CN119998331A - Nanobody-proximity marker enzyme fusion protein and its application - Google Patents

Abstract

The present disclosure belongs to the field of biotechnology, and specifically relates to a nano antibody-proximity marker enzyme fusion protein and its application. Specifically, the present disclosure provides a fusion protein for proximity labeling, wherein the fusion protein is operably linked together by (a) a nano antibody and (b) a proximity marker 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 in the present disclosure is obtained by in vitro expression and purification, and uses antibodies to target target proteins, is independent of overexpression systems, and is compatible with various cell types, tissues, and clinical fixed samples; it can target proteins with post-translational modifications and various organelles, such as membraneless organelles; and the positioning accuracy rate reaches 100%.

Description

Nanobody-proximity marker enzyme fusion protein and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a nanobody-proximity marker enzyme fusion protein and application thereof.

Background

Protein interactions are the basis for various biological processes within cells, including cell signaling, metabolic regulation, gene transcription regulation, protein synthesis, cellular structure assembly, etc., which are of great significance in biology, involving interactions and interactions between proteins. Research on protein interactions helps to reveal complex molecular networks and signaling pathways within cells, understanding the regulatory mechanisms of cellular functions and biological processes.

Proximity labeling provides a complementary option to conventional methods for studying protein interaction networks, organelle proteome and membrane contacts, protein-nucleic acid interactions, subcellular transcriptome analysis, and the like. This technique relies on a tool enzyme with proximity labeling function, which is capable of biotin labeling of a proximity biomolecule at the living cell level. Currently, it has been widely used for macromolecular complex component identification, intracellular protein species analysis, and protein interaction network construction. The strategy of proximity labelling technology is generally to fusion express the protein of interest with enzymes bearing biotin proximity labels, thereby localizing the protein of interest to specific locations within the cell. By adding small molecule substrates such as biotin and derivatives thereof, the proximity marker enzyme can catalyze the biotinylation covalent connection modification of proteins which are spatially adjacent to the target protein. Tool enzymes currently in wide use include biotin ligase A (BirA) and mutants BioID/BioID2 and TurboID thereof, peroxidase APEX (ENGINEERED ASCORBATE PEROXIDASE, APEX) and APEX2, and the like.

However, the current proximity labelling 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-APEX is capable of in situ biotinylation labeling, thereby better preserving the spatial relationship of protein interactions, the function and mechanism of action of these modifications in cells can be further understood by mass spectrometry analysis to determine proteins that react with histone modifications. However, because protein a can interact with a variety of proteins such as endogenous IgG, the orthogonality and reproducibility of the protein A-APEX2 fusion protein with mammalian cells is poor. Although protein A-APEX2 can label the post-nuclear histone modification, protein A-APEX2 has a large labeling radius and cannot label intracellular fine structures such as microtubules, mitochondria and the like.

In conventional methods of studying protein interactions, such as immunoprecipitation methods, antibodies derived from mice and rabbits are an indispensable tool in many basic research techniques and medical diagnostic assays. In general, the method used to detect or immobilize these primary antibodies is the indirect use of polyclonal anti-IgG secondary antibodies. However, to meet the demand for continuous supply of anti-IgG serum, a large number of breeds, immunizations, bloodletting are required, ultimately resulting in slaughter of a large number of goats, sheep, rabbits and donkeys, which is not only costly but also involves significant animal welfare and ethical issues. Current researchers have developed a recombinantly expressed nanosecondary antibody, a single domain antibody from camelid heavy chain antibodies, as a surrogate for polyclonal anti-IgG secondary antibodies. The nanosecondary antibodies can bind to different regions of rabbit or mouse IgG, partially reducing the need to use polyclonal secondary antibodies of animal origin. Nanosecondary antibodies are considered as powerful tools in the fields of cell biology and structural biology, due to their small size, high specificity and the potential of renewable recombinant fusion proteins, as well as excellent biophysical properties.

However, in the technical field of antibody coupling drugs, nanobodies have not been widely used in the field of antibody coupling because of the problem of poor drug efficacy.

Furthermore, protein interaction identification in protein aggregates currently has a difficult-to-break bottleneck, for example, (1) the conventional biochemical scheme cannot effectively separate the aggregates, (2) the formation of protein aggregates is highly dependent on the concentration of the aggregated protein, which leads to the fact that the overexpression system of fusion expression of biotin ligase and target protein cannot represent the true endogenous phase change protein concentration, and (3) partial overexpression of protein can be abnormally located, thus causing false positives. These directly or indirectly affect the detection and analysis of protein interactions.

To date, there has not been disclosed or developed a fusion protein or conjugate of a nanobody integrated with an adjacent labeling tool enzyme in the art, and the space-time labeling property of the tool enzyme and the biological property of the nanobody have not been utilized, so that limitations or problems in protein interaction network research in the conventional immunoprecipitation method and the adjacent labeling technology are broken through or improved. While there is a continuing need in the field of life sciences research, it is desirable 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.

Disclosure of Invention

In order to solve the problems existing in the prior art, the present disclosure integrates the advantages of both nanobody and proximity labeling tool enzyme, provides a fusion protein or conjugate of nanobody and proximity labeling enzyme, and aims to utilize the space-time labeling property of tool enzyme and the biophysical property of nanobody to break through or improve the application of traditional immunoprecipitation method and proximity labeling technology in protein interaction network research.

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 wherein a peptide linker is further included between the nanobody and the proximity labeling enzyme.

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

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

In another aspect, the present disclosure provides a kit comprising the fusion protein as described above, preferably the kit further comprises a primary antibody targeting a protein of interest;

preferably, the kit further comprises a biotinylation reaction solution;

preferably, the kit further comprises a fluorescent agent-coupled streptavidin;

preferably, the kit further comprises a fluorescent agent-conjugated secondary antibody;

Preferably, the biotinylation reaction solution comprises PBS, mgCl ₂, ATP and biotin;

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

In another aspect, the present disclosure provides a proximity labelling method, characterized in that an interaction protein with a target protein is biotin-labelled using the fusion protein or the kit described above, comprising the steps of:

Adding a primary antibody derived from rabbit or mouse species to the cells for incubation, wherein the primary antibody targets the target protein, and combining the primary antibody with the target protein;

Adding fusion proteins of corresponding species into cells according to the first-antibody species, and incubating to form adjacent marker enzyme-nanometer secondary antibody-first-antibody-target protein complexes in the cells;

adding a biotinylation reaction solution into cells, incubating, and carrying out biotin labeling on the interaction protein molecules with the target protein;

after cleaning, adding fluorescent agent coupled streptavidin and fluorescent agent coupled secondary antibody respectively, incubating, cleaning and detecting.

In another aspect, the present disclosure provides a method of intermolecular interaction analysis, the method comprising the steps of:

Biotin labelling of the interacting protein with the protein of interest using the fusion protein described above or the kit described above;

Enrichment or fluorescent localization of biotin-labeled protein molecules using streptavidin-coupled magnetic beads or fluorescent agents;

The enriched biotin-labeled protein molecules were identified by LC-MS/MS.

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

(a) Biotinylated proximity markers and component resolution of cytoskeleton and organelles;

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

(c) Biotinylated proximity markers of different substructures of nucleolus and component resolution;

(d) Biotinylated proximity markers for proteins in FFPE and OCT sections and interacting protein resolution;

(e) Double biotinylated proximity markers of the same sample, or

(F) Biotinylated proximity markers of proteins in model organisms and interacting protein resolution.

The beneficial effects of the present disclosure are at least:

The technical scheme of the method is independent of an over-expression system, the interaction proteins of the post-modified histone are labeled by identifying antibodies modified by the histone, so that a protein interaction network of the post-modified histone is effectively analyzed, protein components in phase-change particles can be accurately analyzed, the proteins with larger molecular weight can be labeled by the targeting antibodies and related interaction proteins are identified independently of construction of cloning vectors, and protein interactions of multiple localization proteins at specific positions can be analyzed at high resolution, such as different protein interaction networks of the same protein in cell nuclei and cytoplasm are distinguished.

The nanobody-proximity tag enzyme fusion proteins provided by the present disclosure can be used to:

(a) Biotinylated proximity markers and component resolution of cytoskeleton and organelles;

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

(c) Biotinylated proximity markers of different substructures of nucleolus and component resolution;

(d) Biotinylated proximity markers for proteins in FFPE and OCT sections and interacting protein resolution;

(e) Double biotinylated proximity markers for the same sample;

(f) Biotinylated proximity markers of proteins in model organisms and interacting protein resolution.

In particular, by designing and providing nanobody-light control proximity labeling enzyme fusion proteins, efficient biotin labeling can be flexibly and controllably achieved. Furthermore, in the future, the AI recognition algorithm can quickly and accurately recognize the interested structure by combining with a full-automatic AI recognition microscope imaging system, so that the positioning and analysis of the target area can be realized. Furthermore, by combining with AI and a microscope, the method is expected to realize high-flux and automatic image acquisition analysis and fixed-point biotinylation marking, and marking specific positions of hundreds of thousands of cells, thereby meeting the requirement of mass spectrum on sample size.

Drawings

FIG. 1 is a schematic diagram of the function of the nanobody-proximity-tagged enzyme fusion proteins of the disclosure in actual sample detection.

FIG. 2 is a schematic diagram showing the action of light-operated nanobody-proximity-labeled enzyme fusion protein in the detection of an actual sample.

