Efficient expression and purification method of vaccinia virus capping enzyme D1D12 compound
By adding His tags and TEV enzyme cleavage sites on the D1 and D12 gene sequences, and using the Bac-to-Bac system and insect cell expression system, the active vaccinia virus capping enzyme D1D12 complex was successfully and efficiently expressed and purified, and the solution was solved The problems of difficult expression and purification in the prior art have achieved efficient and economical protein production.
Patent Information
- Application Number
- CN202411510399.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to efficiently express and purify the active vaccinia virus capping enzyme D1D12 complex, affecting its application prospects in mRNA therapy and other applications.
Using the Bac-to-Bac system, the His tag and TEV cleavage sites were linked at the N-terminals of the D1 and D12 gene sequences, and these gene fragments were linked to the pFastBac expression vector, and the parent rod in DH10Bac E. coli competent cells The plasmid is recombined to form a baculovirus expression plasmid. Then it was expressed in insect cells and purified by nickel column affinity chromatography, Heparin affinity chromatography, gel filtration chromatography.
Successfully and efficiently expressed and purified the cowpox virus capping enzyme D1D12 complex with uniform molecular weight and good properties. It has similar activities as commercial products, can convert SAM into SAH, and is concentration-dependent, laying the foundation for large-scale production technology Base.
Smart Images
Figure CN119391670A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to an efficient method for expressing and purifying a vaccinia virus capping enzyme D1D12 complex. Background Art
[0002] In eukaryotic organisms, mRNA is modified after transcription to form a special structure at the 5′ end, namely the cap structure, which plays an important role in the stability, transport and translation of mRNA. Vaccinia virus capping enzyme is an effective enzyme that catalyzes the formation of the cap structure. It consists of two subunits, D1 and D12, and has RNA triphosphatase activity, guanylyl transferase activity and guanine methyltransferase activity. It can connect the 7-methylguanine cap structure (m7Gppp) to the 5′ end of RNA (m7Gppp5′N).
[0003] The 5' cap structure (m7GpppN) of messenger RNA (mRNA) was discovered in the 1970s. Its existence confers stability to mRNA and enables effective translation. The cap structure of mRNA is closely related to RNA quality control and the body's inherent immunity. Capping enzymes have good application prospects in exogenous mRNA technology, enzymatic synthesis of functional mRNA, mRNA vaccines, self-amplified mRNA and Cappable-Seq RNA sequencing. As mRNA therapy has become a "new star" in the field of biomedical research and development, we need to have a deeper study and exploration of the biological function and application of the 5' cap structure of mRNA. Therefore, it is crucial to express and purify in vitro stable and active vaccinia virus capping enzymes. 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 the vaccinia virus capping enzyme D1D12 complex.
[0005] In order to achieve the above object, the present invention provides the following technical solutions: 1. An efficient expression and purification method for a vaccinia virus capping enzyme D1D12 complex, the method comprising the following steps: based on the Bac-to-Bac system, a 10×His tag and a TEV restriction site are connected to the N-terminus of an optimized D1 gene sequence, and an 8×His tag and a TEV restriction site are connected to the N-terminus of an optimized D12 gene sequence, and the two fragments are respectively connected to the polyhedrin of a pFastBac expression vector and the multiple cloning site behind the P10 promoter, and recombined with the parent bacoid virus plasmid in a DH10Bac Escherichia coli competent cell to form an expression bacoid virus plasmid; then, the expression bacoid virus plasmid is transfected into insect cells to generate recombinant bacoid virus particles; finally, the vaccinia virus capping enzyme D1D12 complex is purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography.
[0006] Preferably, in the present invention, the optimized D1 gene sequence is shown as SEQ ID NO.1.
[0007] Preferably, in the present invention, the optimized D12 gene sequence is shown as SEQ ID NO.3.
[0008] Preferably, the pFastBac expression vector is a pFastBac Dual vector.
[0009] Preferably, the insect cells are Sf9, Sf21 or High Five cells.
[0010] Preferably, during transfection, the density of insect cells is 3×10 6 / mL, and the amount of expression bacmid plasmid added was 0.15% (v / v).
[0011] Preferably, the eluent used in the nickel column affinity chromatography is: 150 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 250 mM imidazole pH 8.0.
[0012] Preferably, the buffer used in Heparin affinity chromatography is: Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 40 mM NaCl; High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1M NaCl.
[0013] Preferably, the buffer used in the gel filtration chromatography is: 100 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT.
