is shown in SEQ ID NO.4.

[0100] In some embodiments, the Nanobody and the proximity labeling enzyme are linked via a click chemistry reaction.

[0101] In some embodiments, the Nanobody and the proximity marker enzyme are linked and fused by any of the following means:

[0102] (a) the C-terminus of the Nanobody is linked to the N-terminus of the adjacent marker enzyme; or

[0103] (b) The N-terminus of the nanobody is linked to the C-terminus of the adjacent marker enzyme.

[0104] In some embodiments, the C-terminus of the Nanobody is fused to the N-terminus of a proximity marker enzyme via a linker.

[0105] In some embodiments, the amino acid sequence of the fusion protein is selected from:

[0106] (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47; or

[0107] (b) a polypeptide homologous to or having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity with any one of the amino acid sequences of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47, which can be used to label neighboring protein molecules that interact with the target protein; or

[0108] (c) A protein or polypeptide derived by inserting, substituting or deleting one or more amino acids in the amino acid sequence of (a) or (b), which can be used to label neighboring protein molecules that interact with the target protein.

[0109] In some embodiments, the fusion protein further comprises a protein tag, and the protein tag is selected from any one of GST, 6x-His, MBP, Flag, HA, cMyc, GFP, eGFP, eYFP, mCherry, AviTag or SUMO tags. In some preferred embodiments, the protein tags are 6xHis and Flag tags.

[0110] In some embodiments, the target protein is any intracellular or extracellular protein that can be recognized by a nanobody or an immunoglobulin.

[0111] In some embodiments, the fusion protein is anti-rabbit nanobody-TurboID, and its amino acid sequence is shown in SEQ ID NO.5. In some embodiments, the fusion protein is anti-rabbit nanobody-APEX2, and its amino acid sequence is shown in SEQ ID NO.6. In some embodiments, the fusion protein is anti-mouse nanobody-TurboID, and its amino acid sequence is shown in SEQ ID NO.12. In some embodiments, the fusion protein is anti-rabbit nanobody-APEX2, and its amino acid sequence is shown in SEQ ID NO.13. In some embodiments, the fusion protein is Protein A-TurboID, and its amino acid sequence is shown in SEQ ID NO.19. In some embodiments, the fusion protein is Protein A-APEX2, and its amino acid sequence is shown in SEQ ID NO.20. In some embodiments, the fusion protein is a nanobody-light-controlled proximity marker enzyme fusion protein, which can accurately start or shut down the enzyme activity of TurboID by controlling light, and realize spatial control of the biotinylation process, and the amino acid sequence is shown in any one of SEQ ID NO.30-39. In some embodiments, the fusion protein is Nb2-ID, and its amino acid sequence is shown as SRQ ID NO.47.

[0112] In another aspect, the present disclosure provides a nucleic acid molecule encoding the aforementioned fusion protein.

[0113] In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.

[0114] On the other hand, the present disclosure provides a kit, which comprises the aforementioned fusion protein: preferably, the kit further comprises a primary antibody targeting the target protein;

[0115] Preferably, the kit further comprises a biotinylation reaction solution;

[0116] Preferably, the kit further comprises fluorescent agent-conjugated streptavidin;

[0117] Preferably, the kit further comprises a secondary antibody coupled to a fluorescent agent;

[0118] Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP and biotin;

[0119] Preferably, the biotinylation reaction solution comprises biotin phenol and hydrogen peroxide.

[0120] On the other hand, the present disclosure provides a proximity labeling method, characterized in that the protein interacting with the target protein is biotin-labeled using the aforementioned fusion protein or the aforementioned kit, comprising the following steps:

[0121] Add a primary antibody of any species targeting the target protein to the cells for incubation to allow the primary antibody to bind to the target protein;

[0122] According to the species of the primary antibody, a fusion protein of the corresponding species is added to the cells, and the cells are incubated to form a proximity marker enzyme-nano secondary antibody-primary antibody-target protein complex in the cells;

[0123] adding a biotinylation reaction solution to the cells, incubating, and biotin-labeling the protein interacting with the target protein;

[0124] After washing, fluorescent agent-conjugated streptavidin and fluorescent agent-conjugated secondary antibody were added, incubated, washed, and detected.

[0125] In another aspect, the present disclosure provides a method for analyzing molecular interactions, the method comprising the following steps:

[0126] Using the aforementioned fusion protein or the aforementioned kit to biotin-label the protein that interacts with the target protein;

[0127] Use magnetic beads or fluorescent agents coupled with streptavidin to enrich or fluorescently locate biotin-labeled protein molecules;

[0128] The enriched biotin-labeled proteins were analyzed and identified by LC-MS / MS.

[0129] In another aspect, the present disclosure provides the use of the aforementioned fusion protein, the aforementioned kit, and the aforementioned method, wherein the use comprises:

[0130] (a) Biotinylation proximity labeling and component analysis of the cytoskeleton and organelles;

[0131] (b) Biotinylation proximity labeling and component analysis of post-modified histones;

[0132] (c) Biotinylation proximity labeling and component analysis of different nucleolar substructures;

[0133] (d) Biotinylation proximity labeling of proteins in FFPE and OCT sections and analysis of interacting proteins;

[0134] (e) Double biotinylation proximity labeling of the same sample;

[0135] (f) Biotinylation proximity labeling of proteins and analysis of interacting proteins in model organisms.

[0136] A further understanding of the present disclosure can be obtained by referring to some specific embodiments given herein, which are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and changes are also within the scope of the protection required by the present application.

[0137] Example 1: Preparation of Nanobody-Proximity Marker Enzyme Fusion Protein

[0138] The nanobodies used in the present disclosure are all specific secondary antibodies. However, the technical solution of the present disclosure is also applicable to primary nanobodies. The working principle diagram of the nanobody-proximity marker enzyme fusion protein is shown in FIG. Figure 1 shown.

