Purification method of glycoprotein

Abstract

The invention relates to the technical field of protein purification, in particular to a purification method of glycoprotein. The invention provides a method for separating and purifying specifically modified proteoglycan only by obtaining a protein sequence and a corresponding sugar modification enzyme sequence. The purification method has high specificity, and glycoprotein formed by specific protein can be purified and separated; the experiment condition consistency is high, and repeated exploration is not needed; the glycoprotein modified by complex or unknown sugar chain glycosylation can be purified and separated, and the sugar chain can be specifically modified.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular to a method for purifying glycoprotein. Background Art

[0002] There are currently several methods for purifying and separating target proteins: including lectin affinity chromatography and anion exchange chromatography. Among them, lectin affinity chromatography is an extraction method based on the interaction between lectin (a protein that can specifically recognize and bind to a specific sugar structure) and glycoprotein. In this method, lectin is immobilized on a chromatographic medium to form a chromatographic column. When a glycoprotein sample containing a target sugar structure passes through this chromatographic column, the specific glycosyl structure in the glycoprotein will bind to the lectin. This binding has a certain specificity, allowing the glycoprotein to be effectively captured from the mixture. Subsequently, by using an elution buffer containing a high concentration of specific sugar molecules, competitive dissociation of glycoproteins and lectins can be achieved, thereby achieving the purification and separation of glycoproteins. Based on the above principle, a lectin that binds to heparan sulfated SDC2 is designed. After forming a specific chromatographic column, the total protein mixture is passed through, and finally the glycoprotein is eluted to complete the purification and separation.

[0003] The principle of anion exchange chromatography is based on the charge interaction between negatively charged molecules (such as certain glycoproteins) and cations on the chromatographic medium. In this method, negatively charged glycoprotein molecules are captured by cations (such as DEAE or Q Sepharose) fixed on the medium. By changing the pH value or salt concentration of the eluent, the target molecules can be specifically eluted and collected. Based on the above principle, after extracting the total protein, it passes through an anion exchange chromatography column, then elutes the non-target proteins, and then changes the pH value or salt concentration of the elution buffer to specifically elute and collect the target glycoprotein.

[0004] These methods have the following limitations: (1) Specificity limitations: The binding capacity of some lectins may cover multiple sugar structures rather than being limited to a single sugar structure, which may reduce the selective purification effect of specific glycoproteins. Similarly, anion exchange chromatography is also limited in separating molecules with similar charge characteristics. (2) Complexity of experimental condition setting: The stability of lectins may be affected by environmental factors such as pH and temperature, and in order to achieve efficient separation effects, the composition of the elution buffer needs to be carefully optimized in the above two methods. (3) Limited usability: The above glycoprotein separation methods require a full understanding of the structure and properties of sugar chains and cannot effectively extract glycoproteins with complex or unknown sugar chains.

[0005] In summary, the existing glycoprotein extraction schemes have poor specificity, complex experimental conditions and limited availability, and are not suitable for specific purification of glycoproteins, especially for the separation and purification of newly discovered or structurally unknown glycosylated glycoproteins. Therefore, it is urgent to develop a new method for protein purification and separation with high specificity and simplicity. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for purifying glycoproteins, which can separate and purify specifically modified proteoglycans by simply obtaining the protein sequence and the corresponding sugar-modifying enzyme sequence.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for purifying a glycoprotein, comprising the following steps:

[0009] (1) Designing plasmid A based on the core protein of glycoprotein to enrich the core protein;

[0010] (2) Designing plasmid B for body sugar chain modification based on sugar modification enzymes;

[0011] (3) co-transfecting plasmid A and plasmid B into target cells;

[0012] (4) Lyse cells to extract total protein;

[0013] (5) Purifying the total protein to obtain the glycoprotein.

[0014] The present invention provides a method for purifying transmembrane proteoglycans modified with sugar chains in vivo. It only needs to obtain the protein sequence and the corresponding sugar-modifying enzyme sequence, and add tags such as His to the protein sequence for subsequent purification, so as to separate and purify the specifically modified proteoglycans. Among them, plasmid A is used for the enrichment of core protein, and plasmid B is used for in vivo sugar chain modification. The design of plasmid A should consider: try to cover the CDS region of the protein sequence encoded by the protein, select and add a tag protein with a small molecular weight, such as 6×His, and the position of the tag protein addition should be adjusted according to the protein structure. Taking SDC2 as an example, since the location of its glycosylation modification is in the extracellular segment, the tag protein is placed on the side of the intracellular segment, that is, the C-terminus of the protein sequence, when designing plasmid A, to prevent the core protein structure from changing and being unable to complete the modification.

