A method for extracting membrane protein from VLPs preparation

Abstract

The present application relates to the field of biotechnology, and in particular to a method for extracting membrane proteins from VLPs preparations; the method comprises: constructing a membrane protein expression plasmid containing a functional gene; mixing the membrane protein expression plasmid containing the functional gene, a gag plasmid and a cationic lipid transfection reagent to obtain an expression complex; causing the expression complex to produce a VLPs precipitate containing a target membrane protein of the functional gene expression product; dissolving the VLPs precipitate using a dissolution buffer system to obtain a membrane protein solution containing the functional gene expression product; performing fluorescence detection on the membrane protein solution to obtain membrane protein expression abundance data; purifying the membrane protein solution based on the membrane protein expression abundance data to obtain membrane proteins; the dissolution buffer system and the equilibrium buffer system both comprise a mixture of DDM and CHS; the method can purify a large amount of active membrane proteins, and the enrichment process is simple and the difference in proteins between different batches is small.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for extracting membrane proteins from VLPs preparations. Background Art

[0002] The primary difficulty in developing innovative drugs targeting transmembrane proteins is the difficulty in obtaining sufficient, stable and active membrane proteins. At present, the technologies used to obtain membrane protein antigens include: (1) preparing fragment antigens, which have the advantages of simple preparation and low cost, but cannot simulate the natural conformation of the protein and have the disadvantages of missing or distorted epitopes; (2) using overexpressed whole cells or cell fragments containing membrane proteins. These overexpressed whole cells or cell fragments can maintain the natural conformation of membrane proteins, but the expression level of membrane proteins is lower than that of total membrane proteins, which makes it easy for antibodies to bind to the membrane or other proteins on the membrane, increasing the difficulty of screening membrane proteins and non-specific binding; (3) preparing antigens by virus-like particle immunization. Compared with the above-mentioned overexpressed whole cells or cell fragments, although the screening background is reduced to a certain extent, this virus-like particle immunization is only suitable for some membrane protein receptors. At present, the most ideal membrane protein antigen is still the full-length membrane protein, which has the advantages of a single component, a sound epitope and a structure close to the natural conformation; and the best expression system for preparing natural conformation membrane proteins is mammalian expression cells, but the expression level of membrane proteins in mammalian expression cells is low. The traditional method of extracting membrane proteins from the plasma membrane is based on differential centrifugation, but the centrifugation speed and time of differential centrifugation need to be optimized for different membrane proteins and cells, and a large number of irrelevant other proteins are released during the differential centrifugation process. These other proteins increase the difficulty of membrane protein purification, making the overall membrane protein extraction process have the disadvantages of long process and cumbersome steps.

[0003] The VLPs model is a new membrane model for studying receptor-ligand interactions in the natural plasma membrane environment; transfection of retroviral core protein gag in mammalian cells can induce mammalian cells to produce VLPs. These VLPs were found to be highly stable and uniform particles with a diameter of about 100nm to 200nm by scanning electron microscopy. Compared with other membrane models (such as isolated cell membranes), the VLPs preparation process does not involve mechanical destruction or the use of detergents, so the natural structure and conformation of membrane proteins can be retained. These VLPs can carry a large number of functional membrane protein receptors due to the characteristics of their natural conformation. The preparation of VLPs mainly involves the co-expression of retroviral gag protein and target membrane protein receptors in mammalian cells. In addition, during the preparation process, VLPs bud from cells as uniform particles and can carry complete cell membrane components, which mainly include membrane-associated proteins from host cells and co-transfected membrane protein receptors. The concentration of the target membrane protein receptor in the VLPs preparation was estimated to be 50 to 250 pmol / mg of total membrane protein, which is approximately 10 to 100 times higher than that in other membrane models.

[0004] Therefore, extracting membrane proteins from VLPs can reduce the extraction process and simplify the steps, and the abundance of target membrane proteins is higher than other methods. However, extracting membrane proteins from VLPs is only suitable for some membrane protein receptors, and the current purification methods cannot effectively purify the extracted membrane proteins, resulting in low purity and low yield of the currently screened membrane proteins, which ultimately cannot be promoted. Summary of the invention

[0005] The present application provides a method for extracting membrane proteins from VLPs preparations to solve the following technical problem: how to improve the yield and activity of membrane proteins extracted from VLPs preparations.

