WO2022077551A1

WO2022077551A1 - Method for separating and detecting protein by means of immuno-precipitation

Info

Publication number: WO2022077551A1
Application number: PCT/CN2020/123690
Authority: WO
Inventors: 曹雪涛; 朱军
Original assignee: 南开大学
Priority date: 2020-10-13
Filing date: 2020-10-26
Publication date: 2022-04-21
Also published as: CN112255061A

Abstract

A method for separating and detecting a protein by means of immuno-precipitation, and a kit for implementing the method.

Description

A method for the isolation and detection of proteins by immunoprecipitation

technical field

The present application relates to the field of protein separation and detection and the field of life sciences. Specifically, the present application provides a method of isolating and detecting proteins by immunoprecipitation, and kits for carrying out the method.

Background technique

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) techniques are powerful and classic molecular experimental techniques for analyzing protein interactions and protein modifications. However, since antibodies are used in IP to capture the target protein of interest, it is inevitable that the antibodies used in IP will be shed together with the captured protein into the eluate for downstream analysis. Typically, antibody denaturation produces a heavy chain of about 55 kD. The human genome encodes about 20,000 proteins, of which the average molecular weight of nearly a quarter of the proteins and most of the important proteins are concentrated around 55kD. Therefore, the detection of proteins with molecular weights in this range by the shed antibodies will cause serious interference signals.

At present, scientists have successively developed a variety of technical means to solve this problem. They can be broadly divided into two broad categories based on the strategies used and the scope of analysis applicable:

1. Eliminate interfering signals by "obfuscation". Such methods need to be used in conjunction with Western blot analysis (Western Blots, WB).

A typical trick is to eliminate interfering signals by using antibodies from different species in IP and in WB. The advantage of this method is that it does not require any additional reagents; the disadvantage is that the elimination effect is very limited, and the antibody heavy and light chain signals are still quite residual. In addition, for some proteins of interest, it is often not easy to obtain antibodies from two different sources.

Another typical trick is to use a secondary antibody that recognizes a specific conformation in WB. This is also the mainstream technology strategy adopted by many companies (such as CST, ProteinTec, biorad, abcam, etc.). However, in practical applications, the elimination effect of this method is still limited, and the antibody heavy and light chain signals are still quite residual.

In addition, it has also been proposed to use HRP-labeled primary antibodies directly in WB without the use of secondary antibodies, thereby eliminating interfering signals. The advantage of this method is that the heavy and light chain signals are completely removed; the disadvantage is that the loss of the signal may be caused by not using the secondary antibody in WB, and the process of labeling the primary antibody is cumbersome and has poor stability. The quantity is also large and the cost is high.

2. Eliminate interfering signals by clearing heavy and light chains. Such methods can subsequently be used in conjunction with WB or mass spectrometry (MS).

The first type of method does not substantially remove heavy and light chains, so it is only suitable for WB analysis and cannot meet the requirements of IP-MS combined analysis. To this end, scientists have further developed several techniques to substantially remove heavy and light chains.

(1) Acid elution method: In the elution step of IP, the antibody is prevented from falling off the magnetic beads by a mild elution method, and only the antigen bound to the antibody is released. However, the acid elution method still results in the dissociation of some antibodies from the magnetic beads, and the stability of different antibodies during IP elution is difficult to control, and the elution efficiency is also limited.

(2) Peptide segment competition elution method: in the IP elution step, the antigen bound to the antibody is released by using the peptide segment that competes for binding to the antibody. This method elution is more efficient and does not cause the antibody to fall off the beads. However, this method is only suitable for IP using exogenous tagged proteins, and in many cases cannot study or reflect the interactions of endogenous proteins under physiological conditions.

(3) Cross-link IP method (cIP): This method adds a cross-linking step (that is, cross-linking the magnetic beads through a chemical cross-linking agent) on the basis of the traditional IP method (traditional IP, tIP). and antibodies), thereby mitigating or avoiding the detachment of antibodies from the beads during acid elution. However, in practical applications, this method cannot completely eliminate the interference signal, but can only partially reduce the interference signal of the heavy chain. In addition, the introduction of cross-linking operation on the basis of tIP results in a cumbersome method and poor stability.

(4) Single-domain antibody IP: This method uses a single-domain antibody derived from alpaca in the IP process, which can fundamentally remove the heavy chain of the antibody, and its eluted product is suitable for both WB analysis and IP-MS analysis. However, unlike antibodies derived from mice and rabbits, single-domain antibodies derived from alpaca are very expensive and have a long production cycle. The quality of the single-domain antibodies obtained requires subsequent verification by experiments, and the stability is difficult to guarantee. Therefore, this method is difficult to be widely used.

To sum up, the various IP methods developed at present still cannot meet the needs of practical applications. There is still an urgent need in the art to develop new IP methods that can effectively remove signal interference from heavy and/or light chains.

SUMMARY OF THE INVENTION

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory procedures of cell culture, nucleic acid chemistry, and immunology used in this paper are all routine procedures widely used in the corresponding fields.

Based on in-depth research, the inventors of the present application proposed a new immunoprecipitation protocol, namely, enzymolysis immunoprecipitation (eIP). In this method, the inventors of the present application proposed for the first time that an enzyme that can specifically degrade antibodies but not common proteins is used in combination in the IP process to eliminate antibodies (such as antibody heavy chains) through enzymatic hydrolysis, thereby substantially removing the Signal interference from antibodies (eg, antibody heavy chains) increases the sensitivity and specificity of protein detection. The method of the present application is easy to operate and low in cost, and the obtained product can be widely used in various downstream analysis methods (eg, WB analysis and IP-MS analysis), and has excellent application prospects.

Therefore, in one aspect, the application provides a method for isolating a protein of interest, comprising: combining a complex containing the protein of interest and an antibody capable of specifically binding to the protein of interest with an antibody capable of specifically degrading the antibody but not degrading the target protein The step of contacting the protein with the enzyme.

In the method of the present application, the protein of interest in the complex is specifically bound to the antibody; and the contact of the complex with the enzyme results in degradation of the antibody and release of the protein of interest .

In certain preferred embodiments, the method comprises the steps of:

(1) Provide a sample containing the target protein;

(2) contacting an antibody that specifically binds to the protein of interest with the sample to form a complex containing the protein of interest and the antibody, and separating the complex; and

(3) contacting the complex with an enzyme capable of specifically degrading the antibody but not degrading the target protein, thereby obtaining the target protein.

In this application, the type and source of the sample is not limited. In certain preferred embodiments, the sample is a solution sample containing the protein of interest, such as, but not limited to, cell lysates, tissue homogenates, bodily fluids (e.g. urine, saliva, blood, lymph, brain, etc.) containing the protein of interest Spinal fluid, bile, gastric fluid, intestinal fluid, tears, etc.), lavage fluids (e.g. alveolar lavage, peritoneal lavage, vaginal lavage, gastric lavage, intestinal lavage, etc.), and buffers (e.g. Phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, etc.).

In certain preferred embodiments, the sample may be derived from any organism, such as, but not limited to, viruses, prokaryotes (eg, bacteria), and eukaryotes (eg, fungi, plants, animals such as invertebrates, vertebrates, mammals (eg, humans). In certain preferred embodiments, the sample may be derived from a non-biological sample, such as a protein library or synthetic product.

In certain preferred embodiments, the sample does not contain components that adversely affect the specific interaction between antigen and antibody. However, it is readily understood that when the sample may contain components that adversely affect the specific interaction between antigen and antibody (eg, protein denaturants), the sample may be pretreated to remove such components. Therefore, in certain preferred embodiments, the method further comprises the step of pre-treating the sample before contacting the sample with the antibody that specifically binds the protein of interest. For example, in certain preferred embodiments, the sample may be subjected to various pretreatment steps, including, but not limited to, centrifugation, concentration, dilution, dialysis, chromatography, electrophoresis, desalting, addition of additional reagents (eg, salt or molecular chaperones), or any combination thereof.

In this application, the protein of interest can be any protein of interest. In certain preferred embodiments, the protein of interest is not an antibody molecule. In certain preferred embodiments, the protein of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein of interest is bound to a protein-binding molecule of interest, and further preferably, the protein-binding molecule of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein-binding molecule of interest is a protein with a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein of interest or protein-binding molecule of interest is a water- or buffer-soluble protein. In certain preferred embodiments, the buffer may be any type of buffer. In certain preferred embodiments, the buffer is one that does not adversely affect the specific interaction between the antigen and antibody. Types of such buffers are well known to those skilled in the art and include, but are not limited to, for example, phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, and the like.

