Mutant protein A and application thereof in field of affinity chromatography

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a mutant protein A and application thereof in the field of affinity chromatography. The amino acid of the mutant protein A is as shown in SEQ ID NO. 2. According to the invention, the 146th glycine (G) of the natural protein A is replaced by the alanine (A), so that the alkali resistance and antibody binding capacity of the obtained mutant protein A are greatly improved; the affinity chromatography medium obtained by coupling the aptamer with a solid phase carrier has the characteristics of selective adsorption of IgG-like protein (containing an Fc structural domain), high antibody binding capacity and sample loading amount, excellent alkali resistance and the like, is suitable for elution at a high pH value, can greatly reduce the aggregation degree and aggregate content of the antibody, and improves the concentration and yield of the purified antibody.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a mutant protein A and its application in the field of affinity chromatography. Background Art

[0002] Protein A is a membrane protein of Staphylococcus aureus, containing 5 homologous domains, each of which is a three-helix bundle composed of 57-60 residues, namely E, D, A, B and C domains, extending from the N-terminus to the C-terminus. These domains can specifically bind to the Fc segment of the antibody to separate and purify antibodies of different subtypes or genetically engineered recombinant proteins containing antibody Fc fragments from ascites, serum and cell culture supernatant or cell extracts. It has the advantages of simple operation, good versatility, strong selectivity and high purification efficiency. Therefore, Protein A has become a widely used antibody affinity chromatography medium.

[0003] Normally, on an affinity chromatography medium with protein A as a ligand, one protein A molecule can bind to at least two IgG molecules at the same time. After IgG binds to protein A, the IgG molecules are eluted from protein A by lowering the pH, and then neutralized with an alkaline buffer. Moreover, for most antibodies, low pH acidic elution conditions often lead to protein denaturation, easily induce the production of polymers and even inactivate the target antibody, ultimately leading to a decrease in antibody purity and a low antibody recovery rate, making this method unsuitable for low pH sensitive proteins. Increasing the pH of the eluent is an effective way to solve this problem. In addition, when regenerating the protein A affinity chromatography medium, a high concentration of alkali (e.g., 0.1-1.0M NaOH solution) is usually used for washing to remove residual contaminants such as endotoxins, protein precipitates, nucleic acids, and viruses attached to the protein A medium. However, in this process, high concentrations of alkali will destroy the structural stability of protein A, greatly reduce its binding capacity, and shorten the service life of the medium. Therefore, it is of great significance to improve the alkali resistance and stability of protein A. Summary of the invention

[0004] In view of the problems in the prior art that protein A has poor alkali resistance, low pH value of the eluent when used as an affinity chromatography medium ligand, and poor stability, the present invention provides a mutant protein A, and further provides the application of the mutant protein A in the field of affinity chromatography.

[0005] The present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a mutant protein A, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] The second aspect of the present invention provides the use of the mutant protein A as described above in the field of affinity chromatography.

[0008] The third aspect of the present invention provides an IgG-like protein affinity chromatography medium, comprising a solid phase carrier and a mutant protein A coupled to the solid phase carrier, wherein the amino acid sequence of the mutant protein A is shown in SEQ ID NO.2.

[0009] Furthermore, the mass volume ratio of the mutant protein A to the solid phase carrier is 2.8:1.

[0010] Furthermore, the solid phase carrier is selected from one or more of agarose gel, dextran gel, agarose and dextran composite gel, cellulose, polyacrylamide gel, polymethyl methacrylate gel, and the IgG-like protein includes IgG antibody or a fusion protein containing the Fc domain of IgG antibody.

[0011] Furthermore, coupling the mutant protein A with the solid phase carrier comprises the following steps: adding the mutant protein A and the activated solid phase carrier to a coupling solution containing sodium bicarbonate and sodium chloride, mixing, reacting at room temperature, removing the supernatant after the reaction is completed, washing the precipitate to neutrality, and obtaining an IgG-like protein affinity chromatography medium.

[0012] Furthermore, the concentration of sodium bicarbonate in the coupling solution is 0.1M, and the concentration of sodium chloride is 0.5M.

[0013] Furthermore, the volume ratio of the coupling liquid to the solid phase carrier is 1:1.

[0014] The fourth aspect of the present invention provides an antibody-immobilized affinity chromatography medium, comprising an antibody and the IgG-like protein affinity chromatography medium as described above, wherein the Fc domain of the antibody is covalently bound to the mutant protein A of the IgG-like protein affinity chromatography medium.

[0015] Furthermore, the mass ratio of the mutant protein A to the antibody is 0.56:4.

[0016] Furthermore, the antibody is an IgG antibody against the Flag tag.

[0017] Furthermore, the Fc domain is covalently bound to the mutant protein A through a cross-linking agent, comprising the following steps: adding the IgG-like protein affinity chromatography medium and the antibody to a PBS solution, incubating at low temperature for 1-10 hours, then removing the reaction solution, adding a solution of dimethyl pimelimidate dihydrochloride, reacting at room temperature for 1-3 hours, and after cross-linking is completed, adding a tris solution for blocking, and then washing to neutrality to obtain the antibody-immobilized affinity chromatography medium.

[0018] Furthermore, the concentration of the dimethyl pimelimidate dihydrochloride solution is 50 mM.

