Preparation method of human PRMT7 protein
Through expression and multi-step purification technology in insect cells, the problem of difficulty in preparing and purification of PRMT7 protein was solved, and a high-purity tagless PRMT7 protein was obtained, meeting the needs of structural function research.
Patent Information
- Application Number
- CN202311250328.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-26
- Publication Date
- 2025-03-28
AI Technical Summary
The lack of a good preparation scheme for PRMT7 protein in the prior art makes it difficult to prepare and purify it.
Using the method of expressing PRMT7 protein in insect systems, high-purity tagless PRMT7 protein was obtained by cloning artificially synthesized nucleotide fragments of human PRMT7 into baculovirus expression vectors, transpossessed into E. coli competent cells, and transfecting insect cells to express PRMT7 proteins. Subsequently, a multi-step purification technique such as affinity chromatography, enzyme cleavage and molecular sieve chromatography were obtained.
It has achieved efficient expression of the complete PRMT7 protein, strong purification process operability, high protein yield, and the final purity of the obtained protein is greater than 90%, meeting the subsequent structural pharmacological research requirements.
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Figure CN119709675A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing a PRMT7 protein, and in particular to a method for preparing a human PRMT7 protein. Background Art
[0002] PRMT7 is called protein arginine N-methyltransferase 7. Protein arginine methyltransferase (PRMT) can methylate a variety of proteins (including histones and non-histones) and affect 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, which can be divided into three categories according to the different ways of catalyzing arginine methylation: PRMT1-4, 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 form is symmetric dimethyl; PRMT7 belongs to type III and can catalyze monomethyl.
[0003] Most PRMT family proteins contain a catalytically active core domain, which is composed of a Rossman fold motif and a β-barrel structure. Previous studies have shown that the active form of this type of enzyme is a homodimer. However, the PRMT7 protein has two consecutive core domains. The first domain at the N-terminus has been confirmed to be catalytically active, and the second domain at the C-terminus is considered to be inactive. However, the activity regulation mechanism and high-level spatial structure of the enzyme are still unclear.
[0004] Previous studies have confirmed that PRMT7 is highly expressed in breast cancer, colorectal cancer, and prostate cancer cells, and its upregulation is associated with high levels of hnRNPA1 arginine methylation and abnormal alternative splicing. At the same time, pharmacological inhibition of PRMT7 can inhibit cancer cell growth, indicating that it can be used as a target for cancer treatment. Since PRMT7 is of great significance in the study of protein methylation and its anti-tumor drugs, the study of the three-dimensional spatial structure and function of PRMT7 has received widespread attention but has progressed slowly. One of the main reasons is that the preparation and purification of PRMT7 is difficult. Summary of the invention
[0005] The purpose of the present invention is to provide a method for expressing PRMT7 protein using an insect system in view of the lack of a good PRMT7 protein preparation solution in the current prior art. The specific technical solution is as follows:
[0006] A method for preparing human PRMY7 protein comprises the following steps:
[0007] S1. Cloning the artificially synthesized nucleotide fragment of human PRMT7 into a baculovirus expression vector to obtain a recombinant expression plasmid; the artificially synthesized nucleotide fragment of human PRMT7 is labeled for easy purification;
[0008] S2, transposing the recombinant expression plasmid obtained in step S1 into competent Escherichia coli cells to obtain recombinant baculovirus, and transfecting insect cells to express PRMT7 protein;
[0009] S3. Purify the PRMT7 protein obtained in step S2 to obtain a tag-free PRMT7 protein, namely the human PRMY7 protein.
[0010] In some embodiments, the recombinant expression plasmid obtained in step S1 is expressed intracellularly, and the PRMT7 protein expressed by the recombinant expression plasmid does not contain a signal peptide.
[0011] In some embodiments, the nucleic acid sequence of the artificially synthesized human PRMT7 nucleotide fragment described in step S1 includes the nucleotide sequence of PRMT7, a His tag sequence and a TEV restriction site sequence; and the PRMT7 protein obtained in step S2 is a fusion protein His-TEV-PRMT7.
