Method for preparing PRMT2 protein by using baculovirus expression system and multi-mode chromatography
Through baculovirus expression system and multi-modal chromatography technology, PRMT2 protein is cloned and expressed, and purified through multiple steps, the problem of low purity of PRMT2 protein in the prior art was solved, and the preparation of high-purity protein was achieved, laying the foundation for structural biology research.
Patent Information
- Application Number
- CN202311261711.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-27
- Publication Date
- 2025-03-28
AI Technical Summary
The PRMT2 protein prepared in the prior art is not purity and is difficult to meet the needs of high-quality proteins.
Using baculovirus expression system and multi-modal chromatography technology, the Gly29-R433 fragment of PRMT2 protein was cloned, expressed by insect cells, and purified by multiple steps such as TEV digestion, Ni column affinity chromatography, molecular sieve chromatography and ion column chromatography to obtain high-purity PRMT2 protein.
Through this method, high purity (greater than 95%) recombinant PRMT2 protein can be obtained in a short time, solving the problem of low protein purity in the prior art and providing high-quality protein materials for structural biology research.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to a method for preparing a recombinant human PRMT2 protein, and specifically relates to a method for preparing a PRMT2 protein using a baculovirus expression system and multi-mode chromatography. Background Art
[0002] PRMT2 is called protein arginine N-methyltransferase 2. Protein arginine methyltransferase can methylate a variety of proteins (including histones and non-histones) and affect a variety of biological processes, such as gene transcription, cell signal transduction, protein stability, cell proliferation, differentiation, apoptosis and tumor formation. At present, 11 members of the PRMT family have been discovered. According to the different ways of catalyzing arginine methylation, they can be divided into three categories: PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8 belong to type I, and the catalytic forms are monomethyl and asymmetric dimethyl; PEMT5 and PRMT9 belong to type II, and the catalytic forms are symmetric dimethyl; PRMT7 belongs to type III and can catalyze monomethyl.
[0003] Since PRMT2 plays an important role in protein methylation, research on PRMT2 has received widespread attention. However, the purity of the PRMT2 protein currently prepared is not high, and there is still much room for improvement. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a method for preparing PRMT2 protein using a baculovirus expression system and multi-mode chromatography. The Gly29-R433 fragment of the PRMT2 protein is cloned for insect cell expression, and only the N-terminal non-structural region is removed, including the complete SH3 domain and the PRMT core domain; the protein after enzyme cleavage is free of any redundant residues by utilizing the characteristics of the recognition sequence of the TEV enzyme; the purification step of two-step affinity chromatography plus molecular sieve chromatography is not only simple, but also can remove foreign proteins to the greatest extent, ensuring that high-quality recombinant PRMT2 protein is obtained in a short time. The specific technical scheme adopted is as follows:
[0005] A method for preparing PRMT2 protein using a baculovirus expression system and multimodal chromatography comprises the following steps:
[0006] S1. The base sequence SEQ ID NO.1 of PRMT2 is cloned into a plasmid by artificial gene synthesis to form a recombinant expression plasmid;
[0007] S2. The recombinant expression plasmid obtained in S1 is transposed into competent E. coli cells to obtain recombinant baculovirus, and then the recombinant baculovirus is transfected into insect cells to obtain baculovirus to express PRMT2 protein SEQ ID NO.2;
[0008] S3. Purified PRMT2 protein SEQ ID NO.3 was obtained by multimodal chromatography.
[0009] The relevant sequences are shown in Table 1 below:
[0010] Table 1 Sequence Listing
[0011]
[0012]
[0013] In some specific embodiments, step S2 is to transpose the recombinant expression plasmid obtained in step S1 into competent Escherichia coli cells to obtain recombinant baculovirus, and transfect insect cells to obtain first-generation baculovirus, and then transfect insect cells to obtain second-generation baculovirus, and infect amplified cultured insect cells to express PRMT2 protein.
