A nanobody specifically binding to maltose binding protein and uses thereof

Abstract

The application provides a kind of nanobody specifically binding maltose binding protein and application, belong to the field of biotechnology.Nanobody can recognize the new epitope on MBP different from maltose binding pocket, so its binding with MBP is not affected by the presence or absence of maltose.This makes the purification process can be carried out in the buffer containing maltose, and elution without adding maltose, significantly simplify the process and avoid foreign sugar pollution, realize the efficient purification without sugar elution, stable protein complex formation and target protein release under mild conditions, significantly improve the applicability and reliability of MBP fusion protein in purification, structure analysis and function research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a nanobody that specifically binds to maltose-binding protein and its applications. Background Technology

[0002] One of the most common methods for purifying proteins or protein complexes in *E. coli* or mammalian cells is to express the target protein as a fusion protein with a proximity affinity tag. An affinity tag is an artificial polypeptide, typically grafted onto the N-terminus or C-terminus of the target protein by inserting the cDNA sequence encoding the tag peptide near the open reading frame. Utilizing the ability of these tags to specifically bind to particular substances, the fused target protein can be pulled down from the complex system, achieving purification. Simultaneously, the tag can optimize the fusion protein in terms of solubility, conformational stability, structural flexibility, and yield. Many types of tags are commonly used for protein purification, including polyhitidine tags, FLAG tags, Strep II tags, glutathione S-transferase (GST) tags, and so on.

[0003] Maltose-binding protein (MBP), a commonly used solubility-enhancing tag, is widely used to improve the expression level, folding efficiency, and water solubility of exogenous proteins. Numerous studies have shown that MBP can not only significantly improve the expression level and correct folding rate of poorly soluble proteins, but also serve as an effective affinity tag for subsequent purification. Traditional MBP purification systems rely on its specific binding ability to maltose and its polymers (such as amylose). Typically, cell lysates are passed through an amylose resin column, thoroughly washed, and then competitively eluted using a maltose-containing buffer to obtain the target fusion protein.

[0004] However, this technical approach has revealed several insurmountable limitations in practical applications. First, the elution process is highly dependent on exogenous small molecule ligands (such as maltose), which not only increases reagent costs and operational steps, but more importantly, residual maltose can severely interfere with downstream experiments—for example, acting as a substrate or inhibitor in enzyme activity assays, affecting results; introducing additional electron density or causing lattice disorder in structural biology studies (such as X-ray crystallography or cryo-electron microscopy); and triggering non-specific signaling pathway activation in cell function experiments. Second, maltose elution is essentially a thermodynamically equilibrium-driven competitive process, and elution efficiency is greatly affected by maltose concentration, temperature, and protein-tag conformational state, often leading to unstable recovery rates or requiring multiple elutions, reducing the overall yield. Third, and more importantly, this system cannot meet the overall purification requirements of protein complexes. When MBP fusion proteins form stable complexes with their interacting partners, maltose elution can release MBP-tagged proteins, but this is often accompanied by complex dissociation and loss of native conformational information, greatly limiting its value in interactionomics and structure-function studies.

[0005] Nanobodies—specifically, the single-domain variable region (VHH) derived from camel heavy chain antibodies (HCAbs)—have become ideal candidates for next-generation molecular recognition tools due to their unique advantages. Compared to traditional IgG antibodies (~150 kDa), nanobodies are only about 15 kDa, exhibiting extremely high thermal stability, tolerance to extreme pH levels (e.g., pH 2–11), and resistance to denaturants and protease degradation. Their complementarity-determining region 3 (CDR3) is typically long and can form a convex ring structure, enabling them to penetrate protein grooves or cryptic epitopes that are inaccessible to traditional antibodies, achieving highly specific recognition. Affinity systems constructed using nanobodies as ligands can effectively circumvent the inherent limitations of small-molecule ligand systems, achieving more flexible, gentler, and more efficient protein manipulation. These characteristics have made them not only a focus of attention in the development of therapeutic antibodies but also demonstrate broad prospects in basic scientific research.

[0006] However, to date, no publicly reported non-competitive nanobody recognition systems targeting MBP tags have been developed. Existing MBP purification techniques still rely entirely on the classic but limited paradigm of maltose-amylose. Therefore, there is an urgent need for a novel binding molecule that can directly, specifically, and with high affinity bind to MBP itself (rather than its ligands). Summary of the Invention

[0007] The purpose of this invention is to provide a nanobody that specifically binds to maltose-binding protein and its application. This nanobody can non-competitively recognize novel epitopes of MBP and maintain high affinity binding in the presence of maltose, thereby achieving efficient purification without sugar elution, stable protein complex formation, and release of the target protein under mild conditions, significantly improving the applicability and reliability of MBP fusion proteins in purification, structural analysis, and functional studies.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a nanobody that specifically binds to maltose-binding protein, the nanobody comprising a complementarity-determining region or a conserved variant of the complementarity-determining region; The complementary determination regions include CDR1, CDR2, and CDR3; The CDR1 includes one or more of the sequences SEQ ID NO.31 to SEQ ID NO.49; The CDR2 includes one or more of the sequences SEQ ID NO.50 to SEQ ID NO.67; The CDR3 includes one or more of the sequences SEQ ID NO.68 to SEQ ID NO.84.

[0009] Preferably, the conservative variants of the complementarity determination region include: Variants that differ from the complementarity-determining region sequence by ≤3 amino acids and maintain a KD ≤100 nM; Or a variant that has 1-5 amino acid substitutions, deletions, or additions compared to the complementarity-determining region sequence and can specifically bind to MBP; Or a polypeptide with 80% or more identity to the complementarity-determining region sequence, and capable of specifically binding to variants of MBP.

[0010] The present invention also provides a nucleic acid molecule encoding the above-mentioned nanobody.

[0011] The present invention also provides a recombinant expression vector comprising the nucleic acid molecules described above.

[0012] The present invention also provides a recombinant host cell containing the recombinant expression vector described above.

[0013] The present invention also provides an affinity chromatography medium comprising a solid matrix and the aforementioned nanobody, wherein the nanobody can be immobilized on the surface of the solid support in a covalent or non-covalent manner, or in a directional or non-directional manner.