FIG. 3 is a SDS-PAGE of purified anti-mouse or anti-rabbit nanobody-TurboID fusion protein, wherein lanes 1-8 represent molecular patterns of samples before and after expression and purification of the constructed anti-mouse nanobody-TurboID fusion protein in host cells, lane 9 is a protein standard (marker), and lanes 10-16 represent molecular weights of the constructed anti-rabbit nanobody-TurboID fusion protein as molecular patterns of samples before and after expression and purification in host cells.

FIG. 4 shows the labeling radii of Nano-ID, nano-APX, pro-ID and Pro-APX in murine cytoskeleton. FIG. 4A shows the fusion expression of six different types of nanobody-adjacent biotin ligase, namely anti-rabbit nanobody-TurboID/APEX 2, anti-mouse nanobody-TurboID/APEX 2, protein A-TurboID/APEX2, FIG. 4b shows the experimental procedure of in vitro biotinylation, cells are first immobilized, blocked and permeabilized, then primary antibodies of a targeted region of interest are added, protein A-TurboID/APEX2 is added or anti-rabbit nanobody-TurboID/APEX 2 is added according to one anti-species, anti-mouse nanobody-TurboID/APEX 2, FIG. 4c-4n shows the fluorescence localization result of different anti-mouse fusion proteins in U-2OS cells, anti-beta-actin primary antibodies are used for labeling actin filaments (4 c,4f,4i,4 l), anti-vin primary antibodies are used for labeling intermediate filaments (4 d,4g,4j,4 m), anti-TubA A primary antibodies are used for labeling proteins (4 e, 4k,4 h) and anti-mouse nanobody-4 c-4n is added, or 4 e-4 h is added to the anti-mouse nanobody-4 k-4 h, 4c-4n is shown in FIG. 4 b. The green label is Alexa Fluor ^TM 488 coupled streptavidin showing biotinylated protein distribution, and the red label is Alexa Fluor ^TM 560 coupled secondary antibody showing primary antibody localization. Scale, 5 μm, enlarged view, 2 μm.

FIG. 5 shows the distribution of fluorescent signals in the white line selection region (upper row) and the exponential fit of fluorescent signals (lower row) in FIG. 4 (4 c,4f,4i,4 l).

FIG. 6 is a graph showing the comparison of the labeling radii of Nano-ID, nano-APX, pro-ID and Pro-APX using different organelle antibodies of murine origin. anti-LAMP1 primary antibody was used for lysosome labeling (6 a,6d,6g,6 j), anti-LaminB1 primary antibody was used for nuclear membrane labeling (6 b,6e,6h,6 k), anti-ATP5A1 primary antibody was used for labeling mitochondria (6 c,6f,6i,6 l), FIG. 6a-6c was added with anti-murine nanobody-TurboID, FIG. 6d-6f was added with anti-murine nanobody-APEX 2, FIG. 6g-6i or FIG. 6j-6l was added with Protein A-TurboID/APEX2, green label was Alexa Fluor ^TM 488-coupled streptavidin showing biotinylated Protein distribution, red label was Alexa Fluor ^TM -coupled secondary antibody showing localization of primary antibodies. Scale, 5 μm, enlarged view, 2 μm.

FIG. 7 is a graph showing the comparison of the labeling radii of Nano-ID, nano-APX, pro-ID and Pro-APX using rabbit derived different organelle antibodies. anti-Paxillin primary antibody was used for focal adhesion labeling (7 a-7 d), anti-PEX14 primary antibody was used for peroxisome labeling (7 e-7 h), anti-EDC4 primary antibody was used for P-mer labeling (7 i-7 l), anti-rabbit nanobody-TurboID was added to FIG. 7a,7e,7i, anti-rabbit nanobody-APEX 2, FIG. 7c,7g,7k or Protein A-TurboID/APEX2 was added to FIG. 7d,7h,7 l. The green label is Alexa Fluor ^TM 488 coupled streptavidin showing the distribution of biotinylated proteins, and the red label is Alexa Fluor ^TM 560 coupled secondary antibody showing the localization of primary antibodies. Scale, 5 μm, enlarged view, 2 μm.

FIG. 8 is a diagram showing the use of Nano-ID for labeling organelles or post-histone modifications. The anti-8F, anti-H3K4me3 (murine antibody) (8 g), anti-H3K27Ac (rabbit antibody) (8H), anti-HAK119ub (rabbit antibody) (8 i), anti-L-LACTYL LYSINE (rabbit antibody) (8 j), anti-rabbit nanobody-32 added to FIGS. 8b,8d, 8H,8i,8j, and anti-mouse nanobody-TurboID added to FIGS. 8a,8c,8f,8g, and the anti-alexin-488-binding protein showed that the anti-alexin was coupled to the alexin-488-binding protein of the alexin-488-binding protein. Scale, 5 μm.

FIG. 9 is a hierarchical analysis of nucleoli using Nano-ID. Fig. 9a is a schematic diagram of the nucleolus structure. Fiber center (Fibrillar Centers, FC), dense fiber component (Dense Fibrillar Component, DFC), particulate component (Granular Component, GC). FIGS. 9b-9d show in situ biotin labeling results for three different substructures of nucleoli. FIG. 9e shows immunoblotting assays for different layers of substructured biotin protein. FIG. 9f is a signal heat map of TCOF1, FBL and NPM1 after detection of different nucleolar substructures by mass spectrometry. FIG. 9g is a heat map of mass spectrum signals of nucleolin at different layers. FIGS. 9h-9j select representative graphs of protein interactions enriched in FC (9 h), DFC (9 i) and GC (9 j) structures. The protein interaction network is shown by StringDb.

Fig. 10 shows the application of Nano-ID in FFPE and OCT slices. FIG. 10a is a representative immunofluorescence image of Nano-ID in vitro biotinylation on FFPE sections of lung adenocarcinoma tissue. FIG. 10b shows the results of total protein biotinylated protein content assays for the experimental group (+PECAM 1 primary antibody) and the control group (-, no PECAM1 primary antibody). FIG. 10c shows the signal intensity of the mass spectrum of PECAM1 after enrichment of total proteins of the experimental and control groups with streptavidin-coupled magnetic beads in the FFPE samples derived from the three patients described above, and N.D., was not detected. FIG. 10d is a Ween diagram showing overlapping interactions of proteins PECAM1 in three patients. FIG. 10e shows the results of an interaction network analysis of the interaction proteins of PECAM1 detected in all three samples. The interaction network information is derived from Stringdb databases, and different colors represent different signal pathways in which proteins participate.

FIG. 11 shows the application of Nano-ID in OCT sections of mouse brain. FIG. 11a is a representative immunofluorescence photograph of Nano-ID biotinylated in vitro on mouse brain OCT sections. After the mouse brain OCT slice is fixed and transparent, a GFAP antibody and a Nano-ID are sequentially added to carry out in-vitro biotinylation reaction, and after the reaction, a sample is incubated to mark GFAP by Alexa Fluor ^TM 560 coupled secondary antibody and mark biotinylation protein by Alexa Fluor ^TM 488 coupled streptavidin. FIG. 11b shows the results of total protein biotinylated protein content assay in the experimental group (+GFAP primary antibody) and control group (-, no GFAP primary antibody). FIG. 11c is a Wen diagram of 689 endogenous wild-type biotin proteins identified after enrichment as well as 795D 4-tagged proteins. FIG. 11d shows the GO analysis of the proteins identified in 11 c. Fig. 11e is a schematic illustration of the experimental procedure.

FIG. 12 shows the application of Nano-ID to the brain of adult mice in HTT disease model. FIG. 12a is a representative immunofluorescence image of the labeling of HTT adjacent proteins in HTT disease model mice. FIG. 12b shows the results of the detection of total protein biotinylated protein content in the experimental group (+HTT primary antibody) and control group (-, HTT primary antibody was not added).

FIG. 13 shows the application of Nano-ID in FFPE and OCT sections and fertilized egg samples. FIGS. 10a and 10b are representative immunofluorescence pictures of in vitro biotinylation of Nano-ID on FFPE sections of lung adenocarcinoma tissue, FIG. 13a with PDL1 antibody added, FIG. 13b with H3K27Ac antibody, alexa Fluor ^TM 560 conjugated secondary antibody labeled PDL1 (a) or H3K27Ac (b), alexa Fluor ^TM 488 conjugated streptavidin labeled biotinylated protein. FIG. 13c shows the biotin-labeled transcription factor PAX 6-adjacent protein on OCT sections of mice brains developed for 12.5 days. FIG. 13d is a schematic representation of the labeling of the top domain with Nano-ID in a mouse 8-cell embryo.

FIG. 14 is a diagram showing a Nano-ID implementation of dual labeling of a sample. Fig. 14a is a schematic illustration of the experimental procedure for dual labeling. Intracellular ROI1 and ROI2 were labeled with rabbit and murine antibodies, respectively. For example, in the first round of reaction, nano-ID (Rb) is used for marking the rabbit primary antibody by identifying the rabbit primary antibody, after the first round of reaction is completed, TEV enzyme is added for cutting, the Nano-ID- (Rb) is cut, free Turbo-ID can be washed, in the second round of reaction, nano-ID (Ms) is added for identifying the mouse primary antibody, biotin in the reaction liquid is marked as D4, and then the second round of reaction is carried out, and the D4 marking is carried out on the mouse primary marked ROI 2. And (3) cell lysis, digestion of the obtained protein precipitate into peptide fragments, and biotinylation enrichment at the peptide fragment level to finally obtain wild type and D4 marked peptide fragments, so as to distinguish the first round of marked proteins from the second round of marked proteins. FIG. 14b shows the first round of labeling mitochondria with anti-TFAM wild type biotin of rabbit origin and the second round of labeling nucleoli with anti-NPM1 isotope D4 of mouse origin, as described in FIG. 14 a. The first and second rounds of labeled biotin proteins were observed with Alexa Fluor ^TM 488 and Alexa Fluor ^TM 560 conjugated streptavidin, respectively. Immunoblotting results for TFAM alone, NPM 1-labeled, and dual TFAM and NPM 1-labeled in fig. 14b are shown in fig. 14c.

