High-affinity monoclonal antibodies against His-tags attached to the C-terminus of membrane proteins

Abstract

To provide a high-affinity monoclonal antibody for a His-tag added to the C terminus of membrane protein.SOLUTION: The present invention provides a monoclonal antibody for a histidine tag (His-tag) or an antigen-binding fragment thereof, wherein the monoclonal antibody recognizes, as an epitope, a His-tag added to the C terminus of a membrane protein.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a high-affinity monoclonal antibody against a His-tag added to the C-terminus of a membrane protein.

Background Art

[0002] A tag is very important for protein purification, detection, etc. in experiments for verifying the structure, function, etc. of a protein to be studied. In particular, tag addition is indispensable in experiments for purifying recombinant proteins.

[0003] A histidine tag (His-tag) is a tag characterized by being composed of 6 to 10 consecutive histidine residues, and is one of the most commonly used tags in protein purification. Since it forms a coordination bond with a divalent transition metal ion, metal affinity purification using a column immobilized with nickel or cobalt is possible.

[0004] However, metal affinity purification often causes problems such as a large amount of non-specific adsorption due to host-derived proteins expressed in addition to the protein to be purified, and denaturation of the protein to be purified due to metal coordination.

[0005] On the other hand, a polypeptide tag antibody system in purification utilizes the property of binding with high specificity, which is a characteristic of an antibody, and enables elution of a target protein under mild conditions by binding an antibody and its epitope peptide. Furthermore, since the antibody protein is stable and the epitope peptide can be released and neutralized by an acidic solution, regeneration (reuse) of the antibody protein is also possible. In this polypeptide tag antibody system, for example, FLAG, HA, Myc, etc. are known as typical tags, and elution of a target protein using an antibody against these has also been performed.

[0006] A plurality of types of antibodies against His-tag, including monoclonal antibodies and polyclonal antibodies, are commercially available.

[0007] Non-patent document 1 investigates the usefulness of four commercially available monoclonal antibodies against His-tag in surface plasmon resonance (SPR) measurements. After immobilizing the four antibodies on a measurement substrate by amine coupling, the binding affinity to 12 human-derived proteins to which His-tags consisting of six histidine residues were attached was measured, and the dissociation constant was investigated in particular. However, these antibodies have only been used to detect His-tag-attached proteins, and the detailed properties of the antibodies have not been clarified.

[0008] Non-patent document 2 discusses purification using anti-His-tag antibodies. However, this involves purification using antibodies on a fraction that has already undergone metal affinity purification, and the nonspecific binding caused by proteins derived from cell (bacterial) lysate, which is a problem in purification, has not been investigated. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] doi:10.1007 / 978-3-642-011-44-3_42 [Non-Patent Document 2] doi:10.1006 / abio.1998.2606. [Overview of the project]

[0010] This invention provides a high-affinity monoclonal antibody or its antigen-binding fragment against a His-tag attached to the C-terminus of a membrane protein.

[0011] The inventors have discovered a monoclonal antibody that exhibits high affinity for His-tags attached to the C-terminus of membrane proteins. This invention is based on these findings.