[0006] In a first aspect, the present application provides a method for extracting membrane proteins from a VLPs preparation, the method comprising:

[0007] Construct membrane protein expression plasmid containing functional genes;

[0008] Mixing the membrane protein expression plasmid containing the functional gene, the gag plasmid and a cationic lipid transfection reagent so that the membrane protein expression plasmid and the cationic lipid transfection reagent form a complex to obtain an expression complex;

[0009] causing the expression complex to produce VLPs precipitating the target membrane protein containing the expression product of the functional gene;

[0010] Using a dissolution buffer system to dissolve the VLPs precipitate to obtain a membrane protein solution containing a functional gene expression product;

[0011] Performing fluorescence detection on the membrane protein solution to obtain membrane protein expression abundance data;

[0012] According to the membrane protein expression abundance data, the optimal purification success rate of the membrane protein solution is obtained;

[0013] Selecting the membrane protein solution with the best purification success rate, and then purifying the membrane protein solution using a balanced buffer system to obtain a membrane protein with biological activity;

[0014] Wherein, the functional gene includes a target gene and a purification tag and / or a reporter gene;

[0015] The dissolution buffer system and the equilibrium buffer system respectively include a mixture of 4,4'-diaminodiphenylmethane and cholesterol succinate.

[0016] Optionally, the reporter gene includes an mCherry reporter gene or a GFP reporter gene; and / or the purification tag includes a his purification tag and / or a strep purification tag.

[0017] Optionally, when the reporter gene includes an mCherry reporter gene, the protein expressed by the mCherry reporter gene has an amino acid sequence as shown in SEQ ID NO.1;

[0018] When the reporter gene includes a GFP reporter gene, the protein expressed by the GFP reporter gene has an amino acid sequence as shown in SEQ ID NO.2.

[0019] Optionally, the target gene includes the transmembrane protein CD20 gene and / or the transmembrane protein CLDN6 gene.

[0020] Optionally, when the target gene includes the transmembrane protein CD20 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.3;

[0021] When the target gene includes the transmembrane protein CLDN6 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.4.

[0022] Optionally, the weight m1 of the 4,4'-diaminodiphenylmethane and the weight m2 of the cholesterol succinate satisfy the relationship: m1:m2≥10:1.

[0023] Optionally, the step of causing the expression complex to produce VLPs precipitation containing the target membrane protein of the functional gene expression product comprises the steps of:

[0024] Using the expression complex to transfect animal cells, and then culturing the transfected cells to obtain a cell culture fluid;

[0025] The cell culture fluid is first centrifuged, and then the centrifugation product is enriched to obtain VLPs precipitate containing the target membrane protein of the functional gene.

[0026] Optionally, the centrifugal force of the first centrifugation is ≥100000 g, and the time of the first centrifugation is ≥2 h.

[0027] Optionally, the method of dissolving the VLPs precipitate using a dissolution buffer system to obtain a membrane protein solution containing a functional gene expression product comprises the steps of:

[0028] Dissolving the VLPs precipitate using a dissolution buffer system to obtain a dissolved protein solution;

[0029] The dissolved protein solution is resuspended and centrifuged a second time, and then the centrifugal supernatant is filtered to obtain a membrane protein solution containing the functional gene expression product.

[0030] Optionally, the rotation speed of the second centrifugation is 10000 rpm to 14000 rpm, and the time of the second centrifugation is ≥10 min.

[0031] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0032] The present application provides a method for extracting membrane proteins from VLPs preparations. First, a membrane protein expression plasmid containing a purification tag, a reporter gene, and a target gene is constructed. Then, the constructed membrane protein expression plasmid, a gag plasmid, and a cationic lipid transfection reagent are used to prepare a VLPs precipitate containing the target membrane protein of the functional gene expression product. Then, a dissolution buffer system is used to dissolve the obtained VLPs precipitate. Since the dissolution buffer system contains a mixture of 4,4'-diaminodiphenylmethane and cholesterol succinate, the target membrane protein can be fully dissolved. In addition, based on the reporter protein expressed by the reporter gene, the target membrane can be constructed in the subsequent fluorescence detection process. The correlation between protein concentration and fluorescence value of fluorescence detection can indicate the expression abundance data of membrane protein solution containing functional gene expression products based on this correlation characteristic. Then, according to the expression abundance data of membrane protein solution, the relative content of expression of membrane protein solution can be determined. The relative content can reflect the purification success rate of membrane protein solution. A membrane protein solution with a relative content of membrane protein close to or greater than 10% is selected. The membrane protein solution has a higher purification success rate. Then, the membrane protein solution is purified in conjunction with a balanced buffer system. The target membrane protein with biological activity can be purified to the maximum extent, thereby improving the yield and activity of membrane protein extracted from VLPs preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0035] Figure 1 A schematic diagram of a method for extracting membrane proteins from VLPs preparations provided in an embodiment of the present application;