[0019] The advantages and positive effects of the present invention are:

[0020] The present invention replaces the 146th glycine (G) of natural protein A with alanine (A), and the alkali resistance and antibody binding ability of the obtained mutant protein A are greatly improved. The affinity chromatography medium obtained by coupling it with a solid phase carrier has the characteristics of selective adsorption of IgG-like proteins (including Fc domain), high antibody binding ability and sample loading capacity, excellent alkali resistance, etc., and can increase the pH of the eluate of wild-type protein A from 2.2 to 3.0, which is beneficial to greatly reduce the aggregation degree and aggregate content of antibodies, and improve the concentration and yield of the purified antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The figure is the SDS-PAGE gel electrophoresis diagram of the protein A purified in Example 1 of the present invention, wherein the left figure is the wild-type protein A and the right figure is the mutant protein A;

[0023] Figure 2 The SDS-PAGE gel electrophoresis diagram of the protein A solution before and after coupling with agarose gel microspheres in Example 2 of the present invention, wherein the left diagram is the wild-type protein A, and the right diagram is the mutant protein A;

[0024] Figure 3 This is an SDS-PAGE gel electrophoresis diagram of the purification of Flag antibody using IgG affinity chromatography medium with protein A as a ligand in Example 3 of the present invention, wherein the left figure is wild-type protein A and the right figure is mutant protein A;

[0025] Figure 4 This is a high performance liquid chromatogram of the Flag antibody purified by IgG affinity chromatography medium using protein A as a ligand in Example 3 of the present invention;

[0026] Figure 5 SDS-PAGE gel electrophoresis diagram for verifying the cross-linking effect between mutant protein A and antibody in the preparation of antibody-immobilized affinity chromatography medium in Example 4 of the present invention;

[0027] Figure 6This is the SDS-PAGE gel electrophoresis diagram of the purification of Flag tag fusion protein by antibody-immobilized affinity chromatography medium in Example 5 of the present invention;

[0028] Figure 7 The SDS-PAGE gel electrophoresis diagrams of the antibody-immobilized affinity chromatography medium of Example 6 of the present invention immersed in different treatment solutions for different time periods, wherein the left diagram is for treatment with 0.3-0.5 M guanidine hydrochloride and 1.5 M urea, and the right diagram is for treatment with 2 M urea and high pH alkali solution. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0031] In order to better understand the present invention rather than limit the scope of the present invention, all the numerals and other numerical values ​​used in this application indicating dosage, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as being obtained according to the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "include", "comprise", "contain", "have" and the like is non-restrictive, and other steps and other ingredients that do not affect the results can be added.

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0033] An embodiment of the present invention provides a mutant protein A, wherein the amino acids of the mutant protein A are shown in SEQ ID NO.2.

[0034] The invention replaces the 146th glycine (G) of natural protein A with alanine (A), and the alkali resistance and antibody binding ability of the obtained mutant protein A are greatly improved. The affinity chromatography medium obtained by coupling the mutant protein A with a solid phase carrier has the characteristics of selective adsorption of IgG-like protein (including Fc domain), high antibody binding ability and sample loading capacity, excellent alkali resistance, etc. The pH of the eluent of wild-type protein A can be increased from 2.2 to 3.0, and the IgG antibody can be eluted from the mutant protein A under the acidic condition of pH 3.0, which greatly reduces the aggregation degree and aggregate content of the antibody. Compared with the wild-type protein A, when the affinity chromatography medium prepared by using the mutant protein A as a ligand is used to purify the IgG antibody, the antibody loading capacity can reach about 20 mg / mL, and the concentration and yield of the purified antibody are increased by about 3 times. In addition, the present invention further reacts the solid phase carrier of coupled protein A with the antibody under the action of a cross-linking agent to obtain an antibody-directed cross-linked affinity chromatography medium, which has the advantages of high affinity, good antigen capture ability, high sample loading capacity and good stability when used to purify antibodies that specifically bind to antigens. Taking the coupled anti-Flag tag antibody as an example, the Flag antibody-directed cross-linked affinity chromatography medium of the present invention has a better purification effect on fusion proteins containing Flag tags than the commercially available DDDDK filler, and has a wide range of application prospects in the field of protein affinity purification. Moreover, the mutant protein A and antibody-directed cross-linked affinity chromatography medium of the present invention have high stability in alkaline environments such as guanidine hydrochloride, urea and NaOH, which is conducive to the removal of impurities during the regeneration of the affinity chromatography medium, prolonging the service life of the affinity chromatography medium, and also indicating that the mutant protein A of the present invention has good structural stability in the aforementioned environment.

[0035] The embodiments of the present invention also provide the use of the mutant protein A as described above in the field of affinity chromatography.

[0036] Based on the same inventive concept as above, another embodiment of the present invention provides an IgG-like protein affinity chromatography medium, comprising a solid phase carrier and a mutant protein A coupled to the solid phase carrier.

[0037] Based on the high affinity and binding activity of mutant protein A to the Fc domain of IgG antibody, the present invention uses it as a ligand and couples it with a solid phase carrier to obtain an IgG-like protein affinity chromatography medium, which can achieve specific binding and efficient recovery of IgG-like proteins in the sample to be purified. The medium of the present invention has the advantages of high sample loading capacity and high elution pH value, which is beneficial to improving the recovery yield and recovery rate of IgG-like proteins, reducing the risk of protein denaturation and aggregation, and improving the purification efficiency of IgG-like proteins.

[0038] The IgG-like protein described above can be selected from IgG antibodies or fusion proteins containing the Fc domain of IgG antibodies. The antibodies described above can be polyclonal antibodies, monoclonal antibodies, bispecific antibodies or multispecific antibodies.

[0039] Optionally, the solid phase carrier is selected from one or more of agarose gel, dextran gel, agarose and dextran composite gel, cellulose, polyacrylamide gel, polymethyl methacrylate gel, preferably agarose gel.