[0012] In some embodiments, the nucleic acid sequence of the artificially synthesized human PRMT7 nucleotide fragment described in step S1 further comprises a linker sequence; the linker sequence comprises a first linker sequence, the amino acid sequence encoded by which is shown in SEQ ID NO:4, and a second linker sequence, the amino acid sequence encoded by which is shown in SEQ ID NO:5; the amino acid sequence encoded by the His tag sequence is shown in SEQ ID NO:6; the amino acid sequence encoded by the TEV cleavage site sequence is shown in SEQ ID NO:7; the first linker sequence, the His tag sequence, the second linker sequence and the TEV cleavage site sequence are connected in sequence, and the TEV cleavage site sequence is connected to the 5' end of the nucleotide sequence of the PRMT7. The addition of the linker sequence is conducive to the smooth progress of affinity chromatography and TEV cleavage, thereby obtaining the target protein after cleavage, namely, human PRMT7 protein.
[0013] In some embodiments, the nucleic acid sequence of the nucleotide fragment of the artificially synthesized human PRMT7 described in step S1 comprises the nucleic acid sequence shown in SEQ ID NO:1; the amino acid sequence of the PRMT7 protein obtained in step S2 comprises the amino acid sequence shown in SEQID NO:2; and the amino acid sequence of the human PRMT7 protein obtained in step S3 comprises the amino acid sequence shown in SEQ ID NO:3.
[0014] In some embodiments, the baculovirus expression vector described in step S1 is pFastBac1.
[0015] In some embodiments, the competent E. coli cells in step S2 are DH10Bac, the insect cells are sf9 insect cells, the transfection reagent is cellfection, and the medium used for insect cell culture is sf-900II SFM medium.
[0016] In some embodiments, the purification described in step S3 is: the transfected insect cells obtained in step S2 are crushed and centrifuged to obtain the supernatant, purified by Ni column affinity chromatography, and after TEV enzyme cleavage to remove the tag, Ni column affinity chromatography is performed to remove the TEV enzyme, His tag and residual His-TEV-PRMT7 fusion protein, and then molecular sieve chromatography purification is performed to obtain human PRMT7 protein.
[0017] In some embodiments, the Ni column affinity chromatography purification medium in step S3 is HisPur TM Ni-NTA Resin.
[0018] In some embodiments, the Ni column affinity chromatography back-sieving medium in step S3 is HisTrap HP; the molecular sieve chromatography medium in step S3 is Hiload Superdex 26-600 200 pg.
[0019] The present invention uses insect cells as hosts for expression and optimizes the purification steps, and obtains a high-purity, tag-free full-length PRMT7 protein through multi-step separation and purification techniques such as affinity chromatography, enzyme digestion, and molecular sieve chromatography. The method adopted by the present invention can efficiently express the complete PRMT7 protein, the purification process is highly operable, and the protein yield is high. The final purity of the obtained protein is greater than 90%, and the ideal concentration is achieved, meeting the requirements of subsequent structural pharmacology research.
[0020] The present invention is the first to prepare unlabeled recombinant human PRMT7 protein through an insect expression system through a purification step, laying the foundation for the subsequent structure-function relationship and biomedical application of PRMT7 protein.
[0021] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the construction map of the pFastBac1-PRMT7 plasmid of the present invention.
[0023] Figure 2This is the affinity purification electrophoresis analysis of the fusion protein His-TEV-PRMT7 before enzyme cleavage. Among them, lane 1: protein standard molecular weight; lane 2: sf9 insect cells after transfection; lane 3: supernatant after sf9 insect cells after transfection; lane 4: precipitate after sf9 insect cells after transfection; lane 5: effluent; lanes 6-12: eluate.
[0024] Figure 3 This is the affinity purification electrophoresis analysis of the fusion protein His-TEV-PRMT7 after digestion. Lane 1: protein standard molecular weight; Lane 2: fusion protein sample after Ni column purification; Lane 3: protein sample after TEV digestion of the fusion protein; Lane 4: effluent; Lanes 5-10: eluate.