[0015] Preferably, the plasmid A contains a tag protein.

[0016] More preferably, the plasmid A contains a His-tagged protein.

[0017] Preferably, the glycoprotein comprises heparan sulfate proteoglycan, and its core protein comprises SDC1, SDC2, SDC3, SDC4, HSPG2, GPC1, GPC2, GPC3, GPC4, GPC5, GPC6, and AGRN.

[0018] Preferably, when the glycoprotein is heparan sulfate proteoglycan and the core protein is SDC2, the nucleotide sequence of plasmid A is as shown in SEQ ID NO.1.

[0019] Preferably, when the glycoprotein is a heparan sulfate proteoglycan, its sugar-modifying enzyme includes HS3ST3B1, HS3ST1, HS3ST2, HS3ST3A1, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS3ST3, HS2ST1, NDST1, NDST2, NDST3, and NDST4.

[0020] Preferably, when the glycoprotein is heparan sulfate proteoglycan, the sugar chain modification includes sulfonation modification.

[0021] Preferably, when the sulfonation modification site is the 3-OS site of the sugar chain, the nucleotide sequence of plasmid B is as shown in SEQ ID NO.2.

[0022] Preferably, in step (3), the target protein comprises HEK293T cells.

[0023] Preferably, in step (5), the purification method includes purification using magnetic beads.

[0024] Preferably, the magnetic beads comprise agarose magnetic beads.

[0025] Preferably, the magnetic bead purification comprises the following steps:

[0026] (a) After the total protein is mixed with magnetic beads and incubated, magnetic separation is performed and the supernatant is removed;

[0027] (b) adding washing solution A and repeating the washing;

[0028] (c) adding eluent B for incubation, performing magnetic separation, and collecting the eluate to obtain the purified glycoprotein.

[0029] Preferably, in step (b), the washing solution A comprises components with the following mass concentrations: 10 mM Tris, 500 mM NaCl, pH=7.4.

[0030] Preferably, in step (c), the washing solution B comprises components with the following mass concentrations: 10 mM Tris, 500 mM NaCl, 500 mM Imidazole, pH=7.4.

[0031] The present invention is beneficial in that:

[0032] The existing glycoprotein extraction schemes are not specific enough, the experimental conditions are complex and the availability is limited, so they are not suitable for specific purification of glycoproteins, especially separation and purification of newly discovered or unknown glycosylated modified glycoproteins. The present invention provides a method for purifying transmembrane proteoglycans modified with sugar chains in vivo, which can separate and purify specific modified proteoglycans by only obtaining the protein sequence and the corresponding sugar modification enzyme sequence.

[0033] The purification method of the present invention has the following advantages:

[0034] (1) High specificity, which can purify and separate glycoproteins formed by specific proteins;

[0035] (2) The experimental conditions are highly consistent and do not require repeated trials;

[0036] (3) It can purify and separate glycoproteins with complex or unknown sugar chain glycosylation modifications, and perform specific modifications on sugar chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The present invention provides a process for purifying glycoproteins.

[0038] Figure 2 The results show the expression levels of SDC2 and HS3ST3B1 in 293T cells after plasmid transfection.

[0039] Figure 3 Flow cytometry was used to detect the expression of heparan sulfate in 293T cells overexpressing SDC2 and HS3ST3B1.

[0040] Figure 4 The results of the silver staining experiment show the purification efficiency of 293T cells overexpressing SDC2 and HS3ST3B1 using IDA-Ni magnetic beads. DETAILED DESCRIPTION

[0041] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0042] Unless otherwise specified, all reagents used in the present invention can be purchased from the market.

[0043] The sources of the reagents used in the present invention are shown in Table 1.

[0044] Table 1

[0045]

[0046] Example 1: Plasmid design and construction (his-SDC2 plasmid and HS3ST3B1 plasmid)

[0047] The technical solution is described in detail by taking the purification of heparan sulfate proteoglycan as an example. Plasmid A is designed to enrich the core protein, and plasmid B is designed to perform sulfonation modification on the sugar chain of the protein.