[0040] Optionally, the mass-to-volume ratio (w / v) of the mutant protein A to the solid phase carrier is 2.8:1.

[0041] In a preferred embodiment, coupling the mutant protein A with the solid phase carrier comprises the following steps: adding the mutant protein A and the activated solid phase carrier to a coupling solution containing sodium bicarbonate and sodium chloride, mixing, reacting at room temperature, removing the supernatant after the reaction is completed, washing the precipitate to neutrality, and obtaining an IgG-like protein affinity chromatography medium.

[0042] The activation method of the solid phase carrier described above includes, but is not limited to, cyanogen bromide, epichlorohydrin or sodium periodate activation method, which is a conventional technique in the art and will not be described in detail here.

[0043] Optionally, the concentration of sodium bicarbonate in the coupling solution is 0.1 M, and the concentration of sodium chloride is 0.5 M. The volume ratio of the coupling solution to the solid phase carrier is 1:1.

[0044] Based on the same inventive concept as above, another embodiment of the present invention provides an antibody-immobilized affinity chromatography medium, comprising an antibody and the IgG-like protein affinity chromatography medium as described above, wherein the Fc domain of the antibody is covalently bound to the mutant protein A of the IgG-like protein affinity chromatography medium.

[0045] At present, the affinity chromatography media on the market mostly adopt random coupling preparation technology, that is, after activating the solid phase carrier with cyanogen bromide, epichlorohydrin or sodium periodate, the antibody is coupled to the solid phase carrier. This coupling mode is non-specific, and the directionality of the antibody binding to the solid phase carrier cannot be controlled, which is bound to affect the efficiency of the affinity chromatography medium binding to the target antigen and increase the risk of non-specific adsorption. Therefore, the present invention uses mutant protein A as a connecting arm to bridge the solid phase carrier and the antibody. Since the mutant protein A binds to the Fc domain of the antibody, the antigen binding region (Fab) of the antibody is fully exposed to the outside, and the antigen binding process is not affected by solidification, which ensures the antigen binding ability and efficiency of the antibody, reduces non-specific adsorption, and is conducive to improving the purification efficiency of the antibody for the target antigen. In addition, the method of the present invention improves the connection stability between the antibody and the mutant protein A, which is conducive to improving the ability to tolerate the washing of acidic eluents and alkaline solutions.

[0046] Optionally, the mass ratio of the mutant protein A to the antibody is 0.56:4.

[0047] Optionally, the Fc domain is covalently bonded to the mutant protein A via a cross-linker, specifically, the cross-linker is selected from dimethyl pimelimidate dihydrochloride (DMP), each of which has an amine-reactive imidate group at each end of the molecule, which can react with primary amines at pH 8-10 to form an amide bond to cross-link the mutant protein A and the Fc domain of the antibody.

[0048] In a preferred embodiment, the cross-linking method described above comprises: adding the IgG-like protein affinity chromatography medium and the antibody to a PBS solution, incubating at low temperature for 1-10 hours, then removing the reaction solution, adding a solution of dimethyl pimelimidate dihydrochloride, reacting at room temperature for 1-3 hours, and after cross-linking is completed, adding a tris solution for blocking, and then washing to neutrality to obtain an antibody-immobilized affinity chromatography medium.

[0049] Optionally, the concentration of the dimethyl pimelimidate dihydrochloride solution is 50 mM, and the DMP solution is obtained by dissolving in 0.1 M borate buffer (pH 8.2).

[0050] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0051] Example 1 Expression and purification of mutant protein A

[0052] When purifying antibodies or other proteins containing Fc domains using affinity chromatography media with protein A as a ligand, elution under lower pH conditions can easily lead to insoluble aggregates of the protein, resulting in a loss in yield, and high-concentration alkali solution is also required for flushing during regeneration of the affinity chromatography medium. Therefore, it is of great significance to improve the alkali resistance of protein A to increase the elution pH during the purification process. The asparagine-glycine sequence in the B binding domain of protein A is more sensitive to alkali treatment. Therefore, the present invention chooses to replace the 146th glycine (G) of natural protein A (protein A, also called wild-type protein A) with alanine (A), and the mutant protein A (protein A-mutant) is obtained. The alkali resistance of the mutant protein A has been greatly improved. Among them, the amino acid sequence of wild-type protein A (protein A-WT) is as follows:

[0053] AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRN G FIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK (see SEQ ID NO.1), wherein the underlined region is the site to be mutated;

[0054] The amino acid sequence of protein A-mutant is as follows:

[0055] AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNAFIQ SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK (see SEQID NO.2), where the area shown in bold gray is the mutation site.

[0056] The wild-type and mutant protein A genes were sent to Wuhan Jinkairui Bioengineering Co., Ltd. for synthesis. The aforementioned genes were constructed between the EcoR I and XhO I restriction sites of the pET28a vector by full gene synthesis. The competent cells of Escherichia coli BL21 (DE3) were transformed by heat shock method to obtain positive clones. The positive clones were tested by colony PCR. After the correct expression of the target gene was detected, the positive clones were inoculated into LB medium and cultured overnight for activation. 2 mL of the activated bacterial solution was added to 300 mL of LB medium and cultured at 37°C until OD 600nmThe concentration of 50 mM IPTG was 0.5-0.55, and then IPTG was added to a final concentration of 0.8 mM, and the expression was induced at 16°C for 16 h.