[0139] 1. Anti-rabbit nanobody-TurboID / APEX2 fusion protein

[0140] The amino acid sequence of the anti-rabbit nanobody is shown in SEQ ID NO.1, the amino acid sequence of TurboID is shown in SEQ ID NO.2, and the amino acid sequence of APEX2 is shown in SEQ ID NO.3. The C-terminus of the nanobody is fused to the N-terminus of the adjacent marker enzyme through a linker, and the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequence of the fused anti-rabbit nanobody-TurboID is shown in SEQ ID NO.5, and the N-terminus of the fusion protein is connected to the 3xFlag tag, and the C-terminus is connected to the 6xHis tag. The amino acid sequence of the anti-rabbit nanobody-APEX2 is shown in SEQ ID NO.6, and the N-terminus of the fusion protein is connected to the 6xHis tag and the 3xFlag tag.

[0141] Table 1. Anti-rabbit nanobody-TurboID / APEX2 fusion protein coding information

[0142]

[0143] Select an expression vector suitable for the E. coli protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, and construct an anti-rabbit nanobody-TurboID fusion protein plasmid as shown in SEQ ID NO.9 and an anti-rabbit nanobody-APEX2 fusion protein plasmid as shown in SEQ ID NO.10. The specific process is as follows:

[0144] The vector is constructed by homologous recombination. The main connection method of homologous recombination is to use the overlapping region of the target fragment and the vector to recombine the two into a new plasmid.

[0145] The synthesized anti-rabbit nanobody-TurboID fusion protein gene and anti-rabbit nanobody-APEX2 fusion protein gene were constructed into an expression vector by homologous recombination.

[0146] (1) PCR of target fragment and vector

[0147] The PCR primers used are shown in Table 2 below:

[0148] Table 2. PCR primers

[0149]

[0150] The PCR system and procedure used are shown in Table 3 below:

[0151] Table 3. PCR reaction system

[0152] Reaction components volume Template (10 ng / μL) 1μL Upstream primer (10 μM) 2.5μL Downstream primer (10 μM) 2.5μL PCR High-Fidelity Enzyme Mix 25μL water Make up to 50 μL

[0153] The PCR program settings are shown in Table 4 below:

[0154] Table 4. PCR program settings

[0155]

[0156] The PCR products were subjected to agarose gel electrophoresis and the bands of the correct size were recovered for later use.

[0157] (2) The system and reaction conditions of homologous recombination are shown in Table 5 below.

[0158] Table 5. Reaction components and reaction conditions

[0159]

[0160]

[0161] The above ligation products were transformed into clonal competent cells, spread onto LB plates containing resistance, and single clones were picked for sequencing the next day. Fusion protein plasmids were extracted from the strains with correct sequencing, transformed into expression competent BL21 (DE3), spread onto LB plates containing resistance, and single clones were picked for protein expression the next day.

[0162] 2. Anti-mouse nanobody-TurboID / APEX2 fusion protein

[0163] The amino acid sequence of the anti-mouse nanobody is shown in SEQ ID NO.11, the amino acid sequence of TurboID is shown in SEQ ID NO.2, and the amino acid sequence of APEX2 is shown in SEQ ID NO.3. The C-terminus of the nanobody is fused to the N-terminus of the adjacent marker enzyme through a linker, and the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequence of the fused anti-mouse nanobody-TurboID is shown in SEQ ID NO.12, and the N-terminus of the fusion protein is connected to the 3xFlag tag, and the C-terminus is connected to the 6xHis tag. The amino acid sequence of the anti-mouse nanobody-APEX2 is shown in SEQ ID NO.13, and the N-terminus of the fusion protein is connected to the 6xHis tag and the 3xFlag tag.

[0164] Table 6. Anti-mouse nanobody-TurboID / APEX2 fusion protein encoding information

[0165]

[0166] Select an expression vector suitable for the E. coli protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, and construct an anti-mouse nanobody-TurboID fusion protein plasmid as shown in SEQ ID NO.16, and an anti-mouse nanobody-APEX2 fusion protein plasmid as shown in SEQ ID NO.17. The specific process is as follows:

[0167] For PCR primer sequences, procedures, connection methods, screening, etc., please refer to the relevant content of anti-rabbit nanobody.

[0168] 3. Protein A-TurboID / APEX2 fusion protein

[0169] The amino acid sequence of Protein A is shown in SEQ ID NO.18, the amino acid sequence of TurboID is shown in SEQ ID NO.2, and the amino acid sequence of APEX2 is shown in SEQ ID NO.3. The C-terminus of Protein A is fused to the N-terminus of the adjacent marker enzyme via a linker, and the amino acid sequence of the linker is shown in SEQ ID NO.4. The amino acid sequence of the fused Protein A-TurboID is shown in SEQ ID NO.19, and the amino acid sequence of Protein A-APEX2 is shown in SEQ ID NO.20. The N-terminus of the fusion protein is connected to a 6xHis tag and a 3xFlag tag.

[0170] Table 7. Anti-mouse nanobody-TurboID / APEX2 fusion protein encoding information

[0171]

[0172] An expression vector suitable for the E. coli protein expression system is selected, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, to construct a Protein A-TurboID fusion protein plasmid as shown in SEQ ID NO.23 or a Protein A-APEX2 fusion protein plasmid as shown in SEQ ID NO.24, and the specific process is as follows:

[0173] The synthesized proteinA-TurboID fusion protein gene and proteinA-APEX2 fusion protein gene were constructed into the expression vector by homologous recombination.

[0174] The PCR primer sequences used are shown in Table 8 below:

[0175] Table 8. PCR primers

[0176]

[0177]

[0178] For PCR procedures, connection methods, screening, etc., please refer to the relevant content of anti-rabbit nanoantibodies.

