Efficient expression and purification method of vaccinia virus mRNA Cap 2 oxygen methyltransferase
By expressing in the E. coli expression system and using a variety of purification techniques to obtain vaccinia virus mRNA Cap 2oxymethyltransferase, the problem of low expression and purification efficiency in the prior art was solved, and efficient and economical protein purification and activity were achieved. Identification.
Patent Information
- Application Number
- CN202411608547.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to efficiently express and purify active vaccinia virus mRNA Cap 2oxymethyltransferase, which affects its use in biotechnology applications.
Using the E. coli expression system, the GST tag and TEV cleavage sites were cloned into the prokaryotic expression vector by connecting the GST tag and TEV cleavage sites at the N-terminus of the mRNA Cap 2oxymethyltransferase gene sequence of the vaccinia virus, and cloned into the prokaryotic expression vector by GS4B affinity chromatography and Heparin proliferative Purification was carried out by chromatography, gel filtration chromatography and other steps.
The mRNA Cap 2oxymethyltransferase with uniform molecular weight and good properties was successfully obtained, with a yield of up to 3mg/L, and its activity is similar to that of commercial products. It is concentration-dependent, providing an economical and affordable synthetic technology route.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to an efficient expression and purification method of vaccinia virus mRNA Cap 2-oxygen methyltransferase. Background Art
[0002] mRNA Cap 2'-O-Methyltransferase, a positive regulator of poly(A) polymerase and transcription elongation activity, participates in the modification of mRNA termini and the positive transcription elongation of intermediate and late genes. It methylates the ribose 2'OH group of the first transcribed nucleotide at the 5' end of the mRNA, thereby generating a 2'-O-methyl purine cap. At the 3' end, it acts as a synthesis factor, stimulating the activity of the viral poly(A) polymerase VP55 to generate the poly(A) tail of the mRNA. In the presence of VP39, VP55 does not dissociate from the RNA, allowing the tail to elongate to approximately 250 adenylate nucleotides.
[0003] The enzyme uses SAM as a methyl donor to methylate capped RNA, thereby forming a Cap1 structure. The Cap1 structure can enhance the translation efficiency of mRNA, and thus can help improve expression in mRNA transfection and microinjection experiments. Therefore, it is of great significance to screen an expression system suitable for efficient expression of active vaccinia virus methyltransferase, optimize the expression conditions, isolate and purify the recombinant protein, and identify its biological activity. Summary of the invention
[0004] In view of this, the object of the present invention is to provide an efficient method for expressing and purifying vaccinia virus mRNA Cap 2-O-methyltransferase.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] 1. An efficient expression and purification method for vaccinia virus mRNA Cap 2-O2 methyltransferase, the method comprising the following steps: based on an Escherichia coli expression system, a GST tag and a TEV restriction site are connected to the N-terminus of an optimized vaccinia virus mRNA Cap 2-O2 methyltransferase gene sequence, and the sequence is cloned into a prokaryotic expression vector; then, the recombinant plasmid is transformed into Escherichia coli cells to induce expression of a target protein, the bacteria are collected by centrifugation and resuspended, and the supernatant is collected by centrifugation after high-pressure crushing to obtain a crude extract; finally, the crude extract is purified by GS4B affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain vaccinia virus mRNA Cap 2-O2 methyltransferase.
[0007] In some embodiments of the present invention, the optimized mRNA Cap 2-O2 methyltransferase gene sequence is shown as SEQ ID NO.1.
[0008] In some embodiments of the present invention, the prokaryotic expression vector is a pGST-parallel2 vector.
[0009] In some embodiments of the present invention, the Escherichia coli cells are BL21 (DE3) or BL21 (DE3, pG-TF2) cells.
[0010] In some embodiments of the present invention, when the target protein is induced to be expressed, the OD of the E. coli cells is 600 =1.0, the amount of tetracycline added was 20 ng / ml, and the concentration of IPTG added was 0.2 mM.
[0011] In some embodiments of the present invention, the buffer used to resuspend E. coli cells is: 250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 1 mM PMSF, 10% Glycerol.
[0012] In some embodiments of the present invention, the washing buffer used in the GS4B affinity chromatography is: 250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP.
[0013] In some embodiments of the present invention, the buffer used in Heparin affinity chromatography is:
[0014] Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol;
[0015] High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol, 1 M NaCl.
[0016] In some embodiments of the present invention, the buffer used in gel filtration chromatography is: 100 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT, 10% Glycerol.