[0025] Figure 4 This is the electrophoresis analysis of molecular sieve purification of human PRMT7 protein. Lane 1: protein standard molecular weight; Lane 2: human PRMT7 protein before molecular sieve; Lanes 3-10: eluent.
[0026] Figure 5 This is the final electrophoresis analysis of human PRMT7 protein. Lane 1: protein standard molecular weight; Lane 2: PRMT7 protein (1μg); Lane 3: PRMT7 protein (2μg); Lane 4: PRMT7 protein (5μg) DETAILED DESCRIPTION
[0027] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described with reference to specific diagrams. However, the invention is not limited to the following implementation cases.
[0028] The human PRMT7 protein sequence was obtained through the UniProt website, sent to a gene synthesis company for codon optimization, label (His tag and TEV restriction site) addition and gene synthesis and cloned into the expression vector pFastBac1 vector to obtain the pFastBac1-PRMT7 plasmid. The pFastBac1-PRMT7 plasmid was transformed into the Escherichia coli competent cell DH10Bac, and the recombinant baculovirus plasmid was obtained by blue-white spot detection, and the recombinant baculovirus plasmid was extracted and transfected into sf9 insect cells to obtain the recombinant baculovirus. The recombinant baculovirus was used to infect new sf9 insect cells to further amplify the recombinant baculovirus, thereby obtaining a sufficient amount of intracellular expression of PRMT7 protein. The infected sf9 insect cells were broken, the supernatant was obtained after centrifugation and purified accordingly, the label was removed, and the target protein-human PRMT7 protein was obtained. Below, the preparation method of the present invention is exemplified.
[0029] 1. Construction of recombinant expression vector of PRMT7 protein
[0030] The amino acid sequence of PRMT7 was obtained from the UniProt protein database, and a His tag (HIStag) and a 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 to obtain SEQ ID NO: 1. SEQ ID NO: 1 was cloned into the pFastBac1 insect expression vector (purchased from Thermo Fisher Scientific) to obtain the pFastBac1-PRMT7 expression plasmid (see Figure 1 ).
[0031] 2. Expression of PRMT7 protein in insect cells
[0032] Preparation of recombinant bacmid: The constructed pFastBac1-PRMT7 expression plasmid was transformed into DH10Bac competent cells (purchased from Thermo Fisher Scientific), and the recombinant bacmid was screened by blue-white screening.
[0033] The recombinant Bacmid was transfected into sf9 insect cells (purchased from Thermo Fisher Scientific), and recombinant baculovirus was obtained after amplification. The second generation (P2) recombinant baculovirus was used to transfect sf9 insect cells to express PRMT7 protein (SEQ ID NO: 2).
[0034] The amplification and expression methods are:
[0035] 1) Spread the cells in a 24-well plate and dilute the cells to a cell count of 1×10 using Sf-900II SFM medium (purchased from Thermo Fisher Scientific). 6 cells / mL, add 2 mL of cells to each well, for each transfection sample, add 4 μL of transfection reagent, add 2 μg of DNA and mix together, and let it stand for 20-30 minutes.
[0036] 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.
[0037] 3) Add 2 mL of Sf-900II SFM medium and culture for 7 days until the cells show pathological changes.
[0038] 4) Collect the supernatant, centrifuge at 3000g for 20 min, aspirate the supernatant and store it at 4°C, which is the first generation (P1) virus.
[0039] 5) Inoculate 200 mL of sf9 insect cells with virus P1 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.
[0040] 6) Inoculate P2 virus to 2.5×106 cells / mL of sf9 insect cells at 27°C and 95rpm, and the cells were harvested after 48h of culture.