[0179] 4. Anti-rabbit nanobody-light-controlled proximity marker enzyme fusion protein

[0180] The photo-controlled proximity marker enzyme uses genetic codon expansion technology to introduce photo-controlled non-natural amino acids into the active pocket of TurboID, thereby inactivating TurboID; the photo-controlled non-natural amino acids can be converted into corresponding natural amino acids under UV irradiation conditions, thereby releasing the enzyme activity of TurboID. Specifically, the photo-controlled proximity marker enzyme replaces lysine or tyrosine at a specific site with corresponding non-natural amino acids such as lysine or tyrosine derivatives, and the site-specific insertion of non-natural amino acids can eliminate the enzyme activity of the proximity biotin ligase; under ultraviolet irradiation with a wavelength of 365, non-natural amino acids such as lysine or tyrosine derivatives at specific sites in the photo-controlled proximity marker enzyme are converted into lysine or tyrosine, thereby restoring the activity of the proximity biotin ligase, thereby specifically biotinylating the proximity protein.

[0181] Therefore, the fusion protein of the nano secondary antibody and the light-controlled proximity labeling enzyme (the photoswitch mutant of TurboID) can precisely activate the enzyme activity of TurboID in a specific area by controlling the illumination area, thereby achieving spatial control of the biotinylation process. The introduction of this photoswitch improves the functional flexibility and controllability of TurboID, enabling efficient biotin labeling at a specific spatial location. The working principle of the fusion protein of the nano secondary antibody and the photoswitch mutant of TurboID is shown in the figure. Figure 2 shown.

[0182] The light-controlled proximity marker enzyme TurboID is a mutant of the biotin ligase BirA derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.65, and its amino acid sequence is shown in SEQ ID NO.66. In the amino acid sequence of the light-controlled proximity marker enzyme, one or more sites of lysine at position 183, tyrosine at position 132, and lysine at position 172 are mutated to non-natural amino acids MNPY-lysine (MNPYK), non-natural amino acids ONB-lysine (ONBK) or non-natural amino acids ONB-tyrosine (ONBK). The light-controlled proximity marker enzymes TurboID-183-MNPYK, TurboID-183-ONBK, TurboID-132-ONBY, TurboID-172-MNPYK, and TurboID-172-ONBK are obtained respectively, and their corresponding amino acid sequences are shown in Table 9. The construction method of the light-controlled proximity marker enzyme, the specific tRNA and tRNA synthetase used refer to patent CN113481173B.

[0183] The construction method of anti-rabbit nanobody-light-controlled proximity marker enzyme fusion protein is shown in Example 2. The nanobody is fused to the light-controlled proximity marker enzyme via a linker, the amino acid sequence of the linker is shown in SEQ ID NO.4, and the amino acid sequence of the fusion protein is shown in Table 9 below.

[0184] Similarly, an anti-mouse nanobody-light-controlled proximity marker enzyme fusion protein can be constructed. Alternatively, a Nb2-ID light-controlled proximity marker enzyme fusion protein suitable for recognizing GFP can be constructed.

[0185] The light-controlled biotin ligase can be activated by irradiating ultraviolet light at a wavelength of 365 nm for 1 min.

[0186] Table 9. Amino acid sequences corresponding to anti-rabbit nanobody-light-controlled proximity marker enzyme fusion proteins

[0187]

[0188] Example 2: Inducible expression and purification of nanobody-proximity marker enzyme fusion protein

[0189] 2.1 Inducible expression of nanobody-proximity marker enzyme fusion protein in prokaryotic system

[0190] The prokaryotic expression recombinant plasmid with the target protein was transformed into the expression competent BL21 (DE3). The next day, a single clone was picked and inoculated into 2 mL LB medium. After shaking, the seed was preserved and expanded into 1 L LB medium. The culture was cultured in a shaker at 37 ° C and 180 rpm. When the OD600 of the bacterial solution reached 0.8-1.0, the bacterial solution was cooled to 16 ° C. For the expression of TurboID fusion protein, IPTG (YEASEN, Cat#10902ES08) was directly added to induce the final concentration of 300 mM; for the expression of APEX2 fusion protein, IPTG with a final concentration of 300 mM and 5-Aminolevulinic acid hydrochloride (Sigma, Cat#1.24802) with a final concentration of 1 mM were added. After 16 hours of induction expression, the bacteria were collected by centrifugation at a speed of 4000 rpm and a time of 20 min.

[0191] 2.2 Expression of Nanobody-Light-Controlled Proximity Marker Enzyme Fusion Protein in Eukaryotic Systems

[0192] (1) According to 1.4x10 6 HEK293-F cells were inoculated into 200 ml of medium in a 1 L shake flask at an inoculum volume of 10 / mL and cultured in a shaker incubator at 37°C, 120 rpm, and 8% CO2. The next day, when the cell concentration reached 2x10 6 pcs / mL, and transfected the plasmid using PEI (Polysciences, Cat#24765-100) as follows:

[0193] (2) Place 600 μL of PEI in a metal bath at 55°C and incubate for 20 min;

[0194] In 10 mL of Opti-MEM, add 100 μg of the plasmid expressing the nanobody-proximity marker enzyme fusion protein and 100 μg of the plasmid expressing tRNA and tRNA synthetase, and shake to mix;

[0195] Add 600 μL PEI to 10 mL Opti-MEM (Gibco, Cat#31985070) and shake to mix;

[0196] Combine the two tubes of Opti-MEM, shake and mix, and incubate at 37°C for 20 min.

[0197] (3) Under light-protected conditions, the above Opti-MEM system was added dropwise to the prepared HEK293-F cells; then, 200 μL of 500 mM light-controlled non-natural amino acids (synthesized by Beijing Okainas Technology Co., Ltd.) were added to a final concentration of 500 μL.

[0198] (4) The cells were wrapped in aluminum foil to avoid light and cultured in a shaking incubator at 37°C, 120 rpm, and 8% CO2 for 72 hours. After the expression was completed, the cells were collected by centrifugation at 2500 rpm for 10 minutes.

[0199] 2.3 Fusion protein purification

[0200] (1) Suspension of bacteria: Add 30 mL of PBS to suspend the bacterial solution, to which protease inhibitors (Target MOI, Cat#C0001) and PMSF (Beyotime, Cat#ST506) are added.