[0012] The present invention provides the following: (1) A monoclonal antibody or antigen-binding fragment against a histidine tag (His-tag), wherein the monoclonal antibody or antigen-binding fragment recognizes the His-tag attached to the C-terminus of a membrane protein as an epitope. (2) The monoclonal antibody or antigen-binding fragment described in (1), wherein the His-tag consists of six or more consecutive histidine residues. (3) A monoclonal antibody or antigen-binding fragment according to (1) or (2), wherein the His-tag consists of 6 to 12 consecutive histidine residues. (4) A monoclonal antibody or antigen-binding fragment according to any of (1) to (3), wherein the membrane protein constitutes a nanodisk. (5) A monoclonal antibody or antigen-binding fragment according to any of (1) to (4), wherein the equilibrium dissociation constant (KD) for interaction with His-tag is 450 nM or less. (6) A monoclonal antibody or antigen-binding fragment according to any of (1) to (5), wherein the equilibrium dissociation constant (KD) for interaction with His-tag is 35 nM or less. (7) A monoclonal antibody or its antigen-binding fragment produced by hybridoma cells having receipt number NITE AP-03635, deposited with the National Institute of Technology and Evaluation (NITE) on April 27, 2022. (8) An antibody or antigen-binding fragment thereof that binds to the epitope to which the monoclonal antibody described in (7) binds. (9) An immobilized antibody comprising an antibody or antigen-binding fragment described in any of (1) to (8) immobilized on a solid support. (10) A method for purifying a membrane protein from a solution containing a membrane protein to which a His-tag has been added to the C-terminus, (a) A step of contacting the membrane protein with an antibody or antigen-binding fragment described in any of (1) to (8) to form a complex composed of the membrane protein and the antibody or antigen-binding fragment. (b) A step of isolating the complex formed in step (a), and (c) Isolating the membrane protein from the complex isolated in step (b) to obtain a filtrate containing the membrane protein A method comprising the above. (11) The method according to (10), wherein the membrane protein is a recombinant protein. (12) The method according to (10) or (11), further comprising, in step (a), a step of purifying the membrane protein using a surfactant. (13) The method according to any one of (10) to (12), wherein the membrane protein constitutes a nanodisc.

[0013] According to the present invention, a high-affinity monoclonal antibody against the His-tag added to the C-terminus of a membrane protein or an antigen-binding fragment thereof can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] FIG. 1 shows the results of a confirmation test for the purification of T4L protein added with His-tag and the results of Western blotting using cHis8 as a primary antibody. [Figure 2] FIG. 2 shows the results of measuring the binding affinity of cHis8 using BLItz. [Figure 3] FIG. 3 shows the results of purifying A2a (GPCR) using cHis8 resin by surfactant purification / nanodisc purification. [Figure 4] FIG. 4 shows the results of purifying hERG (ion channel) using cHis8 resin by surfactant purification / nanodisc purification. [Figure 5] FIG. 5 shows the results of purifying P-glycoprotein (ABC transporter) using cHis8 resin by surfactant purification / nanodisc purification. DETAILED DESCRIPTION OF THE INVENTION

[0015] According to one aspect of the present invention, a monoclonal antibody against a His-tag or an antigen-binding fragment thereof is provided, and the monoclonal antibody or the antigen-binding fragment thereof recognizes the His-tag added to the C-terminus of a membrane protein as an epitope.

[0016] The monoclonal antibody of the present invention can be obtained by known methods. For example, the monoclonal antibody of the present invention can be obtained by immunizing a non-human mammal with an antigen membrane protein having a His-tag added to the C-terminus, or a partial peptide containing the His-tag of the antigen membrane protein, collecting antibody-producing cells (e.g., B cells) from the immunized non-human mammal, fusing the antibody-producing cells with myeloma cells to produce a hybridoma (fused cell line), and collecting the antibody produced from the hybridoma. The His-tag added to the membrane protein is not particularly limited, and for example, a His-tag consisting of 6 to 10 consecutive histidine residues can be used.

[0017] To obtain the monoclonal antibody of the present invention, the full-length protein of the membrane protein having a His-tag added to the C-terminus can be used as an immunizing antigen. The full-length protein of the membrane protein having a His-tag added to the C-terminus is not particularly limited and may be obtained by known methods. Such methods include, but are not limited to, for example, expressing and purifying the target membrane protein having a His-tag added to the C-terminus intracellularly using Escherichia coli or the like, or using a commercially available product. In addition, to obtain the monoclonal antibody of the present invention, a partial peptide containing the His-tag of the membrane protein having a His-tag added to the C-terminus can be synthesized and used as an immunizing antigen.

[0018] To obtain the monoclonal antibody of the present invention, the type of non-human mammal to be immunized is not particularly limited, and examples include mice, rats, guinea pigs, rabbits, dogs, goats, etc., and preferably mice. The non-human mammal to be immunized may be an animal with an autoimmune disease. Immunization is not particularly limited and may be performed, for example, by injection into the vein, subcutaneously, intraperitoneally, intramuscularly, subplantar subcutaneously, etc. The interval between immunizations is not particularly limited and may be performed 1 to 10 times at intervals of several days to several weeks, preferably at intervals of 1 to 2 weeks.