[0041] 3. PRMT7 Protein Purification
[0042] PRMT7 protein is expressed intracellularly, with a His tag and a TEV cleavage site (or TEV cleavage tag) at the N-terminus. After the culture medium containing infected sf9 insect cells is centrifuged to obtain the cells, the cells are broken, the supernatant is obtained by centrifugation, and then purified. The purification method is:
[0043] 1) Breaking the cells
[0044] (1) Use 20mM Tris (purchased from Sigma), 300mM NaCl (purchased from Sigma), 1tab / 100mL completeprotease inhibitor EDTA free (purchased from Roche), 0.2mg / 1L culture Benzonase (purchased from Sigma), 10mM imidazole (purchased from Sigma), 2mM TCEP (purchased from Sigma), 5% Glycerol (purchased from Sigma), 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.
[0045] (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.
[0046] (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.
[0047] 2) Multimodal chromatography protein purification
[0048] (1) Ni column affinity chromatography
[0049] (Ⅰ) Sample source: supernatant after cell disruption and centrifugation
[0050] (Ⅱ) Chromatographic column: HisPur TM Ni-NTA Resin (purchased from Thermo Fisher Scientific)
[0051] (III) Buffer configuration: IMAC-A: 20 mM TRIS, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 10 mM Mimidazole, pH 7.5; IMAC-B: 20 mM TRIS, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 300 mM Mimidazole, pH 7.5.
[0052] (IV) Purification process:
[0053] a. Balance: Balance liquid: IMAC-A, flow rate: 5 mL / min, balance 2CV, stable baseline, UV zero.
[0054] b. Incubation: 4°C, 1h
[0055] c. Wash: Wash buffer: IMAC-A, flow rate: 5 mL / min, 25 CV.
[0056] d. Elution: IMAC-A, IMAC-B, elution gradient: 0-100%, 5CV; UV280: 5mAU, receiving elution peak.
[0057] (V) SDS-PAGE detection, the results are shown in Figure 2 .
[0058] (2) Dialysis enzyme cleavage Ni column back screening
[0059] (I) Sample source: Ni column capture eluate
[0060] (II) Chromatographic column: HisTrap HP (purchased from Cytiva)
[0061] (III) Buffer configuration: DIA: 20 mM TRIS, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, pH 7.5; RIMAC-A: 20 mM TRIS, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 10 mM imidazole, pH 7.5; RIMAC-B: 20 mM TRIS, 300 mM NaCl, 2 mM TCEP, 5% Glycerol, 300 mM imidazole, pH 7.5.
[0062] (IV) Purification process:
[0063] 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.
[0064] b. Transfer the sample and TEV enzyme (purchased from Thermo Fisher Scientific) into a dialysis bag, remove bubbles, seal the bag from top to bottom, place it in a container containing 5 L of dialysis fluid, stir slowly at 2-8°C for 16 hours, and then take it out.
[0065] c. Column equilibration: equilibration liquid: RIMAC-A, flow rate: 5.0 mL / min, equilibration for 5 CV, stable baseline, UV zero adjustment.
[0066] d. Sample loading: flow rate: 2.5 mL / min, UV280: 10 mAU receiving flow-through.
[0067] e.Wash: Wash buffer: RIMAC-A, flow rate: 5 mL / min.
[0068] 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.
[0069] g. SDS-PAGE test, the results are shown in Figure 3 .
[0070] (3) Molecular sieve chromatography
[0071] (I) Sample source: cleaned sample after enzyme digestion and screening
[0072] (II) Chromatographic column: Hiload Superdex 26-600 200 pg (purchased from Cytiva)
[0073] (III) Buffer configuration: SEC buffer: 20 mM TRIS, 150 mM NaCl, 2 mM TCEP, pH 7.5.
[0074] (IV) Purification process:
[0075] a. Column equilibration: equilibration liquid: SEC buffer, flow rate: 2 mL / min, equilibration 1.2 CV, stable baseline, UV zero.
[0076] b. Sample loading: Flow rate: 1 mL / min.
[0077] c. Elution: Elution: SEC buffer, flow rate: 2 mL / min, UV280: 5 mAU to receive the elution peak.