[0014] Preferably, the solid matrix includes one or more of agarose matrix, magnetic particles, and porous polymer carrier.

[0015] This invention also provides a method for purifying MBP or MBP fusion proteins, comprising the following steps: The sample containing MBP or MBP fusion protein is mixed with an affinity chromatography medium to specifically capture the MBP or MBP fusion protein. After washing and elution, high-purity MBP or MBP fusion protein is obtained. The MBP or MBP fusion protein can be released from the affinity medium by changing the elution conditions or cleaving the linker sequence.

[0016] The present invention also provides a stable complex of MBP or MBP fusion protein, wherein the complex is a complex formed by the above-mentioned nanobody and MBP or MBP fusion protein.

[0017] The present invention also provides an immunoprecipitation kit comprising the nanobodies described above.

[0018] The present invention also provides a multi-antibody system that simultaneously binds to MBP, characterized in that it includes two or more of the above-mentioned nanobodies, wherein the nanobodies are linked in the form of tandem single chains, diabody or IgG-Fc fusion.

[0019] This invention also provides the application of the above-mentioned nanobody, nucleic acid molecule, recombinant expression vector, recombinant host cell, affinity chromatography medium, complex for stabilizing MBP or MBP fusion protein, immunoprecipitation kit or multi-antibody system in any of the following aspects, characterized in that it includes: (1) Affinity purification of MBP or MBP fusion protein; (2) Immunoprecipitation or protein capture; (3) Enhanced protein stability; (4) Protein structure analysis; (5) As an alternative to starch- or maltose-based affinity purification systems; (6) X-ray crystallographic analysis; (7) Single-particle cryo-electron microscopy structural analysis; (8) As a structural guide support.

[0020] The beneficial effects of this invention compared to the prior art are as follows: (1) Pioneering a “non-competitive” combination model The nanobody of this invention can recognize novel epitopes on MBP that differ from the maltose binding pocket, thus its binding to MBP is unaffected by the presence or absence of maltose. This allows the purification process to be carried out in a maltose-containing buffer, and elution does not require the addition of maltose, significantly simplifying the process and avoiding exogenous sugar contamination. It overcomes the dependence of traditional MBP affinity systems on competitive elution of small molecules, significantly broadening its application scenarios and solving the problem of dependence on exogenous sugars in traditional MBP affinity systems.

[0021] (2) Excellent physicochemical stability Thanks to the high stability of nanobodies, the affinity media constructed in this invention can maintain activity over a wide pH range (such as pH 2.0) and under harsh conditions such as high salt and reducing agents, supporting more thorough column regeneration and decontamination, significantly extending the service life of the media, and is suitable for the purification of poorly soluble or easily degradable proteins.

[0022] (3) High-efficiency capture and flexible elution The system of this invention exhibits nanomolar-level (≤10 nM) high affinity, with significantly higher capture efficiency for MBP and its fusion proteins than traditional amylose media. Furthermore, the elution method is highly flexible—it can achieve rapid elution using low-pH buffers (e.g., pH 2.2–3.0), or non-destructive co-elution of target proteins (including complexes) by introducing protease-cleavable linkers between the nanobody and the carrier. This adapts to the needs of cutting-edge structural biology research such as cryo-electron microscopy (cryo-EM), achieving high-affinity capture and diverse elution strategies. The affinity media constructed based on the aforementioned nanobody possesses high specificity and high affinity, enabling efficient capture of MBP or MBP fusion proteins with flexible elution methods.

[0023] (4) Structural stability The system described in this invention is suitable for forming stable protein complexes, which helps maintain the conformational integrity of the target protein and provides a powerful tool for structural biology research. Nanobodies and carriers can be gently eluted without destroying the complex, making them particularly suitable for single-particle cryo-electron microscopy (cryo-EM) structural analysis, avoiding the risk of denaturation.