[0036] Figure 2 A detailed schematic diagram of a method for extracting membrane proteins from VLPs preparations provided in an embodiment of the present application;

[0037] Figure 3 The fluorescence standard curve of the membrane protein expressed by mCherry provided in the examples of the present application;

[0038] Figure 4 The fluorescence standard curve of the membrane protein expressed by GFP provided in the examples of the present application;

[0039] Figure 5The purified gel map of the membrane protein expressed by CD20 provided in the embodiment of the present application; in the figure, lane 1 is the stock solution of CD20 ultracentrifugation precipitate dissolved by detergent, lane 2 is the permeate after CD20 is combined with the nickel column, lane 3 is 20mM Tris-HCl, 0.15M NaCl, DDM with a mass concentration of 0.1%, CHS with a mass concentration of 0.01%, 5mM imidazole and an eluent with a pH of 8.0; lane 4 is 20mM Tris-HCl, 0.15M NaCl, DDM with a mass concentration of 0.1%, CHS with a mass concentration of 0.01%, 20mM imidazole and an eluent with a pH of 8.0, and lane 5 is 20mM Tris-HCl, 0.15M NaCl, DDM with a mass concentration of 0.1%, CHS with a mass concentration of 0.01%, 20mM imidazole and an eluent with a pH of 8.0. The concentration is 0.01% CHS, 60mM imidazole and the eluent is pH 8.0, the lane 6 is 20mM Tris-HCl, 0.15M NaCl, 0.1% DDM, 0.01% CHS, 200mM imidazole and the eluent is pH 8.0, the lane 7 is 20mM Tris-HCl, 0.15M NaCl, 0.1% DDM, 0.01% CHS, 500mM imidazole and the eluent is pH 8.0, the markers are 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.4kDa and 14.4kDa from top to bottom, and the arrows indicate the target proteins;

[0040] Figure 6 This is a gel map of the purification of the membrane protein expressed by CLDN6 provided in the examples of the present application; in the figure, lane 1 is the stock solution of the ultracentrifugation precipitate of the membrane protein expressed by CLDN6 dissolved by a detergent, lane 2 is the permeate after the membrane protein expressed by CLDN6 is combined with the Strep column, and lane 3 is the eluate of the membrane protein expressed by CLDN6. The markers are 116.0 kDa, 66.2 kDa, 45.0 kDa, 35.0 kDa, 25.0 kDa, 18.4 kDa and 14.4 kDa from top to bottom, respectively, and the arrows indicate the target protein;

[0041] Figure 7 The ELISA activity test results of the membrane protein expressed by CD20 provided in the examples of the present application;

[0042] Figure 8 This is the ELISA activity test result of the membrane protein expressed by CLDN6 provided in the examples of the present application. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0044] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0045] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions, refers to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0046] Figure 1 A schematic diagram of a method for extracting membrane proteins from a VLPs preparation provided in an embodiment of the present application is shown exemplarily;

[0047] like Figure 1As shown, the present application embodiment provides a method for extracting membrane protein from a VLPs preparation, the method comprising:

[0048] S1. Construction of membrane protein expression plasmid containing functional genes;

[0049] S2. mixing the membrane protein expression plasmid containing the functional gene, the gag plasmid and the cationic lipid transfection reagent so that the membrane protein expression plasmid and the cationic lipid transfection reagent form a complex to obtain an expression complex;

[0050] S3. causing the expression complex to produce VLPs containing functional gene expression products;

[0051] S4. dissolving the VLPs precipitate using a dissolution buffer system to obtain a membrane protein solution containing a functional gene expression product;

[0052] S5. performing fluorescence detection on the membrane protein solution to obtain membrane protein expression abundance data;

[0053] S6. according to the membrane protein expression abundance data, obtaining the optimal purification success rate of the membrane protein solution; and

[0054] S7. selecting the membrane protein solution containing the functional gene with the best purification success rate, and then purifying the membrane protein solution using a balanced buffer system to obtain a membrane protein with biological activity;

[0055] Wherein, the functional gene includes a target gene and a purification tag and / or a reporter gene;

[0056] The dissolution buffer system and the equilibrium buffer system respectively include a mixture of 4,4'-diaminodiphenylmethane and cholesterol succinate.