[0048] The core proteins of heparan sulfate proteoglycans include SDC1, SDC2, SDC3, SDC4, HSPG2, GPC1, GPC2, GPC3, GPC4, GPC5, GPC6, and AGRN; the sugar-modifying enzymes include HS3ST3B1, HS3ST1, HS3ST2, HS3ST3A1, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS3ST3, HS2ST1, NDST1, NDST2, NDST3, and NDST4.

[0049] This example designs and constructs plasmids for the core protein SDC2 of heparan sulfate proteoglycan and the sugar-modifying enzyme HS3ST3B1.

[0050] Uniprot https: / / www.uniprot.org / ) protein sequence, a pair of humanized plasmids were designed for the enrichment of heparan sulfate transmembrane proteoglycans and sulfonation modification of specific sites, including HS3ST3B1 plasmid and his-SDC2 plasmid.

[0051] HS3ST3B1 plasmid: contains a recombinant humanized heparan sulfate-glucosamine 3-sulfotransferase 3B1 (HS3ST3B1) sequence, and the heparan sulfate sugar chain can have sulfonation modifications at multiple sites. The nucleotide sequence of the HS3ST3B1 plasmid designed in this example is shown in SEQ ID NO.1.

[0052] his-SDC2 plasmid: contains a recombinant humanized Syndecan-2 (SDC2) sequence. SDC2 is a transmembrane core protein that binds to heparan sulfate sugar chains. The production of heparan sulfate glycoprotein in cells can be increased by overexpressing the core protein SDC2. At the same time, a His (histidine) tag is fused to the C-terminus of the sequence to facilitate protein purification and detection. The nucleotide sequence of the his-SDC2 plasmid designed in this embodiment is shown in SEQ ID NO.2. Different sulfotransferase sequences can be designed here as needed to complete the enrichment of specific sulfo-modified heparan sulfate proteoglycans.

[0053] Example 2: Plasmid transfection (PEI transfection reagent transient transfection 293T)

[0054] The present invention uses HEK293T cells to enrich glycoproteins, which are tool cells commonly used in commercial production of recombinant proteins. Two plasmids are co-transfected into target cells, and the HS3ST3B1 protein sulfonates the 3-OS site of the sugar chain of heparan sulfate glycoprotein in the target cells.

[0055] The specific process is:

[0056] 1. Treatment one day before transfection: HEK293T cells were cultured at a density of 80% (4.0×10 6 Cells were seeded on plates (10 cm culture dishes) and cultured with 10% FBSDMEM medium without double antibody.

[0057] 2. Treatment on the day of transfection:

[0058] I. Replace the medium of 293T cells in a 10 cm dish with 5 mL of DMEM and starve the cells for 2 h;

[0059] II. Prepare transfection reagent:

[0060] Solution A: Add 500 μL DMEM and 48 μL PEI to a 1.5 mL EP tube (mix by pipetting 20 times).

[0061] Solution B: Add 500 μL DMEM, 8 μg his-SDC2 plasmid, and 8 μg HS3ST3B1 plasmid into a 1.5 mL EP tube;

[0062] Add solution B dropwise to solution A, flick it with your finger after adding each drop, and repeatedly blow and tap 30 times to get a mixed solution after it is completely added. Let it stand at room temperature for 20 minutes.

[0063] III. Slowly add the mixture to the culture medium treated in step (I) to make it evenly distributed;

[0064] IV. Place the culture dish in an incubator and let it stand for 3 hours before changing the medium to 10 mL of 10% FBSDMEM medium without dual antibodies.

[0065] 3. Lyse cells to extract total protein

[0066] Lyse cells 48 hours after transfection to extract proteins:

[0067] Remove the culture medium on ice and gently wash the cells 3 times with pre-cooled phosphate buffer (PBS) to remove residual culture medium and non-adherent cells. Add 1mL IP lysis buffer (Immunoprecipitation Lysis Buffer) to a 10cm culture dish, gently scrape the lysed cells with a cell scraper, and collect them in a 1.5mL EP tube. Lyse the cells on ice for 1h, gently invert the EP tube every 10 minutes to mix and promote full cell lysis. Then centrifuge at 12000g for 15min at 4℃ to separate unlysed cells and cell debris. Carefully transfer the protein-containing supernatant to a clean 1.5mL EP tube to avoid interference from the precipitate.