[0019] The antibody-producing cells for obtaining the monoclonal antibody of the present invention are not particularly limited, but may be prepared from spleen cells or regional lymph nodes of immunized non-human mammals, and are preferably prepared from spleen cells. It is not necessary to perform a specific procedure to separate antibody-producing cells from the collected cell population, but it is preferable to separate only antibody-producing cells from the cell population.

[0020] By fusing the antibody-producing cells described above with myeloma cells, it is possible to create hybridomas that continuously proliferate semi-permanently while producing monoclonal antibodies against His-tag. This fusion may be carried out by known cell fusion methods, but is not limited to them. For example, it can be performed by fusion in the presence of a cell fusion promoter, or by using a commercially available cell fusion device that utilizes electrical stimulation (e.g., electroporation). As the myeloma cells to be fused with the antibody-producing cells, commonly available cell lines from animals such as mice can be used. The cell lines used are not limited to those described above, but those that cannot survive in HAT selective medium (a medium containing hypoxanthine, thymidine, and aminopterin) and can only survive when fused with antibody-producing cells may be used. Examples of myeloma cells, but not limited to those described above, include SP2 / 0, P3U1, NSI, and P3-X63-Ag8.653, with P3U1 being preferred.

[0021] Hybridomas that produce the monoclonal antibody of the present invention can be selected from cells after cell fusion treatment. The method for selecting hybridomas may be a known method and is not limited thereto, but for example, it may be performed by culturing the cells after cell fusion treatment for a certain period of time in a suitable medium such as HAT medium to form colonies, collecting the culture supernatant from each well of the colony-positive culture plate, and confirming the antibody titer against the His-tag attached to the C-terminus of the membrane protein. The method for confirming the antibody titer against the His-tag attached to the C-terminus of the membrane protein is not limited to enzyme immunosorbent assay (ELISA) or radioimmunoassay (RIA), and ELISA is preferred.

[0022] Cloning may be performed on the selected cells in the wells to form single cells. Cloning may be performed by known methods, but is not limited to them. For example, the cell suspension may be appropriately diluted with a suitable medium (e.g., RPMI1640 medium containing 10-20% FCS), the cells may be seeded in each well of a culture plate (preferably so that there is one cell in each well to increase the probability that there is one cell in each well), and after a certain period (e.g., 7-10 days, during which it is preferable to confirm that there are single colonies), the culture supernatant of the colony-positive wells may be collected and the antibody titer of the collected culture supernatant may be checked. Alternatively, cells that produce antibodies showing high affinity for His-tags attached to the C-terminus of membrane proteins may be selected, and the cells in the selected wells may be increased to a certain extent to establish a hybridoma strain. Cloning may also be performed several times as needed. These operations yielded, for example, a hybridoma with receipt number NITE AP-03635, which was deposited with the National Institute of Technology and Evaluation (NITE) on April 27, 2022. This hybridoma produces an anti-His-tag monoclonal antibody called cHis8.

[0023] From the established hybridoma strain, monoclonal antibodies against His-tag according to the present invention can be purified and collected by appropriate methods. This may be done by known methods, and is not limited thereto, but may include, for example, preparing antibodies from culture supernatant cultured in a medium with a reduced serum concentration, preparing antibodies from culture supernatant cultured in a commercially available serum-free medium, or injecting hybridomas into the peritoneal cavity of animals, collecting ascites fluid, and preparing antibodies from that ascites fluid.

[0024] While not limited to specific methods, established hybridoma strains can be cultured using methods such as culture flasks, spinner flasks, shaker flasks, or bioreactors.

[0025] The monoclonal antibody of the present invention may be purified by known methods, and is not limited thereto. Examples include purification by His-tag affinity column, purification by ion exchange chromatography, and purification by affinity column of an immunoglobulin-binding protein such as protein A or protein G. The monoclonal antibody of the present invention can be purified by appropriately selecting or combining these known methods.