[0014] In some specific embodiments, step S3 is to crush the insect cells obtained in step S2 and centrifuge to obtain the supernatant, and then perform Ni column affinity chromatography, dialysis enzyme cleavage Ni column reverse sieving, molecular sieve chromatography, and ion column chromatography to obtain the purified PRMT2 protein.
[0015] In some specific embodiments, the operation method of dialysis enzymatic cleavage Ni column back screening is to perform Ni column affinity chromatography to remove TEV enzyme, His tag and residual His-TEV-PRMT2 fusion protein after TEV enzymatic cleavage to remove the tag.
[0016] In some specific embodiments, in step S1, PRMT2 is expressed intracellularly without adding a signal peptide, and the expression vector used, i.e., the plasmid, is pFastBac1.
[0017] In some specific embodiments, the competent E. coli cells in step S2 are DH10Bac, and the insect cells are sf9 cells.
[0018] In some embodiments, the insect cells are cultured using a cellfection reagent and sf-900II SFM medium.
[0019] In some specific embodiments, the Ni column affinity chromatography medium is HisPur™ Ni-NTA, the molecular sieve chromatography medium is Hiload Superdex 16-600 200 pg, and the ion column chromatography medium is HiTrap™ Q HP.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] Insect cells were used as hosts for expression and the purification steps were optimized. Multi-step separation and purification techniques such as affinity chromatography, enzyme cleavage, molecular sieve chromatography and ion column chromatography were used to solve the problem of low purity of PRMT2 protein.
[0022] The concept and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the construction map of the pFastBac1-PRMT2 plasmid.
[0024] Figure 2 This is an electrophoresis analysis diagram of affinity purification of the fusion protein His-TEV-PRMT2; wherein, Lane 1: protein standard molecular weight; Lane 2: insect cell sample after transfection; Lane 3: precipitate after insect sample crushing; Lane 4: supernatant after insect sample crushing; Lane 5: effluent; Lanes 6-10: eluate.
[0025] Figure 3 This is an electrophoresis analysis diagram of affinity purification of the fusion protein His-TEV-PRMT2 after enzyme cleavage; wherein, Lane 1: protein standard molecular weight; Lane 2: fusion protein sample after NI column purification; Lane 3: protein sample after TEV enzyme cleavage of the fusion protein; Lane 4: effluent; Lane 5: cleaning solution; Lane 6-15: eluent.
[0026] Figure 4 This is the electrophoresis analysis diagram of PRMT2 protein molecular sieve purification; wherein, Lane 1: protein standard molecular weight; Lane 2: PRMT2 protein before molecular sieve; Lane 3-10: eluent.
[0027] Figure 5 This is the electrophoresis analysis diagram of PRMT2 protein ion column purification; wherein, Lane 1: protein standard molecular weight; Lane 2: PRMT2 protein before ion column; Lane 3-10: eluent.
[0028] Figure 6 This is the electrophoresis analysis diagram of the final sample of PRMT2 protein; wherein, Lane 1: protein standard molecular weight; Lane 2: PRMT2 protein (1ug); Lane 3: PRMT2 protein (2ug); Lane 4: PRMT2 protein (5ug). DETAILED DESCRIPTION
[0029] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.
[0030] It should be noted that the drawings and diagrams in the present specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any adjustments without creative work should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.
[0031] All kinds of reagents and equipment involved in the examples are commercially available products, and commercial products can be used.
[0032] (1) Competent cells DH10Bac and cell line sf9 were purchased from Thermo Fisher Scientific.
[0033] (2) Cellfectin transfection reagent, Sf-900II SFM medium, and TEV enzyme were purchased from Thermo Fisher Scientific.
[0034] (3) Complete protease inhibitor Cocktail EDTA free was purchased from Roche.
[0035] (4) Tris, NaCl, Benzonase, imidazole, TCEP, and Glycerol were purchased from Sigma.