[0057] It should be noted that in the co-transfection method of the membrane protein expression plasmid and the gag plasmid, during the fluorescence detection process, a standard curve can be first constructed based on the relationship between the reporter protein expressed by the reporter gene and the fluorescence value, and then the expression abundance of the membrane protein expressed by the membrane protein expression plasmid can be calculated based on the reporter protein expressed by the reporter gene in the membrane protein expression plasmid in combination with the standard curve.

[0058] It should be noted that the membrane protein expression abundance data mainly refers to the relative weight content data of the membrane protein in the membrane protein solution. Generally, if the relative weight content of the membrane protein is close to or greater than 10%, it can be considered that the membrane protein solution contains a high abundance of membrane protein.

[0059] In some optional embodiments, the reporter gene includes an mCherry reporter gene or a GFP reporter gene;

[0060] The purification tag includes a his purification tag and / or a strep purification tag;

[0061] In these embodiments, the reporter gene may include an mCherry reporter gene or a GFP reporter gene, which can promote the production of a sufficient amount of reporter protein in the membrane protein expression plasmid during the fluorescence detection process, so that the expression abundance of the membrane protein expressed by the membrane protein expression plasmid can be accurately calculated in combination with the calibration curve. In addition, the purification tag may include a his purification tag and / or a strep purification tag, and the membrane protein solution can be tagged and purified based on the expression abundance data of the membrane protein in combination with the equilibrium buffer system, so that the membrane protein with good biological activity can be effectively separated and purified from the membrane protein solution.

[0062] In some optional embodiments, when the reporter gene includes an mCherry reporter gene, the protein expressed by the mCherry reporter gene has an amino acid sequence as shown in SEQ ID NO.1;

[0063] When the reporter gene includes a GFP reporter gene, the protein expressed by the GFP reporter gene has an amino acid sequence as shown in SEQ ID NO.2;

[0064] In these embodiments, when the reporter gene includes the mCherry reporter gene, the protein expressed by the mCherry reporter gene generally has the amino acid sequence shown in SEQ ID NO.1, and when the reporter gene includes the GFP reporter gene, the protein expressed by the GFP reporter gene generally has the amino acid sequence shown in SEQ ID NO.2. The gene type corresponding to the reporter gene can be further clarified, so that the expression abundance of the membrane protein expressed by the membrane protein expression plasmid can be accurately calculated in combination with the calibration curve, and the membrane protein solution can be labeled and purified according to the expression abundance data of the membrane protein in combination with the equilibrium buffer system, so that the membrane protein with good biological activity can be effectively separated and purified from the membrane protein solution.

[0065] In some optional embodiments, the target gene includes the transmembrane protein CD20 gene and / or the transmembrane protein CLDN6 gene;

[0066] In these embodiments, the target gene may include the transmembrane protein CD20 gene and / or the transmembrane protein CLDN6 gene, which can promote the expression and translation of the target gene and the reporter gene into membrane protein and reporter protein, so that the expression abundance of the membrane protein expressed by the membrane protein expression plasmid can be accurately calculated in combination with the calibration curve, and the membrane protein solution can be labeled and purified according to the expression abundance data of the membrane protein in combination with the equilibrium buffer system, thereby effectively separating and purifying the membrane protein with good biological activity from the membrane protein solution.

[0067] In some optional embodiments, when the target gene includes the transmembrane protein CD20 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.3;

[0068] When the target gene includes the transmembrane protein CLDN6 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.4;

[0069] In these embodiments, when the target gene includes the transmembrane protein CD20 gene, the protein expressed by the functional gene generally has an amino acid sequence as shown in SEQ ID NO.3. In addition, when the target gene includes the transmembrane protein CLDN6 gene, the protein expressed by the functional gene generally has an amino acid sequence as shown in SEQ ID NO.4, which can prompt the target gene to express a sufficient amount of membrane protein and the reporter gene to express a sufficient amount of reporter protein, so that the expression abundance of the membrane protein expressed by the membrane protein expression plasmid can be accurately calculated in combination with the calibration curve, and the membrane protein solution can be labeled and purified according to the expression abundance data of the membrane protein in combination with the equilibrium buffer system, thereby effectively separating and purifying the membrane protein with good biological activity from the membrane protein solution.