[0026] The epitope (antigenic determinant) of the monoclonal antibody of the present invention is a His-tag attached to the C-terminal side of a membrane protein that exhibits high affinity for the monoclonal antibody of the present invention. Specifically, although not limited, it preferably consists of 6 or more consecutive histidine residues, more preferably 6 to 15 consecutive histidine residues, more preferably 6 to 12 consecutive histidine residues, more preferably 7 to 10 consecutive histidine residues, and more preferably 8 consecutive histidine residues. Furthermore, although not limited, the membrane protein in the present invention preferably forms a nanodisk, and the nanodisk may consist of at least the following components: MSP, lipids, and membrane proteins. The lipids may be, although not limited, phospholipids, lipid bilayers, naturally derived lipids, synthetic lipids, and mixtures thereof.

[0027] The equilibrium dissociation constant (KD) of the interaction between the monoclonal antibody of the present invention and the membrane protein to which His-tag is attached at the C-terminus is, but is not limited, preferably 450 nM or less, more preferably 35 nM or less, more preferably 30 nM or less, and more preferably 25 nM or less.

[0028] The antigen-binding fragment of the monoclonal antibody of the present invention refers to, and is not limited to, a portion of the monoclonal antibody of the present invention, but examples include Fab, Fab', F(ab')2, Fv, diabody (dibodies), dsFv, scFv (single chain Fv), etc. The above antigen-binding fragment is not limited to, but examples include, cleaving the monoclonal antibody of the present invention with various proteolytic enzymes as needed, and the V of the monoclonal antibody of the present invention. H and V L It can be obtained by creating a genetically modified organism from cDNA encoding the antigen. The antigen-binding fragment of the monoclonal antibody of the present invention binds to the same antigen (especially the epitope) to which the monoclonal antibody of the present invention binds.

[0029] As described above, Fab can be obtained, for example, by treating an antibody molecule with papain, and F(ab')2 can be obtained, for example, by treating an antibody molecule with pepsin. Furthermore, Fab' can be obtained, for example, by cleaving the disulfide bond in the hinge region of F(ab')2.

[0030] The above scFv is, for example, the V of the antibody. H and V L This can be obtained by acquiring a cDNA encoding scFv, constructing a DNA encoding scFv, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0031] The above diabody is, for example, the V of an antibody. H and V L This can be obtained by acquiring a cDNA encoding scFv, constructing a DNA encoding scFv such that the amino acid sequence length of the peptide linker is 8 residues or less, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0032] The above dsFv is, for example, the V of the antibody. H and V L It can be obtained by acquiring a cDNA encoding dsFv, constructing a DNA encoding dsFv, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0033] The monoclonal antibodies of the present invention may also be recombinant antibodies or antigen-binding fragments prepared and expressed by recombinant means, for example, chimeric antibodies, humanized antibodies, or fully human antibodies. These antibodies in the present invention may be prepared according to known methods, for example, by recombinant expression of heavy and light chains.

[0034] Furthermore, in the present invention, chimeric antibodies, humanized antibodies, and humanized antibodies may be prepared using hybridoma or DNA or RNA extracted from said hybridoma as raw materials, according to known methods.

[0035] Generally, antibodies can be immobilized, for example, on a solid support made of a water-insoluble substrate using known methods. Based on the specific intermolecular affinity between this immobilized antibody and antigen, it is possible to selectively capture (bind) a target membrane protein from a solution containing various proteins.

[0036] Therefore, by immobilizing the His-tag antibody of the present invention on the surface of a solid support, for example, and capturing the membrane protein with the His-tag attached to its C-terminus with the immobilized anti-His-tag antibody on a column packed with the solid support, it is possible to purify the membrane protein with the His-tag attached to its C-terminus.

[0037] Therefore, according to one aspect of the present invention, an immobilized antibody is provided, wherein the antibody or an antigen-binding fragment of the present invention is immobilized on a solid support.