[0057] The induced bacterial liquid was collected into a centrifuge tube, centrifuged at 12000 rpm and 4°C for 5 min, and the supernatant was discarded to obtain the bacterial cells; 30-50 mL of bacterial disruption solution A (including 50 Mm tris-HCl and 150 Mm NaCl, pH 8.0) was taken to suspend the bacterial cells, and the cells were disrupted using an ultrasonic disruptor.

[0058] First disruption: transfer the suspended bacterial solution to a 50mL centrifuge tube, fix the centrifuge tube with ice, select the No. 6 amplitude changer, power 350W, bacteriostasis time 3s, interval time 3s, time 5min, then place in an ice-water mixture to cool for 5min, repeat the above steps to disrupt the bacteria for 15min. After completion, divide into two centrifuge tubes and centrifuge at 9000rpm for 10min to obtain supernatant 1 and precipitate.

[0059] Second disruption: Pour 30 mL of lysis solution B (including 50 Mm tris-HCl, 150 Mm NaCl and 2 M urea, pH 8.0) into the precipitate, blow the precipitate evenly, transfer it to a 50 mL centrifuge tube, fix the centrifuge tube with ice, select No. 6 amplitude variable rod, power 350 W, lysis time 3 s, interval time 3 s, timing 5 min, and centrifuge at 9000 rpm for 10 min to obtain supernatant 2 and inclusion bodies.

[0060] The supernatant 1 was purified using Ni Seplife FF (NTA) affinity purification matrix (purchased from Lanxiao, product number A4023205). The operation process was as follows: (1) 30 mL of pure water was used to wash away the ethanol in the Ni Seplife FF (NTA) affinity purification matrix (pre-packed column 2 mL) (the commercial filler was stored with 20% ethanol), and the matrix was equilibrated with 30 mL of binding buffer (Binding Buffer), and the flow rate was controlled at 2 mL / min, and the liquid flowed out; (2) The supernatant after bacterial destruction and centrifugation was mixed with the matrix in a 50 mL centrifuge tube, and the mixture was combined in a mixing incubator at 4°C for 45 minutes. The combined mixture was added to the pre-packed column, and the flow rate was controlled at 2 mL / min. The liquid flowed out and the flow-through was collected; (3) 30 mL of washing buffer (Washing Buffer) was used to balance the matrix. (4) Soak the matrix in 4 mL of elution buffer (Elution Buffer A) for 5-8 min. After sufficient reaction, collect the eluate at a flow rate of 0.6 mL / min and use G250 to detect whether there is protein eluted. If G250 turns blue, protein is eluted and the eluate is collected. Otherwise, no protein is eluted. Then use Elution Buffer B as the elution buffer to continue eluting the protein in the same manner as before. Whenever the liquid in the pre-packed column is about to run out, add Elution Buffer A to the pre-packed column in units of 2 mL. Buffer A, collect 4 mL, 6 mL, and 8 mL of the eluate in the EP tube and detect them using G250; (5) wash and recover the matrix. The formula of the buffer mentioned above is as follows:

[0061] Buffer name Tris (mM) NaCl(mM) Imidazole(mM) Glycerol(mM) pH BindingBuffer 50 250 5 / 8.0 Washing Buffer 50 250 50 / 8.0 ElutionBufferA 50 250 250 68.5 8.0 ElutionBufferB 50 250 500 68.5 8.0

[0062] The eluted target protein was subjected to SDS-PAGE gel electrophoresis. Figure 1 As shown, in the left figure, lane 1 is wild-type protein A, lane 2 is 0.4 mg / mL bovine serum albumin, and lane 3 is a molecular marker. In the right figure, lane 1 is a molecular marker, lane 2 is 0.4 mg / mL bovine serum albumin, and lane 3 is a mutant protein A. Figure 1-2It can be seen that the size of the protein after expression is 33kDa. After the supernatant is purified, the concentration of the recombinant wild-type protein A (proteinA-WT) is 0.4mg / mL, the purity is 95%, the total amount is 2.1mg, and the yield is 69mg / 1000mL through BCA quantitative determination; the concentration of the recombinant mutant protein A (proteinA-mutant) is 0.4mg / mL, the purity is 95%, the total amount is 2mg, and the yield is 67mg / 1000mL.

[0063] Example 2 Preparation of IgG Affinity Chromatography Medium

[0064] The mutant protein A of the present invention is used as a ligand and coupled to a solid phase carrier to prepare an affinity chromatography medium, which is suitable for separation and purification of protein molecules containing Fc domains or segments, such as IgG antibodies or Fc fusion proteins.

[0065] In this example, agarose gel microspheres are used as solid phase carriers. Agarose gel microspheres CNBr-activated Singarose 4B are purchased from Huachun (Yishengke), with the product number HC1130702. Protein A is fixed on the CNBr-activated Singarose 4B carrier according to the instructions, including: 1) Assembling the column tube: taking out the sterilized 10mL purification column tube, assembling the gasket, washing the column tube with pure water 1-2 times, observing the water flow rate to prevent the gasket from being improperly installed or damaged, then rewarming at room temperature, inverting and mixing to suspend the matrix; 2) Cleaning the matrix: adding 1mL CNBr-activated Singarose 4B matrix to the column tube, washing 3 column tube volumes with coupling A solution, and then washing 2 column tube volumes with coupling B solution, and finally adding coupling B solution equal to the volume of CNBr-activated Singarose 4B; 3) Coupling of protein A with the matrix: adding 4mg of protein A solution to CNBr-activated Singarose 4B matrix, mix well in the column tube, seal it, and place it on a rotating incubator for 4 hours at room temperature or overnight at 4°C; 4) Blocking: extract and collect the supernatant after coupling, add 8mL of blocking solution, and incubate at room temperature for 2-4 hours; 5) Wash the matrix: use 1×PBS, 0.1M glycine (Gly) solution (pH 4.0), and 1×PBS to wash the matrix, three times each. The formula of the coupling buffer is as follows:

[0066] Reagent name Element pH Coupling A solution 1mM HCl / Coupling Solution B 0.1M sodium bicarbonate and 0.5M sodium chloride 8.3 Blocking solution 0.1MTris 8.3

[0067] The wild-type and mutant protein A solutions before and after coupling were subjected to SDS-PAGE gel electrophoresis. The results were as follows: Figure 2As shown, in the left figure, lane 1 is 0.4 mg / mL bovine serum albumin, lane 2 is a molecular marker, and lanes 3 and 4 are wild-type protein A before and after coupling. In the right figure, lane 1 is 0.4 mg / mL bovine serum albumin, lanes 2 and 3 are mutant protein A before and after coupling, and lane 4 is a molecular marker. It can be seen that the wild-type protein A protein is coupled with 1 mL CNBr-activated Singarose 4B matrix, binding protein 2.4 mg, and the coupling rate is 60%, and the mutant protein A protein is coupled with 1 mL CNBr-activated Singarose 4B matrix, binding protein 2.8 mg, and the coupling rate is 70%.

[0068] Example 3: Purification of Flag Antibody by IgG Affinity Chromatography Medium

[0069] This example uses an antibody against the Flag tag as an example to verify the purification effect of the IgG affinity chromatography medium prepared above. The Flag tag is a short peptide composed of 8 amino acids, and the amino acid sequence is: DYKDDDDK. Its molecular weight is very small, so it will not cover other protein epitopes and domains in the fusion protein, nor will it change the function, secretion or transport of the fusion protein. After the target protein N-terminus is fused with the Flag polypeptide, the C-terminus can also be fused with other modules or different affinity ligands. Therefore, in the field of fusion protein expression, the Flag tag is very commonly used. On the basis of the Flag tag, three Flag tags are connected in series to form a 3×FLAG tag composed of 22 amino acids, which has a molecular weight of only 2.7kDa, is naturally hydrophilic, and contains an enterokinase cleavage site, which is convenient for removing the tag, and the tag has a higher detection sensitivity.

[0070] The vectors containing the anti-Flag tag antibody light chain and heavy chain genes were mixed at a mass ratio of 1:2, and the vector DNA was diluted with Opti MEM (purchased from Thermo Fisher, catalog number 31985070) in a sterile test tube to use 1.3 μg of total DNA per 1 mL of cells; in addition, 1 mg / mL of PEI transfection reagent (PEI MAX TM-Transfection Grade LinearPolyethylenimine Hydrochloride (MW 40000) purchased from polysciences, catalog number 24765-1) was added and diluted into Opti MEM, and the amount of OptiMEM used was one twentieth of the total cell volume; then the PEI solution was added to the carrier solution, PEI (μL): total DNA (ug) = 3:1, mixed immediately, and transfected 293F cells. The transfected cells were cultured with OPM-293CD05Medium (purchased from Aopuma, catalog number 81075-00); the cells were harvested after culturing for 16-20 hours, and the supernatant was collected.

[0071] The IgG affinity chromatography medium prepared in Example 2 was incubated with the harvested cell supernatant to purify the Flag antibody. Specifically, the process includes: 1) transferring the culture supernatant or cell-containing culture medium to a 50 mL centrifuge tube, centrifuging at 400 g, 4°C (or room temperature) for 10 min, discarding the precipitate, and collecting the supernatant; 2) taking 1 mL of the IgG affinity chromatography medium prepared in Example 2, centrifuging at 400 g for 2 min at room temperature, and discarding the supernatant; 3) adding 500 μL of washing buffer to resuspend the IgG affinity chromatography medium, flipping it back and forth 4-5 times, centrifuging at 400 g for 2 min at room temperature, and repeating once; 4) placing the washed IgG affinity Add the chromatography medium to the culture supernatant and incubate at 4°C with shaking for 3-4h or overnight; 5) Centrifuge at 1000g for 10min and carefully aspirate the culture medium; 6) Add a wash buffer that is ten times the volume of the medium to resuspend the medium, centrifuge at 1000g for 10min, carefully aspirate the wash buffer, and repeat twice; 7) Add an elution buffer that is six times the volume of the medium and transfer to an empty centrifuge column, centrifuge at 1000g for 2min to collect the purified IgG antibody, and use the equilibrium buffer to adjust the pH to neutral. The above-mentioned buffer formula is as follows:

[0072] Reagent name Element pH Wash buffer 0.02M phosphate buffer 7.0 Elution buffer 0.1M Glycine 4.0, 3.0 or 2.2 Equilibration buffer 0.1MTris 9.0