[0070] In some optional embodiments, the weight m1 of the 4,4'-diaminodiphenylmethane and the weight m2 of the cholesterol succinate satisfy the relationship: m1:m2≥10:1;

[0071] In these embodiments, the weight m1 of 4,4'-diaminodiphenylmethane and the weight m2 of cholesterol succinate can satisfy the relationship: m1:m2≥10:1, which can promote the presence of sufficient 4,4'-diaminodiphenylmethane and cholesterol succinate in the dissolution buffer system and the equilibrium buffer system, and sufficient 4,4'-diaminodiphenylmethane and cholesterol succinate can effectively purify membrane proteins, so as to facilitate the final screening of membrane proteins with biological activity.

[0072] Figure 2 A detailed schematic diagram of a method for extracting membrane proteins from a VLPs preparation provided in an embodiment of the present application is shown exemplarily;

[0073] In some optional embodiments, the step of causing the expression complex to produce VLPs precipitation containing the target membrane protein of the functional gene expression product comprises the steps of:

[0074] S301. transfecting animal cells using the expression complex, and then culturing the transfected cells to obtain a cell culture fluid;

[0075] S302. The cell culture medium is subjected to a first centrifugation, and then the centrifugation product is enriched to obtain a VLPs precipitate containing a functional gene expression product;

[0076] In these embodiments, the expression complex is introduced into animal cells by transfection and culture, so that the membrane protein expression plasmid and gag plasmid in the expression complex can co-express multiple proteins, thereby promoting sufficient membrane proteins, reporter proteins and tagged purified proteins in the cell culture medium. In addition, the first centrifugation method can be used to separate the target membrane protein, reporter protein and purified tagged protein in the cell culture medium by centrifugation to obtain VLPs precipitate containing functional gene expression products.

[0077] In some optional embodiments, the centrifugal force of the first centrifugation is ≥100000 g, and the time of the first centrifugation is ≥2 h;

[0078] In these embodiments, the centrifugal force of the first centrifugation is ≥100000 g, and the time of the first centrifugation is ≥2 h, which can facilitate the separation of the functional gene expression product from the cell culture fluid by the first centrifugation.

[0079] In some optional embodiments, the step of dissolving the VLPs precipitate using a dissolution buffer system to obtain a membrane protein solution containing a functional gene expression product comprises the following steps:

[0080] S401. Dissolving the VLPs precipitate using a dissolution buffer system to obtain a dissolved protein solution;

[0081] S402. resuspending the dissolved protein solution and performing a second centrifugation, and then filtering the centrifugal supernatant to obtain a membrane protein solution containing a functional gene expression product;

[0082] In these embodiments, the VLPs precipitate is dissolved using a dissolution buffer system, and then resuspended and centrifuged a second time, so that the expression products expressed by the functional genes in the dissolved protein solution can be effectively separated, thereby facilitating the subsequent fluorescence detection process in combination with the standard curve to accurately calculate the expression abundance of various proteins expressed by the membrane protein expression plasmid.

[0083] In some optional embodiments, the speed of the second centrifugation is 10000 rpm to 14000 rpm, and the time of the second centrifugation is ≥ 10 min;

[0084] In these embodiments, the rotation speed of the second centrifugation can be 10000rpm~14000rpm, and the time of the second centrifugation is generally ≥10min. The expression product expressed by the functional gene in the resuspended dissolved protein solution can be effectively separated by the second centrifugation, thereby facilitating the subsequent fluorescence detection process in combination with the calibration curve to accurately calculate the expression abundance of the membrane protein expressed by the membrane protein expression plasmid.

[0085] The rotation speed of the second centrifugation can be 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm.

[0086] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions or conditions recommended by the manufacturer.

[0087] Example 1

[0088] 1. Preparation of experimental materials:

[0089] 1. Construction of various expression plasmids:

[0090] Membrane protein expression plasmids containing only purification tags and membrane protein expression plasmids fused with mCherry or GFP reporter genes were constructed respectively. In addition, expression plasmids containing only mCherry and GFP but not the target gene were constructed as a control group. The specific sequence distribution is shown in Table 1.

[0091] Table 1 Amino acid sequence composition of each expression plasmid

[0092]

[0093]

[0094] 2. Experimental reagents and equipment:

[0095] The reagents and equipment used in the experiment are shown in Table 2.