[0038] The solid support in this invention is not limited to, but examples include resin. The immobilization of the antibody or its antigen-binding fragment on the solid support is not limited to, but may be carried out by, for example, an amine coupling method.

[0039] Furthermore, according to one aspect of the present invention, a method is provided for purifying a membrane protein from a solution containing a membrane protein to which a His-tag has been added to the C-terminus, the method comprising: (a) contacting the membrane protein with an antibody of the present invention or an antigen-binding fragment thereof to form a complex composed of the membrane protein and the antibody or an antigen-binding fragment thereof; (b) isolating the complex formed in step (a); and (c) isolating the membrane protein from the complex isolated in step (b) to obtain a filtrate containing the membrane protein. The method of the present invention may further include a step of purifying the membrane protein using a surfactant in step (a).

[0040] The membrane proteins in this invention are not limited to, but include, for example, GPCRs (G-Protein Coupled Receptors), ion channels, transporters, etc., and are preferably recombinant proteins.

[0041] The membrane proteins in this invention may constitute nanodiscs. The method of this invention makes it possible to improve the yield of membrane proteins constituting nanodiscs compared to conventional methods. [Examples]

[0042] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples. Unless otherwise specified, the content is expressed in mass percent.

[0043] Preparation of purified proteins To express FLAG-mNeonGreen(mNG)-8xHis (SEQ ID NO: 1), mNG-3xFLAG-8xHis (SEQ ID NO: 2), MBP-TEVsite-tagRFP-8xHis (SEQ ID NO: 3), HRV 3C protease (SEQ ID NO: 4), TEV protease (SEQ ID NO: 5), FLAG-BRIL-6xHis (SEQ ID NO: 6), T4L (SEQ ID NOs: 7 and 8, respectively), MSP1D1 (SEQ ID NO: 9), and MSP2N2 (SEQ ID NO: 10), which have a His-tag consisting of 6-10 histidine residues attached to the N-terminus and C-terminus, the genes corresponding to each protein were inserted into the E. coli expression vector pET28, resulting in FLAG-mNeonGreen(mNG)-8xHis, mNG-3xFLAG-8xHis, MBP-TEVsite-tagRFP-8xHis, and HRV. T4L, which has 3C protease, TEV protease, FLAG-BRIL-6xHis, and a His-tag consisting of 6-10 histidine residues attached to its N-terminus / C-terminus, was expressed by transforming E. coli BL21(DE3), while MSP1D1 and MSP2N2 were expressed by transforming E. coli BL21(DE3)RIL. The attached His-tag was purified using a Ni Sepharose 6 Fast Flow column (Cytiva) metal affinity column. After purification, MBP-TEVsite-tagRFP-8xHis was digested with TEV protease and used as MBP and tagRFP-8xHis.

[0044] The genes corresponding to the A2a (SEQ ID NO: 11), hERG (SEQ ID NO: 12), and P-glycoprotein (SEQ ID NO: 13) proteins, with the His-tag added, were designed to become the 3C protease site-mNeonGreen-8xHis and inserted into the animal cell expression vector pEG. Expi293F (Thermo Corporation), which allows for suspension cell culture, was used as the expression host, and shaking culture was performed using HE200CD medium (Gmep Corporation). Transient expression of A2a and P-glycoprotein was performed using polyethyleneimine (PEI), and infection expression of hERG was performed by generating baculovirus according to the method described in the publicly available literature (doi:10.1016 / j.cell.2017.03.048.).

[0045] Establishment, production, and purification of the anti-His-tag antibody cHis8. Purified FLAG-mNG-8xHis was used to immunize autoimmune disease mice (MRL / MpJJmsSLC-lpr / lpr, manufactured by SLC Corporation Japan) five times at 7-day intervals. Splenocytes were collected from the immunized mice and fused with myeloma cells (P3U1) using the PEG method to create hybridomas. Antibodies produced in the hybridoma culture supernatant were used either as stock or after purification. Purification was performed by affinity purification of IgG antibodies by subjecting the hybridoma culture supernatant to Protein G Sepharose Fast Flow (Cytiva). Antibody concentration and buffer replacement were performed using Amicon Ultra 50MWCO (Merck Millipore).