It is well understood that the use of tagged peptides may in some cases facilitate protein isolation and purification. Thus, in certain preferred embodiments, the protein of interest optionally further comprises a tag peptide. In certain preferred embodiments, the protein of interest is a fusion protein comprising a tag peptide and a polypeptide of interest. As used herein, the term "tag peptide" means any peptide segment that can be used as a tag (eg, to facilitate expression, detection, tracking, and purification of a protein of interest). In the present application, any type of tag peptide can be used, such as but not limited to c-Myc, Flag, 6*His, hapten, GST, HA, SUMO, fluorescent proteins (eg GFP, YFP, RFP), etc. According to actual needs, the tag peptide can be linked to the N-terminus or C-terminus of the polypeptide of interest. In addition, the tag peptide can optionally be linked to the polypeptide of interest via a peptide linker and/or a protease cleavage site. Such peptide linkers are known to those skilled in the art and include rigid peptide linkers and flexible peptide linkers such as ( _G4S)3 _. Protease cleavage sites are also known to those skilled in the art, including but not limited to HRV 3C protease cleavage site, TEV protease cleavage site, enterokinase cleavage site, SUMO protease cleavage site, and the like.

In the embodiment where the protein of interest further comprises a tag peptide and a polypeptide of interest, it is easy to understand that the antibody that specifically binds to the protein of interest can be an antibody that specifically binds to the tag peptide or an antibody that specifically binds to the polypeptide of interest. In certain preferred embodiments, the antibody is an antibody that specifically binds a tag peptide. In certain preferred embodiments, the antibody is an IgG, such as animal-derived IgG (eg, human, murine, rabbit, monkey, sheep-derived IgG), and chimeric IgG (eg, human-mouse chimeric IgG), and the like. In certain preferred embodiments, the antibody is not a single domain antibody (e.g., a llama antibody).

As known to those skilled in the art, immunoprecipitation techniques can be used to study protein-binding molecules of interest that interact (eg, bind) with the protein of interest in addition to the protein of interest itself. Thus, in certain preferred embodiments, the protein of interest optionally also binds a protein-binding molecule of interest. The binding between the protein of interest and the protein-binding molecule of interest can be covalent or non-covalent. In certain preferred embodiments, the protein of interest is non-covalently bound to the protein of interest binding molecule. In certain preferred embodiments, the protein of interest is covalently bound to a protein-binding molecule of interest. In certain preferred embodiments, the protein-binding molecule of interest is selected from, but not limited to, proteins, nucleic acids, polysaccharides, lipids, or any combination thereof. In certain preferred embodiments, the protein-binding molecule of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein-binding molecule of interest is a second protein (eg, a second protein having a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD)). In certain preferred embodiments, the protein-binding molecule of interest is not an antibody. In certain preferred embodiments, the complex contains the protein of interest, the antibody, and a protein-of-interest binding molecule (eg, a second protein) bound (eg, covalently or non-covalently) to the protein of interest protein).

In the present application, the antibody may be contacted with the sample by any suitable means. In certain preferred embodiments, the antibody can be mixed and incubated with the sample under conditions that allow specific binding of the antigen to the antibody, thereby forming a complex comprising the protein of interest and the antibody. Conditions that allow specific binding of an antigen to an antibody are well known to those skilled in the art. For example, at a suitable temperature (eg, 0-4°C, 4-10°C, 10-20°C, 20-30°C, 30-37°C, or 37-40°C), in a suitable buffer (eg, without in a buffer that would be unfavorable for the specific binding of the antigen to the antibody), mix the antibody with the sample and incubate for an appropriate time (e.g. 5-30min, 0.5-1h, 1-2h, 2-5h, 5- 12h or 12-24h), thereby forming a complex containing the target protein and antibody. Types of such buffers are well known to those skilled in the art and include, but are not limited to, for example, phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, and the like.

In certain preferred embodiments, the antibody is immobilized or attached to a support. Typically, the support used to attach or immobilize the antibody is in a solid phase for ease of manipulation. Accordingly, in this disclosure, "support" is also sometimes referred to as "solid support" or "solid support." It should be understood, however, that the "support" referred to herein is not limited to solids, but can also be semi-solids (eg, gels).

The use of a support is particularly advantageous in many situations, for example, to facilitate separation of complexes containing the protein of interest and the antibody. For example, in certain embodiments, after contacting the sample solution with the antibody immobilized or attached to the support, the complexes can be conveniently separated by solid-liquid separation (ie, phase separation of the insoluble support from the sample solution). thing. In certain embodiments, after contacting the sample solution with the antibody immobilized or attached to the support, the complex can be conveniently separated by centrifugation or filtration or the action of a magnetic field to separate the insoluble support from the sample solution.

In certain preferred embodiments, the antibody is not attached to a support. In such embodiments, the complex can be isolated using an antibody binding molecule capable of specifically binding the antibody. Such antibody binding molecules include, but are not limited to, anti-antibodies, protein A, protein G, and protein L, among others. As is well known to those skilled in the art, an anti-antibody can be an antibody capable of specifically recognizing an antibody (eg, an Fc region of an antibody). Protein A (Protein A) is a cell wall protein of Staphylococcus aureus, which can be specific to mammalian IgG antibodies (such as mouse IgG2a, IgG2b, IgA; rabbit IgG; human IgG1, IgG2 and IgG4 antibodies, etc.) through the Fc region Binds sexually, but not to other miscellaneous proteins. Protein G is a cell wall protein derived from Streptococcus group G, which can specifically bind to the Fc region of IgG. Protein L is a protein derived from Peptostreptococcus Magnus that binds specifically to immunoglobulins through light chain interactions. Therefore, anti-antibodies, protein A, protein G and protein L can all be used as antibody binding molecules for the isolation and purification of antibodies. In certain preferred embodiments, the antibody binding molecule is selected from the group consisting of protein A, protein G and protein L.

As will be appreciated, antibody binding molecules (eg, anti-antibodies, protein A, protein G, or protein L) can be immobilized or linked to a support to facilitate separation of the complexes. Thus, in certain preferred embodiments, the antibody-binding molecule is immobilized or linked to a support. For example, in certain embodiments, the antibody-binding molecule (eg, anti-antibody, protein A, protein G, or protein L) can be coupled to an agarose matrix as an affinity ligand to obtain antibodies capable of specific binding of agarose (eg anti-antibody agarose, protein A agarose, protein G agarose or protein L agarose). In certain preferred embodiments, the antibody binding molecule is selected from the group consisting of protein A, protein G and protein L.

In certain embodiments, in step (2), the antibody can be contacted with the sample first to form a complex containing the protein of interest and the antibody; then, the complex is immobilized or linked to a support The complexes (eg, which can be attached to the insoluble support by means of antibody binding molecules) are then conveniently isolated by solid-liquid separation (ie, separating the insoluble support from the solution phase). In certain embodiments, the complex (eg, which can be attached to the insoluble support by means of an antibody binding molecule) can be conveniently isolated by separating the insoluble support from the solution phase by centrifugation or filtration or the action of a magnetic field.

In certain embodiments, in step (2), the antibody that specifically binds the target protein can be first contacted with the antibody-binding molecule immobilized or linked to the support, and then contacted with the sample to form a protein containing the target protein and A complex of antibodies (eg, which can be attached to an insoluble support by means of antibody binding molecules); the complex is then conveniently isolated by solid-liquid separation (ie, separation of the insoluble support from the solution phase). In certain embodiments, the complex (eg, which can be attached to the insoluble support by means of an antibody binding molecule) can be conveniently isolated by separating the insoluble support from the solution phase by centrifugation or filtration or the action of a magnetic field.

Thus, in some embodiments, an antibody that specifically binds a protein of interest can be contacted with the sample and then contacted with an antibody-binding molecule to form a complex comprising the protein of interest and the antibody. In some embodiments, an antibody that specifically binds a protein of interest can be contacted with an antibody binding molecule and then contacted with the sample to form a complex comprising the protein of interest and the antibody.