[0078] d. Fraction collection: SDS-PAGE detection ( Figure 4) and collect the components from lanes 4 to 12, and finally concentrate the purified product to 12 mg / ml using an ultrafiltration tube (purchased from Millipore). The final SDS-PAGE analysis showed that the protein purity was greater than 90% ( Figure 5 ).
[0079] in conclusion
[0080] The present invention provides a method for expressing PRMT7 recombinant protein in an insect system, and uses a multimodal chromatography method to obtain a label-free and highly pure PRMT7 protein, laying a foundation for future related functional and structural research.
[0081] 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 human PRMY7 protein, characterized in that: The following steps are involved: S1. Cloning the artificially synthesized nucleotide fragment of human PRMT7 into a baculovirus expression vector to obtain a recombinant expression plasmid; the artificially synthesized nucleotide fragment of human PRMT7 is labeled for easy purification; S2, transposing the recombinant expression plasmid obtained in step S1 into competent Escherichia coli cells to obtain recombinant baculovirus, and transfecting insect cells to express PRMT7 protein; S3. Purify the PRMT7 protein obtained in step S2 to obtain a tag-free PRMT7 protein, namely the human PRMY7 protein.
2. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The recombinant expression plasmid obtained in step S1 is expressed intracellularly, and the PRMT7 protein expressed by the plasmid does not contain a signal peptide.
3. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The nucleic acid sequence of the artificially synthesized human PRMT7 nucleotide fragment described in step S1 includes the nucleotide sequence of PRMT7, His tag sequence and TEV restriction site sequence; the PRMT7 protein obtained in step S2 is a fusion protein His-TEV-PRMT7.
4. The method for preparing human PRMY7 protein according to claim 3, characterized in that: The nucleic acid sequence of the nucleotide fragment of the artificially synthesized human PRMT7 described in step S1 also includes a linker sequence; the linker sequence includes a first linker sequence, whose amino acid sequence is shown in SEQ ID NO:4, and a second linker sequence, whose amino acid sequence is shown in SEQ ID NO:5; the amino acid sequence encoded by the His tag sequence is shown in SEQ ID NO:6; the amino acid sequence encoded by the TEV cleavage site sequence is shown in SEQ ID NO:7; the first linker sequence, the His tag sequence, the second linker sequence and the TEV cleavage site sequence are connected in sequence, and the TEV cleavage site sequence is connected to the 5' end of the nucleotide sequence of the PRMT7.
5. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The nucleic acid sequence of the nucleotide fragment of the artificially synthesized human PRMT7 described in step S1 includes the nucleic acid sequence shown in SEQ ID NO:1; the amino acid sequence of the PRMT7 protein obtained in step S2 includes the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence of the human PRMT7 protein obtained in step S3 includes the amino acid sequence shown in SEQ ID NO:
3.
6. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The baculovirus expression vector described in step S1 is pFastBac1.
7. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The competent E. coli cells in step S2 are DH10Bac, the insect cells are sf9 insect cells, the transfection reagent is Cellfection, and the medium used for insect cell culture is sf-900II SFM medium.
8. The method for preparing human PRMY7 protein according to claim 1, characterized in that: The purification described in step S3 is as follows: the transfected insect cells obtained in step S2 are crushed and centrifuged to obtain the supernatant, which is purified by Ni column affinity chromatography, and after TEV enzyme cleavage to remove the tag, Ni column affinity chromatography is performed to remove the TEV enzyme, His tag and residual His-TEV-PRMT7 fusion protein, and then molecular sieve chromatography purification is performed to obtain human PRMT7 protein.
9. The method for preparing human PRMY7 protein according to claim 8, characterized in that: The Ni column affinity chromatography purification medium in step S3 is HisPur TM Ni-NTA Resin.
10. The method for preparing human PRMY7 protein according to claim 8, characterized in that: The Ni column affinity chromatography back-sieving medium in step S3 is HisTrap HP; the molecular sieve chromatography medium in step S3 is Hiload Superdex 26-600 200 pg.