[0036] (5) HisPurTM Ni-NTA Resin was purchased from Thermo Fisher Scientific.
[0037] (6) HisTrap HP and Hiload Superdex 26-600 200 pg chromatography columns were purchased from Cytiva.
[0038] (7) Ultrafiltration tubes were purchased from Millipore.
[0039] The human PRMT2 protein sequence was obtained through the Uniprot website, sent to a gene synthesis company for codon optimization and gene synthesis, and cloned into the expression vector pFastBac1 vector to obtain the pFastBac1-PRMT2 plasmid. The pFastBac1-PRMT2 plasmid was transformed into the DH10Bac competent state, and the bacmid was extracted. After obtaining the bacmid, the recombinant baculovirus was obtained by transfecting sf9 insect cells, and the sf9 insect cells were infected with the virus to express the PRMT2 protein intracellularly. After crushing and centrifugation, the supernatant was obtained and purified accordingly to obtain the PRMT2 protein. The following is an exemplary description of the preparation method of the present invention.
[0040] Experimental Example 1 Construction of recombinant expression vector of PRMT2 protein
[0041] The amino acid sequence of PRMT2 was obtained from uniprot, and a HIS tag and TEV restriction site were added to the N-terminus of the sequence. The sequence was sent to a gene synthesis company for sequence optimization and gene synthesis, and finally cloned into the pFastBac1 insect expression vector.
[0042] Experimental Example 2 Expression of PRMT2 protein in insect cells
[0043] Preparation of recombinant bacmids: The constructed pFastBac1-PRMT2 expression plasmid was transformed into DH10Bac competent cells, and the recombinant Bacmid was screened by blue-white screening.
[0044] The recombinant Bacmid was transfected into sf9 cells, and the recombinant baculovirus was obtained after amplification. The P2 recombinant baculovirus was used to transfect sf9 cells to express PRMT2 protein.
[0045] The amplification and expression methods are:
[0046] 1) Spread the cells in a 24-well plate, dilute the cells with sf-900II SFM medium to a cell count of 1*10^6 cells / mL, add 2 mL of cells to each well, add 4 μL of transfection reagent to each transfection sample, add 2 μg of DNA and mix together, and let stand for 20-30 minutes.
[0047] 2) Add the transfection reagent and DNA complex to the wells containing cells and culture medium, shake gently to mix evenly, and incubate at 27°C for 3-5 hours.
[0048] 3) Add 2 mL of sf-900II SFM medium and culture for 7 days until the cells show pathological changes.
[0049] 4) Collect the supernatant, centrifuge at 3000g for 20 min, aspirate the supernatant and store at 4°C, which is the P1 virus.
[0050] 5) Inoculate virus P1 into 200 mL of Sf9 cells at a ratio of 1:200, culture for 5-7 days, centrifuge at 3000 g for 20 min, aspirate the supernatant, and store at 4°C to obtain the P2 virus.
[0051] 6) Inoculate P2 virus into SF9 cells at a concentration of 2.5×106 cells / mL, culture at 27°C, 95 rpm, and harvest the cells after 48 h of culture.
[0052] Experimental Example 3 PRMT2 protein purification
[0053] PRMT2 protein is expressed intracellularly, with a His tag and a TEV restriction tag at the N-terminus. After obtaining the cells by centrifugation, the cells are broken, the supernatant is obtained by centrifugation, and then purified. The purification method is:
[0054] 1. Breaking bacteria
[0055] (1) Use 20mM HEPES, 300mM NaCl, complete protease inhibitor EDTA free 1tab / 100mL, 0.2mg Benzonase / 1L culture, 10mM imidazole, 2mM TCEP, 5% Glycerol, pH7.5 disruption buffer to resuspend the cells and homogenize with a T25 disperser. The homogenization is qualified when there is no obvious solid or filamentous matter.