[0096] Table 2 Reagents and equipment used

[0097] Column material model Nickel pillar Qiagen, 30210 Streptactin-XT Column Fitness, KTSM1345 ELISA reader SPARK Ultracentrifuge Thermo, WX90+

[0098] 2. Experimental methods:

[0099] 1. Enrichment of membrane proteins:

[0100] HEK293F cells in the exponential growth phase with a viability of >98% were used for transfection;

[0101] The cell density of HEK293F cells was diluted to 3 × 10 6 / mL, and then prepare the membrane protein plasmid and gag plasmid for cotransfection according to Table 1, using ExpiFectamine TM 293Reagent cationic lipid transfection reagent, after each expression plasmid forms a complex with the cationic lipid transfection reagent, add it to the prepared cells to obtain transfected cells.

[0102] Then the transfected cells were placed in a CO2 shaker with a volume fraction of 8%, and the transfected cells were cultured at a constant temperature and shaking at 37°C and 120rpm to allow the expression plasmid to express sufficient protein. The cell culture fluid was collected after 5 days of culture.

[0103] The cell culture medium was then transferred to a 38 mL open thin-walled tube, placed in an ultracentrifuge (Thermo, WX90+), the centrifugal force of the ultracentrifuge was set to 100,000 g, and centrifuged at 4°C for 2 h to obtain a VLPs precipitate enriched with the target membrane protein.

[0104] 2. Dissolution of membrane proteins:

[0105] Weigh the VLPs precipitate enriched with the target membrane protein, and then resuspend it in a dissolution buffer containing a detergent (formula composition: 20mM Tris-HCl, 0.15M NaCl, 1% DDM, 0.1% CHS, pH 8.0); 1mL of the dissolution buffer is used to dissolve every 100mg of membrane precipitate. After the VLPs precipitate is resuspended, place it at 4°C and oscillate for 2h. Then centrifuge it at 4°C and 12000rpm for 10min to obtain the supernatant; the obtained supernatant is filtered using a 0.45μm filter, and the obtained membrane protein filtrate is ready for fluorescence detection or tag purification.

[0106] 3. Fluorescence detection of membrane proteins:

[0107] The membrane protein filtrate was diluted with PBS buffer to dilute the membrane protein filtrate to 0.1 mg / mL, 0.02 mg / mL, 0.01 mg / mL and 0.004 mg / mL. Then, each dilution was added with 100 μL of membrane protein filtrate of different concentrations in the Elisa assay strip (enzyme-labeled well strip, Y8), with 3 replicate wells per group of concentrations and 3 measurements per well. At the same time, a blank plate group and a buffer group were set as controls. After the wells were spotted, fluorescence detection was performed in an ELISA reader (SPARK). The determination conditions of GFP protein were set as: excitation wavelength Ex was 485 nm and emission wavelength Em was 530 nm; the determination conditions of mCherry protein were set as: excitation wavelength Ex was 580 nm and emission wavelength Em was 610 nm. The measured membrane protein expression abundance data were then processed.

[0108] 4. Tag purification of membrane proteins:

[0109] Select membrane proteins with higher expression abundance for membrane protein purification:

[0110] When using the HIS tag of membrane protein for purification, a nickel column (Qiagen, 30210) was balanced using a balancing buffer system (formula composition: 20 mM Tris-HCl, 0.15 M NaCl, 0.1% DDM, 0.01% CHS, pH 8.0), and the membrane protein filtrate was appropriately diluted with a washing buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) and then bound to the nickel column.

[0111] After the membrane protein filtrate has been combined for at least 2 times, the target membrane protein is washed and eluted with a balance buffer containing different imidazole concentrations to obtain an eluate.

[0112] The obtained eluate was concentrated using a 100 kDa ultrafiltration tube (Millipore), and then the concentration was measured by BCA.

[0113] When using Twin-Strep tag purification of proteins, the compositions of the equilibrium buffer system and the buffer system for washing impurities of proteins are: 20mM Tris-HCl, 0.15M NaCl, 0.1% DDM and 0.01% CHS, all configured into a buffer solution with a pH of 8.0.

[0114] 50 mM biotin was added to each elution buffer, and the eluted membrane protein was dialyzed against the equilibration buffer twice to remove biotin. The protein was then concentrated using a 100 kDa ultrafiltration tube (Millipore) and the concentration was determined by BCA.