[0073] The flow-through, washing buffer and elution buffers of different pH values ​​during the purification of IgG affinity chromatography medium using wild-type and mutant protein A as ligands were collected and subjected to SDS-PAGE gel electrophoresis. The results are shown in FIG. Figure 3As shown, in the left figure, lane 1 is 0.4 mg / mL bovine serum albumin, lane 2 is cell culture supernatant, lane 3 is flow-through, lane 4 is washing buffer, lane 5 is molecular marker, lane 6 is 0.1 M glycine eluent (pH 4.0), lanes 7-8 are 0.1 M glycine eluent (pH 3.0), and lanes 9-10 are 0.1 M glycine eluent (pH 2.0); in the right figure, lane 1 is 0.4 mg / mL bovine serum albumin, lane 2 is cell culture supernatant, lane 3 is flow-through, lane 4 is washing buffer, lane 5 is 0.1 M glycine eluent (pH 4.0), lanes 6-7 are 0.1 M glycine eluent (pH 3.0), and lane 8 is molecular marker. It can be seen that the wild-type proteinA was coupled to CNBr-activated Singarose 4B to purify Flag antibody, and the obtained antibody concentration was 0.22 mg / mL, the total amount was 1.8 mg, and the antibody yield was 62.1 mg / 1000 mL; the mutant proteinA was coupled to CNBr-activated Singarose 4B to purify Flag antibody, and the obtained antibody concentration was 0.64 mg / mL, the total amount was 5 mg, and the antibody yield was 172.5 mg / 1000 mL. Comparing the antibody purification process of the mutated and unmutated proteinA media, the elution pH of the mutated proteinA purified antibody is greatly improved, and the present invention improves the affinity and binding efficiency of proteinA to the antibody Fc segment through mutation, which is conducive to improving the antibody yield.

[0074] In addition, SEC-HPLC was used to detect the purity of the antibody, using a TSK superSW3000 column (purchased from TOSOHBIOSCIENCE, product number 18675) and a mobile phase (pH 6.4) of 50 mM PB and 500 mM NaCl; the antibody eluted with wild-type protein A at pH 2.2 and the antibody eluted with mutant protein A at pH 3.0 were dialyzed and filtered, and loaded onto HPLC with a loading volume of 20 μL and a concentration of 0.1 μg / mL; the peak area percentage at the A280 wavelength in the HPLC detection was used to reflect the aggregation degree of the antibody, and the results are shown in Figure 4. Among them, the horizontal axis is the elution time, the unit is minutes (min), and the vertical axis is the electrical signal, the unit is mAU, which corresponds to the peak height. It can be seen from the figure that the antibody purified by mutant protein A as a ligand has high purity, basically no impurity peaks, the antibody purity reaches 100%, and no aggregate peak is seen, while the antibody purity and aggregate content obtained by wild-type protein A as a ligand purification are 65% and 35% respectively. It is confirmed that the mutant protein A can significantly reduce the risk of antibody aggregation and improve the stability of the antibody when eluted as a ligand.

[0075] Example 4 Preparation of antibody-immobilized affinity chromatography medium

[0076] In this embodiment, the IgG affinity chromatography medium prepared in Example 2 is combined with the Fc segment of a specific antibody, and then a cross-linking agent is added to react, so that the antibody is connected to the microsphere with the Fc region and the Fab region extends outward, and the variable region retained outside can achieve efficient binding with the antigen, thereby achieving affinity purification of the specific antigen. In the aforementioned method, the protein A of the present invention plays a bridging role to ensure that the antibody is cross-linked to the solid phase medium in a directional manner, and the antigen binding domain of the antibody extends outside the solid phase medium. The obtained antibody-immobilized affinity chromatography medium is suitable for efficient purification of proteins, which is beneficial to improve the binding efficiency and activity of the affinity chromatography medium with the target antigen and reduce non-specific adsorption. Moreover, the mutant protein A of the present invention has good affinity and high binding strength with the Fc segment of the antibody. After cross-linking, it can ensure that the antibody and the affinity ligand are firmly fixed, which is beneficial to greatly improve the stability of the affinity chromatography medium in the acidic eluent, improve the purity of the purified protein and reduce the risk of antibodies mixing into the purified protein. Compared with affinity chromatography media that use cyanogen bromide, epichlorohydrin or sodium periodate to activate agarose gel solid phase carrier and then couple antibodies, the antibody-immobilized affinity chromatography media prepared by the present invention has obvious application advantages.

[0077] Taking the Flag antibody of coupling Example 3 as an example, the preparation method of the antibody-immobilized affinity chromatography medium includes: 1) matrix balance: take 0.2 mL of the IgG affinity chromatography medium prepared in Example 2, wash it with pure water for 4-6 column volumes, then wash it with 1×PBS for 4-6 column volumes, and store it with 1×PBS; 2) antibody binding: add 4 mg of Flag antibody and mix it thoroughly with 0.2 mL of IgG affinity chromatography medium, wherein the mass ratio of antibody to protein A protein is 4:0.56, and then incubate it at 4°C for 3 hours on a rotating incubator. After the binding is completed, collect the effluent, and determine the A280 value by spectrophotometry to detect the concentration of unbound antibody in the effluent and determine the binding efficiency; 3) wash the Flag-protein A mixture: wash the Flag-protein A mixture with 1×PBS and collect the washing buffer; 4) antibody crosslinking with protein A: add 10 mL of 50 mM dimethyl pimelimidate dihydrochloride to the Flag-protein A mixture. Dihydrochloride (DMP) solution (obtained by dissolving DMP in 0.1M borate buffer (pH8.2)), mixed well, reacted at room temperature for 2h, replaced with fresh DMP solution in the middle, after cross-linking, collected the flow-through after antibody cross-linking, and washed the cross-linked product with 10mL 50mM sodium tetraborate buffer (pH 8.2); 5) blocking: blocking with 10mL 50mM tris solution (pH 8.3) at room temperature for 2h, and collecting the effluent; 6) washing: washing with 10mL 50mM sodium tetraborate buffer, and collecting the washing buffer (wash); 7) acid washing: washing the cross-linked product with 0.1M glycine solution at pH3.0 and 2.2, respectively, and collecting the acid washing solution; 8) neutralization: washing the cross-linked product with 1×PBS solution until neutral, and obtaining the Flag antibody immobilized affinity chromatography medium, named Flag-proteinA cross-linking matrix.