[0014] The beneficial effects of the present invention are as follows: the present invention provides a method for efficiently expressing an active vaccinia virus capping enzyme D1D12 complex by using an insect baculovirus expression system, connecting a 10×His tag and a TEV restriction site to the N-terminus of an optimized D1 gene sequence, connecting an 8×His tag and a TEV restriction site to the N-terminus of an optimized D12 gene sequence, and connecting the two fragments to the polyhedrin of a pFastBacDual expression vector and the multiple cloning site after a P10 promoter, respectively, and recombining with a parent baculovirus plasmid in a DH10Bac Escherichia coli competent cell to form an expression baculovirus plasmid; then, the expression baculovirus plasmid is transfected into insect cells to generate recombinant baculovirus particles; and finally, the vaccinia virus capping enzyme D1D12 complex with uniform molecular weight and good properties is successfully purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography. The vaccinia virus capping enzyme D1D12 complex prepared by the present invention has similar activity to commercial vaccinia virus capping enzyme, 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 the vaccinia virus capping enzyme D1D12 complex, laying a technical foundation for large-scale production of vaccinia virus capping enzyme. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] 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: Figure 1 The experimental flow chart is shown in Figure 2.
[0016] Figure 2 Expression and purification results of the vaccinia virus capping enzyme D1D12 complex; a: His 10 -TEV CS -D1(1-844) and His 8 -TEV CS a: Gel filtration chromatogram of the D12(1-287) complex; b: SDS-PAGE image of the sample at positions 14-19 of the elution peak in a.
[0017] Figure 3 Schematic diagram of the MTase-Glo™ Methyltransferase Assay principle.
[0018] Figure 4 Schematic diagram of the detection process of the ELISA instrument.
[0019] Figure 5 It is the luminescent signal of the vaccinia virus capping enzyme D1D12 complex with different concentrations (d247 is the recombinant protein prepared by the present invention, and D1D12 is the commercial vaccinia virus capping enzyme). DETAILED DESCRIPTION
[0020] 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.
[0021] The experimental flow chart of the present invention for in vitro expression and purification of the vaccinia virus capping enzyme D1D12 complex and identification of its activity is as follows: Figure 1 shown.
[0022] Example 1. Construction of expression vector The optimized vaccinia virus capping enzyme gene large subunit D1 (1-844, SEQ ID NO.2) (UniProtID MCEL_VACCW, P04298) full length (SEQ ID NO.1) and small subunit D12 (1-287, SEQ ID NO.4) (UniProt ID MCES_VACCW, P04318) full length (SEQ ID NO.3) were amplified, and a 10×His tag and a TEV restriction site were connected to the N-terminus of the D1 gene, and an 8×His tag and a TEV restriction site were connected to the N-terminus of the D12 gene, and the two fragments were ligated to pFastBac TM Dual expression vector Polyhedrin and P10 promoter after the multiple cloning site, to obtain the recombinant plasmid pFastBacDual-pH-His 10 -TEV CS -D1(1-844)-p10-His 8 -TEV CS -D12(1-287).
[0023] Example 2. Preparation of recombinant baculovirus Refer to the Bac-to-Bac Baculovirus Expression System Operation Manual to recombinant with the parental bacmid in DH10Bac E. coli competent cells to form an expression bacmid for expressing recombinant protein.
[0024] Example 3. Expression of recombinant protein The density of Sf9 cells used for protein expression was 3×10 6 / ml, the amount of expression bacmid added was 0.15% (v / v), the temperature was 27°C, and the rotation speed was 120 rpm. Generally, after 60 hours of expression, the cell pellet was collected by centrifugation at 3000 rpm for 15 minutes at 4°C.
[0025] Example 4. Purification of recombinant protein 4.1 Resuspend the cell pellet in Buffer A (150 mM NaCl, 25 mM Tris pH 8.0, 0.5 mM TCEP, 1mM PMSF, 0.8 µM Aprotinin, 1 µM Pepstatin, 10 µM Leupeptin).
[0026] 4.2 Disrupt cells using a high pressure disruptor at 500 bar, 2 min, 4°C.
[0027] 4.3 Centrifuge at 17,000 rpm, 4°C for 40 min and collect the supernatant.
[0028] 4.4 Ni-NTA affinity chromatography (1) Incubate the supernatant with the Ni-NTA column at 4°C for 40 minutes.
[0029] (2) Wash for 10 column volumes with Wash buffer (150 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 20 mM imidazole pH 8.0).
[0030] (3) Add appropriate amount of Elute buffer (150 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 250 mM imidazole pH 8.0) in small amounts and multiple times.