FIG. 15 shows the labeling results of Nb2-ID in cell lines and model organisms stably expressing GFP fusion proteins. FIG. 15a shows a cell line overexpressing mEmerald-SRSF3, FIG. 15b shows a cell line knocked-in GFP using CRISPR at the SC35 locus, FIG. 15c shows a cell line overexpressing mEmerald-ensconsin, and FIG. 15d shows a nematode with stable ajm-1-GFP cut development at 1.8 fold. After incubation of the samples with Nb2-ID, in vitro biotinylation reactions were performed, and Alexa Fluor ^TM 560-coupled streptavidin localized biotinylated proteins.

FIG. 16 shows the labeling results of nanobody-light-operated proximity labeling enzyme in U-2OS cells. Fig. 16a incubates rabbit antibody anti-PEX14 anti-ibody, fig. 16b incubates mouse antibody anti-Tubulin anti-ibody, and nanobody-light-operated proximity marker enzymes of the corresponding species are added respectively for biotinylation reaction. Purple label is Alexa Fluor ^TM 647 coupled secondary antibody, showing primary antibody localization, and green label is Alexa Fluor ^TM 488 coupled streptavidin, showing biotinylated protein distribution. Scale, 5 μm.

Detailed Description

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the description below are only examples of the application and that other embodiments can be obtained according to these drawings by a person skilled in the art.

Definition of the definition

The following definitions are provided herein 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 one of ordinary skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and such definitions contained herein should not be construed as representing substantial differences from the definition of terms commonly understood in the art. In case of ambiguity, chemical, pharmaceutical and medical dictionary may be used as a further source of supplemental information to the extent consistent with the present invention.

The terms "a" or "an" mean, unless the context indicates otherwise, the substance or ingredient, but is not limited to the number/amount thereof, and may be used in the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, reference to "a pharmaceutical combination" is to be understood as a combination of two or more components.

In this document, the terms "comprises," "comprising," and "includes" or equivalents thereof, unless otherwise specified, are open ended and mean that other unspecified elements, components, and steps are contemplated in addition to those listed.

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

As used herein, the terms "about", "substantially" and "substantially" refer to deviations from the stated values in the range of-10% to +10%. When the term "about" is used herein to refer to a number, it is to be understood that another embodiment of the invention includes the number as modified by the absence of the term "about".

As used herein, the term "antibody" includes any immunoglobulin that binds to a particular antigen, including monoclonal antibodies, polyclonal antibodies, multivalent antibodies, bivalent antibodies, monovalent antibodies, multispecific antibodies, or bispecific antibodies. Natural intact antibodies comprise two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain comprising a variable region (VH) and a first constant region, a second constant region, a third constant region and optionally a fourth constant region (CH 1, CH2, CH3, CH4, respectively), mammalian light chains are classified as λ or κ, and each light chain comprises a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of the Y-shaped structure comprises a second constant region and a third constant region of two heavy chains that are joined together by disulfide bonds. Each arm of Y comprises a variable region and a first constant region of a single heavy chain in combination with a variable region and a 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 typically contains three hypervariable regions, known as Complementarity Determining Regions (CDRs), wherein the light chain CDRs comprise LCDR1, LCDR2, LCDR3 and the heavy chain CDRs comprise HCDR1, HCDR2, HCDR3. The three CDRs are separated by flanking segments called Framework Regions (FR) which are more highly conserved than the CDRs and form scaffolds to support highly variable loops, wherein the light chain FR comprises LFR1, LFR2, LFR3 and LFR4 and the heavy chain FR comprises HFR1, HFR2, HFR3 and HFR 4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies can be classified into several classes based on the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, igD, igE, igG and IgM, which are characterized by the presence of the alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain) or IgA2 (alpha 2 heavy chain).

As used herein, the term "monoclonal antibody" refers to an antibody that is highly homogeneous and directed against only a particular epitope, produced by cloning from a single B cell (the gene of which encodes only one antibody). The hybridoma (hybridoma) antibody technology is generally prepared by adopting a hybridoma technology, and is based on a cell fusion technology, sensitized B cells with the capability of secreting specific antibodies and myeloma cells with unlimited reproductive capability are fused into a B cell hybridoma. By culturing a cell population with a single hybridoma cell having such characteristics, a specific antibody against an epitope, i.e., a monoclonal antibody, can be prepared.

As used herein, the term "polyclonal antibody" refers to a group of immunoglobulins secreted by plasma cells of an organism that stimulate the organism to produce an immune response by heterologous antigens (macromolecular antigens, hapten conjugates). Typically comprising at least 2 or more different antibodies, which typically recognize multiple epitopes, may cause precipitation reactions. The preparation cost is low, the preparation speed is high, the preparation process is simpler than that of monoclonal antibodies, and the preparation method is widely applied to research and diagnosis.

As used herein, the term "primary antibody" or "primary antibody" refers to a protein that specifically binds to a non-antibody antigen (specific antigen). Classes include monoclonal antibodies and polyclonal antibodies. I.e. antibodies which bind specifically to antigens.

As used herein, the term "secondary antibody" or "secondary antibody" refers to an antibody that is capable of binding to, i.e., an antibody, and whose primary function is to detect the presence of an antibody and amplify the signal of the primary antibody. The secondary antibody is an immunoglobulin against an antibody which is produced by the immune system of a xenogeneic animal by using the antigenic property of the antibody, which is a macromolecular protein. The secondary antibody is reactive against all antibodies (e.g., igG, igM, igA, etc.) of a particular species (e.g., mouse).

As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from an antibody fragment comprising one or more CDRs, or any other antibody moiety that binds to an antigen but does not comprise the complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fab ', F (ab ') 2, fd, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibody fragments, camelylated single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen binding fragment may comprise one or more CDRs from a particular antibody.

As used herein, the term "Fab" refers to a monovalent antigen binding fragment of an antibody consisting of a single light chain (variable and constant regions) linked via disulfide bonds to the variable and first constant regions of a single heavy chain. Fab can be obtained by papain digestion of residues near the N-terminus of disulfide bonds between heavy chains of the antibody hinge region.

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-terminus of the disulfide bond between the heavy chains of the antibody hinge region, and thus a small number of residues (comprising one or more cysteines) in the hinge region are different from Fab.

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

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

As used herein, the term "Fv" refers to the smallest antibody fragment with an intact antigen binding site. Fv fragments consist of the variable region of a single light chain combined with the variable region of a single heavy chain. "dsFv" refers to disulfide stabilized Fv fragments in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is disulfide.

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 linked to each other either directly or through a peptide linker sequence. "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, the "scFv dimer" is a bivalent diabody or a bivalent scFv (BsFv) comprising a VH-VL (linked by a peptide linker) dimerized with another VH-VL moiety such that the VH of one moiety coordinates with the VL of the other moiety 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) bound to VL1-VH2 (also linked by a peptide linker) such that VH1 coordinates VL1 and VH2 coordinates VL2 and each coordination pair has a different antigen specificity.

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

As used herein, the term "nanobody" or "nanobody" has the same meaning as "camelized single domain antibody", "heavy chain antibody (singledomain antibody, sdAb)" or "HCAb", used interchangeably, refers to the variable region of a cloned antibody heavy chain, constructing a nanobody consisting of only one heavy chain variable region. Typically, the naturally deleted light chain and heavy chain constant region 1 (CH 1) antibodies are obtained first, and then the variable region of the heavy chain of the antibody is cloned to construct nanobodies consisting of only one heavy chain variable region. Heavy chain antibodies were originally obtained from Camelidae (CAMELIDAE) (camel, dromedary and llama), and while free of light chains, camelized antibodies have a reliable antigen-binding repertoire. The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen binding unit produced by the innate immune response.

As used herein, the term "bifunctional antibody" comprises a small antibody fragment having two antigen-binding sites, wherein the fragment comprises a VH domain (VH-VL or VL-VH) linked to a VL domain in a single polypeptide chain. Because the linker is too short, the two domains on the same strand cannot be paired, thus forcing the domains to pair with the complementary domains of the other strand, thereby creating two antigen binding sites. The antigen binding sites may target the same or different antigens (or epitopes).

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 enzymes depends on the primary and spatial structural integrity of the enzyme molecule. Enzyme activity may be lost if the enzyme molecule is denatured or the subunits depolymerized. The enzyme belongs to biological macromolecules, and the molecular mass is at least over 1 ten thousand and can reach millions. Enzymes are a very important class of biocatalysts (biocatalyst). The chemical reaction in the living body can be efficiently and specifically performed under extremely mild conditions due to the action of the enzyme. Enzymes can be classified into simple enzymes and bound enzymes according to their chemical composition. Simple enzyme molecules are enzymes that have only an amino acid component after hydrolysis. The conjugated enzyme molecule is composed of protein part and non-protein part, such as metal ion, iron porphyrin or small molecule organic matter containing B vitamins.

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, substance metabolism, etc.