In the present application, supports for attaching or immobilizing antibodies or antibody-binding molecules can be made of various suitable materials. Such materials include, for example, inorganics, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to: cellulose, cellulose derivatives (eg, nitrocellulose), acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, Cross-linked polystyrene such as vinylbenzene (see, eg, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, metal material (eg, magnetic metal materials), cross-linked dextran (eg, Sephadex ^™ ), agarose gels (eg, Sepharose ^™ ), and other supports known to those skilled in the art. In certain preferred embodiments, the support used to attach or immobilize the antibody or antibody-binding molecule is agarose beads.

In certain preferred embodiments, the support used to attach or immobilize the antibody or antibody-binding molecule may be a solid support comprising an inert substrate or matrix (eg, glass slide, polymer beads, etc.) that Or the matrix has been functionalized, eg by applying an intermediate material containing reactive groups that allow covalent attachment of biomolecules such as antibodies or antibody-binding molecules. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on inert substrates such as glass. In such embodiments, the biomolecule (eg, antibody or antibody-binding molecule) can be directly covalently attached to the intermediate material (eg, the hydrogel), which itself can be non-covalently attached to the substrate or Substrates (eg, glass substrates). In certain preferred embodiments, the support is a glass slide or silicon wafer whose surface is modified with a layer of avidin, amino, acrylamide silane or aldehyde-based chemical groups. In certain preferred embodiments, the support is magnetic (eg, magnetic beads). Thereby, the magnetic support and the molecular entities attached or bound thereto can be separated by means of a magnet or magnetic force.

In this application, the support or solid support is not limited to its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip, and/or array. The surface of such a support may be in the form of a planar layer.

In some embodiments, the support or surface thereof is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the support or solid support includes microspheres or beads. "Microspheres" or "beads" or "particles" or grammatical equivalents herein refer to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thorium dioxide sol, carbon graphite, titanium dioxide, latex, agarose, dextran, Cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles, and teflon, as well as any of the other materials outlined herein for preparing solid supports. Additionally, the beads can be spherical or non-spherical. In some embodiments, spherical beads may be used. In some embodiments, irregular particles may be used. In addition, the beads can also be porous.

In certain preferred embodiments, the support used to attach or immobilize the antibody or antibody-binding molecule is an array of beads or wells (also referred to as a chip). The arrays can be prepared using any of the materials outlined herein for preparing solid supports, and preferably, the surfaces of the beads or wells on the array are functionalized to facilitate attachment of antibodies. The number of beads or wells on the array is not limited. For example, each ^array may ^contain ^10-102 , ^102-103 , ^103-104 , ^104-105 ^, ^105-106 ^, ^106-107 ^, ^107-108 ^, ¹⁰⁸ -10 ⁹ or more beads or holes. In certain exemplary embodiments, one or more antibodies or antibody-binding molecules can be attached to the surface of each bead or well. Accordingly, each array can connect 10-10 ² , 10 ² -10 ³ , 10 ³ -10 ⁴ , 10 ⁴ -10 ⁵ , 10 ⁵ -10 ⁶ , 10 ⁶ -10 ⁷ , 10 ⁷ -10 ⁸ , 10 ^8-10 ⁹ or more antibodies or antibody-binding molecules.

Supports can be fabricated using a variety of techniques, as generally known in the art. Such techniques include, but are not limited to, photolithography, stamping techniques, lamination techniques, and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition, structure and shape of the support.

In the present application, the antibody or antibody-binding molecule can be attached to the support (eg, covalently or non-covalently) by any method known to those of ordinary skill in the art. For example, the antibody or antibody-binding molecule can be attached to the support by covalent attachment, or by irreversible passive adsorption, or by intermolecular affinity (eg, between biotin and avidin). Preferably, however, the linkage between the antibody or antibody-binding molecule and the support is sufficiently strong that the antibody or antibody-binding molecule does not detach from the support due to the conditions used in the various reactions and washing with water or buffer solutions.

For example, in certain preferred embodiments, the free amino or carboxyl group (eg, N-terminus or C-terminus) of an antibody or antibody-binding molecule carries a device capable of covalently attaching the antibody or antibody-binding molecule to a support, such as a chemical modified functional groups. Examples of such functional groups include, but are not limited to, carboxylic acid molecules, aldehyde molecules, thiols, hydroxyl groups, dimethoxytrityl (DMT), or amino groups.

In addition, cross-linking agents can also be used to link the antibody or antibody-binding molecule to the support. Such cross-linking agents include, for example, succinic anhydride, phenyldiisothiocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dithiocyanate) Methylaminopropyl)-carbodiimide hydrochloride (EDC), m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl[4 -Iodoacetyl]aminobenzoic acid (SIAB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleyl Imidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB), and the corresponding thio compounds (water soluble of).

In addition, supports can also be derivatized with bifunctional crosslinkers (eg, homobifunctional crosslinkers and heterobifunctional crosslinkers) to provide modified functionalized surfaces. The antibody or antibody-binding molecule with a thiol or amino group is then able to interact with the functionalized surface to form a covalent linkage between the antibody or antibody-binding molecule and the support. Numerous bifunctional crosslinking agents and methods for their use are known in the art (see, eg, Pierce Catalog and Handbook, pp. 155-200).

In certain preferred embodiments, the method further comprises, prior to performing step (3), the step of washing the complex. Washing steps are particularly advantageous in many cases, for example, to reduce or remove molecules that are non-specifically bound to the antibody or antibody-binding molecule. In certain preferred embodiments, various suitable buffers can be used to wash the complexes. Types of such buffers are well known to those skilled in the art and include, but are not limited to, for example, phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, and the like.

In certain preferred embodiments, the complex may be washed one or more times, eg, 1, 2, or 3 washes.

In the method of the present application, various enzymes capable of specifically degrading antibodies can be used as long as they do not degrade the protein of interest. In certain preferred embodiments, the enzyme is an immunoglobulin-degrading enzyme or an endo-immunoglobulinase, or any combination thereof. In certain preferred embodiments, the enzyme is an immunoglobulin G degrading enzyme or an immunoglobulin G endonuclease or any combination thereof. In certain preferred embodiments, the enzyme is a hydrolase capable of specifically cleaving the hinge region of an immunoglobulin molecule (eg, IgG). In certain embodiments, the enzyme is IdeS (Immunoglubulin G-degrading enzyme of Streptococcus pyogenes, IdeS). IdeS is a cysteine hydrolase produced by Streptococcus pyogenes and secreted to the outside of the cell. It has extremely high substrate specificity, can only recognize IgG, and is specific in the hinge region of the antibody. The site is digested to hydrolyze IgG into complete F(ab') ₂ fragment and Fc fragment. IdeS can recognize IgG of human and other animal sources, such as mouse, rabbit, monkey, sheep and human animal chimeric IgG. In certain preferred embodiments, the enzyme is IdeZ. IdeZ is a highly specific IgG protease isolated from Streptococcus equi ssp equi that can specifically cleave IgG under native conditions below the hinge region of IgG, leaving intact Fab and Fc fragments. This unique substrate specificity of IdeS and IdeZ makes them particularly suitable for use in the methods of the present application for degrading antibodies in the complexes. IdeS and IdeZ are commercially available, eg from

(IdeS).

In the methods of the present application, the complex can be contacted with the enzyme by any suitable means. In certain preferred embodiments, the complexes can be mixed and incubated with the enzymes under the working conditions of the enzymes to degrade the antibodies in the complexes. The working conditions of enzymes are well known to those skilled in the art. For example, at a suitable temperature (eg, 30-40°C, eg, 37°C) and a suitable pH (eg, pH 6-8, eg, pH 6.6 or pH 7), in a suitable buffer (eg, phosphate buffered saline) or Tris-HCl buffer), incubate the complex with IdeS for an appropriate time (eg, 5-30 min, 0.5-1 h, or 1-2 h) to degrade the antibody in the complex.

In certain preferred embodiments, the complex is linked to the support via an antibody or antibody-binding molecule. In such embodiments, degradation of the antibody can result in the release of the protein of interest (ie, the protein of interest is separated from the support). Thus, protein samples isolated or obtained by the methods of the present application are substantially free of antibodies (eg, antibody heavy chains) and can be used for a variety of desired uses. For example, a protein of interest isolated or obtained by the methods of the present application can be used for WB analysis or MS analysis (eg, to determine its structure or content); or can be used for functional analysis (eg, to determine its activity).