[0115] 5. Enzyme-linked immunosorbent assay (ELISA) detection:

[0116] CD20 membrane protein and CLDN6 membrane protein were diluted to 2μg / mL~5μg / mL with CB buffer and then coated. When coating, set up blank control group, negative control group and positive control group respectively. Among them, the blank control group was directly added with protein-free CB buffer; the negative control group and each experimental group were coated with proteins CD20 and CLDN6; the positive control group was coated with CD20 and CLDN6 recombinant antibodies (1mg / mL). Coat at 4°C overnight and incubate in a 37°C incubator for 2h with blocking solution (4% skim milk powder by mass concentration) for blocking. Then incubate with CD20 and CLDN6 recombinant antibodies, use TMB for color development for 15 minutes, and then add the stop solution to terminate the experiment. After the absorbance value is read, perform data analysis in the Graphpad software and read the EC 50 data and draw a graph.

[0117] 3. Experimental Results

[0118] 1. Fluorescence detection results and data processing:

[0119] As shown in Table 3, according to the fluorescence values ​​measured under the concentration conditions of 0.1 mg / mL, 0.02 mg / mL and 0.01 mg / mL of the membrane protein expressed by mCherry, the corresponding average values ​​were calculated to obtain Figure 3 The standard curve is shown in Figure 2. The fluorescence value of the membrane protein expressed by CD20-mCherry at 0.1 mg / mL is 2058. Figure 3 The standard curve shown can obtain the concentration value corresponding to the fluorescence value of 0.00882 mg / mL. This concentration is 8.82% of the total protein concentration of 0.1 mg / mL, which shows that compared with the membrane protein expressed by mCherry, the expression abundance of the membrane protein expressed by CD20-mCherry accounts for 8.82% of the total protein of 0.1 mg / mL. The CD20 (C-terminal 10xHis-tagged) protein was prepared according to the same steps and then purified using a nickel column.

[0120] Table 3 Fluorescence data of membrane proteins expressed by mCherry and CD20-mCherry

[0121]

[0122] As described in Table 4, according to the fluorescence values ​​measured by the GFP control at 0.1 mg / mL, 0.02 mg / mL, 0.01 mg / mL and 0.004 mg / mL concentration conditions, the corresponding average values ​​were calculated to obtain Figure 4The standard curve is shown in Figure 2. The fluorescence value of the membrane protein expressed by CLDN6-GFP at 0.1 mg / mL is 5273. Figure 4 The standard curve shown can obtain the concentration value corresponding to the fluorescence value as 0.009678 mg / mL. This concentration is 9.68% of the total protein concentration of 0.1 mg / mL, which shows that compared with the membrane protein expressed by GFP, the expression abundance of the membrane protein expressed by CLDN6-GFP accounts for 9.68% of the total protein of 0.1 mg / mL. The sample was directly selected and purified using the Strep antibody column.

[0123] Table 4 Fluorescence data of membrane proteins expressed by GFP and CLDN6-GFP

[0124]

[0125] 2. Membrane protein purification results:

[0126] like Figure 5 As shown, after the co-expressed membrane proteins of VLPs and CD20 were precipitated, they were dissolved with detergent and loaded onto the column. Figure 5 In the figure, lane 1 is the stock solution, lane 2 is the penetration, and lanes 3 to 6 are the eluates containing different imidazole concentrations. It can be seen that the target protein CD20 was eluted at a concentration of 200 mM imidazole, and the obtained membrane protein had good purity.

[0127] In addition, Figure 6 As shown, after obtaining the co-expressed membrane protein of VLPs and CLDN6, the protein was precipitated and dissolved with a detergent and loaded onto the column. Figure 6 In the figure, lane 1 is the stock solution, lane 2 is the penetration, and lane 3 is the eluate. It can be seen that the target protein CLDN6 was eluted at a biotin concentration of 50 mM, and then dialyzed using a buffer system of 20 mM Tris-HCl, 0.15 M NaCl, 0.1% DDM, 0.01% CHS, and pH 8.0.

[0128] 3. Test results:

[0129] The activity of the membrane proteins expressed by CD20 and CLDN6 was detected, and the results are shown in Table 5, Table 6, Figure 7 and Figure 8 shown.

[0130] Table 5 Activity test results of membrane proteins expressed by CD20

[0131]

[0132] From Table 5 and Figure 7As shown, the activity of the membrane protein expressed by CD20 provided in the examples of the present application is good.