As used herein, the term "Protein-interaction network (PPI)" is composed of proteins that are involved in various links of life processes such as biological signaling, regulation of gene expression, energy and substance metabolism, and regulation of cell cycle by interactions with each other. The system analyzes the interaction relation of a large amount of proteins in a biological system, and has important significance for understanding the working principle of the proteins in the biological system, understanding the reaction mechanism of biological signals and energy substance metabolism under special physiological states such as diseases and the like, and understanding the functional relation among the proteins. Currently, the most commonly used research means for searching protein interaction are mainly yeast two-hybrid (Y2H) screening and co-immunoprecipitation combined mass spectrometry (IP-MS) analysis.

As used herein, the term "proximity labeling technique (Proximity labeling, PL)" refers to a technique in which a protein spatially adjacent to a target protein is labeled by fusion expression of certain enzymes to the target protein by gene fusion, and by specific reaction under the catalytic action of the enzymes. Proximity labelling techniques use genetically engineered enzymes such as peroxidases or biotin ligases which catalyze the conversion of inert substrates to 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), the extent of the labeling being dependent on the half-life of the active substance and the concentration of the quencher. Covalently labeled biomolecules are enriched by streptavidin magnetic beads and identified by mass spectrometry or nucleic acid sequencing. Proximity labeling techniques are widely used to identify protein interaction networks and to study protein interactions with RNA and protein interactions with DNA in living cells. It is also suitable for constructing the interaction network of insoluble proteins, and can detect the proteins with transient or dynamic interactions. In addition, it is also suitable for analyzing protein components of subcellular organelle localization, or for studying interaction networks in living organisms. In the last decade, proximity marking technology has evolved rapidly. Currently, spatial resolution on the order of nanometers, temporal resolution on the order of minutes have evolved and are used to construct molecular interaction maps in living organisms.

As used herein, the term "proximity labeling enzyme" or "proximity labeling tool enzyme" refers to a tool enzyme having a proximity labeling function, including peroxidase (e.g., APEX, HRP) and biotin ligase (e.g., bioID, turboID), etc., to biotin-label a proximity biomolecule at the living cell level. The proximity labeling technology has high spatial specificity because of the short lifetime of the highly reactive small molecules produced by the enzymatic catalysis. Combining mass spectrometry-based proteomics techniques with high throughput sequencing techniques, one can achieve large-scale analysis of neighboring biomolecules.

The tool enzymes with linking activity used can be divided into two types, namely complete type and split type, in terms of structure, the complete type of proximity marker enzyme is mainly used for researching potential interaction proteins of single target proteins, and the split type of proximity marker enzyme is used for researching proteins associated with the existence of known protein complexes or interaction proteins. Proximity labelling assays based on intact proximity labelling enzymes are those in which biotin ligase or ascorbate peroxidase (APEX enzyme) is fused to the protein of interest and expressed in living cells, and after addition of a substrate, such as biotin or biotin-phenol and hydrogen peroxide (H ₂O₂), to the medium, the proteins or RNAs in the vicinity of the protein of interest can be labelled 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 for identifying protein complex composition is a fusion of N-terminal and C-terminal portions of the proximity marker enzyme with a pair of known interacting proteins, respectively. When the pair of proteins interact in the cell, the two halves of the proximity marker enzyme are pulled close together and reconstituted to complete proximity marker enzyme, marking the proteins in the vicinity of the protein complex, and by lysing the cells and incubating with streptavidin magnetic beads, the biotin-labeled proteins can be enriched for subsequent LC-MS/MS sequencing analysis.

There are many proximity marker enzymes used for protein interaction identification, among which commonly used proximity marker tool enzymes are mutants of E.coli biotin ligase BirA (BioID) and Ascorbate Peroxidase (APEX). Based on this optimization and many other proximity marker enzymes have been developed, such as the APEX2 optimizing enzyme of APEX series, the BioID optimizing enzymes BioID, air id, BASU, etc., and some small-scale tool enzymes such as HRP, EXCELL, PUP-IT, NEDDylation, which have been developed to expand the application range of proximity marker technology.

As used herein, the term "fusion protein" has the ordinary and customary meaning as understood by those of ordinary skill in the art from the specification and the specification of the present application. In the present application, a "fusion protein" is an expression product of two recombinant genes obtained by a DNA recombination technique, and two different proteins can be linked into one macromolecule by fusion of the genes. The fusion proteins of the application may include, in addition to the nanobody listed herein, an optional tag sequence (e.g., 6xHis tag, GGGS sequence, FLAG tag) that facilitates expression and/or purification, or an optional polypeptide molecule or fragment having therapeutic functions, or an optional protein domain that facilitates physicochemical or pharmaceutical functions (e.g., a molecule capable of extending the half-life of the nanobody in vivo, such as an Fc fragment, HLE, ABD).

In the application, the Nanobody-adjacent marker enzyme fusion protein is formed by coupling a Nanobody (Nano) and an adjacent marker enzyme. When the proximity marker enzyme is TurboID, it may 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 may be referred to as "Nano-APEX (Ms/Rb)", "Nanobody-APEX (Ms/Rb)", or "anti-rabbit/anti-mouse Nanobody-APEX 2". The fusion proteins formed by coupling protein A and the adjacent marker enzyme may be referred to as "ProteinA-TurboID", "Pro-ID", "ProteinA-APEX2", "Pro-APX". In some embodiments, "Nb2-ID", "nanobody-photo proximity marker enzyme" is also one of nanobody-proximity marker enzyme fusion proteins.

As used herein, the term "Post protein modification", post-translational Modifications, PTMs for short, refers to the chemical modification of a protein after translation of the protein. These modifications include acetylation, methylation, phosphorylation, ubiquitination, ADP ribosylation, etc., which can occur at amino acid residues of histones, thereby altering chromatin structure and function and thereby regulating gene expression. "post-modified histone" refers to post-translationally modified histone.

Detailed description of the embodiments

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 wherein a peptide linker is further included between the nanobody and the proximity labeling enzyme.

In some embodiments, a peptide linker is also included between the nanobody and the adjacent marker enzyme.

In some embodiments, the proximity marker 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 biological ligase is an engineered light control TurboID. In some embodiments, the amino acid sequence of light control TurboID is set forth in any one of SEQ ID NO. 25-29.

In some embodiments, the nanobody is a primary antibody targeted to a target protein or a secondary antibody targeted to an immunoglobulin. In some embodiments, the immunoglobulin is a rabbit immunoglobulin, a human immunoglobulin, or a murine 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 that targets IgG that specifically binds to a primary antibody that targets a protein of interest. In some embodiments, the nanobody is a secondary antibody having an amino acid sequence as set forth in SEQ ID NO.1, SEQ ID NO.11, or SEQ ID NO. 18.

In some embodiments, the nanobody is a nanobody that recognizes GFP. In some embodiments, the amino acid sequence of the GFP-recognizing nanobody is shown as SEQ ID No. 46.

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 as set forth in SEQ ID NO. 4.

In some embodiments, the nanobody and the proximity labeling enzyme are linked by a click chemistry reaction.

In some embodiments, the nanobody and proximity tag enzyme are linked and fused by any of the following means:

(a) The C-terminal of nanobody and N-terminal of adjacent marker enzyme are linked, or

(B) The N-terminus of the nanobody is linked to the C-terminus of the adjacent tag enzyme.

In some embodiments, the nanobody C-terminus is fused to the N-terminus of the adjacent marker enzyme by a linker.

In some embodiments, the amino acid sequence of the fusion protein is selected from the group consisting of:

(a) A polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO.5, 6, 12, 13, 19, 20, 30-39, 47, or

(B) A polypeptide homologous or having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity to the amino acid sequence as set forth in any one of SEQ ID NO.5, 6,12, 13, 19, 20, 30-39, 47, which can be used to label a contiguous protein molecule that interacts with a protein of interest, or

(C) Proteins or polypeptides derived from insertion, substitution or deletion of 1 or more amino acids in the amino acid sequence of (a) or (b) can be used to label adjacent protein molecules that interact with the protein of interest.

In some embodiments, the fusion protein further comprises a protein tag 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.

In some embodiments, the protein of interest is any intracellular or extracellular protein that can be recognized by a nanobody or immunoglobulin.

In some embodiments, the fusion protein is an anti-rabbit nanobody-TurboID, the amino acid sequence of which is shown in SEQ ID NO. 5. In some embodiments, the fusion protein is an anti-rabbit nanobody-APEX 2, the amino acid sequence of which is shown in SEQ ID No. 6. In some embodiments, the fusion protein is an anti-murine nanobody-TurboID, the amino acid sequence of which is shown in SEQ ID NO. 12. In some embodiments, the fusion protein is an anti-rabbit nanobody-APEX 2, the amino acid sequence of which is shown in SEQ ID No. 13. In some embodiments, the fusion Protein is Protein A-TurboID, the amino acid sequence of which is shown in SEQ ID NO. 19. In some embodiments, the fusion Protein is Protein A-APEX2, the amino acid sequence of which is shown in SEQ ID NO. 20. In some embodiments, the fusion protein is a nanobody-light-operated proximity marker enzyme fusion protein, which can accurately start or shut down TurboID enzyme activity by controlling illumination, so as to realize space control on 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, the amino acid sequence of which is shown as SRQ ID No. 47.