[0201] (2) Ultrasonic disruption: 40% power, on for 3 seconds, off for 20 seconds, and ultrasonic for 30 minutes.

[0202] (3) Centrifuge to obtain the protein supernatant: Centrifuge at 14,000 rpm, 4°C for 1 h and obtain the supernatant.

[0203] (4) Affinity chromatography: Add 1 mL of nickel column filler (Qiagen, Cat#30210) to the affinity chromatography column and equilibrate the column with 10 mL of PBS before use. Pour the collected protein supernatant into the column and let it flow out by gravity (repeat this operation twice).

[0204] (5) Elution of impurities: Add 10 mL of washing solution (PBS, 30 mM imidazole) to the nickel column and allow the column to flow out by gravity.

[0205] (6) Elution of target protein: Add 10 mL of elution solution (PBS, 300 mM imidazole) to the nickel column, allow gravity to flow out, and collect the eluate.

[0206] (7) Concentration: Use a 50 mL concentrator tube with a molecular weight cutoff of 10 kD and centrifuge at 3500 rpm and 4°C until the eluate is concentrated to 500 μL.

[0207] (8) Dialysis: To remove McAc from the protein solution, add 10 mL of PBS to 500 μL of the concentrate to make it concentrated to 500 μL. Repeat this process once to obtain 500 μL of protein.

[0208] Figure 3 The SDS-PAGE image of the purified anti-rabbit and anti-mouse nanobody-TurboID fusion proteins is shown.

[0209] Example 3: Preparation of biotinylated cell samples

[0210] (1) Prepare cells: One day in advance, plate the required amount of cells (e.g., human osteosarcoma U-2OS cells) in a 6 cm cell culture dish. The next day, fix the cells. Different fixation methods can be selected according to different antibodies: 4% PFA / -20°C methanol / -20°C ethanol.

[0211] (2) Blocking and permeabilization: Use PBST + 5% BSA solution to block and permeabilize the cells for 1 hour (methanol and ethanol fixation does not require PBST permeabilization, just replace it with PBS).

[0212] (3) Incubation with primary antibody: dilute the primary antibody with PBS + 5% BSA solution at a ratio of 1:200, add to the cells, and incubate at room temperature for 2 hours or at 4°C overnight. Wash three times with PBS.

[0213] (4) Incubation of nanobody-proximity marker enzyme fusion protein: Dilute the fusion protein with PBS solution at a ratio of 1:200, add it to the cells, and incubate at room temperature for 1 hour. Wash with PBS three times.

[0214] (5) Biotinylation reaction: Add biotinylation reaction solution (1 mL PBS + 20 mM MgCl2 + 100 mM ATP + 500 μM biotin) to the cells. Incubate at 37°C for 16 h. After the reaction, stain to detect the biotinylation effect, or lyse the cells for preparation of mass spectrometry samples.

[0215] (6) Cell staining: After the biotinylation reaction, wash the cells three times with PBS and add AlexaFluor at a ratio of 1:1000. TM 488 or 560 conjugated streptavidin, and Alexa Fluor TM The secondary antibody conjugated with 560 or 488 was incubated at room temperature for 1 h, washed three times with PBS, and the biotinylation reaction effect was detected under a fluorescence microscope.

[0216] (6) Cell lysis: After the biotinylation reaction, wash three times with PBS, add 500 μL of lysis buffer 1 (300 mM Tris-HCl, 2% SDS, 0.2 M glycine, pH 9.0), scrape the cells into a low adsorption tube, and heat the lysis sample in a metal bath: 98°C for 20 min, 80°C for 2 h. After the lysis is completed, centrifuge at 14,000 rpm, 4°C for 20 min, and take the supernatant.

[0217] Example 4: Biotinylated protein enrichment and mass spectrometry sample preparation

[0218] (1) Add an equal volume of water to the sample to dilute it by half. Dilute Lysis Buffer 1 by half with water to make Lysis Buffer 2.

[0219] (2) Take 50 μL of streptavidin C1 magnetic beads (Invitrogen, Cat#65002), add 200 μL of lysis buffer 2, gently pipette and fully resuspend, place on a magnetic stand for separation for 10 seconds, remove the supernatant, and repeat this step three times. Add the sample from step 1 to the magnetic beads and incubate at 4°C with shaking for 1 hour.

[0220] (3) After the incubation, place the beads on a magnetic rack and discard the supernatant; add 200 μL of lysis buffer 2 to wash the beads, repeat three times. Then, wash once with 200 μL of buffer A (1M KCL), buffer B (0.1M Na2CO3), buffer C (50mM Tris-HCl, 2M urea, pH=7.5), and once with 200 μL of 100mM Tris-HCl (pH=8.0).

[0221] (4) Add 40 μL of 100 mM Tris-HCl (pH 8.0) to the magnetic beads, add DTT to a final concentration of 1 mM and 0.4 μL of trypsin, and incubate at 25°C with shaking for 1 h.

[0222] (5) After the incubation, place the beads on a magnetic rack, transfer the supernatant to a new tube, and wash the beads twice with 30 μL of 100 mM Tris (pH 8.0). Combine the wash solutions and the volume of the eluate is now 100 μL.

[0223] (6) Add dithiothreitol to a final concentration of 4 mM and incubate at 25°C with shaking for 30 min.

[0224] (7) Add iodoacetamide to a final concentration of 10 mM and incubate at 25°C with shaking for 45 min.

[0225] (8) Add 0.4 μg of trypsin and incubate at 25°C with shaking overnight.

[0226] (9) After overnight digestion, the pH of the sample was adjusted to less than 3 using 10% trifluoroacetic acid for desalting.

[0227] Example 5: Biotinylated peptide enrichment and mass spectrometry sample preparation

[0228] (1) Reductive alkylation: Add 30 μL of 200 mM TCEP to the cell lysate and incubate at 55°C for 1 h.

[0229] Add 30 μL of 375 mM iodoacetamide and incubate at 25°C in the dark for 30 min.