Accordingly, in certain preferred embodiments, the method further comprises the step of: (4) analyzing the protein of interest (eg, WB analysis, MS analysis, functional analysis, or any combination thereof). In certain preferred embodiments, the method further comprises the step of performing WB analysis on the protein of interest. In certain preferred embodiments, the method further comprises the step of performing MS analysis on the protein of interest. In certain preferred embodiments, the method further comprises the step of functionally analyzing the protein of interest.

In another aspect, the present application provides a kit for separating a target protein or a target protein-binding molecule in a sample, comprising: (1) an antibody that specifically binds to the target protein, and/or, can specifically and, (2) an enzyme that specifically degrades the antibody but not the protein of interest.

In another aspect, the application provides, (1) an antibody capable of specifically binding a protein of interest and/or an antibody-binding molecule capable of specifically binding the antibody and (2) an antibody capable of specifically degrading the antibody but not the protein of interest Use of a combination of enzymes for isolating a protein of interest or a protein-binding molecule of interest in a sample. In another aspect, the application provides, (1) an antibody capable of specifically binding a protein of interest and/or an antibody-binding molecule capable of specifically binding the antibody and (2) an antibody capable of specifically degrading the antibody but not the protein of interest Use of the combination of enzymes for the preparation of a kit for isolating a protein of interest or a protein-binding molecule of interest in a sample.

In certain preferred embodiments, the sample is a solution sample containing the protein of interest, such as, but not limited to, cell lysates, tissue homogenates, bodily fluids (e.g. urine, saliva, blood, lymph, brain, etc.) containing the protein of interest Spinal fluid, bile, gastric fluid, intestinal fluid, tears, etc.), lavage fluids (e.g. alveolar lavage, peritoneal lavage, vaginal lavage, gastric lavage, intestinal lavage, etc.), and buffers (e.g. Phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, etc.).

In this application, the protein of interest can be any protein of interest. In certain preferred embodiments, the protein of interest is not an antibody molecule. In certain preferred embodiments, the protein of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein of interest is bound to a protein-binding molecule of interest, and further preferably, the protein-binding molecule of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein-binding molecule of interest is a protein with a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein of interest or protein-binding molecule of interest is a water- or buffer-soluble protein.

In certain preferred embodiments, the protein of interest optionally further comprises a tag peptide. In certain preferred embodiments, the protein of interest is a fusion protein comprising a tag peptide and a polypeptide of interest. In certain preferred embodiments, the tag peptide is, for example, but not limited to, c-Myc, Flag, 6*His, hapten, GST, HA, SUMO, fluorescent proteins (eg, GFP, YFP, RFP), and the like. According to actual needs, the tag peptide can be linked to the N-terminus or C-terminus of the polypeptide of interest. In addition, the tag peptide can optionally be linked to the polypeptide of interest via a peptide linker and/or a protease cleavage site. Such peptide linkers are known to those skilled in the art and include rigid peptide linkers and flexible peptide linkers such as ( _G4S)3 _. Protease cleavage sites are also known to those skilled in the art, including but not limited to HRV 3C protease cleavage site, TEV protease cleavage site, enterokinase cleavage site, SUMO protease cleavage site, and the like.

In certain preferred embodiments, the antibody that specifically binds to the target protein may be an antibody that specifically binds to a tag peptide, or an antibody that specifically binds to the target polypeptide. In certain preferred embodiments, the antibody is an antibody that specifically binds a tag peptide. In certain preferred embodiments, the antibody is an IgG, such as animal-derived IgG (such as human, murine, rabbit, monkey, sheep-derived IgG), and chimeric IgG (such as human-mouse chimeric IgG), and the like. In certain preferred embodiments, the antibody is not a single domain antibody (eg, an alpaca antibody).

In certain preferred embodiments, the protein of interest optionally also binds a protein-binding molecule of interest. The binding between the protein of interest and the protein-binding molecule of interest can be covalent or non-covalent. In certain preferred embodiments, the protein of interest is non-covalently bound to the protein of interest binding molecule. In certain preferred embodiments, the protein of interest is covalently bound to a protein-binding molecule of interest. In certain preferred embodiments, the protein-binding molecule of interest is selected from proteins, nucleic acids, polysaccharides, lipids, or any combination thereof. In certain preferred embodiments, the protein-binding molecule of interest has a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD). In certain preferred embodiments, the protein-binding molecule of interest is a second protein (eg, a second protein having a molecular weight of 30-80 kD (eg, 40-70 kD, 45-65 kD)). In certain preferred embodiments, the protein-binding molecule of interest is not an antibody.

In certain preferred embodiments, the kit comprises, an antibody that specifically binds to the protein of interest, and an enzyme that specifically degrades the antibody but not the protein of interest. In certain preferred embodiments, the kit comprises an antibody binding molecule capable of specifically binding the antibody, and an enzyme that specifically degrades the antibody but not the protein of interest. In certain preferred embodiments, the kit comprises, an antibody that specifically binds the protein of interest, an antibody binding molecule capable of specifically binding the antibody; and an enzyme that specifically degrades the antibody but not the protein of interest.

In certain preferred embodiments, the antibody is immobilized or attached to a support. In certain preferred embodiments, the antibody is not attached to a support.

In certain preferred embodiments, the antibody binding molecules include, but are not limited to, anti-antibodies, protein A, protein G, protein L, and the like. In certain preferred embodiments, the antibody binding molecule is immobilized on or linked to a support.

Supports for attaching or immobilizing antibodies or antibody-binding molecules can be made of a variety of suitable materials. Such materials include, for example, inorganics, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to: cellulose, cellulose derivatives (eg, nitrocellulose), acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, Cross-linked polystyrene such as vinylbenzene (see, eg, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, metal materials ( such as magnetic metal materials), cross-linked dextran (eg, Sephadex ^™ ), agarose gels (eg, Sepharose ^™ ), and other supports known to those skilled in the art. In certain preferred embodiments, the support used to attach or immobilize the antibody or antibody-binding molecule may be a solid support comprising an inert substrate or matrix (eg, glass slide, polymer beads, etc.) that Or the matrix has been functionalized, eg by applying an intermediate material containing reactive groups that allow covalent attachment of biomolecules such as antibodies or antibody-binding molecules.

In certain preferred embodiments, the support is magnetic. In certain preferred embodiments, the kit further comprises a magnet. Such magnets can be used, for example, to adsorb magnetic supports.

In certain preferred embodiments, the enzyme is an immunoglobulin-degrading enzyme or an endo-immunoglobulinase. In certain preferred embodiments, the enzyme is an immunoglobulin G degrading enzyme or an immunoglobulin G endonuclease. In certain preferred embodiments, the enzyme is a hydrolase capable of specifically cleaving the hinge region of an immunoglobulin molecule (eg, IgG). In certain embodiments, the enzyme is IdeS or IdeZ.

In certain preferred embodiments, the kit further comprises a component selected from the group consisting of: one or more buffers (such as, but not limited to, buffers for solubilizing the protein of interest, for antigen and antibody specificity Binding buffers, buffers for specific binding of antibodies to antibody-binding molecules, buffers for washing, working buffers for the enzyme, etc.), reagents for lysing cells (e.g., cell lysates), with Reagents for protein electrophoresis (polyacrylamide gels, loading buffers, running buffers, reducing agents), reagents for WB analysis (eg, cellulose membranes, blocking solutions, capable of specifically binding the protein of interest or A primary antibody (which is optionally labeled) to the protein-binding molecule of interest, a secondary antibody (which is optionally labeled) capable of specifically binding to the primary antibody, a chromogenic reagent, etc.) for performing MS Analytical reagents, and any combination thereof.

It is easy to understand that the kit of the present application is used to carry out the method as described above. Thus, above for various reagents or materials (including but not limited to, the antibody, enzyme, sample, protein of interest, tag peptide, peptide linker, protease cleavage site, protein of interest binding molecule, support, antibody binding molecule, Buffers, etc.), including descriptions of various preferred and exemplary features, also apply here.

Beneficial Effects of Invention

Compared with the prior art, the technical scheme of the present invention has the following beneficial effects:

(1) The eIP method of the present application skillfully combines enzymes that degrade antibodies specifically to remove antibodies (eg, antibody heavy chains) by enzymatic hydrolysis, which can substantially eliminate the signal interference of antibodies (eg, antibody heavy chains).

(2) The eIP method of the present application is easy to operate and has low cost. For example, the methods of the present application may eliminate the need for complex competitive elution steps (eg, using competing peptides to elute the protein of interest from antigen-antibody complexes), and may eliminate the need for additional cross-linking steps (eg, The antibody is cross-linked to a solid support to avoid elution of the antibody), eliminating the need for expensive special antibodies (eg, antibodies recognizing specific conformations or single domain antibodies). Therefore, the eIP method of the present application is more convenient for promotion and use.