[0031] 4.5 Heparin affinity chromatography (impurity removal) Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 40 mM NaCl.
[0032] High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1M NaCl.
[0033] Collect the corresponding tubes of target protein and concentrate to 2 ml using a 100 KDa cut-off tube.
[0034] 4.6 Gel filtration chromatography (Superdex 200 10 / 300 GL) Buffer B: 100 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT.
[0035] The expression and purification results of the vaccinia virus capping enzyme D1D12 complex are shown in Figure 2 As shown in the figure, it can be seen that the cowpox virus capping enzyme pFastBacDual- pH-His 10 -TEV CS -D1(1-844)-p10-His 8 -TEV CS -D12(1-287) was expressed in the eukaryotic insect cell Sf9, and finally purified successfully by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain a vaccinia virus capping enzyme D1D12 complex with uniform molecular weight and good properties. The final yield of 1 liter of sf9 cells was greater than or equal to 2 mg.
[0036] 5. Identification of enzyme activity of recombinant protein 5.1 Detection Principle MTase-Glo™ Methyltransferase Assay is a bioluminescence-based detection system that can monitor the activity of multiple methyltransferases (MTases) by monitoring the formation of the reaction product S-adenosyl homocysteine (SAH). When the methyltransferase reaction is completed, MTase-Glo™ Reagent is added to convert SAH to ADP, and then MTase-Glo™ Detection Solution is added to convert ADP to ATP, and then ATP is detected 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™ Methyltransferase Assay Instructions).
[0037] 5.2 Detection steps (1) Prepare 2× reaction substrate Component Volume Concentration 4×Reaction Buffer 22.5 µL 1× 1mM SAM 18 µL 0.2 mM 10mM GTP 9 µL 1 mM RNase-free ddH2O 40.5 µL Total 90 µL (2) Prepare serial two-fold dilutions of the capping enzyme in 1× reaction buffer.
[0038] (3) Add 2× substrate (4 µL) and different concentrations of capping enzyme (4 µL) to a 384-well plate and react at 37°C for 1 hour.
[0039] (4) Add 5× MTase-Glo™ Reagent (2 µL) and react at 25°C for 30 min.
[0040] (5) Add 1× MTase-Glo™ Detection Solution (10 µL) and incubate at 25°C for 30 min.
[0041] (6) Detect the luminescent signal using an enzyme reader.
[0042] The detection process diagram is as follows Figure 4 (From MTase-Glo™ Methyltransferase AssayInstructions).
[0043] The results of the enzymatic activity identification of the vaccinia virus capping enzyme D1D12 complex are as follows Figure 5 As shown in the figure, it can be seen that the activity of d247 (vaccinia virus capping enzyme D1D12 complex) expressed and purified by the present invention is similar to that of commercial vaccinia virus capping enzyme (manufacturer NEB), and has the activity of converting SAM into SAH in a concentration-dependent manner.
[0044] 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 the vaccinia virus capping enzyme D1D12 complex, characterized in that: The method comprises the following steps: based on the Bac-to-Bac system, a 10×His tag and a TEV restriction site are connected to the N-terminus of the optimized D1 gene sequence, an 8×His tag and a TEV restriction site are connected to the N-terminus of the optimized D12 gene sequence, and the two fragments are respectively connected to the polyhedrin of the pFastBac expression vector and the multiple cloning site behind the P10 promoter, and recombined with the parent bacoid virus plasmid in the DH10Bac Escherichia coli competent cells to form an expression bacoid virus plasmid; then, the expression bacoid virus plasmid is transfected into insect cells to generate recombinant bacoid virus particles; finally, the vaccinia virus capping enzyme D1D12 complex is purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography.
2. The method according to claim 1, characterized in that The optimized D1 gene sequence is shown as SEQ ID NO.
1.
3. The method according to claim 1, characterized in that The optimized D12 gene sequence is shown in SEQ ID NO.
3.
4. The method according to claim 1, characterized in that: The pFastBac expression vector is a pFastBac Dual vector.
5. The method according to claim 1, characterized in that The insect cells are Sf9, Sf21 or High Five cells.
6. The method according to claim 1, characterized in that During transfection, the density of insect cells was 3 × 10 6 / mL, and the amount of expression bacmid plasmid added was 0.15% (v / v).
7. The method according to claim 1, characterized in that The eluent used for nickel column affinity chromatography was: 150 mM NaCl, 25 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 250 mM imidazole pH 8.
0.
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, 40 mM NaCl; High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1M 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.