[0230] (2) Methanol-chloroform protein precipitation: Add 500 μL methanol and 125 μL chloroform to the sample after reduction and alkylation, vortex and oscillate for 30 seconds to mix the sample thoroughly. Centrifuge at 14000 rpm for 10 minutes at room temperature, and discard the supernatant. Add 500 μL methanol, vortex and oscillate for 30 seconds to mix the sample thoroughly. Centrifuge at 14000 rpm for 10 minutes at room temperature, discard the supernatant, and dry the sample.

[0231] (3) Trypsin digestion: Add 100 μL PTS buffer (100 mM Tris-HCl, pH 8.0, 12 mM SDC, 12 mM SLS) to the dried protein precipitate and heat in a 90°C metal bath for 10 min. Dissolve 20 μg of trypsin in 400 μL 100 mM Tris-HCl (pH 8.0), add to the above sample, and incubate at 25°C with shaking overnight.

[0232] (4) Protein G beads coupled with antibodies: Add 100 μL 300 mM NaAc (pH=3.0) to 50 μL protein G beads (NEB, Cat#S1430S) and wash twice. Add 10 μL anti-biotin antibody and 400 μL 300 mM NaAc, incubate at 4°C with shaking for 2 hours. Wash twice with 200 μL 300 mM NaAc.

[0233] (5) Biotinylated peptide enrichment: Heat the peptides digested overnight in a 90°C metal bath for 10 min to inactivate the trypsin. Add 15 μL of NaAc and adjust the sample pH to about 6.5. Add the acidified peptides to the protein G beads coupled to the antibody and incubate at 4°C with shaking overnight.

[0234] (6) Elution: After discarding the supernatant of the above sample, add 200 μL H2O, wash 3 times, discard the supernatant each time. Add 25 μL 0.2% TFA, wash 2 times, 10 min each time, take the supernatant, and combine the eluate.

[0235] (7) Desalination

[0236] Example 6: Labeling radius of nanobody-proximity marker enzyme fusion protein in mouse skeleton and different organelles

[0237] The experimental procedure for in vitro biotinylation is shown in Figure 4 b, cells were first fixed, blocked, and permeabilized; then the primary antibody targeting the target area was added; Protein A-TurboID / APEX2 or anti-rabbit nanobody-TurboID / APEX2 or anti-mouse nanobody-TurboID / APEX2 was added according to the species of the primary antibody; U-2OS cells were cultured according to Figure 4b, first incubated with anti-β-actin primary antibody (marking actin filaments) (c, f, i, l), anti-vimentin primary antibody (marking intermediate filaments) (d, g, j, m), or anti-TubA4A primary antibody (marking tubulin) (e, h, k, n); then added anti-mouse nanobody-TurboID (ce), anti-mouse nanobody-APEX2 (fh), or Protein A-TurboID (ik) / APEX2 (ln); finally added biotinylation reaction solution for reaction. The specific experimental process refers to Example 3.

[0238] Test results are shown in Figure 4 c-4n, where the green color is labeled with Alexa Fluor TM 488-conjugated secondary antibody shows the localization of the primary antibody; the red marker is Alexa Fluor TM 560 conjugated streptavidin, showing the distribution of biotinylated proteins.

[0239] Select Figure 4 The dotted areas in c, f, i, and l are measured to characterize the distribution of fluorescence signals, and the fluorescence signal index is fitted. The results are shown in Figure 5 .

[0240] Similarly, the labeling radius of Nano-ID, Nano-APX, Pro-ID and Pro-APX was compared using mouse antibodies against different organelles in U-2OS cells. For the specific experimental process, refer to Example 3. First, incubate with anti-LAMP1 primary antibody (lysosome marker) (a, d, g, j), anti-LaminB1 primary antibody (nuclear membrane marker) (b, e, h, k), or anti-ATP5A1 primary antibody (mitochondrial marker) (c, f, i, l); then add anti-mouse nano antibody-TurboID (ac), anti-mouse nano antibody-APEX2 (df), or Protein A-TurboID (gi) / APEX2 (jl); finally, add biotinylated in vitro reaction solution for reaction. See the test results. Figure 6 .

[0241] Depend on Figure 4-Figure 6 It can be seen that when using mouse primary antibodies, Nano-ID can effectively mark the target area and form a mark with a high signal-to-noise ratio. Although Pro-ID, Pro-APX or Nano-APX can also mark the target area, the marking radius is too large and cannot be effectively marked.

[0242] Example 7: Labeling radius of nanobody-proximity marker enzyme fusion protein in rabbit skeleton and different organelles

[0243] U2OS cells were first incubated with anti-Paxillin primary antibody (focal adhesion marker) (ad), anti-PEX14 primary antibody (peroxisome marker) (eh), or anti-EDC4 primary antibody (P body marker) (il) according to the process described in Example 3; then anti-rabbit nanobody-TurboID (a, e, i), anti-rabbit nanobody-APEX2 (b, f, j), or ProteinA-TurboID (c, g, k) / APEX2 (d, h, l) were added; finally, biotinylated in vitro reaction solution was added for reaction.

[0244] Test results are shown in Figure 7 , where the green one is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa Fluor TM 560 conjugated secondary antibody to show the localization of the primary antibody. Figure 7 It can be seen that when using rabbit primary antibodies, Nano-ID and Pro--ID can effectively mark the target area and form a high signal-to-noise ratio mark. The Pro--APX or Nano-APX marking radius is too large and cannot effectively mark the target area.

[0245] Example 8: Nano-ID is used to label more types of organelles or histone post-modifications

[0246] U-2OS cells were first incubated with anti-GOLG2 primary antibody (mouse antibody, Golgi apparatus marker) (a), anti-CD98 primary antibody (rabbit antibody, cell membrane marker) (b), anti-SC35 primary antibody (mouse antibody, nuclear plaque marker) (c), anti-PDI primary antibody (rabbit antibody, endoplasmic reticulum marker) (d), anti-CEP250 primary antibody (rabbit antibody, centriole marker), anti-FUS primary antibody (mouse antibody, f), anti-H3K4me3 (mouse antibody, g), anti-H3K27Ac (rabbit antibody, h), anti-HAK119ub (rabbit antibody, i), and anti-L-Lactyl Lysine (j) according to the process described in Example 3; then anti-rabbit (b, d, e, h, i, j) or anti-mouse (a, c, f, g) nanoantibody-TurboID was added; finally, biotinylated in vitro reaction solution was added for reaction.