Furthermore, in certain preferred embodiments, protein samples obtained by the eIP methods of the present application, since they do not contain antibody components (eg, antibody heavy chains), can be conveniently used in a variety of subsequent analytical methods, including, for example, WB analysis, MS analysis and functional analysis. Therefore, the eIP method of the present application provides a powerful analytical strategy for the isolation, detection and study of proteins.

The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Description of drawings

Figure 1 shows an exemplary scheme of the enzymatic immunoprecipitation (eIP) of the present application. In this exemplary embodiment, an antibody that specifically binds a protein of interest M is contacted with a sample (eg, a cell lysate) containing the protein of interest, thereby forming a complex containing the protein of interest and the antibody. Subsequently, the sample is incubated with protein A or protein G-linked agarose beads, whereby, by virtue of the specific binding between the antibody and protein A or protein G, the complexes in the sample are Capture onto the agarose beads. The agarose beads are isolated and washed, and then contacted with enzymes that specifically degrade antibodies, thereby degrading the antibodies in the complexes and releasing the M of interest. Subsequently, the target protein M can be used for subsequent analysis, such as WB analysis.

Figure 2 shows the WB detection results of Myc-METTL3 protein obtained by traditional IP and eIP methods, wherein the sample in lane 1 is the Myc-METTL3 protein obtained by traditional IP method (traditional IP group); the samples in lanes 2-4 is the Myc-METTL3 protein obtained by the eIP method of the present application (eIP-0.5U/μl group, eIP-1U/μl group and eIP-2U/μl group); wherein, the FabRICATOR enzyme concentrations used in lanes 2-4 are respectively 0.5, 1, and 2 U/µl.

Fig. 3 shows the detection results of WB analysis of Myc-METTL3 protein obtained by traditional IP method using homologous antibody (Fig. 3A) or heterologous antibody (Fig. 3B); wherein, in Fig. 3A-3B, lane 1 The sample is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc-METTL3 protein obtained by the traditional IP method.

Figure 4 shows the detection results of WB analysis of Myc-METTL3 protein obtained by traditional IP method using common secondary antibody (Figure 4A) or conformation-specific secondary antibody (Figure 4B) or light chain-specific secondary antibody (Figure 4C). ; Among them, in Figure 4A-4C, the sample in lane 1 is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc-METTL3 protein obtained by the traditional IP method, and the primary antibodies used in the WB analysis are all Homologous antibodies.

Figure 5 shows the comparison of anti-Myc antibodies labeled by commercial HPR (Figure 5A) or by Novus HRP kit (Figure 5B) or by Thermo HRP kit (Figure 5C) by conventional The detection results of Myc-METTL3 protein obtained by IP method by WB analysis; wherein, in Figure 5A-5C, the sample in lane 1 is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc obtained by traditional IP method - METTL3 protein.

Figure 6 shows the detection results of silver staining analysis of the proteins in the polyacrylamide gel after electrophoretic separation in Example 4, wherein the sample in lane 1 is the supernatant sample obtained from the negative control group; the sample in lane 2 is obtained by Myc-METTL3 protein obtained by traditional IP method.

Figure 7 shows the detection results of silver staining analysis of A2B1 protein obtained by tIP method (Figure 7A), cIP method (Figure 7B) or eIP (Figure 7C) method, wherein 1 is a protein sample obtained by immunoprecipitation of virus-infected cells, and the sample in lane 2 is a protein sample obtained by immunoprecipitation of cells infected with HSV-1 virus.

Detailed ways

The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the invention.

Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method described in F.M. Ausubel et al., The Experiment Guide for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995 was performed; the enzymes were used according to the conditions recommended by the product manufacturer. Those skilled in the art appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: Immunoprecipitation of Myc-METTL3 protein by eIP method and traditional IP method

1. Recovery of 293T cells

Take out the cryopreserved 293T cells from the liquid nitrogen tank, thaw the cryopreserved cells quickly in a 37°C water bath, and then centrifuge at 500 × g for 5 minutes, discard the cryopreservation solution, and add 1 ml of DMEM medium containing 10% fetal bovine serum. Mix gently, then transfer it to a petri dish with a diameter of 10 cm, add 9 ml of DMEM medium, mix gently and place it in a cell culture incubator.

2. Cell Passaging

The next day, after the cells recovered in the above step 1 grow to about 80% confluence, discard the medium, add 3 ml of PBS to wash the cells once, and then add trypsinization for 1 minute. Subsequently, the trypsin was discarded, and 2 ml of DMEM medium containing 10% fetal bovine serum was added to terminate the digestion, and then 1 ml of the cell suspension was transferred to a petri dish with a diameter of 10 cm, supplemented with 9 ml of DMEM medium, and placed in a cell incubator nourish.

3. Cell Transfection

On the third day, after the cells recovered in step 1 above were grown to a confluence of about 80%, a plasmid containing the Myc-METTL3 encoding gene was transfected. Gene ID: 56335). Briefly, 10 μg of plasmid containing the gene encoding Myc-METTL3 was added to 500 μl

Buffer (purchased from

In the centrifuge tube of SA Company), mix by pipetting repeatedly, centrifuge briefly, and then add 20 μl

(purchased from

SA Company), mix thoroughly, and after standing at room temperature for 10 minutes, drop it evenly into a petri dish with a diameter of 10 cm, and put it into a cell culture incubator after mixing. 24h after transfection, the protein can be collected for related experiments.

4. Extraction of Cell Total Protein

Cells were washed twice with pre-chilled PBS buffer and the buffer was aspirated a final time. Add 1 ml of pre-chilled lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, PMSF, Cocktail 100*) to the dish. Scrape the cells from the culture dish with a pre-cooled cell scraper, transfer the suspension to a clean 1.5ml centrifuge tube, and place the centrifuge tube at 4°C for 15 minutes with slow shaking. The supernatant was then collected by centrifugation at 16,000 xg for 15 minutes at 4°C (transfer the supernatant to a new centrifuge tube immediately after the centrifugation was completed).

The supernatant protein concentration was determined with BCA protein quantification kit (purchased from Thermo Company), and the protein concentration was adjusted to 1 μg/μl with lysis buffer.

5. Immunoprecipitation (IP)

5.1 Take 4 0.6ml centrifuge tubes, add 500μl of the cell lysate with a total cell protein concentration of 1μg/μl obtained in the above step 4 to each tube, and then add 5μl of anti-Myc antibody (purchased from CST) to each tube. The antigen-antibody mixture was shaken slowly overnight.

5.2 The protein G-coupled magnetic beads (purchased from Thermo Company) were washed three times with lysis buffer. Add 50 μl of magnetic bead suspension to each of the 4 tubes of antigen-antibody mixture described in step 5.1 above, and shake the antigen-antibody-magnetic bead mixture slowly for 2 h at 4°C.

5.3 After separating the magnetic beads from the supernatant with a magnetic stand, wash the magnetic beads three times with lysis buffer to obtain magnetic beads-antibody-antigen complexes.

5.4 Label the above 4 tubes of magnetic bead-antibody-antigen complexes as traditional IP group, eIP-0.5U/μl group, eIP-1U/μl group and eIP-2U/μl group respectively. In the traditional IP group, add 20 μl of 2*SDS loading buffer and cook at 100°C for 10 minutes. In the eIP-0.5U/μl group, eIP-1U/μl group and eIP-2U/μl group, add 20μl of FabRICATOR enzyme (0.5, 1, or 2U/μl) diluted in PBS, respectively, and incubate at 37°C with slow shaking for 10 -30 minutes.

5.5 Use a magnetic stand to separate the magnetic beads from the solution, wherein the solution supernatant of the traditional IP group is directly reserved for future use; 5 μl of 5*SDS loading buffer is added to the solution supernatant of each eIP group, and heated at 100°C for 5 After a few minutes, set aside for backup.