[0046] The binding specificity of antibodies produced in hybridomas was verified. Verification was performed using the ELISA method. Purified antigen proteins were immobilized on immunoplates (Thermo) at concentrations of 1-10 μg / mL. Blocking was then performed with 1% BSA / PBST solution. As the primary antibody, either the stock solution of hybridoma culture supernatant or 10 μg / mL of purified IgG antibody was used. As the secondary antibody, an HRP-labeled anti-mouse IgG (Fc-specific) antibody was used at a 10,000-fold dilution. In each step, the reaction was carried out at 37°C for 1 hour, followed by washing using a plate washer. Subsequently, color development was performed using a 1-step Ultra TMB ELISA (Thermo), and the target protein was detected. Using this ELISA method, antibodies that specifically react with mNG-8xHis were selected, and to rule out the possibility of anti-mNG antibodies, binding to multiple proteins with the His-tag antigen was confirmed, thereby establishing cHis8, an anti-His-tag antibody.

[0047] The binding specificity of the established cHis8 was verified. Verification was performed by Western blotting. First, T4L, in which a His-tag consisting of 6-10 histidine residues was added to the N-terminus / C-terminus, was expressed using the method described above and purified. To confirm that the purified protein was uniform, a His-tag specific staining solution was used, followed by Western blotting using cHis8 as the primary antibody. The SDS-PAGE-treated polyacrylamide gel was transferred to a PVDF membrane using a tris-glycine-based buffer. Blocking was then performed using Blocking One (Nacalai Tesque). A 1 μg / mL purified antibody IgG was used as the primary antibody. As the secondary antibody, an HRP-labeled anti-mouse IgG (Fc-specific) antibody was used at a 5000-fold dilution. In each step, the reaction was carried out at 37°C for 1 hour, followed by washing with PBST. After that, color development was performed using Chemi-Lumi One Ultra (Nacalai Tesque) to detect the target protein. As a result, cHis8 showed little reactivity to His-tags attached to the N-terminus of T4L, but showed good reactivity to His-tags consisting of 6 to 10 histidine residues attached to the C-terminus (Figure 1).

[0048] Measurement of cHis8 binding affinity using BLItz To perform a more detailed binding affinity analysis, we measured the binding affinity using BLItz (Sartorius). After capturing purified cHis8 IgG on the BLItz AMC (anti-mouse IgG-Fc) sensor chip, T4L, which had a His-tag consisting of 6-10 histidine residues attached to its N-terminus / C-terminus, was measured as the analyte (Figure 2).

[0049] The results are shown in Table 1. cHis8 showed low binding affinity or was unmeasurable for His-tags attached to the N-terminus, while it showed high affinity of approximately 400 nM for His-tags consisting of 6 histidine residues attached to the C-terminus, and approximately 30 nM for His-tags consisting of 7 to 10 histidine residues.

[0050] [Table 1]

[0051] On-column purification of membrane proteins using cHis8-immobilized resin First, cHis8 immobilized resin was prepared for packing the column. Purified IgG antibody, buffered in PBS, was used to prepare cHis8 immobilized resin using an NHS-activated Sepharose Fast Flow (Cytiva) according to the provided protocol. Considering the amount of membrane protein bound per 1 mL of resin and the antibody activity rate for each IgG immobilization concentration, the condition was set to 5 mg IgG / mL resin.

[0052] On-column purification using cHis8-immobilized resin was performed using the GPCR A2a, the ion channel hERG, and the ABC transporter P-glycoprotein as model proteins. HEK cells were expressed with A2a, hERG, and P-glycoprotein, respectively, and solubilized by directly adding a surfactant. All operations after solubilization were performed at 4°C or on ice. A2a was solubilized at a final concentration of 1% (w / v) DDM / 0.2% (w / v) CHS, hERG was solubilized according to the method described in the publicly available literature (doi:10.1016 / j.cell.2017.03.048.), and P-glycoprotein was solubilized according to the method described in the publicly available literature (doi:10.1116 / science.aav7102.). After solubilization, the solubilized supernatant was separated by centrifugation, and cHis8-immobilized resin was added and slowly mixed by inversion for 2-4 hours. Subsequently, the sample was passed through an empty column, washed with 10-30 column volumes (CV) of buffer, and then recovered with approximately 2 CV of buffer. On-column purification was then performed using two methods: surfactant purification and nanodisc purification.