[0024] (5) High versatility This invention provides a highly versatile and scalable MBP-related separation and analysis platform that can be widely applied in basic research, biotechnology, and protein engineering. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1-1 The following are the screening results of MBP nanobodies in this embodiment of the invention. A represents the results of ELISA after three rounds of screening of the alpaca-derived nanobodies library against MBP-Tdp43. The absorbance at 450 nm was measured after color development (all values ​​were subtracted from the average value of the negative controls E12-H12). The red color scale indicates the relative size. B represents the experimental results of BLI testing after sequencing to remove repetitive sequences, expressing a small amount of 8 antibodies, purifying them with Ni-NTA, and using MBP as the immobilizer. Nb5 and Nb6 are repetitive sequences, hence the names Nb1-5 and 7-9. Figure 1-2 The results of MBP nanobody screening in this embodiment of the invention are shown in C, where C is the predicted structure of the top 10 antigen-antibody complexes at the average ipTM after three repetitions of Alphafold3 prediction, all of which are bound to the same epitope; D is the predicted structure of the 11th-22nd positions of the average ipTM and the predicted structure of the MBPNb4&Nb8 complex, excluding MBPNb4 bound to 4 different epitopes. Figure 2 The electrophoresis results of Ni-NTA purification of MBPNb4 and MBPNb8 in the embodiments of the present invention are shown. In this embodiment, A is SUMO-MBPNb4, B is SUMO-MBPNb8, and lanes 1-7 are, in order, whole bacteria, supernatant, flow-through, Ulp1 digestion followed by flow-through 1-3, and NiB elution (the main band is Ulp1). Figure 3 The following are the electrophoresis results of Ni-NTA purification of HME1-LC3B and SUMO-MBP using anion exchange in the embodiments of the present invention. In lane A, lanes 1-5 show the whole bacteria, supernatant, flow-through, and NiB elution of HME1-LC3B (Ni-NTA purification process) in sequence, and lanes 6-14 show the elution of anion exchange column; lane B shows the whole bacteria, supernatant, flow-through, NiB elution 1, NiB elution 2, Ulp1 digestion (smaller bands correspond to the digested SUMO), Ni column reverse coating, and flow-through 1, flow-through 2, flow-through 3, and flow-through 4 in lanes 1-10; lane C shows the NaCl gradient elution of anion exchange column in lanes 1-10. Note that lanes 6-9 have smaller bands, which are the residual SUMO. Figure 4 This is a BLI curve diagram of HME1-LC3B and MBPNb4&8 in an embodiment of the present invention; Figure 5 The figures show the SPR curves of MBPNb4 and 8 binding to MBP in embodiments of the present invention, where A is the Nb4 binding dissociation curve with concentration gradients of 0.3, 0.9, 2.7, 8.1, and 24.3 (nM); and B is the Nb8 binding dissociation curve with concentration gradients of 0.3, 0.9, 2.7, 8.1, and 24.3 (nM). Figure 6 This document describes parallel purification experiments using Amylose, MBPNb4, and MBPNb8 media in this invention. A represents the electrophoresis results of the supernatant before and after gel coupling of MBPNb8 with NHS, with the order within the group being before loading and after overnight coupling. B represents the electrophoresis results of parallel purification of HME1-LC3B using Amylose and MBPNb8, with lane 1 representing before loading, and the order within the group being flow-through, washing, elution 1, and elution 2. C represents the structure of MBP-THAP11 predicted by AlphaFold 3, with orange representing MBP, green representing the 8xHis tag and linker, and blue and red representing THAP11, where red indicates the location of 48Q, and yellow-green represents the alpha and Avi tag. D represents the electrophoresis results of parallel purification of MBP-THAP11 using Amylose and MBPNb8, with lanes 2, 4, and 6 representing Amylose media before loading, flow-through, and elution, respectively, and lanes 3, 5, and 7 representing MBPNb8 media before loading, flow-through, and elution, respectively. The black arrows indicate the location of the target band; lanes E represent HME1-LC3B before loading (bacterial supernatant), Amylose elution, Nb4 medium elution, and Nb8 medium elution, respectively; lanes 6-9 represent MBP-ATXN3 before loading (bacterial supernatant), Amylose elution, Nb4 medium elution, and Nb8 medium elution, respectively. Figure 7This is an example of an experiment involving MBPNb4 & 8 and the MBP monomer & HME1-LC3B complex in this invention. A shows the structural diagram of the MBP complex with MBPNb4 and MBPNb8 predicted by AlphaFold 3, with magenta representing MBPNb4 and orange representing MBPNb8; B shows the electrophoresis results of samples collected from tubes after overnight incubation of the MBP monomer with MBPNb4 and MBPNb8 and passing through a Superdex 75 molecular sieve, with tubes 4-10 in the group; C shows the electrophoresis results of samples collected from tubes after passing HME1-LC3B with MBPNb4 and MBPNb8 through a molecular sieve, with tubes 5-9, 12-13, 27-31, and 33-34 in the group; D is a schematic diagram of a system where the nanobody and fusion protein are cleaved together; E is a schematic diagram of a nanobody tandem system with different binding sites; F shows the crystallization of the HME1-LC3B+MBPNb4 complex under Crystallization conditions. ScreenB6;G: Crystals formed from the HME1-LC3B+MBPNb8 complex, crystallized under Crystal Screen B8 conditions. Figure 8 This is an anion exchange column experiment of MBPNb4&8 and MBP monomer & HME1-LC3B complex in an embodiment of the present invention. Figure A shows the SDS-PAGE diagram of the separation experiment of MBP, HME1-LC3B and MBPNb4&Nb8 complex anion exchange column; Figure B shows the SDS-PAGE diagram of the separation experiment of MBP and MBPNb4 complex anion exchange column; Figure C shows the left peak of lanes 1-3 in Figure A (HME1-LC3B+Nb4). (Collected samples from tubes 3, 4, and 5), lanes 4-8 correspond to the right-side peaks (collected samples from tubes 7, 9, 11, and 13). Figure D shows the collected samples from tubes 3-13 corresponding to lanes 1-11 in Figure B, where lanes 1-5 correspond to the nanobody peaks and lanes 6-11 correspond to the complex peaks; Figure E shows the left-side peaks of (MBP+MBPNb8) corresponding to lanes 9-11 in Figure A (collected samples from tubes 3, 4, and 5), and lanes 12-15 correspond to the right-side peaks (collected samples from tubes 9, 11, 13, and 15). Figure 9 In the embodiments of the present invention, the horizontal axis for detecting the thermal stability of the MBPNb4&8 and MBP monomer complex is temperature, and the vertical axis is the first derivative of the fluorescence intensity ratio (350 nm / 330 nm). The peak / valley points correspond to the inflection points of the fluorescence intensity ratio, which are also the unfolding temperatures. The temperature corresponding to the first inflection point is marked in the figure. Figure 10 This is an immunoprecipitation electrophoresis image of the MBPNb4&8 and MBPHTSSSA complex in an embodiment of the present invention, where lane 1 corresponds to MBPNb4 and lane 2 corresponds to MBPNb8. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] In this invention, the "nanobody" is not limited to the specific amino acid sequences listed in the specification or sequence listing. As long as the antibody molecule can specifically bind to MBP and thereby produce the technical effects described in this invention, it should be considered to fall within the protection scope of this invention.

[0033] In some embodiments, the nanobody may comprise a variant with one or more amino acid substitutions, deletions, or additions compared to a reference sequence, preferably a conserved amino acid substitution. The technical effects of the present invention can be achieved as long as the variant can still specifically bind to MBP.

[0034] In some embodiments, the nanobody may have at least about 80%, 85%, 90%, 95% or higher amino acid sequence identity with the reference sequence and retain its ability to bind to MBP. Those skilled in the art can determine whether the variant still has MBP binding ability using methods known in the art, including but not limited to immunoadsorption assays, biolayer interference, surface plasmon resonance, and enzyme-linked immunosorbent assays.

[0035] The nanobodies described in this invention also include their derived forms or equivalent forms, such as multivalents, fusion proteins, tandem structures, forms linked to functional domains or markers, or multispecific molecules containing multiple binding domains capable of recognizing MBP. Any of the above forms that can achieve specific binding to MBP and produce the corresponding technical effect should be considered equivalent technical solutions of this invention.

[0036] Those skilled in the art should understand that any modifications, substitutions, or combinations made to the sequence, structure, or expression form of nanobodies without departing from the overall concept and technical effect of the present invention are equivalent transformations of the present invention and should be included within the scope of protection of the present invention.