[0247] Test results are shown in Figure 8 Among them, the green mark is Alexa Fluor TM 488-coupled streptavidin shows the distribution of biotinylated proteins; the red marker is Alexa Fluor TM560 conjugated secondary antibody to show the localization of the primary antibody. Figure 8 It can be seen that the Nano-ID prepared in the present disclosure can identify more types of rabbit-derived or mouse-derived organelles or histone post-modifications, and compared with the Pro-ID in the prior art, it expands the range of antibodies that can be effectively identified.

[0248] Example 9: Hierarchical analysis of nucleoli by Nano-ID

[0249] The nucleolus can be divided into three layers from the inside to the outside: the Fibrillar Center (FC, which is highly related to the transcription of POLI), the Dense Fibrillar Component (DFC, which is the main area for ribosomal RNA shearing), and the Granular Component (GC, which is used for ribosome biosynthesis). For a schematic diagram, see Fig. 9 a. U-2OS cells were incubated with anti-TCOF1 (rabbit antibody, labeled FC region), anti-FBL (rabbit antibody, labeled DFC region), and anti-NPM1 (mouse antibody, labeled GC region) according to the process described in Example 3, and then anti-rabbit or anti-mouse nanoantibody-TurboID was added, and the biotinylation reaction solution was added for reaction. The biotinylation reaction was then detected and Alexa Fluor was added to the cells. TM 488-coupled streptavidin visualizes biotinylated protein localization, AlexaFluor TM 560 coupled secondary antibody to show the localization of the primary antibody, the detection results are as follows Fig. 9 As shown in b-9d, the layered structure of the nucleolus can be clearly distinguished, and the positioning accuracy of Nano-ID is high. Subsequently, U-2OS cells were treated in the same way, and the cells were lysed after the biotinylation reaction was completed, and the content of biotinylated proteins was detected by immunoblotting. Fig. 9 As shown in e, compared with the control group, the biotinylated protein in the experimental group was significantly enriched. The total protein of the experimental group and the control group was enriched by coupling streptavidin and then detected by mass spectrometry. Fig. 9 f shows the signal heat map of TCOF1, FBL and NPM1. Fig. 9 g is the heat map of mass spectrometry signals of nucleolar proteins in different layers. Fig. 9 h-9j selected representative protein interaction networks enriched in FC structure (9h), DFC structure (9i) and GC structure (9j), which are highly related to the functions of the nucleolus layered regions. Based on the above in situ biotin labeling of the three substructures of the nucleolus, Nano-ID achieved the hierarchical analysis of the nucleolus structure.

[0250] Example 10: Nano-ID is applicable to various types of sections such as FFPE and OCT

[0251] FFPE sections of lung adenocarcinoma tissue were selected for in vitro biotinylation labeling. Paraffin-embedded lung adenocarcinoma sections were dewaxed, rehydrated, and then added with PECAM1 antibody and Nano-ID for in vitro biotinylation reaction. TM 560 conjugated antibody labeled with PECAM1, Alexa Fluor TM 488 coupled to streptavidin to label biotinylated proteins for immunofluorescence detection, and images were collected by PANNORAMIC 1000 scanner. The results of immunofluorescence detection are shown in Fig.10 a, The results of the detection of biotinylated protein content of total protein in the experimental group (+, with PECAM1 primary antibody) and the control group (-, without PECAM1 primary antibody) are shown in Fig.10 b. In the FFPE samples from three patients, the total proteins in the experimental and control groups were enriched by streptavidin coupling. The mass spectrometry signal intensity of PECAM1 is shown in Figure 2. Fig.10 c, ND, not detected. A Venn diagram was constructed for the overlap of PECAM1 protein interactions in the three patients. Fig.10 d, the interacting proteins of PECAM1 detected in all three samples were input into Stringdb for analysis, see Fig.10 e, Different colors represent different signaling pathways in which proteins participate.

[0252] Similarly, mouse brain OCT slices were biotinylated in vitro. After fixation and permeabilization, GFAP antibody and Nano-ID were added to the slices for in vitro biotinylation reaction. After the reaction, the samples were incubated with Alexa Fluor TM 560 conjugated secondary antibody labeled with GFAP, Alexa Fluor TM 488 coupled with streptavidin to label biotinylated protein, immunofluorescence detection results are shown in Fig.11 a. Fig.11 The schematic diagram of e is Fig.11 a The biotin in the in vitro biotinylation reaction solution of the sample is replaced with D4 isotope labeling. After the reaction is completed, the protein is precipitated and digested. After the digested peptides are enriched with anti-biotin antibodies, the biotinylation sites and biotin label types are identified by mass spectrometry, that is, whether it is wild-type biotin (endogenous biotin protein) or D4 isotope biotin labeling (D4 isotope biotin label added by Nano-ID), so as to distinguish endogenous biotinylated proteins from target biotinylated proteins. The test results of the total protein biotinylated protein content in the experimental group (+, with GFAP primary antibody) and the control group (-, without GFAP primary antibody) are shown in Fig.11b, Venn diagram of 689 endogenous wild-type biotin proteins and 795 D4-labeled proteins identified after enrichment. Fig.11 c. GO analysis was further performed on the identified proteins, and the results are shown in 11d.

[0253] In HTT disease model mice, HTT neighboring proteins were biotinylated. Representative immunofluorescence images are shown in Fig.12 a. The biotinylated protein content of total protein in the experimental group (+, with HTT primary antibody) and the control group (-, without HTT primary antibody) is shown in 12b.