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

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

In another aspect, the present disclosure provides a kit comprising the fusion protein as described above, preferably the kit further comprises a primary antibody targeting a protein of interest;

preferably, the kit further comprises a biotinylation reaction solution;

preferably, the kit further comprises a fluorescent agent-coupled streptavidin;

preferably, the kit further comprises a fluorescent agent-conjugated secondary antibody;

Preferably, the biotinylation reaction solution comprises PBS, mgCl ₂, ATP and biotin;

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

In another aspect, the present disclosure provides a proximity labelling method, characterized in that a protein interacting with a target protein is biotin-labelled using the fusion protein described above or the kit described above, comprising the steps of:

Adding a primary antibody of any species targeting the target protein to the cells for incubation, so that the primary antibody is combined with the target protein;

Adding fusion proteins of corresponding species into cells according to the first-antibody species, and incubating to form adjacent marker enzyme-nanometer secondary antibody-first-antibody-target protein complexes in the cells;

adding a biotinylation reaction solution into cells, incubating, and carrying out biotin labeling on proteins interacted with the target proteins;

after cleaning, adding fluorescent agent coupled streptavidin and fluorescent agent coupled secondary antibody respectively, incubating, cleaning and detecting.

In another aspect, the present disclosure provides a method of intermolecular interaction analysis, the method comprising the steps of:

biotin labelling of proteins interacting with the target protein using the fusion protein described above or the kit described above;

Enrichment or fluorescent localization of biotin-labeled protein molecules using streptavidin-coupled magnetic beads or fluorescent agents;

the enriched biotin-labeled protein was identified by LC-MS/MS.

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

(a) Biotinylated proximity markers and component resolution of cytoskeleton and organelles;

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

(c) Biotinylated proximity markers of different substructures of nucleolus and component resolution;

(d) Biotinylated proximity markers for proteins in FFPE and OCT sections and interacting protein resolution;

(e) Double biotinylated proximity markers for the same sample;

(f) Biotinylated proximity markers of proteins in model organisms and interacting protein resolution.

A further understanding of the present disclosure may be obtained by reference to the specific examples presented herein which are intended to be illustrative of the present disclosure and are not intended to limit the scope of the present disclosure in any way. It will be apparent that various modifications and variations can be made to the present disclosure without departing from the spirit of the disclosure, and therefore, such modifications and variations are also within the scope of the claimed application.

Example 1 preparation of nanobody-proximity-tagged enzyme fusion proteins

The nanobodies used in the present disclosure are all specific secondary antibodies. The technical scheme of the present disclosure is equally applicable to a primary anti-nanobody. The working principle of the nanobody-adjacent marker enzyme fusion protein is schematically shown in figure 1.

1. Anti-rabbit nanobody-TurboID/APEX 2 fusion proteins

The amino acid sequence of the anti-rabbit nanometer antibody is shown as SEQ ID NO.1, the amino acid sequence of TurboID is shown as SEQ ID NO.2, and the amino acid sequence of APEX2 is shown as SEQ ID NO. 3. The C end of the nano antibody is fused with the N end of the adjacent marker enzyme through a linker, and the amino acid sequence of the linker is shown as SEQ ID NO. 4. The amino acid sequence of the fused anti-rabbit nanometer antibody-TurboID is shown as SEQ ID NO.5, the N end of the fusion protein is connected with a 3xFlag label, and the C end is connected with a 6xHis label. The amino acid sequence of the anti-rabbit nanobody-APEX 2 is shown as SEQ ID NO.6, and the N end of the fusion protein is connected with a 6XHis tag and a 3xFlag tag.

TABLE 1 coding information for anti-rabbit nanobody-TurboID/APEX 2 fusion protein

Selecting an expression vector suitable for an escherichia coli protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, and constructing an anti-rabbit nanobody-TurboID fusion protein plasmid shown as SEQ ID NO.9, wherein the specific process is as follows:

the carrier is constructed through homologous recombination, and the connection mode of the homologous recombination mainly utilizes the overlapping region of the target fragment and the carrier to reconstruct the target fragment and the carrier into a new plasmid.

The synthesized anti-rabbit nanobody-TurboID fusion protein gene and the anti-rabbit nanobody-APEX 2 fusion protein gene are constructed on an expression vector by a homologous recombination method.

(1) PCR of fragments of interest and vectors

The PCR primers used are shown in table 2 below:

TABLE 2 PCR primers

The PCR system and the procedure used are shown in Table 3 below:

TABLE 3 PCR reaction System

Reaction components	Volume of
		Stencil (10 ng/. Mu.L)	1μL
Upstream primer (10. Mu.M)	2.5μL
		Downstream primer (10. Mu.M)	2.5μL
PCR high-fidelity enzyme mixture	25μL
		Water and its preparation method	Supplement to 50 mu L

The PCR program settings are shown in table 4 below:

TABLE 4 PCR Programming

And (3) carrying out agarose gel electrophoresis on the PCR product, and recovering the band with the correct size for later use.

(2) The system and reaction conditions for homologous recombination are shown in Table 5 below.

TABLE 5 reaction Components and reaction conditions

The ligation products were transformed into clonotype competent cells, plated onto LB plates containing resistance, and the monoclonal colonies were picked the next day for sequencing. Fusion protein plasmids were extracted from correctly sequenced strains, transformed into expression competent BL21 (DE 3), plated onto LB plates containing resistance, and monoclonal colonies were picked the next day for protein expression.

2. Anti-mouse nanobody-TurboID/APEX 2 fusion proteins

The amino acid sequence of the anti-mouse nano antibody is shown as SEQ ID NO.11, the amino acid sequence of TurboID is shown as SEQ ID NO.2, and the amino acid sequence of APEX2 is shown as SEQ ID NO. 3. The C end of the nano antibody is fused with the N end of the adjacent marker enzyme through a linker, and the amino acid sequence of the linker is shown as SEQ ID NO. 4. The amino acid sequence of the fused anti-mouse nano antibody-TurboID is shown as SEQ ID NO.12, the N end of the fusion protein is connected with a 3xFlag tag, and the C end of the fusion protein is connected with a 6xHis tag. The amino acid sequence of the anti-mouse nanobody-APEX 2 is shown as SEQ ID NO.13, and the N end of the fusion protein is connected with a 6XHis tag and a 3xFlag tag.

TABLE 6 coding information for anti-murine nanobody-TurboID/APEX 2 fusion protein

Selecting an expression vector suitable for an escherichia coli protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, and constructing an anti-mouse nanobody-TurboID fusion protein plasmid shown as SEQ ID NO.16, wherein the specific process is as follows:

PCR primer sequences, procedures, ligation, screening, etc. see anti-rabbit nanobody related content.

Protein A-TurboID/APEX2 fusion protein

The amino acid sequence of Protein A is shown as SEQ ID NO.18, the amino acid sequence of TurboID is shown as SEQ ID NO.2, and the amino acid sequence of APEX2 is shown as SEQ ID NO. 3. The C-terminal of ProteinA is fused with the N-terminal of 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 Protein A-TurboID is shown as SEQ ID NO.19, the amino acid sequence of the Protein A-APEX2 is shown as SEQ ID NO.20, and the N end of the fusion Protein is connected with a 6xHis tag and a 3xFlag tag.

TABLE 7 coding information for anti-murine nanobody-TurboID/APEX 2 fusion protein

Selecting an expression vector suitable for an escherichia coli Protein expression system, including but not limited to any one of pET15b, pET28a, pGEX4T1 or pGEX-6p-1, and constructing a Protein A-TurboID fusion Protein plasmid shown as SEQ ID NO.23, and a Protein A-APEX2 fusion Protein plasmid shown as SEQ ID NO.24, wherein the specific process is as follows:

The synthesized protein A-TurboID fusion protein gene and proteinA-aPEX fusion protein gene are constructed on an expression vector by a homologous recombination method.

The PCR primer sequences used are shown in table 8 below:

TABLE 8 PCR primers

See anti-rabbit nanobody related content for PCR procedures, ligation, screening, etc.

4. Anti-rabbit nanobody-light-operated proximity marker enzyme fusion protein

The light-controlled adjacent marker enzyme is prepared by introducing light-controlled unnatural amino acid into TurboID active pocket position to deactivate TurboID, and converting the light-controlled unnatural amino acid into corresponding natural amino acid under UV irradiation to release TurboID enzyme activity. Specifically, the light-operated adjacent marking enzyme replaces lysine or tyrosine at a specific position with corresponding unnatural amino acids such as lysine or tyrosine derivatives, and the fixed-point insertion of the unnatural amino acids can eliminate the enzyme activity of adjacent biotin ligase, and the unnatural amino acids such as lysine or tyrosine derivatives at the specific position in the light-operated adjacent marking enzyme are converted into lysine or tyrosine under the irradiation of ultraviolet with 365 wavelengths so as to restore the activity of adjacent biotin ligase, thereby specifically labeling the adjacent proteins by biotinylation.

Therefore, the fusion expression protein of the nanometer secondary antibody and the light-controlled adjacent marker enzyme (TurboID light switch mutant) can accurately start the enzyme activity of TurboID in a specific area by controlling the illumination area, so that the space control of the biotinylation process is realized. The introduction of such an optical switch improves the functional flexibility and controllability of TurboID, enabling efficient biotin labeling at specific spatial locations. The working principle schematic diagram of the fusion expression protein of the nanometer secondary antibody and TurboID optical switch mutant is shown in figure 2.