[0068] 4. The expression levels of SDC2 and HS3ST3B1 in 293T cells were detected by WB experiment after transfection.

[0069] The results are as follows Figure 2 As shown, SDC2 and HS3ST3B1 were highly expressed after plasmid transfection into 293T cells.

[0070] 5. Heparan sulfate glycoprotein will bind to KIR2DL4 protein. In this example, KIR2DL4-Fc recombinant protein was co-incubated with 293T cells transfected with plasmid at 37° C. for 1 hour, and the Fc segment of the recombinant protein was detected by flow cytometry.

[0071] The results are as follows Figure 3 As shown, flow cytometry results can determine that 293T cells overexpressing SDC2 and HS3ST3B1 have more heparan sulfate.

[0072] Example 3: Purification of proteoglycan

[0073] In this example, agarose magnetic beads (IDA-Ni magnetic beads) were used to purify the His-tagged protein.

[0074] 1. Prepare buffer according to Table 2:

[0075] Table 2

[0076]

[0077] 2. Preparation of His-tagged protein purification agarose magnetic beads (referred to as IDA-Ni magnetic beads):

[0078] I. Use a pipette to gently blow and resuspend the IDA-Ni magnetic beads, take 200μL of IDA-Ni magnetic beads and add them to a clean 1.5mL EP tube, remove the supernatant by magnetic separation; add 200μL Binding / Wash Buffer to wash the magnetic beads;

[0079] II. Use a pipette to gently resuspend the IDA-Ni magnetic beads. Place on a magnetic stand for 10 seconds and remove the supernatant. Repeat the above steps twice.

[0080] 3. Binding of target protein to magnetic beads:

[0081] I. Incubate the treated magnetic beads with the extracted total protein on a rotating mixer at room temperature for 20-30 minutes;

[0082] II. Magnetic separation: After incubation, place on a magnetic stand for 10 seconds and remove the supernatant;

[0083] III. Washing: Add 400 μL Binding / Wash Buffer, gently pipette to resuspend the IDA-Ni magnetic beads, place on a magnetic stand for 10 seconds, remove the supernatant, and repeat washing three times.

[0084] 4. Elution: Add 100 μL Elution Buffer, gently flip the centrifuge tube several times, incubate the magnetic beads for 5 minutes, separate magnetically, and collect the eluate into a new centrifuge tube to obtain the purified heparan sulfated SDC2 protein.

[0085] 5. Silver staining to detect protein purification efficiency:

[0086] (1)Fixed:

[0087] After SDS-PAGE electrophoresis, the gel was placed in about 100 mL of fixative solution and shaken on a shaker at room temperature for 20 minutes at a speed of 60-70 rpm.

[0088] Preparation of the fixative: Add 50 mL of ethanol, 10 mL of acetic acid and 40 mL of Milli-Q grade water in sequence and mix well to make 100 mL of the fixative.

[0089] (2) 30% ethanol washing:

[0090] Discard the fixative solution, add 100 mL of 30% ethanol, and shake on a shaker at room temperature for 10 minutes at a speed of 60-70 rpm.

[0091] Preparation of 30% ethanol: Add 30 mL of ethanol to 70 mL of Milli-Q grade pure water and mix well to obtain 100 mL of 30% ethanol.

[0092] (3) Water washing:

[0093] Discard the 30% ethanol, add 200 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm.

[0094] (4) Sensitization:

[0095] Discard the water, add 100 mL of silver staining sensitizer (1X), and shake on a shaker at room temperature for 2 minutes at a speed of 60-70 rpm.

[0096] Preparation of silver staining sensitizer (1X): Add 1 mL of silver staining sensitizer (100X) to 99 mL of Milli-Q pure water and mix well to obtain the silver staining sensitizer (1X).

[0097] (5) Water washing (2 times in total):

[0098] Discard the liquid, add 200 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 1 minute at a shaking speed of 60-70 rpm.

[0099] Discard the water, add 200 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 1 minute at a speed of 60-70 rpm.

[0100] (6) Silver staining:

[0101] Discard the water, add 100 mL of silver solution (1X), and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm.

[0102] Preparation of silver solution (1X): Add 1 mL of silver solution (100X) to 99 mL of Milli-Q pure water and mix well to obtain the silver solution (1X).