[0056] (2) Use a disruptor to disrupt the cells at a pressure of 600 bar. First, rinse the system with purified water for more than 2 dead volumes, then rinse 1 dead volume with disruption buffer, add the sample and disrupt, 800 mL / cycle, number of cycles: 2, and then rinse the system with 15 mL of disruption buffer.
[0057] (3) The bacterial suspension was centrifuged in a BECKMAN Avanti J-26S XP centrifuge at 35,000 g and 4°C for 45 min to obtain the supernatant.
[0058] 2. Multimodal chromatography protein purification
[0059] (1) Ni column affinity chromatography
[0060] (Ⅰ) Sample source: supernatant after cell disruption and centrifugation.
[0061] (Ⅱ) Chromatographic column: HisPurTM Ni-NTA Resin.
[0062] (III) Buffer configuration: IMAC-A: 20 mM HEPES, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 10 mM Mimidazole, pH 7.5; IMAC-B: 20 mM HEPES, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 300 mM Mimidazole, pH 7.5.
[0063] (IV) Purification process:
[0064] a. Balance: Balance liquid: IMAC-A, flow rate: 5 mL / min, balance 2CV, stable baseline, UV zero.
[0065] b. Incubation: 4°C, 1h
[0066] c. Wash: Wash buffer: IMAC-A, flow rate: 5 mL / min, 25 CV.
[0067] d. Elution: IMAC-A, IMAC-B, elution gradient: 0-100%, 5CV; UV280: 5mAU, receiving elution peak.
[0068] (V) SDS-PAGE detection Figure 2 )
[0069] (2) Dialysis enzyme digestion Ni column back screening
[0070] (I) Sample source: Ni column captures the eluate.
[0071] (II) Chromatographic column: HisTrap HP.
[0072] (III) Buffer configuration: DIA: 20 mM HEPES, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, pH 7.5; RIMAC-A: 20 mM HEPES, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 10 mM imidazole, pH 7.5; RIMAC-B: 20 mM HEPES, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 300 mM imidazole, pH 7.5.
[0073] (IV) Purification process:
[0074] a. Take out the dialysis bag, wash it with purified water for more than three times, and soak it in dialysis buffer for more than 5 minutes after finding all leaks.
[0075] b. Transfer the sample and TEV enzyme into a dialysis bag, remove bubbles, seal the bag from top to bottom, place it in a container containing 5L of dialysis fluid, stir slowly at 2-8℃ for 16h, and then take it out.
[0076] c. Column equilibration: equilibration liquid: RIMAC-A, flow rate: 5.0 mL / min, equilibration for 5 CV, stable baseline, UV zero adjustment.
[0077] d. Sample loading: flow rate: 2.5 mL / min, UV280: 10 mAU receiving flow-through.
[0078] e.Wash: Wash buffer: RIMAC-A, flow rate: 5 mL / min.
[0079] f. Elution: RIMAC-A, RIMAC-B, elution gradient: 0-50%, 20CV; elution gradient: 50%, 10CV; elution gradient: 50-100%, 1CV; elution gradient: 100%, 10CV; UV280: 10mAU receives the elution peak.
[0080] g.SDS-PAGE detection ( Figure 3 )
[0081] (3) Molecular sieve chromatography
[0082] (I) Sample source: cleaned sample after enzyme digestion and counter-screening.
[0083] (II) Chromatographic column: Hiload Superdex 16-600 200 pg.
[0084] (III) Buffer configuration: SEC: 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, pH 7.5.
[0085] (IV) Purification process:
[0086] a. Column balance: Balance liquid: SEC, flow rate: 1 mL / min, balance 1.2 CV, stable baseline, UV zero.
[0087] b. Sample loading: Flow rate: 1 mL / min.
[0088] c. Elution: eluent: SEC, flow rate: 1 mL / min, UV280: 5 mAU to receive the elution peak.
[0089] d.SDS-PAGE detection ( Figure 4 ).
[0090] (4) Ion column chromatography
[0091] (I) Sample source: molecular sieve elution sample.