The light-operated proximity marker enzyme TurboID is a mutant of biotin ligase BirA derived from escherichia coli, the amino acid sequence of the light-operated proximity marker enzyme is shown as SEQ ID NO.65, and the amino acid sequence of the light-operated proximity marker enzyme is shown as SEQ ID NO. 66. One or more of the 183 rd lysine, 132 th tyrosine and 172 th lysine in the amino acid sequence of the optically controlled proximity marker enzyme are mutated into unnatural amino acid MNPY-lysine (MNPYK), unnatural amino acid ONB-lysine (ONBK) or unnatural amino acid ONB-tyrosine (ONBK). Light-operated proximity marker enzymes TurboID-183-MNPYK, turboID-183-ONBK, turboID-132-ONBY, turboID-172-MNPYK, turboID-172-ONBK were obtained, respectively, and the corresponding amino acid sequences thereof are shown in Table 9. Methods for construction of optically controlled proximity markers, tRNA's and tRNA synthetases for specific use are described in patent CN113481173B.

The construction method of the anti-rabbit nanobody-light control proximity marker enzyme fusion protein is described in example 2. The nanobody is fused with light-operated proximity marker enzyme through a linker, the amino acid sequence of the linker is shown as SEQ ID NO.4, and the amino acid sequence of the fusion protein is shown in Table 9 below.

Similarly, an anti-mouse nanobody-light control proximity marker enzyme fusion protein can be constructed. Or Nb2-ID photo-controlled proximity marker enzyme fusion proteins suitable for recognizing GFP.

The photo-controlled biotin ligase can be activated by ultraviolet irradiation with a wavelength of 365nm for 1 min.

TABLE 9 amino acid sequences corresponding to anti-Rabbit nanobody-light control proximity marker enzyme fusion proteins

Example 2 Induction expression and purification of nanobody-proximity marker enzyme fusion proteins

2.1 Induction of expression of nanobody-proximity marker enzyme fusion proteins in prokaryotic systems

The prokaryotic expression recombinant plasmid with the target protein is transformed into expression competent BL21 (DE 3), the monoclonal is picked up the next day, the monoclonal is connected into 2mL LB culture medium, the seed is preserved after shaking, the prokaryotic expression recombinant plasmid is amplified into 1L LB culture medium, and the prokaryotic expression recombinant plasmid is cultured in a shaking table at 37 ℃ and the rotating speed of 180 rpm. And cooling the bacterial liquid to 16 ℃ when the OD600 of the bacterial liquid reaches 0.8-1.0. For TurboID fusion protein expression, IPTG (YEASEN, cat#10902ES 08) was added directly to a final concentration of 300mM for induction, and for APEX2 fusion protein expression, IPTG was added to a final concentration of 300mM and 5-Aminolevulinic acid hydrochloride (Sigma, cat# 1.24802) to a final concentration of 1 mM. After 16h of induction expression, the cells were collected by centrifugation at 4000rpm for 20min.

2.2 Expression of nanobody-light control proximity marker enzyme fusion proteins in eukaryotic systems

(1) HEK293-F cells were inoculated into 200mL of medium in 1L flasks at an inoculum size of 1.4X10 ⁶/mL and cultured in a shaker incubator at 37℃and a rotational speed of 120rpm at 8% CO ₂. The next day, when the cell concentration reached 2x10 ⁶/mL, plasmids were transfected with PEI (Polysciences, cat# 24765-100) by the following method:

(2) 600 μl PEI was placed in a metal bath, 55℃and incubated for 20min;

Adding 100 mug of nanobody-adjacent marker enzyme fusion protein expression plasmid and 100 mug of plasmid for expressing tRNA and tRNA synthetase into 10mL of Opti-MEM, and shaking and mixing uniformly;

Adding 600 mu L PEI into 10mL Opti-MEM (Gibco, cat# 31985070), shaking and mixing;

Mixing the two tubes Opti-MEM, shaking and mixing, and standing at 37deg.C for 20min;

(3) Under dark conditions, the Opti-MEM system was added drop-wise to prepared HEK293-F cells, after which 200. Mu.L of 500mM optically controlled unnatural amino acid (synthesized by Beijing Kaiser technology Co., ltd.) was added at a final concentration of 500. Mu.L.

(4) Cells were wrapped in aluminum foil and protected from light and incubated for 72 hours in a shaker incubator at 37℃and 120rpm at 8% CO ₂. After the completion of the expression, the cells were collected by centrifugation at 2500rpm for 10 min.

2.3 Fusion protein purification

(1) Suspension of bacteria the bacterial solution was suspended by adding 30mL of PBS, to which protease inhibitors (TargetMOI, cat#C0001) and PMSF (Beyotime, cat#ST506) were added.

(2) Ultrasonic crushing, namely, 40% of power, opening for 3s, closing for 20s, and ultrasonic treating for 30min.

(3) The protein supernatant was collected by centrifugation at 14000rpm and 4℃for 1 hour.

(4) Affinity chromatography 1mL of nickel column packing (Qiagen, cat# 30210) was added to the affinity chromatography column and the column was equilibrated with 10mL of PBS prior to use. The collected protein supernatant was poured into a column and gravity flowed out (this procedure was repeated twice).

(5) Eluting the hybrid protein, adding 10mL of the hybrid washing solution (PBS, 30mM imidazole) into the nickel column, and flowing out by gravity.

(6) Elution of target protein 10mL of elution solution (PBS, 300mM imidazole) was added to the nickel column, followed by gravity flow out, and the eluate was collected.

(7) Concentration 50mL of a 10kD cut-off was used, centrifuged at 3500rpm at 4℃until the eluate was concentrated to 500. Mu.L.

(8) Dialysis, in order to remove McAc of protein solution, 10mLPBS was added to 500. Mu.L of the concentrated solution, concentrated to 500. Mu.L, and repeated once to obtain 500. Mu.L of protein.

FIG. 3 shows SDS-PAGE patterns of purified anti-rabbit and anti-mouse nanobody-TurboID fusion proteins.

Example 3 preparation of biotinylated cell samples

(1) Preparation of cells the cells were plated a day in advance in 6cm cell culture dishes with the desired amount of cells (e.g., human osteosarcoma cells U-2 OS) and the next day, the cells were fixed, and different fixing modes were selected according to different antibodies, 4% PFA/-20℃methanol/-20℃ethanol.

(2) Blocking permeabilization, namely, blocking permeabilized cells for 1h by using PBST+5% BSA solution (methanol ethanol fixation is carried out without PBST permeabilization and PBS is used instead).

(3) Primary antibody was diluted 1:200 with pbs+5% bsa solution, added to cells, incubated at room temperature for 2h or at 4 ℃ overnight. The PBS was washed three times.

(4) Incubating the nanobody-adjacent marker enzyme fusion protein by diluting the fusion protein with PBS solution at a ratio of 1:200, adding into cells, and incubating at room temperature for 1h. The PBS was washed three times.

(5) Biotinylation reaction A biotinylation reaction solution (1mL PBS+20mM MgCl ₂ +100mM ATP+500. Mu.M biotin) was added to the cells. Incubate for 16h at 37 ℃. After the reaction is finished, the staining is used for detecting the biotinylation effect, or the lysed cells are used for preparing a mass spectrum sample.

(6) Cell staining, namely washing three times by PBS after the biotinylation reaction is finished, adding Alexa Fluor ^TM 488 or 560 coupled streptavidin according to the proportion of 1:1000, simultaneously adding Alexa Fluor ^TM or 488 coupled secondary antibody according to the proportion of 1:1000, incubating for 1h at room temperature, washing three times by PBS, and detecting the biotinylation reaction effect by a fluorescence microscope.

(6) Cell lysis after the end of the biotinylation reaction, the cells were scraped off in a low adsorption tube and lysed by heating in a metal bath at 98℃for 20min at 80℃for 2h by adding 500. Mu.L of lysis buffer 1 (300 mM Tris-HCl,2% SDS,0.2M glycine, pH 9.0) three times with PBS. After completion of the lysis, the mixture was centrifuged at 14000rpm at 4℃for 20min, and the supernatant was collected.

Example 4 enrichment of biotinylated protein and preparation of Mass Spectrometry samples

(1) An equal volume of water was added to the sample and diluted one-fold. Lysis buffer 1 was diluted twice with water to give lysis buffer 2.

(2) 50. Mu.L of streptavidin C1 beads (Invitrogen, cat # 65002) were added to 200. Mu.L of lysis buffer 2, gently blotted with a pipette and resuspended sufficiently, placed on a magnetic rack for 10s separation, the supernatant removed, and this step was repeated three times. The sample from step 1 was added to the beads and incubated for 1h with shaking at 4 ℃.

(3) After the incubation, the supernatant was discarded on a magnetic rack, and the beads were washed three times by adding 200. Mu.L of lysate 2. Thereafter, 200. Mu.L of buffer A (1M KCL), buffer B (0.1M Na ₂CO₃), buffer C (50 mM Tris-HCl,2M urea, pH=7.5) were washed once, and 200. Mu.L of 100mM Tris-HCl (pH=8.0) were washed once, respectively.

(4) To the beads, 40. Mu.L of 100mM Tris-HCl (pH 8.0) was added, 1mM DTT and 0.4. Mu.L trypsin were added, and incubated at 25℃for 1 hour with shaking.

(5) After incubation, the supernatant was transferred to a new tube and the beads were washed twice with 30. Mu.L of 100mM Tris (pH 8.0) and the washes were pooled and the eluent volume at this time was 100. Mu.L.

(6) Final concentration 4mM dithiothreitol was added and incubated for 30min with shaking at 25 ℃.

(7) The final concentration of 10mM iodoacetamide was added and incubated for 45min with shaking at 25 ℃.