[0053] For surfactant purification, first, HRV3C protease was added to the recovered cHis8 solid-phase resin suspension and slowly mixed by inversion for 2 to 16 hours. Subsequently, the fraction passed through an empty column and the fraction washed with 1 to 3 CV of buffer were collected and designated as the surfactant-purified fraction. This fraction was concentrated using Amicon Ultra as needed, and the surfactant was purified by gel filtration using Superdex 200 Increase 10 / 300 GL or Superose 6 Increase 10 / 300 GL (Cytiva).

[0054] For nanodisc purification, first, the desired purified MSP and lipid mixture were added to the recovered cHis8 solid-phase resin suspension and slowly mixed by inversion for 1 to 3 hours. The mixed cHis8 solid-phase resin suspension was passed through an empty column and washed with approximately 10 to 20 CV of buffer. After washing, Bio-Beads SM2 (Bio-Rad) was added to the recovered cHis8 solid-phase resin suspension (approximately 2 CV) and slowly mixed by inversion overnight. Subsequently, after recovering the Bio-Beads SM2, HRV3C protease was added to the recovered cHis8 solid-phase resin suspension (approximately 2 CV) and slowly mixed by inversion for 2 to 16 hours. Next, the fraction passed through the empty column and the fraction washed with 1 to 3 CV of buffer were collected and used as the nanodisc purified fraction. This was concentrated using Amicon Ultra as needed, and then purified into nanodiscs by gel filtration using Superdex 200 Increase 10 / 300 GL or Superrose 6 Increase 10 / 300 GL (Cytiva).

[0055] The results are shown in Figures 3-5. It was demonstrated that high-purity surfactant purification and nanodisk purification were possible for all membrane proteins used as model proteins (A2a, hERG, and P-glycoprotein) by using cHis8-immobilized resin (Figure 3: Results for A2a, Figure 4: Results for hERG, Figure 5: Results for P-glycoprotein). In particular, the nanodisk purification method of the present invention yielded approximately 2-5 times more yield compared to conventional nanodisk purification.

Claims

1. A monoclonal antibody or antigen-binding fragment against a histidine tag (His-tag), wherein the monoclonal antibody or antigen-binding fragment recognizes the His-tag attached to the C-terminus of a membrane protein as an epitope, His-tag consists of seven or more consecutive histidine residues. A monoclonal antibody or its antigen-binding fragment, in which membrane proteins constitute a nanodisk.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the His-tag consists of 7 to 12 consecutive histidine residues.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the equilibrium dissociation constant (KD) for interaction with His-tag is 35 nM or less.

4. A monoclonal antibody or its antigen-binding fragment produced by hybridoma cells with receipt number NITE AP-03635, deposited with the National Institute of Technology and Evaluation (NITE) on April 27, 2022.

5. An immobilized antibody comprising an antibody or antigen-binding fragment according to any one of claims 1 to 4, immobilized on a solid support.

6. A method for purifying a membrane protein from a solution containing a membrane protein to which a His-tag has been added to the C-terminus, His-tag consists of seven or more consecutive histidine residues. Membrane proteins constitute the nanodiscs. (a) A step of contacting the membrane protein with an antibody or antigen-binding fragment according to any one of claims 1 to 4 to form a complex composed of the membrane protein and the antibody or antigen-binding fragment. (b) A step of isolating the complex formed in step (a), and (c) A step of isolating the membrane protein from the complex isolated in step (b) and obtaining a filter containing the membrane protein. Methods that include...

7. The method according to claim 6, wherein the membrane protein is a recombinant protein.

8. The method according to claim 6, further comprising the step of purifying a membrane protein using a surfactant in step (a).

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