[0254] In summary, Nano-ID is compatible with FFPE sections. If the endothelial marker PECAM1 antibody is used, PECAM1 and its interacting proteins in FFPE sections can be identified. In addition, Nano-ID is also compatible with OCT sections. If the astrocyte intermediate filament skeleton protein marker GFAP antibody is used, GFAP and its interacting proteins can be identified. In addition to being part of the cell intermediate filament skeleton, these interacting proteins are also involved in the maintenance of synapses, axons and dendrites.

[0255] In addition, Nano-ID is also suitable for the localization of immune-related proteins and post-modification of histones in FFPE sections and OCT sections. In vitro biotinylation labeling on FFPE sections of lung adenocarcinoma tissue. Paraffin-embedded lung adenocarcinoma sections were dewaxed, rehydrated, and subjected to in vitro biotinylation reaction after adding PDL1 antibody (a) or H3K27Ac antibody (b) and Nano-ID. Alexa560-conjugated antibody labeled PDL1 (a) or H3K27Ac (b), Alexa Fluor TM 488-coupled streptavidin labeled biotinylated protein. The image was collected by PANNORAMIC 1000 scanner, and the fluorescence localization results are shown in Fig.13 a and 13b.

[0256] Biotinylated transcription factor PAX6 neighboring protein on mouse brain OCT slices at 12.5 days of development. After fixation and permeabilization, PAX6 antibody and Nano-ID were added to the OCT slices for in vitro biotinylation reaction. After the reaction, the samples were incubated with Alexa Fluor TM 560 conjugated antibody labeled with PAX6, Alexa Fluor TM 488 coupled streptavidin labeled biotinylated protein, fluorescence localization results are shown in Fig.13 c. In the mouse 8-cell embryo, the fluorescence results of the apical domain labeled by Nano-ID are shown in Fig.13 d.

[0257] Example 11: Nano-ID can achieve dual labeling of a sample

[0258] The schematic diagram of the double labeling experimental process is shown in Fig.14 a. ROI1 and ROI2 in the cell are labeled with rabbit and mouse antibodies, respectively. For example, in the first round of reaction, Nano-ID (Rb) recognizes the rabbit primary antibody and labels the rabbit primary antibody labeled ROI1 with wild-type biotin; after the first round of reaction, TEV enzyme is added for cleavage, Nano-ID-(Rb) is cleaved, and the free TurboID will be washed; in the second round of reaction, Nano-ID (Ms) is added to recognize the mouse primary antibody, and then the biotin in the reaction solution is labeled with D4, and the second round of reaction is carried out to label the mouse-labeled ROI2 with D4. The cells are lysed, and the obtained protein precipitate is digested into peptides, which are then biotinylated and enriched at the peptide level, and finally the wild-type and D4-labeled peptides are obtained, thereby distinguishing the proteins labeled in the first and second rounds.

[0259] As described above, the first round of mitochondrial labeling was performed with rabbit primary antibody anti-TFAM wild-type biotin, and the second round of nucleoli was labeled with mouse primary antibody anti-NPM1 isotope D4 biotin. TM 488 and Alexa Fluor TM 560 coupled streptavidin for observation, the test results are shown in Fig.14 b. The immunoblotting results of single TFAM, NPM1 labeling and double TFAM and NPM1 labeling are shown in Fig.14 c.

[0260] In summary, the Nano-ID disclosed in the present invention can achieve double labeling of the same sample.

[0261] Example 12: Nano-ID can be used to label cell lines or model organisms expressing GFP fusion proteins

[0262] In order to better adapt to GFP-overexpressing or GFP-gene knock-in cell lines and model animals such as nematodes and fruit flies, we replaced the nanoantibodies that recognize rabbit or mouse IgG in Nano-ID with nanoantibodies that recognize GFP, and obtained Nb2-ID. Nb2 is a nanoantibody that specifically recognizes GFP, and its amino acid sequence is shown in SEQ ID NO.46. The amino acid sequence of Nb2-ID is shown in SEQ ID NO.47, and the nucleotide sequence of Nb2-ID is shown in SEQ ID NO.48.

[0263] Fig.15ac show the in vitro biotin labeling results of cell lines overexpressing mEmerald-SRSF3 (a) or mEmerald-ensconsin (c) or GFP knocked in at the SC35 locus using CRISPR (b) after incubation with Nb2-ID, using Alexa Fluor TM Biotinylated proteins were detected by 560-conjugated streptavidin. Fig.12 d is a nematode stably expressing ajm-1-GFP with a developmental stage of 1.8 fold. After incubation with Nb2-ID, an in vitro biotinylation reaction was performed. The above samples were stained with Alexa Fluor TM Biotinylated proteins were detected by 560-conjugated streptavidin.

[0264] In summary, Nano-ID can also be used to label cell lines or model organisms that express GFP.

[0265] Example 13: Verification of the biotinylation labeling ability of the nanobody-light-controlled proximity labeling enzyme fusion protein

[0266] In this example, the biotinylation labeling ability of the nanobody-light-controlled proximity labeling enzyme fusion protein was further verified.

[0267] After fixing, permeabilizing and blocking, U-2OS cells were incubated with rabbit anti-PEX14 antibody (a) or mouse anti-Tubulin antibody (b), and the corresponding species of nanoantibodies-photosensitive proximity marker enzymes were added. The experimental group and the control group were treated with or without illumination, and then biotinylation reaction was performed. After the reaction, immunofluorescence staining and imaging were performed.

[0268] Results Fig.15 In the control group without light treatment, the biotin signal is almost invisible, which means that the fusion protein is in a state of inhibited enzyme activity. TM 647-conjugated secondary antibody signal and Alexa Fluor TM The colocalization of the biotinylated protein signal labeled with 488-coupled streptavidin demonstrated the localization accuracy of the fusion protein and the recovery of enzyme activity after illumination.