(8) 0.4. Mu.g trypsin was added and incubated overnight with shaking at 25 ℃.

(9) After overnight digestion, the pH of the sample was adjusted to less than 3 using 10% trifluoroacetic acid and desalted.

Example 5 enrichment of biotinylated peptide fragments and preparation of Mass Spectrometry samples

(1) Reductive alkylation 30. Mu.L of 200mM TCEP was added to the cell lysate and incubated at 55℃for 1h.

30. Mu.L 375mM iodoacetamide was added and incubated at 25℃for 30min in the dark.

(2) Methanol-chloroform to precipitate protein, 500. Mu.L methanol and 125. Mu.L chloroform were added to the sample after reductive alkylation, and the sample was thoroughly mixed by vortexing for 30 s. And 14000rpm, centrifuging at room temperature for 10min, and discarding the supernatant. 500. Mu.L of methanol was added and vortexed for 30s to mix the sample thoroughly. And (3) centrifuging at 14000rpm at room temperature for 10min, discarding the supernatant, and airing the sample.

(3) Pancreatin digestion the dried protein pellet was added with 100. Mu.L PTS buffer (100 mM Tris-HCl, pH8.0,12mM SDC,12mM SLS) and heated in a metal bath at 90℃for 10 min. mu.L of 100mM Tris-HCl (pH 8.0) was used to dissolve 20. Mu.g of pancreatin, added to the above samples and incubated overnight with shaking at 25 ℃.

(4) ProteinG beads coupling to antibody 50 μ L protein G beads (NEB, cat#S1430S) was washed 2 times with 100 μl of 300mM NaAc (pH=3.0). mu.L of anti-biotin antibody was added, 400. Mu.L of 300mM NaAc and incubated at 4℃for 2 hours with shaking. 200 μL 300mM NaAc was washed twice.

(5) Enrichment of biotinylated peptide fragments by heating the peptide fragments after overnight digestion in a metal bath at 90℃for 10min to inactivate the pancreatin. 15. Mu.L NaAc was added to adjust the pH of the sample to about 6.5. The acidified peptide fragment was added to protein G beads-coupled antibody and incubated overnight at 4 ℃.

(6) Eluting, namely, after the supernatant is discarded from the sample, 200 mu L H ₂ O is added, and the sample is washed for 3 times, and the supernatant is discarded each time. mu.L of 0.2% TFA was added, washed 2 times for 10min each, the supernatants were pooled.

(7) Desalting.

Example 6 labeling radii of nanobody-proximity marker enzyme fusion proteins in murine scaffolds and different organelles

The experimental procedure for in vitro biotinylation is shown in FIG. 4b, cells are first immobilized, blocked and permeabilized, then primary antibody targeting the target region is added, protein A-TurboID/APEX2 or anti-rabbit nanobody-TurboID/APEX 2 is added according to the primary antibody species, anti-mouse nanobody-TurboID/APEX 2, U-2OS cells are first incubated with anti- β -actin primary antibody (labeled actin filaments) (c, f, i, l), anti-vimentin primary antibody (labeled intermediate filaments) (d, g, j, m) or anti-TubA A primary antibody (labeled tubulin) (e, h, k, n), then anti-mouse nanobody-TurboID (c-e), anti-mouse nanobody-APEX 2 (f-h), or Protein A-TurboID (i-k)/APEX 2 (l-n) is added according to the procedure described in FIG. 4b, and finally the biotinylation reaction solution is added. For specific experimental procedures reference is made to example 3.

The detection results are shown in FIGS. 4c-4n, wherein the green label is Alexa Fluor ^TM 488 coupled secondary antibody, which shows the localization of the primary antibody, and the red label is Alexa Fluor ^TM 560 coupled streptavidin, which shows the distribution of biotinylated proteins.

The dotted line area measurement in c, f, i, l of fig. 4 is selected to characterize the distribution of the fluorescence signal, and the fluorescence signal index is fitted, and the result is shown in fig. 5.

Similarly, the labeling radii of Nano-ID, nano-APX, pro-ID and Pro-APX were compared using different organelle murine antibodies in U-2OS cells. For specific experimental procedures, reference is made to example 3, which is followed by incubation with anti-LAMP1 primary antibody (lysosome label) (a, d, g, j), anti-LaminB1 primary antibody (nuclear membrane label) (b, e, h, k), or anti-ATP5A1 primary antibody (mitochondrial label) (c, f, i, l), then addition of anti-murine nanobody-TurboID (a-c), anti-murine nanobody-APEX 2 (d-f), or Protein A-TurboID (g-i)/APEX 2 (j-l), and finally addition of biotinylated in vitro reaction solution. The detection results are shown in FIG. 6.

From FIGS. 4 to 6, it is understood that when the murine primary antibody is used, the Nano-ID can effectively label the target region, resulting in a high signal to noise ratio label, and Pro-ID, pro-APX or Nano-APX can also label the target region, but the label radius is too large to effectively label.

Example 7 labelling radii of nanobody-proximity marker enzyme fusion proteins in rabbit-derived scaffold and different organelles

U2OS cells were incubated with anti-Paxillin primary antibody (focal adhesion marker) (a-d), anti-PEX14 primary antibody (peroxisome marker) (e-h), or anti-EDC4 primary antibody (P-small marker) (i-l) first, then anti-rabbit nanobody-TurboID (a, e, i), anti-rabbit nanobody-APEX 2 (b, f, j), or Protein A-TurboID (c, g, k)/APEX 2 (d, h, l) were added and finally biotinylated in vitro reaction solution was added according to the procedure described in example 3.

The detection results are shown in FIG. 7, wherein green label is Alexa Fluor ^TM 488 coupled streptavidin, which shows distribution of biotinylated protein, and red label is Alexa Fluor ^TM 560 coupled secondary antibody, which shows localization of primary antibody. As can be seen from FIG. 7, when rabbit primary antibodies are used, the Nano-ID and Pro-ID can effectively label the target region, resulting in a high signal to noise ratio label, and Pro-APX or Nano-APX labels have too large a radius to effectively label the target region.

Example 8 Nano-ID is used to tag more types of organelle or post-histone modifications

U-2OS cells were incubated with anti-GOLG primary antibody (murine antibody, golgi apparatus tag) (a), anti-CD98 primary antibody (rabbit antibody, cell membrane tag) (b), anti-SC35 primary antibody (murine antibody, nuclear plaque tag) (c), anti-PDI primary antibody (rabbit antibody, endoplasmic reticulum tag) (d), anti-CEP250 primary antibody (rabbit antibody, centromere tag), anti-FUS primary antibody (murine antibody, f), anti-H3K4me3 (murine antibody, g), anti-H3K27Ac (rabbit antibody, H), anti-HAK119ub (rabbit antibody, i), anti-L-LACTYL LYSINE (j), then anti-rabbit (b, d, e, H, i, j) or anti-mouse (a, c, f, g) nanobody-TurboID, and finally the biotinylated in vitro reaction solution was added.

The detection results are shown in FIG. 8. The green label is Alexa Fluor ^TM 488 coupled streptavidin, which shows distribution of biotinylated protein, and the red label is Alexa Fluor ^TM 560 coupled secondary antibody, which shows localization of primary antibody. As can be seen from fig. 8, the Nano-ID prepared by the present disclosure is capable of recognizing more types of rabbit or mouse-derived organelle or histone post-modifications, and expands the range of antibodies that can be recognized efficiently compared to Pro-IDs in the prior art.

EXAMPLE 9 hierarchical resolution of Nano-ID on nucleoli

The nucleolus can be morphologically divided into three layers, fiber center (Fibrillar Center, FC, highly correlated with transcription of POLI), dense fiber component (Dense Fibrillar Component, DFC, the main region where ribosomal RNA cleavage occurs), particle component (Granular Component, GC, biosynthesis of ribosomes occurs), and the pattern is shown in fig. 9a. U-2OS cells were first incubated with anti-TCOF1 (rabbit antibody, labeled FC region), anti-FBL (rabbit antibody, labeled DFC region), anti-NPM1 (mouse antibody, labeled GC region) and then anti-rabbit or anti-mouse nanobody-TurboID was added and the biotinylated reaction solution was added according to the procedure described in example 3. And then detecting the biotinylation reaction condition, adding Alexa Fluor ^TM 488 coupled streptavidin into the cells to display biotinylation protein localization, adding Alexa Fluor ^TM 560 coupled secondary antibody to display primary antibody localization, wherein the detection result is shown in figures 9b-9d, the nucleolus layered structure can be clearly distinguished, and the Nano-ID localization accuracy is higher. Subsequently, U-2OS cells were treated in the same manner, lysed after the end of the biotinylation reaction, and immunoblotted experiments were performed to examine the content of biotinylated proteins as shown in FIG. 9e, which showed a significant enrichment of biotinylated proteins in the experimental group compared to the control group. After enrichment of total proteins of the experimental group and the control group by coupled streptavidin, mass spectrometry detection is carried out, and fig. 9f shows signal heat maps of TCOF1, FBL and NPM1, and fig. 9g shows mass spectrum signal heat maps of nucleolin at different layers. FIGS. 9h-9j select representative protein-interacting networks enriched in FC (9 h), DFC (9 i) and GC (9 j) structures, which are highly correlated with the function of nucleolar layering regions. The layering analysis of Nano-ID on nucleolus structure is realized by labeling nucleolus three-layer substructure in situ biotin.