[0078] The flow-through, washing buffer and acid washing solution of the preparation process of the affinity chromatography medium immobilized with the wild-type and mutant protein A as ligands were collected and subjected to SDS-PAGE gel electrophoresis. The results are as follows: Figure 5As shown, lane 1 is 0.4 mg / mL bovine serum albumin, lane 2 is Flag antibody before binding, lane 3 is flow-through after Flag antibody binding, lane 4 is molecular marker marker, lane 5 is washing buffer after Flag antibody binding, lanes 6-7 are flow-through after Flag antibody cross-linking, lane 8 is flow-through after blocking, lane 9 is washing buffer after blocking, lane 10 is pH3.0 acid wash solution, lane 11 is pH2.2 acid wash solution, and lane 12 is washing buffer after acid wash. Calculate the antibody concentration of each lane, and the results are shown in the following table:

[0079] Sample name Concentration (mg / mL) Volume (mL) Total amount (mg) Flow through after Flag antibody binding 0.041 6.4 0.262 After cross-linking of the Flag antibody, the pH value was 3.0. 0.07 0.92 0.0644 After cross-linking of the Flag antibody, the pH was washed at 2.2 0.032 0.85 0.0272

[0080] The above results show that the mutant protein A of the present invention has a high binding efficiency with the Flag antibody. When 4 mg Flag antibody is added, the antibody cross-linking rate is as high as 93.45%. Moreover, in the acid solution process, almost no antibody is eluted, indicating that the antibody has a strong binding strength with the mutant protein A and high stability, which is conducive to ensuring that it resists the destructive effect of the low pH acidic eluent during protein purification and improves the purity and specificity of the purified protein.

[0081] Example 5 Purification of Flag-tagged fusion protein using antibody-immobilized affinity chromatography medium

[0082] The amino acid sequence of the fusion protein with a flag tag in this example is as follows:

[0083] DYKDDDDKGSMSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEE IKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE (see SEQ ID NO. 3).

[0084] Take 2 mL of the supernatant of the fusion protein with the flag tag (verify in advance that the concentration of the target protein is 0.8 mg / mL), incubate the protein supernatant with the Flag-proA cross-linking matrix, and then purify it. (1) Incubation: Take out the sterilized 10 ml purification column tube and rinse the column tube with pure water 1-2 times. Add the antibody-immobilized affinity chromatography medium to the purification column tube, wash with pure water for 6 column volumes, and balance with Binding Buffer for 6 column volumes. After balance, add the fusion protein supernatant to the medium at a ratio of protein: medium = 2 mg: 1 mL. Seal with sealing film and place on a rotating incubator at 20 rpm and 4°C overnight; (2) Collect the flow-through: After the incubation is completed, centrifuge at 4°C and 600 rpm for 10 min, and pour the supernatant after centrifugation into a new centrifuge tube, which is the flow-through; (3) Washing: Add 3 mL of Washing Buffer to the purification column tube to wash away unbound impurities, and repeat elution 5 times until the impurities are eluted completely; (4) Elution: a. Add 0.5 mL of Elution Buffer 1 to wash the column, collect the effluent in a 1.5 mL endotoxin-free EP tube, and detect with G250 until G250 does not turn blue, ending the pre-elution with Elution Buffer 1; b. Add 0.5 mL of Elution Buffer 2. Wash the column, collect the effluent with a 1.5mL endotoxin-free EP tube, and detect it with G250 until the G250 does not turn blue, and end the elution with Elution Buffer 2; c. Add 0.5mL Elution Buffer 3 to wash the column, and collect the effluent with a 1.5ml endotoxin-free EP tube. The aforementioned EP tubes must be placed on an ice box to keep low temperature, and 0.5mL 1MTris solution (pH 8.0) must be added in advance to adjust the pH of the protein solution. The buffer formula mentioned above is as follows:

[0085] Buffer name Element pH BindingBuffer PBS 7.4 Washing Buffer PBS 7.4 ElutionBuffer1 0.1M glycine and 150mM NaCl 4.0 ElutionBuffer2 0.1M glycine and 150mM NaCl 3.0 ElutionBuffer3 0.1M glycine and 150mM NaCl 2.2

[0086] The collected flow-through and eluate were sampled and subjected to SDS-PAGE electrophoresis. Figure 6 , where lane 1 is 0.4 mg / mL bovine serum albumin, lane 2 is flow-through, lane 3 is washing buffer, lane 4 is pH 3.0 eluent, lane 5 is pH 2.2 eluent, lane 6 is molecular marker, and lane 7 is Flag-protein A cross-linked matrix after purification of fusion protein. The protein concentration in the pH 3.0 eluent is calculated to be 0.2 mg / mL, the volume is 1.15 mL, and the total amount of protein obtained is 0.23 mg.

[0087] Compared with the commercial DDDDK filler (purchased from Changzhou Tiandi Renhe, catalog number SA042001) with a matrix loading capacity of 0.5 mg protein / mL, 1 mL of the IgG affinity chromatography medium of the present invention can bind to approximately 20 mg of Flag antibody, with an antibody loading capacity of 20 mg / mL of matrix. The Flag-proteinA cross-linked matrix obtained thereby can purify 1.15 mg of Flag tag fusion protein, with a Flag tag fusion protein loading capacity of 1.15 mg / mL of matrix, which is far superior to commercial fillers.