[0037] Example This invention provides an example of preparing a nanobody that specifically binds to maltose-binding protein. The specific steps are as follows: Table 1 Ni-NTA Affinity Chromatography Buffer

[0038] Table 2 Media Coupling Buffer

[0039] Table 3 Amylose gel-related buffers

[0040] Table 4. Crystallization conditions of Hampton Crystal Screen 1 / 2

[0041] 1. Construction and amplification of plasmids: Based on the complete sequences shown in SEQ ID NO:1 to SEQ ID NO:30, complete sequences of nanobodies were designed, as shown in Table 5. Table 5. Complete sequence of nanobodies

[0042] Continued from Table 5: Complete sequence of nanobodies

[0043] Continued from Table 5: Complete sequence of nanobodies

[0044] Continued from Table 5: Complete sequence of nanobodies

[0045] Continued from Table 5: Complete sequence of nanobodies

[0046] Based on the sequences in Table 5, complete nanobody sequences were synthesized from the whole genome and ligated into pSUMO plasmid vectors containing 10xHis-SUMO. Codons were optimized for *E. coli*. Synthesis was provided by Genewiz. The synthesized plasmid powder was diluted to 100 ng / μL. 100 μL of competent DH5α cells were thawed on ice for 10 min, 1 μL of plasmid solution was added, and the mixture was incubated on ice for 30 min. After heat shock at 42°C for 60 s, the mixture was placed on ice for 1 min. 200 μL of LB medium was added in a clean bench, and the mixture was incubated at 37°C in a shaker at 220 rpm for 45 min. After centrifugation at 6000 rpm for 1 min, 200 μL of supernatant was aspirated, and the mixture was resuspended and plated onto LB solid medium containing 0.1 mg / mL kanamycin. The mixture was incubated overnight at 37°C inverted mode. Single colonies were picked and cultured overnight at 220 rpm in a shaker at 37°C in 4 mL of LB medium containing 0.1 mg / mL ampicillin. Plasmids were extracted using the ABclonal plasmid extraction kit.

[0047] 2. Expression of the target protein Add 1 μL of the plasmid extracted in step 1 to 100 μL of thawed competent BL21(DE3) cells on ice and incubate for 20 min. Then, incubate in a 42℃ metal bath for 1 min, add 200 μL of LB medium, and incubate at 37℃ for 45 min on a shaker at 220 rpm. Centrifuge at 6000 rpm for 60 s, collect 200 μL of the supernatant, mix thoroughly by pipetting, and plate. Incubate inverted at 37℃ overnight. Use a 10 μL pipette tip to pick a single colony and transfer it to 4 mL of antibiotic-treated LB medium. Incubate overnight at 37℃ for 220 rpm. Add 1 mL of the bacterial culture to 1 L of antibiotic-treated LB medium and incubate at 37℃ for 4–7 h. Transfer to 20℃ at 180 rpm and add 400 μL of 1M IPTG for induction for 20 h. Centrifuge at 5000 rpm for 15 min to collect the precipitate, dilute with NiA to 50 mL, break up using an autoclave, centrifuge at 17000 rpm for 1 h to collect the supernatant.

[0048] 3. Ni-NTA purification On-column digestion: Equilibrate the Ni-NTA column with 3 column volumes of NiA, add the supernatant after lysis and mix thoroughly. Add 3 column volumes of NiA, NiC, and NiA sequentially for washing. Add 2 mg of Ulp1 enzyme diluted with NiA and digest overnight. At this point, the target protein is in the liquid phase. Collect the effluent and use Nanodrop to determine the protein concentration. Add sufficient NiA until the target protein is fully washed. Add 3 column volumes of NiB to elute the SUMO tag and Ulp1 enzyme bound to the medium. Then, perform column regeneration.

[0049] NiB direct elution: Equilibrate the Ni-NTA column with 3 column volumes of NiA, add the supernatant after lysis and mix thoroughly, then add 3 column volumes of NiA, NiC, and NiA sequentially for washing, add NiB directly in portions, collect the eluent and use Nanodrop to determine the protein concentration, elute thoroughly until the eluted protein concentration is extremely low, and then perform column regeneration.

[0050] Column regeneration: Add 5 column volumes of 0.1 M EDTA to remove nickel ions, add 5 column volumes of deionized water to remove EDTA, add 5 column volumes of 0.1 M NaOH and soak for 1 hour to further remove EDTA and residual protein, add 5 column volumes of deionized water to remove NaOH, add 5 column volumes of 0.1 M nickel sulfate to allow nickel ions to recombine in the medium, and add 5 column volumes of deionized water to remove nickel sulfate.

[0051] 4. Molecular sieve purification Connect the Superdex 200 molecular sieve column to the AKTA chromatography system, set the pressure limit to 1.5 MPa, and the flow rate to 0.5 mL / min. Wash with GF buffer until UV and conductivity remain constant. Before loading, concentrate the target protein solution to 1 mL and replace it with GF buffer. Centrifuge at 12000 rpm for 10 min. Carefully aspirate the supernatant using a 1 mL syringe, expel all gas from the syringe, and load the solution into the AKTA system well. After loading, zero the UV value and continue washing with GF buffer. Use peak collection. When the UV value exceeds 20 mAU, start collecting the eluent in 1 mL tubes.

[0052] 5. Medium coupling The purified protein (ligand) solution environment was changed to GF buffer using a concentration tube. 10 mL of NHS agarose medium was poured onto the column, and the isopropanol preservation solution was washed away with 10 volumes of 1 mM HCl in an ice bath followed by hydrochloric acid. The column was washed twice with 10 volumes of Buffer S, and the ligand solution was added and thoroughly mixed. The column was incubated at room temperature for 4 h. The ligand solution was removed by washing with 10 volumes of deionized water, and 10 volumes of Buffer A were added for passivation and blocking for 6 h, followed by washing with deionized water. 10 volumes of Buffer B were added, followed by washing with deionized water. 10 volumes of Buffer A were added again, followed by washing with deionized water. The column was equilibrated with 10 volumes of GF buffer (20 mM HEPES, 150 mM NaCl) and then added to the preservation solution.