6. Western Blot (WB) Analysis

Proteins in the solution supernatant sample obtained in the above step 5.5 were separated by electrophoresis in SDS (10%) polyacrylamide gel, and then transferred to nitrocellulose membrane (purchased from GE). Membranes were blocked in 5% nonfat dry milk in TBST (0.1% Tween-20 in TPBS) for 1 hour at room temperature. The membrane was then incubated with anti-Myc antibody (1:1000) overnight at 4°C. Membranes were washed 3 times (5 min each) with TBST. For standard WB detection, membranes were incubated with HRP-conjugated anti-mouse IgG antibody (purchased from abcam) for 1 hour at room temperature. Afterwards, the membrane was washed 3 times (5 min each) with TBST at room temperature and then developed with ECL Plus (available from ThermoFisher).

The experimental results are shown in Figure 2. Figure 2 shows the WB detection results of Myc-METTL3 protein obtained by traditional IP and eIP methods, wherein the sample in lane 1 is the Myc-METTL3 protein obtained by traditional IP method (traditional IP group); the samples in lanes 2-4 is the Myc-METTL3 protein obtained by the eIP method of the present application (eIP-0.5U/μl group, eIP-1U/μl group and eIP-2U/μl group). The results showed that there was substantial contamination of antibody heavy and light chains in lane 1 (as indicated by the black arrows). In contrast, in lanes 2-4, essentially no antibody heavy chain contamination was observed. This indicates that in the eIP method of the present application, the enzymatic hydrolysis treatment of FabRICATOR completely eliminates the contamination of the antibody heavy chain. In addition, it was also observed that the amount of Myc-METTL3 protein released gradually increased with increasing concentrations of the FabRICATOR enzyme used (lanes 2-4). These experimental data show that the eIP method of the present application can indeed solve the problem of antibody heavy chain contamination well and thoroughly.

In order to further demonstrate the advantages of the eIP method over the existing methods for removing antibody heavy chain contamination, the inventors also explored the experimental effects of several current mainstream strategies for removing antibody heavy chain contamination under the same experimental conditions.

Example 2: Detection of Myc-METTL3 Protein Immunoprecipitated by Traditional IP Methods Using Homologous Primary Antibodies or Heterologous Primary Antibodies

1. The preparation method of the cell lysate containing Myc-METTL3 protein is as described in steps 1-4 in Example 1.

2. Immunoprecipitation (IP)

2.1 Take two 0.6ml centrifuge tubes, add 500μl of the cell lysate obtained in step 1 above (total protein concentration is 1μg/μl) into each tube, and add 5μl of rabbit-derived anti-Myc antibody (purchased from sigma) to each tube, 4 The antigen-antibody mixture was shaken slowly overnight at °C.

2.2 The protein G-coupled magnetic beads (purchased from Thermo Company) were washed three times with lysis buffer. Add 50 μl of magnetic bead suspension to the two tubes of antigen-antibody mixture described in step 2.1 above, and shake the antigen-antibody-magnetic bead mixture slowly for 2 h at 4°C.

2.3 After separating the magnetic beads from the supernatant with a magnetic stand, wash the magnetic beads three times with lysis buffer to obtain magnetic beads-antibody-antigen complexes.

2.4 Add 20 μl 2*SDS loading buffer to the magnetic bead-antibody-antigen complex obtained in step 2.3 above, and cook at 100°C for 10 minutes.

2.5 Use a magnetic stand to separate the magnetic beads from the solution, and reserve the supernatant of the solution for future use.

3. Western blot (WB) analysis

The supernatants of the above two tubes were marked as homologous antibody IP group and heterologous antibody IP group respectively, and then WB analysis was performed as described in step 6 of Example 1; wherein, the WB used to analyze homologous antibody IP group samples The primary and secondary antibodies used were rabbit-derived anti-Myc antibody (1:1000; purchased from abcam company) and HRP-conjugated anti-rabbit IgG antibody (purchased from abcam company); used to analyze heterologous antibody IP group samples The primary and secondary antibodies used in WB were mouse-derived anti-Myc antibody (1:1000; purchased from CST company) and HRP-conjugated anti-mouse IgG antibody (purchased from abcam company).

In addition, set up a parallel control experiment as follows: replace the rabbit-derived anti-Myc antibody in the above step 2.1 with an irrelevant antibody, and repeat the above steps 1-3.

The experimental results are shown in Figure 3. Fig. 3 shows the detection results of WB analysis of Myc-METTL3 protein obtained by traditional IP method using homologous antibody (Fig. 3A) or heterologous antibody (Fig. 3B); wherein, in Fig. 3A-3B, lane 1 The sample is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc-METTL3 protein obtained by the traditional IP method. As shown in Figure 3A, both

lanes

1 and 2 were heavily contaminated with antibody heavy and light chains (as indicated by black arrows). In Figure 3B, the antibody heavy chain contamination in

lanes

1 and 2 was significantly reduced compared to Figure 3A, however, the antibody heavy chain contamination signal remained. These experimental results show that WB using heterologous antibodies cannot completely solve the problem of antibody heavy chain contamination after immunoprecipitation of the target protein using the traditional IP method.

Example 3: Detection of Myc-METTL3 protein immunoprecipitated by traditional IP methods using homologous primary antibodies and secondary antibodies that recognize specific conformations or common secondary antibodies

2. Immunoprecipitation (IP)

2.1 Take three 0.6ml centrifuge tubes, add 500μl of the cell lysate obtained in step 1 above (total protein concentration is 1μg/μl) into each tube, and add 5μl of rabbit-derived anti-Myc antibody (purchased from sigma) to each tube, 4 The antigen-antibody mixture was shaken slowly overnight at °C.

2.2 The protein G-coupled magnetic beads (purchased from Thermo Company) were washed three times with lysis buffer. Add 50 μl magnetic bead suspension to the 3 tubes of antigen-antibody mixture described in step 2.1 above, and shake the antigen-antibody-magnetic bead mixture slowly for 2 h at 4°C.

3. Western blot (WB) analysis

The supernatants of the above three tubes were marked as ordinary secondary antibody group, conformation-specific antibody group and light chain-specific antibody group, respectively, and then WB analysis was performed as described in step 6 of Example 1; wherein, the three groups were used to analyze The primary antibodies used in the WB of the samples were all rabbit-derived anti-Myc antibodies (1:1000; purchased from sigma company); the secondary antibodies used were HRP-conjugated anti-rabbit IgG antibodies (purchased from abcam company; common secondary antibodies) group), conformation-specific secondary antibody (purchased from CST company; conformation-specific antibody group), light chain-specific secondary antibody (purchased from proteintec company; light chain-specific antibody group).

The experimental results are shown in Figure 4. Figure 4 shows the detection results of WB analysis of Myc-METTL3 protein obtained by traditional IP method using common secondary antibody (Figure 4A) or conformation-specific secondary antibody (Figure 4B) or light chain-specific secondary antibody (Figure 4C). ; Among them, in Figure 4A-4C, the sample in lane 1 is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc-METTL3 protein obtained by the traditional IP method, and the primary antibodies used in the WB analysis are all Homologous antibodies. As shown in Figure 4A, both

lanes

1 and 2 were heavily contaminated with antibody heavy and light chains (as indicated by black arrows). In Figures 4B and 4C, the antibody heavy chain contamination in

lanes

1 and 2 was significantly reduced compared to Figure 4A, however, the antibody heavy chain contamination signal still remained. These experimental results show that, after immunoprecipitation of the target protein using the traditional IP method, WB using a homologous primary antibody and a secondary antibody that recognizes a special conformation cannot completely solve the problem of antibody heavy chain contamination.

Example 4: Detection of Myc-METTL3 protein immunoprecipitated by traditional IP method using HRP-labeled primary antibody

2. Immunoprecipitation (IP): As described in step 2 in Example 3, immunoprecipitation was performed to obtain 3 tubes of solution supernatant containing Myc-METTL3 protein.

3. Western blot (WB) analysis

The supernatants of the above 3 tubes were labeled as commercial antibody group, Novus labeling group and Thermo labeling group, respectively, and then WB analysis was performed; wherein, for the commercial antibody group, commercial HPR-labeled anti-Myc antibody (purchased from CST Company) was used. ) directly detected; for the Novus labeling group, use the anti-Myc antibody (purchased from CST company) labeled by Novus HRP kit (purchased from Novus company) for direct detection; The detection was carried out directly with the labeled anti-Myc antibody (purchased from CST Company) from Thermo Company. The HRP labeling operation was carried out according to the kit instructions.

In addition, a parallel control experiment was set up as follows: the rabbit-derived anti-Myc antibody in the above step 2 was replaced with an irrelevant antibody, and the above steps 1-3 were repeated.