[0103] (7) Water washing:

[0104] Discard the liquid, add 100 mL of Milli-Q grade pure water or double distilled water, and shake on a shaker at room temperature for 1.5 minutes at a shaking speed of 60-70 rpm.

[0105] (8) Color rendering:

[0106] Discard the water, add 100 mL of silver staining solution, and shake on a shaker at room temperature for 3-10 minutes until the expected ideal protein band appears. The shaking speed is 60-70 rpm.

[0107] Preparation of silver staining solution: Add 20 mL of silver staining basic solution (5X) to 80 mL of Milli-Q pure water or double distilled water, then add 0.05 mL of silver staining acceleration solution (2000X), and mix well to obtain the silver staining solution.

[0108] (9) Termination:

[0109] Discard the solution, add 100mL silver staining stop solution (1X), and shake on a shaker at room temperature for 10 minutes at a speed of 60-70rpm. It is normal for gas to be generated during the stop, and the generated gas is carbon dioxide.

[0110] Preparation of Silver Staining Stop Solution (1X): Add 5 mL of Silver Staining Stop Solution (20X) to 95 mL of Milli-Q grade pure water or double distilled water, and mix well to obtain Silver Staining Stop Solution (1X). Silver Staining Stop Solution (1X) should be used on the same day after preparation.

[0111] (10) Water washing:

[0112] Discard the liquid, add 100 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 2-5 minutes at a shaking speed of 60-70 rpm.

[0113] The results are as follows Figure 4 The protein marker used in SDS-PAGE electrophoresis is 26616. The marked position in the figure is the location of the glycosylated his-SDC2 purified protein, and the results of his-SDC2 protein purification.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for purifying glycoprotein, characterized in that: The steps include: (1) Designing plasmid A based on the core protein of glycoprotein to enrich the core protein; (2) Designing plasmid B for body sugar chain modification based on sugar modification enzymes; (3) co-transfecting plasmid A and plasmid B into target cells; (4) Lyse cells to extract total protein; (5) Purifying the total protein to obtain the glycoprotein.

2. The method for purifying glycoprotein according to claim 1, characterized in that: The glycoprotein includes heparan sulfate proteoglycan, and its core protein includes SDC1, SDC2, SDC3, SDC4, HSPG2, GPC1, GPC2, GPC3, GPC4, GPC5, GPC6, and AGRN.

3. The method for purifying glycoprotein according to claim 2, characterized in that: When the glycoprotein is heparan sulfate proteoglycan and the core protein is SDC2, the nucleotide sequence of plasmid A is shown in SEQ ID NO.

1.

4. The method for purifying glycoprotein according to claim 1, characterized in that: When the glycoprotein is a heparan sulfate proteoglycan, its sugar-modifying enzymes include HS3ST3B1, HS3ST1, HS3ST2, HS3ST3A1, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS3ST3, HS2ST1, NDST1, NDST2, NDST3, and NDST4.

5. The method for purifying glycoprotein according to claim 1, characterized in that: When the glycoprotein is heparan sulfate proteoglycan, the sugar chain modification includes sulfonation modification.

6. The method for purifying glycoprotein according to claim 5, characterized in that: When the sulfonation modification site is the 3-OS site of the sugar chain, the nucleotide sequence of plasmid B is shown as SEQ ID NO.

2.

7. The method for purifying glycoprotein according to claim 1, characterized in that: In the step (5), the purification method includes purification using magnetic beads.

8. The method for purifying glycoprotein according to claim 7, characterized in that: The magnetic bead purification comprises the following steps: (a) After the total protein is mixed with magnetic beads and incubated, magnetic separation is performed and the supernatant is removed; (b) adding washing solution A and repeating the washing; (c) adding eluent B for incubation, performing magnetic separation, and collecting the eluate to obtain the purified glycoprotein.

9. The method for purifying glycoprotein according to claim 8, characterized in that: In the step (b), the washing solution A comprises components with the following mass concentrations: 10 mM Tris, 500 mM NaCl, pH=7.

4.

10. The method for purifying glycoprotein according to claim 8, characterized in that: In the step (c), the washing solution B comprises the following components in mass concentrations: 10 mM Tris, 500 mM NaCl, 500 mM Imidazole, pH=7.4.

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