[0053] 6. Nanobody Screening 6.1 Antigen Biotin Labeling: Prepare a reaction system with a final concentration of 100 μM of the protein to be labeled (with an avi tag), 1 μM BirA, 0.15 mM biotin, 5 mM magnesium chloride, and 1 mM ATP. React in a mixer for 1 h, then add equal amounts of biotin and BirA, and react at room temperature for 1 hour. Remove impurities such as BirA enzyme and biotin by loading the sample onto a molecular sieve. After incubating the labeled protein with streptavidin, perform electrophoresis. If the molecular weight increases by 17 kDa compared to before labeling, indicating that streptavidin has been bound, the labeling is considered successful. The labeling efficiency can be judged by the band ratio.

[0054] 6.2 Host bacterial amplification: After thawing the TG1 strain preserved in glycerol, spread it onto antibiotic-free LB agar plates and incubate overnight. Pick one or more TG1 Escherichia coli colonies from the plate and transfer them to 2 mL of antibiotic-free 2YT medium. Incubate at 37°C and 250 rpm until slightly turbid, approximately 3 hours.

[0055] 6.3 Phage Display Using Magnetic Beads: Prepare a 3% skim milk solution (MPBS) using PBS. In a 1.5 mL EP tube, prepare a mixture containing 330 μL MPBS, 620 μL PBS, and 50 μL of phage library. Mix at room temperature for 0.5 h to reduce non-specific binding. Add the mixture to an EP tube containing 5 μg (3 μg for the second round and 2 μg for the third round) of biotin-labeled antigen. Mix at room temperature for 0.5 h to ensure sufficient antigen-antibody binding and complex formation. Add 20 μL of Streptavidin magnetic beads and mix at room temperature for 1.5 h. The antigen-antibody complex is immobilized on the magnetic beads by the antigen-labeled biotin. Place the EP tube on a magnetic rack for 1-2 minutes, aspirate the supernatant, add 1 mL of PBST, gently blow down the magnetic beads to mix and wash, then place the tube on the magnetic rack again and aspirate the supernatant. Repeat this process multiple times, increasing the number of washes with each round, to thoroughly remove non-specific phages bound to the magnetic beads. After the final wash, add PBS to remove the PBST, aspirate the PBS, and then add 100 μL of PBS to resuspend the magnetic beads. Add the magnetic bead suspension to 2 mL of TG1 bacterial culture and incubate at 37°C for 45 minutes. After infection, aspirate 2 μL of the bacterial culture to dilute and plate the tube to determine if nanobodies have been screened and to estimate nanobodies abundance. In the third round, dilute to 10³, 10⁴, and 10⁵ and plate with ampicillin for final ELISA validation.

[0056] 6.4 Phage amplification: Add glucose solution (final concentration 2%) and ampicillin (final concentration 0.1 g / L) to the phage-infected bacterial culture, and incubate at 37℃ and 250 rpm for 1 h 45 min to amplify the bacterial count. Add 10 μL of M13 helper phage to assemble with the phage plasmid to form a complete phage, which can then be released. Incubate at 37℃ for 45 min. Aliquot the bacterial culture into EP tubes and place them on a magnetic rack. Transfer the supernatant to a new EP tube, centrifuge the supernatant at 10000 rpm for 10 min, then remove the supernatant. Resuspend the bacteria in 2YT medium. Add the bacterial culture to a 50 mL shaker containing 10 mL of 2YT (containing 0.1 g / L ampicillin and 0.05 g / L kanamycin), and incubate overnight at 30℃ and 250 rpm to allow for sufficient bacterial amplification and phage assembly and release.

[0057] 6.5 Phage Extraction: Centrifuge the cultured bacterial solution at 4000 rpm for 20 min at 4°C. Pour the supernatant into a 50 mL centrifuge tube containing 2.5 mL of PEG-8000. Incubate on ice for 45 min to precipitate the phage. Then centrifuge at 4000 rpm for 20 min at 4°C. Carefully discard the supernatant; a distinct white precipitate should be present at the bottom of the tube. Centrifuge again at 4000 rpm for 5 min at 4°C to remove the supernatant from the tube wall. After aspirating the supernatant, gently resuspend the phage in 500 μL of PBS. Transfer the solution to a 1.5 mL centrifuge tube, determine the concentration, and calculate the amount of phage solution to be added in the next round using the formula: 1000 / (measured concentration * 2.33) (μL). Repeat this process up to the third round.

[0058] 6.6 nanoparticle antibody expression and antigen coating: For the third round of plating, first prepare a 96-well shake plate containing 100 μL of 2YT medium (containing 0.2% glucose and 0.1 g / L ampicillin) per well. Select a plate with suitable colony density, pick one single colony from each well, and thoroughly pipette it into the medium. Incubate at 37°C and 250 rpm for 7 hours to allow bacterial amplification. Transfer 15 μL of bacterial culture from the shake plate to each well of a shake plate containing 150 μL of 2YT medium (containing only 0.1 g / L ampicillin). Incubate at 37°C and 250 rpm for 1 hour. Then add 15 μL of 1M IPTG to each well and incubate overnight at 30°C and 180 rpm to induce bacterial expression of phages carrying nanobodies. Simultaneously, take a 96-well ELISA plate, add 50 μL of 2 ng / μL antigen to each well, and incubate at 4°C overnight to allow the antigen to coat the bottom of the wells through hydrophobic interactions.

[0059] 6.7 Enzyme-linked immunosorbent assay (ELISA): The next day, discard the antigen and wash 5 times with PBST to remove unfixed antigen. The washing volume depends on the well volume of the ELISA plate. (For all operations involving discarding liquid, tap the plate – invert it onto clean absorbent paper and gently tap it on a work surface lined with multiple layers of paper to thoroughly remove the supernatant). Then, add 100 μL of 3% MPBS to each well and block at 37°C for 1 hour to reduce subsequent non-specific binding. Discard the MPBS and wash 5 times with PBST. In a clean bench, add 50 μL of bacterial culture from the shake plate to each well of a 96-well plate, according to the corresponding positions. Aspirate the bacterial culture before adding it, but do not aspirate during addition. Incubate at 37°C for 1.5 hours (a negative control is required, i.e., wells without bacterial culture, as the baseline for subsequent ELISA). Discard the bacterial culture and wash 5 times with PBST. Add 50 μL of 1 / 10000 anti-HA-tag (secondary antibody) to each well and bind at 37°C for 45 minutes. Discard the liquid and wash 5 times with PBST. Add 50 μL of chromogenic solution to each well and react at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL of 2M HCl. Measure the absorbance at 450 nm using a SpectraMax microplate reader. The absorbance can be used to preliminarily infer the binding ability of the nanobody to the antigen. Results are as follows: Figure 1-1 , 1-2 As shown.