[0133] Table 6 Results of the activity test of membrane proteins expressed by CLDN6

[0134]

[0135]

[0136] In summary, the embodiment of the present application provides a method for extracting membrane proteins from VLPs preparations, based on the characteristic that the concentration of the target membrane protein in the VLPs preparation is significantly higher than that in cells or other membrane models due to the effective enrichment of membrane protein receptors, combined with the characteristic that GFP or mCherry reporter proteins indicate the relative enrichment of the target membrane protein, and membrane protein receptors with a relative content of about 10% or higher are selected for purification, and during the purification process, mild detergent-like membrane environments 4,4'-diaminodiphenylmethane (DDM) and cholesterol succinate (CHS) are added, so that active membrane proteins can be purified; this method has a simple enrichment process for membrane proteins and small protein differences between different batches.

[0137] In addition, the method for extracting membrane proteins from VLPs preparations provided in the embodiments of the present application is not only applicable to HEK293F cells expressing transmembrane proteins, but can also be applied to other mammalian cells expressing transmembrane proteins, such as CHO or stable cell lines that have been constructed to express membrane proteins.

[0138] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by the present application.

Claims

1. A method for extracting membrane proteins from VLPs preparations, characterized in that: The method comprises: Construct membrane protein expression plasmid containing functional genes; Mixing a membrane protein expression plasmid containing a functional gene, a gag plasmid and a cationic lipid transfection reagent so that the membrane protein expression plasmid and the cationic lipid transfection reagent form a complex to obtain an expression complex; causing the expression complex to produce VLPs precipitation containing the target membrane protein of the functional gene expression product; Using a dissolution buffer system to dissolve the VLPs precipitate to obtain a membrane protein solution containing a functional gene expression product; Performing fluorescence detection on the membrane protein solution to obtain membrane protein expression abundance data; According to the membrane protein expression abundance data, obtaining the optimal purification success rate of the membrane protein solution; and Selecting the membrane protein solution containing the functional gene with the best purification success rate, and then purifying the membrane protein solution using a balanced buffer system to obtain a membrane protein with biological activity; Wherein, the functional gene includes a target gene and a purification tag and / or a reporter gene; The dissolution buffer system and the equilibrium buffer system respectively include a mixture of 4,4'-diaminodiphenylmethane and cholesterol succinate.

2. The method according to claim 1, characterized in that: The reporter gene includes an mCherry reporter gene or a GFP reporter gene; and / or The purification tag includes a his purification tag and / or a strep purification tag.

3. The method according to claim 1, characterized in that When the reporter gene includes an mCherry reporter gene, the protein expressed by the mCherry reporter gene has an amino acid sequence as shown in SEQ ID NO.1; When the reporter gene includes a GFP reporter gene, the protein expressed by the GFP reporter gene has an amino acid sequence as shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that The target gene includes the transmembrane protein CD20 gene and / or the transmembrane protein CLDN6 gene.

5. The method according to claim 1, characterized in that When the target gene includes the transmembrane protein CD20 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.3; When the target gene includes the transmembrane protein CLDN6 gene, the protein expressed by the functional gene has an amino acid sequence as shown in SEQ ID NO.

4.

6. The method according to claim 1, characterized in that The weight m1 of the 4,4'-diaminodiphenylmethane and the weight m2 of the cholesterol succinate satisfy the relationship: m1:m2≥10:

1.

7. The method according to claim 1, characterized in that The method of causing the expression complex to produce VLPs precipitation containing the target membrane protein of the functional gene expression product comprises the steps of: Using the expression complex to transfect animal cells, and then culturing the transfected cells to obtain a cell culture fluid; The cell culture fluid is first centrifuged, and then the centrifugation product is enriched to obtain VLPs precipitate containing functional gene expression products.

8. The method according to claim 7, characterized in that The centrifugal force of the first centrifugation is ≥100000 g, and the time of the first centrifugation is ≥2 h.

9. The method according to claim 1, characterized in that: The method of dissolving the VLPs precipitate using a dissolution buffer system to obtain a membrane protein solution containing a functional gene expression product comprises the following steps: Dissolving the VLPs precipitate using a dissolution buffer system to obtain a dissolved protein solution; The dissolved protein solution is resuspended and centrifuged a second time, and then the centrifugal supernatant is filtered to obtain a membrane protein solution containing the functional gene expression product.

10. The method according to claim 9, characterized in that The rotation speed of the second centrifugation is 10000 rpm to 14000 rpm, and the time of the second centrifugation is ≥10 min.

Images