The experimental results are shown in Figure 5. Figure 5 shows the comparison of anti-Myc antibodies labeled by commercial HPR (Figure 5A) or by Novus HRP kit (Figure 5B) or by Thermo HRP kit (Figure 5C) by conventional The detection results of Myc-METTL3 protein obtained by IP method by WB analysis; wherein, in Figure 5A-5C, the sample in lane 1 is the supernatant sample obtained from the negative control group; the sample in lane 2 is the Myc obtained by traditional IP method - METTL3 protein.

As shown in Figure 5, direct WB detection of Myc-METTL3 protein obtained by traditional IP method using HRP-labeled anti-Myc antibody can effectively reduce the contamination signal of antibody light and heavy chains. However, there are differences in the labeling effect of labeling kits from different companies (Fig. 5A-5C), which makes the assay less robust.

In addition, proteins in polyacrylamide gels after electrophoretic separation were also detected by conventional silver staining methods. The experimental results are shown in Figure 6. Figure 6 shows the detection results of silver staining analysis of proteins in polyacrylamide gels after electrophoretic separation, wherein the samples in lane 1 are the supernatant samples obtained from the negative control group; the samples in lane 2 are obtained by traditional IP methods Myc-METTL3 protein. As shown in Figure 6, distinct IgG heavy chain bands were observed in both

lanes

1 and 2. This shows that, in this example, the solution supernatant obtained by the traditional IP method still contains a large amount of IgG heavy chains, which cannot be used for mass spectrometry detection subsequently. Similarly, it can be determined that in Examples 2-3, the solution supernatant obtained by the traditional IP method still contains a large amount of IgG heavy chains, which cannot be subsequently used for mass spectrometry detection. The methods described in Examples 2-4 do not substantially remove antibody heavy chains from the sample, but only selectively reduce the coloration of the antibody heavy chains.

Example 5: Silver staining detection of A2B1 protein obtained by eIP method

In order to verify that the immunoprecipitation of the target protein by eIP method can substantially remove the antibody heavy chain in the sample, we immunoprecipitated the DNA virus sensor protein A2B1 (the coding sequence of which is produced by the eIP method after HSV-1 virus stimulated cells) (the coding sequence of which is shown in NCBI database Gene ID : 53379), and silver-stained the obtained samples to detect the antibody heavy chain content in the samples. At the same time, we also set up parallel experiments (ie, immunoprecipitation of protein A2B1 by tIP and cIP methods) as a control group.

1. Recovery of RAW264.7 cells

Take out the cryopreserved RAW264.7 cells from the liquid nitrogen tank, quickly thaw the cryopreserved cells in a 37°C water bath, and then centrifuge at 500 × g for 5 minutes, discard the cryopreserved solution, and add 1 ml of DMEM containing 10% fetal bovine serum to culture Then, it was transferred to a petri dish with a diameter of 10 cm, supplemented with 9 ml of DMEM medium, gently mixed, and then placed in a cell incubator for cultivation.

2. Cell Passaging

The next day, after the cells recovered in step 1 above grow to about 80% confluence, discard the medium, add 3 ml of PBS to wash the cells once, then add 3 ml of PBS, and gently scrape the cells with a cell scraper. Transfer to a 15ml centrifuge tube, centrifuge at 500 × g for 5 minutes, discard PBS, add 1ml DMEM medium containing 10% fetal bovine serum, mix gently, count the cells, and then inoculate 5*10 ⁶ cells with a diameter of A 10 cm petri dish (6 petri dishes in total) was supplemented with 10 ml of DMEM medium, and cultured in a cell incubator.

3. Cell Transfection

On the third day, the mice were infected with HSV-1 virus at a multiplicity of infection of MOI=10 (three dishes were not added with virus, and the other three dishes were infected with HSV-1 virus), and the culture was continued for 2 h.

4. Extraction of Cell Total Protein

Cells were washed twice with pre-chilled PBS buffer and the buffer was aspirated a final time. Add 1 ml of pre-chilled lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, PMSF, Cocktail 100*) to the dish. Scrape the cells from the culture dish with a pre-cooled cell scraper, transfer the suspension to a clean 1.5ml centrifuge tube, and place the centrifuge tube at 4°C for 15 minutes with slow shaking. Then centrifuge at 16000×g for 15 minutes at 4°C, collect the supernatant (transfer the supernatant to a new centrifuge tube immediately after the centrifugation), and label the control group (not infected with virus) and virus-treated group accordingly.

5. Immunoprecipitation (IP)

5.1 Take six 0.6ml centrifuge tubes, label them as follows, and add 500μl of cell lysate with a total protein concentration of 1μg/μl obtained in step 4 above to each tube.

5.2 Immunoprecipitation

The

samples

① and ② of the tIP group were processed as follows:

A. 5 μl of anti-A2B1 antibody (purchased from Santa Cruz Biotechnology) was added to each tube, and the antigen-antibody mixture was slowly shaken at 4°C overnight.

B. Protein G-coupled magnetic beads (purchased from Thermo) were washed three times with lysis buffer. Add 50 μl of magnetic bead suspension to the two tubes of antigen-antibody mixture described in step 5.1 above, and shake the antigen-antibody-magnetic bead mixture slowly at 4°C for 2 h.

C. After separating the magnetic beads from the supernatant with a magnetic stand, wash the magnetic beads three times with lysis buffer to obtain magnetic beads-antibody-antigen complexes.

D. Add 20μl 2*SDS loading buffer and cook at 100°C for 10 minutes. The magnetic beads were separated from the solution with a magnetic stand, and the supernatant of the solution was directly reserved for future use.

The

cIP group samples

③ and ④ were processed according to the Dynabeads Protein G (ThermoFisher, 10004D) manufacturer’s instructions.

The eIP group samples ⑤, ⑥ are processed as follows:

D. Add 20 μl 2U/μl of FabRICATOR enzyme diluted in PBS, and incubate at 37°C with gentle shaking for 10-30 minutes.

E. Use a magnetic stand to separate the magnetic beads from the solution, add 5 μl of 5*SDS loading buffer to the supernatant of each of the above solutions, heat at 100°C for 5 minutes, and set aside for later use.

6. Silver staining analysis

Proteins in polyacrylamide gels after electrophoretic separation were detected by conventional silver staining methods.

The experimental results are shown in Figure 7. Figure 7 shows the detection results of silver staining analysis of A2B1 protein obtained by tIP method (Figure 7A), cIP method (Figure 7B) or eIP (Figure 7C) method, wherein 1 is a protein sample obtained by immunoprecipitation of virus-infected cells, and the sample in lane 2 is a protein sample obtained by immunoprecipitation of cells infected with HSV-1 virus. As shown in Fig. 7, obvious IgG heavy chain bands were observed in both Fig. 7A and Fig. 7B, while no obvious IgG heavy chain bands were seen in Fig. 7C. This indicates that, compared with tIP and cIP methods, the eIP method can substantially remove antibody heavy chains from the samples, and the obtained protein samples can be used for subsequent mass spectrometry detection.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been disclosed, and that these changes are all within the scope of the present invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

A method for isolating a target protein, comprising the step of contacting a complex containing the target protein and an antibody capable of specifically binding the target protein with an enzyme capable of specifically degrading the antibody but not degrading the target protein.
The method of claim 1, comprising the steps of:

(1) Provide a sample containing the target protein;

(2) contacting an antibody that specifically binds to the protein of interest with the sample to form a complex containing the protein of interest and the antibody, and separating the complex; and

(3) contacting the complex with an enzyme capable of specifically degrading the antibody but not degrading the target protein, thereby obtaining the target protein.
The method of claim 2, wherein the method has one or more features selected from the group consisting of:

(a) The sample is derived from a biological sample or a non-biological sample; for example, the sample is derived from a virus, a prokaryote (eg, bacteria), a eukaryote (eg, a fungus, a plant, an animal such as an invertebrate, a vertebrate, a mammal animal (eg, human), protein library, or synthetic product;

(b) The sample is a solution sample containing the target protein; for example, the sample is selected from: cell lysate containing the target protein, tissue homogenate, body fluids (such as urine, saliva, blood, lymph, cerebrospinal fluid) , bile, gastric, intestinal, tears, etc.), lavage fluids (such as alveolar lavage, peritoneal lavage, vaginal lavage, gastric lavage, intestinal lavage, etc.), and buffers (such as phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, etc.);

(c) the target protein has a molecular weight of 30-80kD (eg, 40-70kD, 45-65kD);