[0060] 7. Biomembrane Layer Interference (BLI) This invention uses biotin-labeled MBP-Tdp43 as the antigen. After three rounds of phage display screening, a series of nanobodies were obtained, with OD values ​​exceeding the negative control (0.05) by up to 16 times after ELISA colorimetry. After obtaining the antibody coding sequences through bacterial sequencing, duplicate nanobodies were removed. Parallel small-scale expression was performed using BL21, followed by Ni-NTA purification and affinity determination with MBP using BLI assays. MBP and Tdp43 were immobilized separately using SA probes, and BLI experiments were conducted using the nanobodies as analytes.

[0061] For cured products without Avi tags, biotin labeling is first performed using the NHS-Biotin kit, utilizing the coupling reaction of the primary amino group in the NHS domain. The reagent:target molar ratio is 1.5:1. The reaction is carried out at room temperature for 1 hour. Excess reagent is removed using a desalting column, and the labeled target product is washed off with PBS. The sample with the highest concentration is then used for subsequent curing.

[0062] Prepare a PBS + 0.02% Tween buffer solution. Dilute the solidified material to 500 mM with the buffer solution. Perform serial dilutions of the analyte: 1000 mM, 500 mM, 250 mM, 125 mM, 62.5 mM, 31.25 mM, etc. For SA and HIS1K sensors, use 10 mM pH=2.0 glycine as the regeneration condition. Add each sample to a BLI-specific 96-well plate according to the program settings, generally in the following order: buffer solution (baseline 1), solidified material, buffer solution (baseline 2, dissociation environment), various concentrations of analyte, regeneration solution, buffer solution. Immerse the sensor tip in the buffer solution for at least 10 min to pre-wet it. Place the sensor housing and 96-well plate in their respective positions on the instrument and start the program. The program design generally performs binding detection from low to high analyte concentrations. The results are shown in Table 6.

[0063] Table 6. BLI results of MBP nanobody binding to MBP and Tdp43

[0064] Table 6 shows that the seven different nanobodies (Nb1-5, 7, 8) exhibited binding to MBP (fitted R0). 2 >0.95) rather than binding with Tdp43, the equilibrium dissociation constant (KD) is 10. -9 M level.

[0065] Table 7. Alphafold3 prediction results of MBP nanobody (second batch) and MBP complex.

[0066] As shown in Table 7, the first 20 bits of the ipTM sequence have multiple different binding methods with MBP, binding in 4 different table positions.

[0067] The two antibodies with the lowest KD values ​​were selected: MBPNb4 (KD = 0.588 ± 0.009 nM, R...). 2 =0.9813), MBPNb8 (KD=1.070±0.007 nM, R 2 =0.9679), as a representative example, subsequent experiments were conducted.

[0068] Similar to the MBP tag, ubiquitin-like small molecule modified protein (SUMO) can also be used as a fusion tag to improve the solubility and stability of the target protein. When fused to the N-terminus of the target protein, it can be cleaved from the fusion protein by the natural yeast protease Ulp1. Therefore, before expressing the MBP nanobody, a plasmid containing 10His-SUMO-MBPNb was designed, synthesized by a third party, and expressed in E. coli BL21(DE3). During the Ni-NTA purification process, after the loading and washing steps, the on-column enzyme digestion method was directly used, adding 2 mg of Ulp1 and digesting at room temperature for 3 h. Since Ulp1 has a His tag, it will be immobilized on the Ni gel, so the target protein can be obtained by collecting the flow-through. Since there is no need to use high concentration of imidazole to elute the sample, the impurities that are non-specifically bound to the medium will not be washed down with the target protein, and the target protein with high purity can be obtained directly.

[0069] MBPNb4 and MBPNb8 purified by Ni-NTA (on-column enzyme digestion) in step 3 were subjected to electrophoresis, and the results are as follows: Figure 2 As shown.

[0070] Figure 2 The results showed that both MBPNb4 and MBPNb8 had good soluble expression capabilities, and samples with high concentration and purity could be obtained after Ni-NTA purification.

[0071] 8. Expression and purification of HME1-LC3B and MBP To further determine the affinity between the nanobody and the fusion protein, HME1-LC3B was selected as the test subject. HME1 is a modified MBP variant designed in previous studies. By adding a rigid linker to the C-terminus of the MBP mutant, the structural stability of the fusion protein is improved, thus facilitating crystallization. It is a commonly used MBP alternative tag. LC3B is a subtype of microtubule-associated protein 1 light chain 3 (LC3). Because previous purification processes revealed that eluting HME1-LC3B using a gravity chromatography column yielded a significant amount of contaminating proteins, an AKTA system with UV monitoring was used to ensure thorough washing, and an imidazole gradient elution was designed to better remove non-specifically bound contaminating proteins. Figure 3 Electrophoresis results showed that HME1-LC3B had good soluble expression capacity in E. coli, and after one-step purification, it already had high purity. After replacing the buffer with GF buffer using an ultrafiltration tube, it could be used for subsequent analysis. The results are as follows... Figure 3 As shown.

[0072] 9. Affinity test of MBPNb4 & MBPNb8 with HME1-LC3B and MBP The affinity of MBPNb4&8 for MBP was determined using surface plasmon resonance (SPR). All SPR experiments were performed on a Cytiva Biacore™ 8K system using the CM5 sensor chip (Cytiva). The run buffer was HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20), and all samples were dialyzed or diluted with this buffer to avoid matrix effects. Experiments were conducted at a constant temperature of 25 °C.