[0088] Example 6 Study on the stability of antibody-immobilized affinity chromatography medium

[0089] Protein A is usually cleaned by using guanidine hydrochloride, urea or low-concentration alkali solution to regenerate the medium. In this embodiment, 30 μL of the antibody-immobilized affinity chromatography medium (Flag-protein A cross-linked matrix) is soaked in 60 μL of different buffers for different treatment times. The buffer includes: 0.3M guanidine hydrochloride, 0.5M guanidine hydrochloride, 1.5M urea, 2M urea, 0.1M Tris solution (pH10.91) and 0.1M NaOH solution (pH 12.76). Samples are taken in turn after soaking for 20min, 40min, and 60min, and the samples are used for SDS-PAGE electrophoresis to verify the binding stability of the antibody and Protein A.

[0090] Figure 7The SDS-PAGE gel electrophoresis diagrams of the medium immersed in different treatment solutions for different time periods are shown. For distinction, the lanes marked with matrix in the figure are all Flag-proteinA cross-linked matrices that are immersed and heated at 95°C for 5 minutes before loading, and the supernatants of the other lanes are directly loaded after the immersion treatment; among them, the left figure is 0.3-0.5M guanidine hydrochloride and 1.5M urea treatment, and the right figure is 2M urea and high pH alkali solution treatment. In the left figure, lane 1 is 0.4mg / mL bovine serum albumin, lane 2 is a molecular marker marker, and lanes 3-5 are respectively The samples were immersed in 0.3M guanidine hydrochloride for 20 min, 40 min and 60 min, lanes 7-10 were immersed in 0.5M guanidine hydrochloride for 20 min, 40 min and 60 min, lanes 11-14 were immersed in 1.5M urea for 20 min, 40 min and 60 min, in the right figure, lane 1 was 0.4 mg / mL bovine serum albumin, lanes 2 and 4-6 were immersed in 2.0M urea for 20 min, 40 min and 60 min, lane 3 was 0.4 mg / mL bovine serum albumin, lanes 7-10 were immersed in 0.1M Tris (pH 10.91) for 20 min, 40 min and 60 min, and lanes 11-14 were immersed in 0.1M NaOH for 20 min, 40 min and 60 min. As can be seen from the figure, immersion in guanidine hydrochloride (0.3M, 0.5M) and urea (1.5M, 2M) for 60 minutes does not affect the stability of the mutant protein A and the antibody binding, but the Flag tag fusion protein remaining on the matrix cannot be effectively eluted; after the matrix was immersed in 0.1M Tris (pH 10.91) for 60 minutes, no obvious antibody (heavy and light chain 50KD, 27KD) and protein A bands were found, and a small amount of residual Flag tag fusion protein could be washed off, which shows that the matrix can withstand 0.1M Tris (pH 10.91) treatment for 60 minutes; when treated with 0.1M sodium hydroxide for 20-60 minutes, a very small amount of antibody shedding occurred, but the residual Flag protein on the matrix had a good removal effect, so the antibody-immobilized affinity chromatography medium of the present invention is suitable for high pH alkali solution regeneration treatment.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A mutant protein A, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. Use of the mutant protein A as claimed in claim 1 in the field of affinity chromatography.

3. An IgG-like protein affinity chromatography medium, characterized in that It comprises a solid phase carrier and a mutant protein A coupled to the solid phase carrier, and the amino acid sequence of the mutant protein A is shown as SEQ ID NO.

2.

4. The IgG-like protein affinity chromatography medium according to claim 3, characterized in that The mass volume ratio of the mutant protein A to the solid phase carrier is 2.8:

1.

5. The IgG-like protein affinity chromatography medium according to claim 3, characterized in that The solid phase carrier is selected from one or more of agarose gel, dextran gel, agarose and dextran composite gel, cellulose, polyacrylamide gel, polymethyl methacrylate gel, and the IgG-like protein includes IgG antibody or a fusion protein containing the Fc domain of IgG antibody.

6. The IgG-like protein affinity chromatography medium according to claim 3, characterized in that The solid phase carrier is coupled to the mutant protein A comprising the following steps: The mutant protein A and the activated solid phase carrier are added to a coupling solution containing sodium bicarbonate and sodium chloride, mixed, reacted at room temperature, and after the reaction is completed, the supernatant is removed, and the precipitate is washed to neutrality to obtain an IgG-like protein affinity chromatography medium; Wherein, the concentration of sodium bicarbonate in the coupling liquid is 0.1 M, and the concentration of sodium chloride is 0.5 M; the volume ratio of the coupling liquid to the solid phase carrier is 1:

1.

7. An antibody-immobilized affinity chromatography medium, characterized in that: It comprises an antibody and an IgG-like protein affinity chromatography medium as described in any one of claims 3 to 6, wherein the Fc domain of the antibody is covalently bound to the mutant protein A of the IgG-like protein affinity chromatography medium.

8. The antibody-immobilized affinity chromatography medium according to claim 7, characterized in that: The mass ratio of the mutant protein A to the antibody is 0.56:

4.

9. The antibody-immobilized affinity chromatography medium according to claim 7, characterized in that: The antibody is an IgG antibody against the Flag tag.

10. The antibody-immobilized affinity chromatography medium according to claim 7, characterized in that: The Fc domain is covalently bonded to the mutant protein A via a cross-linking agent, comprising the following steps: The IgG-like protein affinity chromatography medium and the antibody are added to a PBS solution, incubated at low temperature for 1-10 hours, then the reaction solution is removed, 50 mM dimethyl pimelimidate dihydrochloride solution is added, and the reaction is carried out at room temperature for 1-3 hours. After cross-linking is completed, a tris solution is added for blocking, and then washed to neutrality to obtain the antibody-immobilized affinity chromatography medium.

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