[0017] The beneficial effects of the present invention are as follows: the present invention provides a method for efficiently expressing active vaccinia virus mRNA Cap 2-oxygen methyltransferase using an Escherichia coli expression system, connecting a GST tag and a TEV restriction site to the N-terminus of the optimized vaccinia virus mRNA Cap 2-oxygen methyltransferase gene sequence, cloning it into the expression vector pGST-parallel2, and then transforming the recombinant plasmid into Escherichia coli BL21; finally, the vaccinia virus mRNA Cap 2-oxygen methyltransferase with uniform molecular weight and good properties is successfully purified by GS4B affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography. The vaccinia virus mRNA Cap 2-oxygen methyltransferase prepared by the present invention has similar activity to commercial vaccinia virus methyltransferase, has the activity of converting SAM into SAH, and is concentration-dependent. The present invention provides a more economical and practical technical route for synthesizing vaccinia virus mRNA Cap 2-oxygen methyltransferase, which lays a technical foundation for large-scale production of vaccinia virus methyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:
[0019] Figure 1 The experimental flow chart is shown in Figure 2.
[0020] Figure 2 The expression and purification results of vaccinia virus mRNA Cap 2 O-methyltransferase; a: gel filtration chromatogram of vaccinia virus mRNA Cap 2 O-methyltransferase (1-333); b: SDS-PAGE image.
[0021] Figure 3 MTase-Glo TM Schematic diagram of the Methyltransferase Assay principle.
[0022] Figure 4 Schematic diagram of the detection process of the ELISA instrument.
[0023] Figure 5 It is the luminescent signal of different concentrations of vaccinia virus mRNA Cap 2 oxygen methyltransferase (d195T is the recombinant protein prepared by the present invention, and OMT is the commercial vaccinia virus methyltransferase). DETAILED DESCRIPTION
[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0025] The experimental flow chart of the present invention for in vitro expression and purification of vaccinia virus mRNA Cap 2-oxygen methyltransferase and identification of its activity is as follows: Figure 1 shown.
[0026] Example 1. Construction of expression vector
[0027] The full length (SEQ ID NO.1) of the optimized vaccinia virus mRNA Cap 2-oxygen methyltransferase gene (1-333, SEQ ID NO.2) (UniProt ID MCEL_VACCW, P07617) was amplified, and the GST tag and TEV restriction site were connected to the N-terminus of the mRNA Cap 2-oxygen methyltransferase gene, and cloned into the expression vector pGST-parallel2 to obtain the recombinant plasmid pGST-parallel2-GST-TEV CS -OMT(1-333).
[0028] Example 2. Preparation of BL21 (DE3, pG-TF2) competent cells and transformation of recombinant plasmids
[0029] The pG-TF2 plasmid was transferred into BL21 (DE3) cells to form BL21 (DE3, pG-TF2) competent cells that can induce the expression of molecular chaperones, and then the recombinant plasmid pGST-parallel2-GST-TEVCS-OMT (1-333) was transferred to express the recombinant protein.
[0030] Example 3. Expression of recombinant protein
[0031] When inducing expression, the OD of the E. coli cells containing the recombinant plasmid prepared in Example 2 was 600 =1.0, the amount of tetracycline added was 20 ng / ml, and the concentration of IPTG added was 0.2 mM. The expression temperature was 16°C and the rotation speed was 220 rpm. Generally, after 16 hours of expression, the cell pellet was collected by centrifugation at 4°C and 3800 rpm for 15 minutes.
[0032] Example 4. Purification of recombinant protein
[0033] 4.1 The cell pellet obtained in Example 3 was resuspended in Buffer A (250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 1 mM PMSF, 10% Glycerol).
[0034] 4.2 Use a high pressure disruptor to disrupt the cells, 800 bar, 2 min, 4 °C.
[0035] 4.3 Centrifuge at 17000 rpm, 4°C for 40 minutes and collect the supernatant.
[0036] 4.4 GS4B affinity chromatography
[0037] (1) Incubate the supernatant with GS4B column at 4°C for 40 minutes.
[0038] (2) Wash with Wash buffer (250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP) for 10 column volumes.
[0039] (3) Add one column volume of wash buffer and an appropriate amount of TEV, mix well, and digest at 4°C overnight.
[0040] (4) After the tag is cut off, the protein is collected after passing through the gravity column, and then washed with wash buffer for 2 column volumes and collected.
[0041] 4.5 Heparin affinity chromatography (excluding TEV enzyme and GST tag)
[0042] Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol;
[0043] High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol, 1 M NaCl.
[0044] Collect the corresponding number of tubes of target protein and concentrate to 2 ml using a 30KDa cut-off tube.
[0045] 4.6 Gel filtration chromatography (Superdex 200 10 / 300GL)
[0046] Buffer B: 100mM NaCl, 20mM Tris-HCl pH 8.0, 1mM DTT, 10% Glycerol.