[0092] (II) Chromatographic column: HiTrap TM Q HP.
[0093] (III) Buffer configuration: AEX-A: 20 mM HEPES, 50 mM NaCl, 2 mM TCEP, 5% Glycerol, pH 7.5; AEX-B: 20 mM HEPES, 1 M NaCl, 2 mM TCEP, 5% Glycerol, pH 7.5.
[0094] (IV) Purification process:
[0095] a. Column equilibration: equilibration liquid: AEX-A, flow rate: 5.0 mL / min, equilibration for 5 CV, stable baseline, UV zero adjustment.
[0096] b. Sample loading: Flow rate: 2.5 mL / min, UV280: 10 mAU to receive the flow-through.
[0097] c. Wash: Wash buffer: AEX-A, flow rate: 5 mL / min, UV280 5 mAU.
[0098] d. Elution: AEX-A, AEX-B, elution gradient: 0-45%, 30CV; elution gradient: 45%, 5CV; elution gradient: 45-100%, 1CV; elution gradient: 100%, 5CV; UV280: 10mAU receives the elution peak.
[0099] e.SDS-PAGE detection ( Figure 5 ) The results showed that the protein purity was greater than 95%. Finally, the purified product was ultrafiltered and concentrated using a Millpore ultrafiltration tube. The final SDS-PAGE analysis of protein purity ( Figure 6 ).
[0100] The present invention provides a method for expressing PRMT2 recombinant protein in an insect system, and uses a multimodal chromatography method to obtain a label-free and highly pure PRMT2 protein, laying a foundation for future related structural biology research.
[0101] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for preparing PRMT2 protein using a baculovirus expression system and multimodal chromatography, characterized in that: The following steps are involved: S1. The base sequence SEQ ID NO.1 of PRMT2 is cloned into a plasmid by artificial gene synthesis to form a recombinant expression plasmid; S2. The recombinant expression plasmid obtained in S1 is transposed into competent E. coli cells to obtain recombinant baculovirus, and then the recombinant baculovirus is transfected into insect cells to obtain baculovirus to express PRMT2 protein SEQ ID NO.2; S3. Purified PRMT2 protein SEQ ID NO.3 was obtained by multimodal chromatography.
2. The method according to claim 1, characterized in that The step S2 is to transpose the recombinant expression plasmid obtained in the step S1 into competent Escherichia coli cells to obtain recombinant baculovirus, transfect insect cells to obtain first-generation baculovirus, then transfect insect cells to obtain second-generation baculovirus, and infect amplified cultured insect cells to express PRMT2 protein.
3. The method according to claim 1, characterized in that The step S3 is to crush the insect cells obtained in the step S2 and centrifuge to obtain the supernatant, and then perform Ni column affinity chromatography, dialysis enzyme cleavage Ni column reverse sieving, molecular sieve chromatography, and ion column chromatography to obtain the purified PRMT2 protein.
4. The method according to claim 3, characterized in that The operation method of the dialysis enzyme digestion Ni column back screening is to perform Ni column affinity chromatography to remove the TEV enzyme, the His tag and the residual His-TEV-PRMT2 fusion protein after the TEV enzyme digestion removes the tag.
5. The method according to claim 1, characterized in that In the step S1, PRMT2 is not added with a signal peptide, and is expressed intracellularly. The expression vector used, i.e., the plasmid, is pFastBac1.
6. The method according to any one of claims 1 or 2, characterized in that: The competent E. coli cells in step S2 are DH10Bac, and the insect cells are sf9 cells.
7. The method according to any one of claims 1 or 2, characterized in that: Use cellfection reagent and culture insect cells in sf-900II SFM medium.
8. The method according to claim 3, characterized in that The Ni column affinity chromatography medium was HisPurTM Ni-NTA, the molecular sieve chromatography medium was Hiload Superdex 16-600 200 pg, and the ion column chromatography medium was HiTrapTM Q HP.