(d) the target protein is not an antibody molecule;

(e) the target protein further comprises a tag peptide; preferably, the target protein is a fusion protein containing a tag peptide and a target polypeptide; preferably, the tag peptide is selected from c-Myc, Flag, 6*His, half Antigen, GST, HA, SUMO, fluorescent protein (eg GFP, YFP, RFP) or any combination thereof; preferably, the tag peptide is optionally linked to the N-terminus of the polypeptide of interest via a peptide linker and/or a protease cleavage site or C-terminal; preferably, the antibody is an antibody that specifically binds to a tag peptide or an antibody that specifically binds to a target polypeptide;

(f) the antibody is an IgG, such as an animal-derived IgG (eg, human, murine, rabbit, monkey, or sheep-derived IgG), or a chimeric IgG (eg, human-mouse chimeric IgG);

(g) the target protein is bound to a target protein-binding molecule (eg, covalently or non-covalently); preferably, the target protein-binding molecule is selected from proteins, nucleic acids, polysaccharides, lipids, or any combination thereof Preferably, the target protein-binding molecule has a molecular weight of 30-80kD (eg, 40-70kD, 45-65kD); preferably, the target protein-binding molecule is a protein (eg, with 30-80kD (eg, 40-70kD) , 45-65kD) molecular weight protein); preferably, the protein-binding molecule of interest is not an antibody; and

(h) the enzyme is selected from immunoglobulin degrading enzymes, endo-immunoglobulinases, or any combination thereof; preferably, the enzyme is selected from immunoglobulin G-degrading enzymes, immunoglobulin G endonucleases, or any combination thereof; preferably, the enzyme is a hydrolase capable of specifically cleaving the hinge region of an immunoglobulin molecule (eg, IgG); preferably, the enzyme is selected from IdeS or IdeZ or a combination thereof.
The method of claim 2 or 3, wherein the method has one or more features selected from the group consisting of:

(a) The method further includes, before step (2), the step of pre-processing the sample; preferably, the pre-processing step is selected from the group consisting of centrifugation, concentration, dilution, dialysis, chromatography, electrophoresis, desalting, adding Additional reagents (such as salts or molecular chaperones), or any combination thereof;

(b) in step (2), mixing and incubating the antibody with the sample under conditions that allow specific binding of the antigen to the antibody, thereby forming a complex containing the protein of interest and the antibody;

(c) The method further comprises, prior to performing step (3), the step of performing one or more washes on the complex; and

(d) The method further comprises the step of (4) analyzing the protein of interest (eg, WB analysis, MS analysis, functional analysis, or any combination thereof).
The method of any one of claims 2-4, wherein the antibody is immobilized on or linked to a support;

Preferably, the support is solid or semi-solid;

Preferably, in step (2), after contacting the sample solution with the antibody immobilized or attached to the support, the complex is separated by solid-liquid separation (eg, by centrifugation or filtration or the action of a magnetic field);

Preferably, the support is agarose beads or magnetic particles.
The method of any one of claims 2-4, wherein the antibody is not attached to a support;

Preferably, in step (2), the complex is isolated using an antibody-binding molecule capable of specifically binding to the antibody;

Preferably, the antibody binding molecule is selected from, anti-antibody, protein A, protein G and protein L;

Preferably, the antibody-binding molecule is immobilized on or linked to a support;

Preferably, in step (2), the antibody is first contacted with the sample to form a complex containing the protein of interest and the antibody; then, the complex is contacted with an antibody-binding molecule immobilized or linked to a support The complex is then separated by solid-liquid separation (for example, by centrifugation or filtration or the action of a magnetic field); or, in step (2), the antibody that specifically binds the protein of interest is first immobilized or linked to the support. The antibody binding molecule is contacted and then contacted with the sample to form a complex containing the protein of interest and the antibody; the complex is then separated by solid-liquid separation (eg, by centrifugation or filtration or the action of a magnetic field);

Preferably, the support is agarose beads or magnetic particles.
A test kit for separating a target protein or target protein-binding molecule in a sample, comprising: (1) an antibody that specifically binds to the target protein, and/or, an antibody-binding molecule that can specifically bind to the antibody and, (2) an enzyme that specifically degrades the antibody but does not degrade the protein of interest;

Preferably, the kit has one or more features selected from the group consisting of:

(a) The sample is derived from a biological sample or a non-biological sample; for example, the sample is derived from a virus, a prokaryote (eg, bacteria), a eukaryote (eg, a fungus, a plant, an animal such as an invertebrate, a vertebrate, a mammal animal (eg, human), protein library, or synthetic product;

(b) The sample is a solution sample containing the target protein; for example, the sample is selected from: cell lysate containing the target protein, tissue homogenate, body fluids (such as urine, saliva, blood, lymph, cerebrospinal fluid) , bile, gastric, intestinal, tears, etc.), lavage fluids (such as alveolar lavage, peritoneal lavage, vaginal lavage, gastric lavage, intestinal lavage, etc.), and buffers (such as phosphate buffer, citrate buffer, Tris-HCl buffer, MOPS buffer, Tris-Gly buffer, etc.);

(c) the target protein has a molecular weight of 30-80kD (eg, 40-70kD, 45-65kD);

(d) the target protein is not an antibody molecule;

(e) the target protein further comprises a tag peptide; preferably, the target protein is a fusion protein containing a tag peptide and a target polypeptide; preferably, the tag peptide is selected from c-Myc, Flag, 6*His, half Antigen, GST, HA, SUMO, fluorescent protein (eg GFP, YFP, RFP) or any combination thereof; preferably, the tag peptide is optionally linked to the N-terminus of the polypeptide of interest via a peptide linker and/or a protease cleavage site or C-terminal; preferably, the antibody is an antibody that specifically binds to a tag peptide or an antibody that specifically binds to a polypeptide of interest;

(f) the antibody is an IgG, such as an animal-derived IgG (such as human, murine, rabbit, monkey, or sheep-derived IgG), or a chimeric IgG (such as a human-mouse chimeric IgG);

(g) the target protein is bound to a target protein-binding molecule (eg, covalently or non-covalently); preferably, the target protein-binding molecule is selected from proteins, nucleic acids, polysaccharides, lipids, or any combination thereof Preferably, the target protein-binding molecule has a molecular weight of 30-80kD (eg, 40-70kD, 45-65kD); preferably, the target protein-binding molecule is a protein (eg, with 30-80kD (eg, 40-70kD) , 45-65kD) molecular weight protein); preferably, the target protein binding molecule is not an antibody;

(h) the enzyme is selected from immunoglobulin degrading enzymes, endo-immunoglobulinases, or any combination thereof; preferably, the enzyme is selected from immunoglobulin G-degrading enzymes, immunoglobulin G endonucleases, or any combination thereof; preferably, the enzyme is a hydrolase capable of specifically cleaving the hinge region of an immunoglobulin molecule (eg, IgG); preferably, the enzyme is selected from IdeS or IdeZ or a combination thereof;

(i) the antibody or antibody-binding molecule is immobilized on or linked to a support; preferably, the support is solid or semi-solid; preferably, the support is agarose beads or magnetic particles; and

(j) The antibody binding molecule is selected from anti-antibody, protein A, protein G and protein L.
The kit of claim 7, wherein the kit further comprises a component selected from the group consisting of: one or more buffers (such as, but not limited to, buffers for dissolving the protein of interest, for antigens and antibodies) Buffers for specific binding, buffers for specific binding of antibodies to antibody-binding molecules, buffers for washing, working buffers for the enzymes, etc.), reagents for lysing cells (e.g., cell lysates) , Reagents for performing protein electrophoresis (polyacrylamide gel, loading buffer, running buffer, reducing agent), Reagents for performing WB analysis (eg, cellulose membrane, blocking solution, capable of specifically binding to the target A primary antibody (which is optionally labeled) to a protein or protein of interest binding molecule, a secondary antibody capable of specifically binding to the primary antibody (which is optionally labeled), a chromogenic reagent, etc.) for use in Reagents for MS analysis, and any combination thereof.
Use of a combination of (1) an antibody capable of specifically binding a protein of interest and/or an antibody-binding molecule capable of specifically binding the antibody and (2) an enzyme capable of specifically degrading the antibody but not the protein of interest, for separation The target protein or target protein-binding molecule in the sample, or used to prepare a kit for separating the target protein or target protein-binding molecule in the sample.