[0047] The expression and purification results of vaccinia virus mRNA Cap 2-O-methyltransferase are shown in Figure 2 As shown, each lane in Figure b corresponds to the sample of the elution peak marked at position 19-24 in Figure a. As can be seen from the figure, the recombinant plasmid pGST-parallel2-GST-TEV for constructing vaccinia virus mRNA Cap 2-oxygen methyltransferase CS -OMT(1-333) was expressed in Escherichia coli BL21 and finally successfully purified by GS4B affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain vaccinia virus mRNA Cap 2-O-methyltransferase with uniform molecular weight and good properties, with a yield of up to 3 mg / L.
[0048] Example 5. Identification of enzyme activity of recombinant protein
[0049] 5.1 Detection Principle
[0050] MTase-Glo TM Methyltransferase Assay is a bioluminescent detection system that can monitor the activity of various methyltransferases (MTases) by monitoring the formation of the reaction product S-adenosyl homocysteine (SAH). TM Reagent converts SAH into ADP, then adds MTase-Glo TM The Detection Solution converts ADP into ATP, and then detects ATP through a pair of luciferase reactions. The luminescent signal can be detected by a plate reader with luminescence detection function, and then correlated with the SAH concentration using the SAH standard curve. The principle diagram is shown in the figure below. Figure 3 (From MTase-Glo TM Methyltransferase AssayInstructions).
[0051] 5.2 Detection steps
[0052] (1) Prepare 2× reaction substrate
[0053]
[0054] (2) Prepare two-fold serial dilutions of vaccinia virus mRNA Cap 2-O-methyltransferase in 1× reaction buffer.
[0055] (3) 2× substrate (4 μL) and different concentrations of vaccinia virus mRNA Cap 2-O methyltransferase (4 μL) were added to a 384-well plate and reacted at 37°C for 1 hour.
[0056] (4) Add 5×MTase-Glo TM Reagent (2 μL) and react at room temperature (25°C) for 30 minutes.
[0057] (5) Add 1×MTase-Glo TM Detection Solution (10 μL) was added and reacted at room temperature (25°C) for 30 minutes.
[0058] (6) Detect the luminescent signal using an enzyme-labeled instrument.
[0059] The detection process diagram is as follows Figure 4 (From MTase-Glo TM Methyltransferase AssayInstructions).
[0060] The results of the enzymatic activity identification of vaccinia virus mRNA Cap 2-O-methyltransferase are as follows Figure 5 As shown in the figure, it can be seen that the d195T (vaccinia virus mRNA Cap 2 oxygen methyltransferase) expressed and purified by the present invention has similar activity to the commercial vaccinia virus methyltransferase (OMT) purchased from NEB, has the activity of converting SAM into SAH, and is concentration-dependent.
[0061] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims
1. An efficient method for expressing and purifying vaccinia virus mRNA Cap 2-oxygen methyltransferase, characterized in that: The method comprises the following steps: based on an Escherichia coli expression system, a GST tag and a TEV restriction site are connected to the N-terminus of an optimized vaccinia virus mRNA Cap 2-O2 methyltransferase gene sequence, and the sequence is cloned into a prokaryotic expression vector; then, the recombinant plasmid is transformed into Escherichia coli cells to induce the expression of a target protein; the bacteria are collected by centrifugation and resuspended; after high-pressure crushing, the supernatant is collected by centrifugation to obtain a crude extract; finally, the crude extract is purified by GS4B affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain the vaccinia virus mRNA Cap 2-O2 methyltransferase.
2. The method according to claim 1, characterized in that The optimized mRNA Cap 2-O2 methyltransferase gene sequence is shown in SEQ ID NO.
1.
3. The method according to claim 1, characterized in that The prokaryotic expression vector is a pGST-parallel2 vector.
4. The method according to claim 1, characterized in that The Escherichia coli cells are BL21 (DE3) or BL21 (DE3, pG-TF2) cells.
5. The method according to claim 1, characterized in that When the target protein is induced to express, the OD of E. coli cells is 600 =1.0, the amount of tetracycline added was 20 ng / ml, and the concentration of IPTG added was 0.2 mM.
6. The method according to claim 1, characterized in that The buffer used to resuspend E. coli cells was: 250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 1 mM PMSF, 10% Glycerol.
7. The method according to claim 1, characterized in that The washing buffer used for GS4B affinity chromatography was: 250 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP.
8. The method according to claim 1, characterized in that The buffer used for Heparin affinity chromatography is: Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol; High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 10% Glycerol, 1 M NaCl.
9. The method according to claim 1, characterized in that: The buffer used for gel filtration chromatography was: 100 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT, 10% Glycerol.