[0269] The technical solution disclosed in the present invention can reveal the protein interaction networks and modification patterns in cells and tissues in a more detailed and comprehensive manner, which will help to deeply understand the regulatory mechanisms of biological processes and provide more precise targets and strategies for drug development and disease treatment; at the same time, this technology also has broad application prospects and can play an important role in the field of spatial proteomics.

[0270] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A fusion protein for proximity labeling, wherein: The fusion protein is operably linked by (a) a nanobody and (b) a proximity labeling enzyme; a peptide linker is also included between the nanobody and the proximity labeling enzyme.

2. The fusion protein according to claim 1, wherein The proximity labeling enzyme comprises peroxidase and / or biotin ligase; Preferably, the peroxidase is horseradish peroxidase HRP or ascorbate peroxidase; Preferably, the ascorbate peroxidase is APEX or APEX2; Preferably, the biotin ligase is selected from any one of Mini TurboID, TurboID, AirID, BioID, BASU or BirA; Preferably, the biotin ligase is TurboID; Preferably, the biological ligase is an engineered light-controlled TurboID; Preferably, the amino acid sequence of the light-controlled TurboID is as shown in any one of SEQ ID NOs. 25-29.

3. The fusion protein according to claim 1 or 2, wherein The nanobody is a primary antibody targeting a target protein or a secondary antibody targeting an immunoglobulin; Preferably, the immunoglobulin is rabbit immunoglobulin or mouse immunoglobulin; Preferably, the immunoglobulin is selected from IgG, IgM, IgD, IgE, IgA or IgY; Preferably, the nanobody is a secondary antibody targeting IgG, and the secondary antibody specifically binds to a primary antibody targeting a target protein; Preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO.1, SEQ ID NO.11 or SEQ ID NO.

18.

4. The fusion protein according to claim 1 or 2, wherein The nanobody is a nanobody that recognizes GFP; Preferably, the amino acid sequence of the Nanobody that recognizes GFP is as shown in SEQ ID NO.

46.

5. The fusion protein according to any one of claims 1 to 4, wherein The peptide linker is a flexible linker or a rigid linker; Preferably, the peptide linker is a flexible linker; Preferably, the amino acid sequence of the flexible linker is shown as SEQ ID NO.

4.

6. The fusion protein according to any one of claims 1 to 5, wherein The nanobody and the proximity labeling enzyme are linked via a click chemistry reaction.

7. The fusion protein according to any one of claims 1 to 6, wherein The nanobody and the proximity marker enzyme are connected and fused by any of the following methods: (a) the C-terminus of the Nanobody is linked to the N-terminus of the adjacent marker enzyme; or (b) The N-terminus of the nanobody is linked to the C-terminus of the adjacent marker enzyme.

8. The fusion protein according to any one of claims 1 to 7, wherein The amino acid sequence of the fusion protein is selected from: (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47; or (b) a polypeptide homologous to or having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity with any one of the amino acid sequences of SEQ ID NO. 5, 6, 12, 13, 19, 20, 30-39, 47, which can be used to label neighboring protein molecules that interact with the target protein; or (c) A protein or polypeptide derived by inserting, substituting or deleting one or more amino acids in the amino acid sequence of (a) or (b), which can be used to label neighboring protein molecules that interact with the target protein.

9. The fusion protein according to any one of claims 1 to 8, wherein The fusion protein further comprises a protein tag, and the protein tag is selected from any one of GST, 6x-His, MBP, Flag, HA, cMyc, GFP, eGFP, eYFP, mCherry, AviTag or SUMO tags; Preferably, the protein tags are 6xHis and Flag tags.

10. The fusion protein according to any one of claims 1 to 9, wherein The target protein is any intracellular or extracellular protein that can be recognized by nanobodies or immunoglobulins.

11. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A kit comprising the fusion protein according to any one of claims 1 to 10; Preferably, the kit further comprises a primary antibody targeting the target protein; Preferably, the kit further comprises a biotinylation reaction solution; Preferably, the kit further comprises fluorescent agent-conjugated streptavidin; Preferably, the kit further comprises a secondary antibody coupled to a fluorescent agent; Preferably, the biotinylation reaction solution comprises PBS, MgCl2, ATP and biotin; Preferably, the biotinylation reaction solution comprises biotin phenol and hydrogen peroxide.

14. A proximity marking method, characterized in that: Using the fusion protein according to any one of claims 1 to 10 or the kit according to claim 13 to biotin-label a protein that interacts with a target protein comprises the following steps: Add a primary antibody targeting the target protein from rabbit or mouse species to the cells for incubation to allow the primary antibody to bind to the target protein; According to the species of the primary antibody, a fusion protein of the corresponding species is added to the cells, and the cells are incubated to form a proximity marker enzyme-nano secondary antibody-primary antibody-target protein complex in the cells; adding a biotinylation reaction solution to the cells, incubating, and biotin-labeling the protein interacting with the target protein; After washing, fluorescent agent-conjugated streptavidin and fluorescent agent-conjugated secondary antibody were added, incubated, washed, and detected.

15. A method for analyzing molecular interactions, comprising the following steps: Using the fusion protein according to any one of claims 1 to 10 or the kit according to claim 13 to biotin-label a protein that interacts with the target protein; Use magnetic beads or fluorescent agents coupled with streptavidin to enrich or fluorescently locate biotin-labeled protein molecules; The enriched biotin-labeled proteins were analyzed and identified by LC-MS / MS.

16. Use of the fusion protein according to any one of claims 1 to 10, the kit according to claim 13, or the method according to claim 14 or claim 15, wherein: The applications include: (a) Biotinylation proximity labeling and component analysis of the cytoskeleton and organelles; (b) Biotinylation proximity labeling and component analysis of post-modified histones; (c) Biotinylation proximity labeling and component analysis of different nucleolar substructures; (d) Biotinylation proximity labeling of proteins in FFPE and OCT sections and analysis of interacting proteins; (e) Dual biotinylation proximity labeling of the same sample; or (f) Biotinylation proximity labeling of proteins and analysis of interacting proteins in model organisms.

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