[0073] Antigens or target proteins are immobilized on the detection channel surface of the CM5 chip via standard EDC / NHS chemical coupling. The specific steps are as follows: First, the chip surface is activated by sequentially injecting a 0.2 M EDC / 0.05 M NHS mixture at a flow rate of 10 μL / min for 7 min. Then, the ligand is dissolved in 10 mM sodium acetate buffer (pH 4.0–5.5, optimized according to isoelectric point) at a flow rate of 5–10 μL / min until the target immobilization level is reached (typically 200–2000 RU, depending on molecular weight and binding characteristics). Finally, unreacted active groups are blocked with 1 M ethanolamine-HCl (pH 8.5) for 7 min. One or more reference channels undergo the same activation and blocking treatment but are not coupled with ligands to eliminate non-specific binding and system drift.

[0074] Combined analysis employs single-cycle kinetics (SCK) or multi-cycle modes. Analytes (such as antibodies or interacting proteins) are arranged in concentration gradients (typically 5–6 concentrations, covering the estimated Kc). D The buffer was injected sequentially at a flow rate of 30 μL / min at 0.1–10 times the original concentration, with a binding time of 120–180 s, followed by dissociation monitoring at 300–600 s. After each binding round, pulse elution (10–30 s) was performed using an optimized regeneration buffer (e.g., 10 mM glycine-HCl pH 1.5–3.0, 10–50 mM NaOH, or a solution containing 0.5–2 M MgCl2) to restore the response signal to baseline and ensure the chip surface is reusable.

[0075] Raw sensor data were processed using Biacore™ Insight Evaluation software, including dual-reference subtraction (subtraction of the reference channel signal and the buffer blank injection signal). Kinetic parameters (binding rate constant k) were also analyzed. on Dissociation rate constant k off and equilibrium dissociation constant (K) D=k off / k on The result was obtained by globally fitting a 1:1 junction, as shown below. Figure 4 , 5 As shown in Tables 8 and 9.

[0076] The results showed that the dissociation equilibrium constant KD was in the nM range, and this result remained essentially unchanged after the addition of 10 mM maltose, indicating that maltose could not competitively inhibit the binding of nanobodies to MBP.

[0077] Furthermore, SPR and biofilm layer interference (BLI) were used to further detect the affinity of the two nanobodies for HME1-LC3B. The results showed that the KD was in the nM level, which was close to the KD value of the nanobodies-MBP, indicating that the fused LC3B had no significant effect on the antigen-antibody interaction between MBPNb4&8 and HME1. This nanobodies can be used to develop a purification system for MBP fusion proteins.

[0078] Table 8 BLI results of HME1-LC3B and MBPNb4&8

[0079] Table 9 SPR results of MBPNb4 & 8 combined with MBP

[0080] 8. Media performance testing To ensure adequate coupling, two coupling concentrations of 2 mg / mL and 4 mg / mL were used for the nanobodies, with 3 mg of coupling gel medium prepared for each concentration. Electrophoresis results showed that both groups were fully coupled after overnight reaction. However, a new band at 30 kDa was observed in the lane after coupling at 4 mg / mL, indicating that the nanobodies may have polymerized. Given the background on nanobodies tandem coupling discussed earlier, this polymerization, depending on the polymerization mode, may have a positive effect on purification efficiency. Furthermore, considering that at high concentrations, the cross-linking reaction between NHS and the antibody may reach equilibrium, potentially solidifying more nanobodies, 4 mg / mL was used as the medium for subsequent testing.

[0081] Take multiple small centrifuge columns with a capacity of 0.8 mL, add 700 μL of GF buffer, centrifuge at 12000 rpm for 1 min to fully wet the column, and after the liquid in the column has drained, add 100 μL of buffer solution and mark the outside of the liquid level for subsequent accurate filling of 100 μL of different types of media. Wash three times with 700 μL of GF buffer to remove the 20% ethanol used for storage. Then add an equal volume of sample solution to each centrifuge column, mix thoroughly, and incubate at room temperature for 1 h.

[0082] For Amylose gels, washing was performed three times with 700 μL of Amylose Wash Buffer, followed by fractional elution with 200 μL of Amylose Elu Buffer. For nanobody media, washing was performed three times with 700 μL of NiC buffer. 22 μL of elution protection buffer was added to the EP tube beforehand to prevent protein denaturation due to prolonged exposure to low pH. Then, 200 μL of 10 mM glycine was added for fractional elution and collection. The purification efficiency was evaluated by SDS-PAGE after sample collection.

[0083] In previous purification processes in the laboratory, it was found that the Amylose medium had a weak binding ability to MBP-THAP11 (48Q) and the purification results contained many impurities. Therefore, we tried to use nanobody media for purification testing.

[0084] from Figure 6 Electrophoresis results showed that with only 5 repeated loadings, the binding capacity of HME1-LC3B in the MBPNb8 medium was low. However, after incubation for 1 hour following loading, followed by washing and elution, the purification effect of the nanobody medium on the target protein was similar to that of the Amylose medium in terms of loading capacity and purity. For MBP-THAP11, a direct 1-hour incubation was used, indicating that the nanobody medium can capture MBP-THAP11 and can serve as an alternative to the Amylose system.

[0085] 9. MBPNb4&8 can form complexes with MBP monomer &HME1-LC3B. To explore the application potential of the nanobodies of this invention in structural biology, this invention verified whether MBPNb4 and MBPNb8 can form stable antigen-antibody complexes with MBP monomers and the MBP fusion protein HME1-LC3B. Previously, researchers designed a scaffold structure called "Legobody" based on the composite structure of MBP, Protein A, and nanobodies. Some proteins whose structures are difficult to resolve due to their small molecular weight can be analyzed using single-particle cryo-electron microscopy after forming a complex with this scaffold. Furthermore, due to the unique structural shape of the scaffold, the target protein is easily located in the cryo-electron microscopy images. In other words, by forming a complex with a relatively stable structural protein, the molecular weight of the target protein is increased, which facilitates the structural resolution of the target protein under cryo-electron microscopy. Simultaneously, anion exchange columns were also used to verify the complex.

[0086] Electrophoresis and UV monitoring show that after the antigen-antibody mixture at a ratio of 1:1.5 binds to the chromatography column, if NaCl gradient elution is performed, the nanobody will wash down before the complex, and the UV value shows a double peak. In the electrophoresis results, the nanobody band first increases, then decreases, and then increases again from left to right, proving that the chromatography column does not simply separate the antigen and antibody, but rather separates the antibody and the complex. This indirectly proves that the antibody can form a complex with the antigen.