EXAMPLE 10 Nano-ID is applicable to various types of sections such as FFPE and OCT

FFPE slices of lung adenocarcinoma tissues are selected for in-vitro biotinylation labeling, and paraffin-embedded lung adenocarcinoma slices are subjected to dewaxing, rehydration, PECAM1 antibody and Nano-ID adding and in-vitro biotinylation reaction. Alexa Fluor ^TM coupled antibody labeled PECAM1, alexa Fluor ^TM 488 coupled streptavidin labeled biotinylated protein, immunofluorescence detection was performed and pictures were taken by a PANNORAMIC 1000 scanner. The immunofluorescence assay results are shown in FIG. 10a, and the assay results for total protein biotinylated protein content in the experimental group (+PECAM 1 primary antibody) and control group (-, without PECAM1 primary antibody) are shown in FIG. 10b. In three patient-derived FFPE samples, the total protein of the experimental and control groups was enriched in coupled streptavidin, and the mass spectrum signal intensity of PECAM1 was shown in fig. 10c, n.d., and was undetectable. And for the overlapping condition of PECAM1 protein interactions in three patients, see figure 10d, PECAM1 interaction proteins detected in all three samples were input into Stringdb for analysis, see figure 10e, with different colors representing different signaling pathways in which the proteins participated.

Similarly, in vitro biotinylation labeling is carried out on the mouse brain OCT slice, after the mouse brain OCT slice is fixed and transparent, a GFAP antibody and a Nano-ID are sequentially added to carry out in vitro biotinylation reaction, after the reaction, a sample is incubated, alexa Fluor ^TM is coupled with a secondary antibody labeled GFAP, alexa Fluor ^TM 488 is coupled with streptavidin labeled biotinylated protein, and an immunofluorescence detection result is shown in figure 11a. FIG. 11e is a schematic diagram showing that in vitro biotinylation reaction solution biotin of the sample shown in FIG. 11a is changed into D4 isotope labeling, proteins are precipitated and digested after the reaction is completed, and after peptide fragments obtained by digestion are enriched by an anti-biotin antibody, biotinylation sites and biotin label types, namely whether wild-type biotin (endogenous biotin protein) or D4 isotope biotin labeling (D4 isotope biotin label added by Nano-ID) are identified by mass spectrometry, so that the endogenous biotinylation proteins and target biotinylation proteins are distinguished. The results of the detection of total protein biotinylated protein content of the experimental group (+gfap primary antibody) and control group (-, no GFAP primary antibody) are shown in fig. 11b, and a wien diagram of 689 endogenous wild-type biotin proteins and 795D 4-labeled proteins identified after enrichment is shown in fig. 11c. Further GO analysis was performed on the identified proteins, see 11d.

Representative immunofluorescence pictures of biotinylated markers for HTT-adjacent proteins in HTT disease model mice are shown in fig. 12a. Total protein biotinylated protein content of the experimental group (+HTT primary antibody) and control group (-, HTT primary antibody not added) is shown in 12b.

Taken together, nano-ID is compatible with FFPE sections, and can identify PECAM1 and its interacting proteins in FFPE sections if using vascular endothelial marker PECAM1 antibody, and also with OCT sections, and can identify GFAP and its interacting proteins if using astrocyte intermediate filament skeleton protein marker GFAP antibody, and these interacting proteins are involved in the maintenance of synapses, axons and dendrites in addition to being partially cellular intermediate filament skeletons.

In addition, nano-ID is also suitable for localization markers for post-immune related proteins and histones modification in FFPE and OCT sections. In vitro biotinylation labeling is carried out on FFPE slices of lung adenocarcinoma tissues, and paraffin embedded lung adenocarcinoma slices are subjected to dewaxing, rehydration, and in vitro biotinylation reaction after PDL1 antibody (a) or H3K27Ac antibody (b) and Nano-ID are added. Alexa560 conjugated antibodies labeled PDL1 (a) or H3K27Ac (b), alexa Fluor ^TM 488 conjugated streptavidin labeled biotinylated proteins. Pictures were taken with PANNORAMIC 1000,1000 scanner and the fluorescent positioning results are shown in figures 13a and 13b.

After 12.5 days of development, the biotin-labeled transcription factor PAX6 adjacent protein is on a mouse brain OCT section, the PAX6 antibody and the Nano-ID are sequentially added after the mouse brain OCT section is fixed and penetrated, in-vitro biotinylation reaction is carried out, after the reaction, a sample is incubated, alexa Fluor ^TM is 560 coupled with the antibody to label the PAX6, alexa Fluor ^TM 488 is coupled with the streptavidin to label the biotinylation protein, and a fluorescence positioning result is shown in figure 13c. In the mouse 8-cell embryo, the fluorescence results using the Nano-ID tag top domain (ApicalDomain) are shown in FIG. 13d.

EXAMPLE 11 Nano-ID can enable double labeling of a sample

The schematic of the experimental procedure for dual labelling is shown in figure 14a. Intracellular ROI1 and ROI2 were labeled with antibodies of rabbit and murine species, respectively. For example, in the first round of reaction, nano-ID (Rb) is used for marking the rabbit primary antibody by identifying the rabbit primary antibody, after the first round of reaction is completed, TEV enzyme is added for cutting, then the Nano-ID- (Rb) is cut, the free TurboID can be washed, in the second round of reaction, nano-ID (Ms) is added for identifying the mouse primary antibody, biotin in the reaction liquid is marked as D4, and then the second round of reaction is carried out, and the D4 marking is carried out on the mouse primary marked ROI 2. And (3) cell lysis, digestion of the obtained protein precipitate into peptide fragments, and biotinylation enrichment at the peptide fragment level to finally obtain wild type and D4 marked peptide fragments, so as to distinguish the first round of marked proteins from the second round of marked proteins.

As previously described, the first round uses rabbit primary anti-TFAM wild-type biotin to label mitochondria and the second round uses murine primary anti-NPM1 isotope D4 biotin to label nucleoli. The first and second rounds of labelled biotin proteins were observed with Alexa Fluor ^TM 488 and Alexa Fluor ^TM 560 coupled streptavidin, respectively, and the results of the assays are shown in FIG. 14b. Immunoblotting results for TFAM alone, NPM 1-labeled, and dual TFAM and NPM 1-labeled are shown in fig. 14c.

Taken together, the Nano-ID of the present disclosure can achieve dual labeling of the same sample.

EXAMPLE 12 Nano-ID allows the labelling of cell lines or model organisms expressing GFP fusion proteins

In order to better adapt to GFP-over-expression or GFP-gene knock-in cell lines and model animals such as nematodes, drosophila and the like, a Nano antibody for recognizing rabbit or mouse IgG in Nano-ID is replaced by a Nano antibody for recognizing GFP, nb2-ID is obtained, nb2 is a Nano antibody for specifically recognizing GFP, the amino acid sequence of the Nano antibody is shown as SEQ ID NO.46, the amino acid sequence of Nb2-ID is shown as SEQ ID NO.47, and the nucleotide sequence of Nb2-ID is shown as SEQ ID NO. 48.

FIGS. 15a-c show in vitro biotin labeling results of cell lines (b) expressing mEmerald-SRSF3 (a) or mEmerald-enscondin (c) or SC35 gene loci knocked in GFP using CRISPR after incubation with Nb2-ID, with Alexa Fluor ^TM 560 coupled streptavidin for detection of biotinylated proteins. FIG. 12d shows that nematodes stably expressing ajm-1-GFP, which had a development stage of 1.8fold, were incubated with Nb2-ID and subjected to in vitro biotinylation, and that the samples were assayed for biotinylated protein using Alexa Fluor ^TM 560 conjugated streptavidin, respectively.

Taken together, nano-ID can also enable the labelling of GFP-expressing cell lines or model organisms.

Example 13 verification of biotinylated Capacity of nanobody-light operated proximity marker enzyme fusion protein

In this example, the biotinylated ability of nanobody-light control proximity marker enzyme fusion proteins was further demonstrated.

After fixing, penetrating and blocking U-2OS cells, incubating rabbit antibody anti-PEX14 anti-ibody (a) or mouse antibody anti-Tubulin anti-ibody (b), adding nanobody-light-operated proximity marking enzyme of corresponding species, and performing light irradiation and non-light irradiation treatment on experimental groups and control groups, and performing biotinylation reaction. After the reaction is finished, immunofluorescent staining is carried out, and imaging is carried out.

As a result, as shown in FIG. 15, in the control group which was not subjected to light treatment, the biotin protein signal was hardly seen, i.e., the fusion protein was in a state in which the enzyme activity was inhibited. After the light treatment, the secondary antibody signal coupled with Alexa Fluor ^TM 647 and the biotinylated protein signal coupled with Alexa Fluor ^TM 488 are co-localized, which proves the localization precision of the fusion protein and the recovery of the enzyme activity after the light treatment.

The technical scheme provided by the disclosure can more fully and fully reveal protein interaction networks and modification modes in cells and tissues, which is helpful for in-depth understanding of a regulation mechanism of a biological process, provides more accurate targets and strategies for drug development and disease treatment, and simultaneously has wide application prospects and can play an important role in the field of space proteomics.

The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.

Claims (16)

1. 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. 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 marker 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, MgCl ₂ , ATP and biotin; Preferably, the biotinylation reaction solution comprises biotin phenol and hydrogen peroxide. 14. A proximity labeling method, characterized in that a protein interacting with a target protein is biotin-labeled using the fusion protein according to any one of claims 1 to 10 or the kit according to claim 13, comprising 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 use comprises: (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.