[0087] Therefore, a system was designed: gel-directed coupling sequence-cleavable linker-nanobody-MBP-target protein. After the nanobody is bound to the MBP fusion protein, a protease is added to cleave the cleavable linker, allowing the nanobody-fusion protein complex to be directly washed off. This increases the protein molecular weight, facilitating structural resolution, while avoiding the extreme pH elution environment required for antigen-antibody binding. To preliminarily verify the feasibility of this system, complex experiments were conducted with the nanobody, MBP monomer, and HME1-LC3B, respectively. The antibody-antigen molar ratio was set to 1.5:1. After overnight incubation at 4°C, excess nanobody was removed by separation using a molecular sieve.

[0088] Electrophoresis showed that the final effluent from the molecular sieve consisted of nanobodies, while the samples collected from the previous peaks all showed two bands in electrophoresis: antigen and antibody. This result indicates that both nanobodies formed complexes with MBP monomers and HME1-LC3B.

[0089] According to AlphaFold 3's predictions, MBPNb4 and MBPNb8 have different binding sites with MBP, which means that heterogeneous antibody tandems can be designed to improve both affinity and specificity. Therefore, to confirm the predicted binding sites, after obtaining the complex, further crystallization of the complex was attempted to resolve its structure via X-ray diffraction, providing a reference for subsequent modification and optimization. Microscopically visible protein crystals have now been obtained, demonstrating that this nanobody can be used to form stable complexes and for structural analysis (see...). Figure 7 , 8 ).

[0090] 10. Thermal stability analysis of nanobody-MBP complex The melting temperatures (Tm) of the two MBP nanobodies, MBP, and antigen-antibody complexes were determined using nanoDSF (label-free differential scanning fluorescence) technology. Figure 9 As shown in Table 10.

[0091] Table 10 Thermal stability test results of MBPNb4&8 and MBP monomer complex

[0092] The results showed that the Tm value of the MBP-nanobody complex was significantly increased compared to that of the monomeric MBP, indicating improved thermal stability.

[0093] 11. Immunoprecipitation Mix 20 μg of antigen with 20 μg of specific antibody, and bring the total volume to 100 μL with appropriate buffer. Incubate on ice for 45 minutes to form antigen-antibody complexes. Then add approximately 20 μL of Strep-Tactin® resin (premixed in GF buffer, final volume approximately 40 μL), and incubate at 4°C on a rotary mixer for 1 hour to capture the immune complexes. After the reaction, wash the resin four times with 1 mL of GF buffer, resuspending thoroughly each time and centrifuging to remove the supernatant to remove non-specifically bound proteins. Finally, add 50 μL of Strep Elution Buffer (containing desthiobiotin) and incubate at room temperature for 5 minutes for specific elution. Add 12 μL of 5× SDS-PAGE loading buffer to the eluent, mix well, denature at 95°C for 5 minutes, and load 20 μL for SDS-PAGE analysis. Figure 10 As shown.

[0094] SDS-PAGE results showed that the antibody band and the antigen band appeared in the same lane, proving that the antibody can bind to the MBP fusion protein.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanobody that specifically binds to maltose-binding protein, characterized in that, The nanobody includes a complementarity-determining region or a conserved variant of the complementarity-determining region; The complementary determination regions include CDR1, CDR2, and CDR3; The CDR1 includes one or more of the sequences SEQ ID NO.31 to SEQ ID NO.49; The CDR2 includes one or more of the sequences SEQ ID NO.50 to SEQ ID NO.67; The CDR3 includes one or more of the sequences SEQ ID NO.68 to SEQ ID NO.

84.

2. The nanobody that specifically binds to maltose-binding protein according to claim 1, characterized in that, Conservative variants of the complementarity determination region include: Variants that differ from the complementarity-determining region sequence by ≤3 amino acids and maintain a KD ≤100 nM; Or a variant that has 1-5 amino acid substitutions, deletions, or additions compared to the complementarity-determining region sequence and can specifically bind to MBP; Or a polypeptide with 80% or more identity to the complementarity-determining region sequence, and capable of specifically binding to variants of MBP.

3. A nucleic acid molecule encoding a nanobody as described in any one of claims 1 to 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3.

5. A recombinant host cell, characterized in that, The recombinant host cell contains the recombinant expression vector as described in claim 4.

6. An affinity chromatography medium, characterized in that, The affinity chromatography medium comprises a solid matrix and the nanobody according to any one of claims 1 to 2.

7. The affinity chromatography medium as described in claim 6, characterized in that, The solid matrix includes one or more of agarose matrix, magnetic particles, and porous polymer carriers.

8. A method for purifying MBP or MBP fusion protein, characterized in that, Includes the following steps: The sample containing MBP or MBP fusion protein is mixed with the affinity chromatography medium described in claim 6 or 7 to specifically capture the MBP or MBP fusion protein. After washing and elution, high-purity MBP or MBP fusion protein is obtained.

9. A stable complex of MBP or an MBP fusion protein, characterized in that, The complex is a complex formed by the nanobody of any one of claims 1 to 2 and MBP or MBP fusion protein.

10. An immunoprecipitation kit, characterized in that, Including nanobodies as described in any one of claims 1 to 2.

11. A multi-antibody system that simultaneously binds to MBP, characterized in that, The invention comprises two or more nanobodies according to any one of claims 1 to 2, wherein the nanobodies are linked in the form of tandem single chains, Diabody or IgG-Fc fusion.

12. The use of any of the following aspects of a nanobody according to claim 1-2, a nucleic acid molecule according to claim 3, a recombinant expression vector according to claim 4, a recombinant host cell according to claim 5, an affinity chromatography medium according to claim 6 or 7, a complex for stabilizing MBP or MBP fusion protein according to claim 9, an immunoprecipitation kit according to claim 10, or a multi-antibody system according to claim 11, characterized in that, include: (1) Affinity purification of MBP or MBP fusion protein; (2) Immunoprecipitation or protein capture; (3) Enhanced protein stability; (4) Protein structure analysis; (5) As an alternative to starch- or maltose-based affinity purification systems; (6) X-ray crystallographic analysis; (7) Single-particle cryo-electron microscopy structural analysis; (8) As a structural guide support.

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