MRNA (messenger ribonucleic acid) combined capping enzyme, preparation method of mRNA combined capping enzyme and mRNA capping kit
By combining TPase-GTase and MTase domains to express proteins, the problem of low expression and purification efficiency of existing mRNA capping enzymes is solved, efficient capping and thermal stability are achieved, and production costs are reduced.
Patent Information
- Application Number
- CN202310983687.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-07
- Publication Date
- 2025-02-14
AI Technical Summary
Existing mRNA capping enzymes have low expression and purification efficiency, and are difficult to stabilize the control of the synergistic effects and expression ratios of large subunit D1 and small subunit D12, resulting in low capping efficiency and stability.
A combination of TPase-GTase domain-expressing protein and MTase domain-expressing protein was used to efficiently express the protein through recombinant vectors, and the molar ratio of the two domains was controlled to enhance the activity and stability of the capping enzyme.
The efficient expression and purification of mRNA capping enzymes is achieved, which significantly improves capping efficiency and thermal stability and reduces production costs.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological enzymes, and in particular to an mRNA combined capping enzyme and a preparation method thereof and an mRNA capping kit. Background Art
[0002] The mRNA of eukaryotic organisms and many viruses contains an inverted 7-methylguanosine (m7G) cap, which is attached to the nucleotide at the 5' end through a 5'-5' triphosphate. The formation of the cap involves the following three enzymatic reactions: 1) RNA triphosphatase (TPase) hydrolyzes the γ-phosphate of the 5' end mRNA to produce diphosphate RNA (ppNp-RNA); 2) RNA guanylyltransferase (GTase) transfers GMP from GTP to the 5' end of diphosphate RNA to form GpppNp-RNA cap; 3) cap-specific RNA (guanine-N 7 ) methyltransferase (MTase) adds a methyl group to the guanine of the GpppNp-RNA cap to form m 7 GpppNp-RNA cap 0 structure. The cap has multiple biological functions, including: in the cell nucleus, it can support the splicing and excretion of mRNA from the nucleus; prevent 5' to 3' exonuclease-mediated degradation and promote the stability of mRNA; bind to eukaryotic translation initiation factor 4E (eIF4E) to form an initiation complex and recruit ribosomes to mRNA, thereby promoting protein translation; help cells distinguish between their own RNA and foreign RNA, and uncapped RNA is recognized by cell recognition receptors, subsequently inducing the expression of a series of innate immune response cytokines and antiviral effectors. Because it is widely used in life sciences, biotechnology and medicine, the raw enzymes used for mRNA capping are also particularly important.
[0003] The existing capping enzymes are mainly capping enzymes derived from vaccinia virus, which contain a large subunit D1 and a small subunit D12. D1 is a multifunctional 844 amino acid polypeptide, composed of N-terminal TPase, central GTase and C-terminal MTase domains. The MTase activity of D1 itself is very low, and D12 stimulation is required to promote activity enhancement. The expression and purification efficiency of vaccinia virus capping enzymes is relatively low, and it is difficult to stably control the synergistic effect and expression ratio of large subunit D1 and small subunit D12 through existing processes. In addition to vaccinia virus capping enzymes, capping enzymes from other species also encode separate components of TPase, GTase and MTase, or encode multifunctional capping enzymes composed of cis-linked catalytic modules, but the capping efficiency is only 50%-80% of that of vaccinia virus capping enzymes, and the stability is low. Therefore, the research on mRNA capping enzymes that are easy to express and purify, have high capping efficiency and good thermal stability is of great significance to the development of life sciences. Summary of the invention
[0004] In order to overcome the above-mentioned defects of existing mRNA capping enzymes, the present invention proposes an mRNA combination capping enzyme and a preparation method thereof and an mRNA capping kit, wherein the mRNA combination capping enzyme can achieve the advantages of easy expression and purification, high mRNA capping efficiency and good thermal stability.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] An mRNA combination capping enzyme comprises a TPase-GTase domain expression protein and a MTase domain expression protein, wherein the molar ratio of the MTase domain expression protein to the TPase-GTase domain expression protein is ≥1.
[0007] Preferably, the mRNA combination capping enzyme comprises a TPase-GTase domain expression protein and a MTase domain expression protein in a molar ratio of 1:(1-2).
[0008] Preferably, the molar ratio of the TPase-GTase domain expression protein to the MTase domain expression protein is 1:(1.5-2).
[0009] Preferably, the TPase-GTase domain expression protein includes at least one of an expression protein having a sequence identity of more than 80% with the TPase-GTase domain expression protein of the vaccinia virus capping enzyme as shown in the amino acid sequence SEQ ID NO:1, an expression protein having a sequence identity of more than 80% with the TPase-GTase domain expression protein of the amoeba virus capping enzyme as shown in the amino acid sequence SEQ ID NO:2, and an expression protein having a sequence identity of more than 80% with the TPase-GTase domain expression protein of the African swine fever virus capping enzyme as shown in the amino acid sequence SEQ ID NO:3;
[0010] The MTase domain expression protein includes at least one of an expression protein whose sequence identity is more than 80% with the African swine fever virus capping enzyme MTase domain expression protein as shown in the amino acid sequence SEQ ID NO:4, and an expression protein whose sequence identity is more than 80% with the amoeba virus capping enzyme MTase domain expression protein as shown in the amino acid sequence SEQ ID NO:5.
[0011] Preferably, the TPase-GTase domain expression protein includes at least one of the TPase-GTase domain expression protein of vaccinia virus capping enzyme, the TPase-GTase domain expression protein of amoeba virus capping enzyme, and the TPase-GTase domain expression protein of African swine fever virus capping enzyme;
[0012] The MTase domain expression protein includes at least one of an African swine fever virus capping enzyme MTase domain expression protein and an amoeba virus capping enzyme MTase domain expression protein.
[0013] The capping enzyme composed of the TPase-GTase domain expression protein and the MTase domain expression protein in the above molar ratio has significant advantages and improvements in capping efficiency compared to the existing vaccinia virus capping enzyme and capping enzymes from other sources.
[0014] The mRNA combination capping enzyme in the present invention includes a TPase domain, a GTase domain and a MTase domain; the three domains may be capping enzymes from the same species; or they may be capping enzymes from different species. The TPase-GTase domain expression protein of the mRNA combination capping enzyme may form a fusion protein with the MTase domain; or it may be two independent proteins. When it is a fusion protein, the molar ratio of the two is regulated by controlling the fragment ratio of the TPase-GTase domain and the MTase domain. When the two are independent proteins, the molar ratio of the two can be controlled by their respective addition amounts; compared with the lower expression and purification efficiency of the existing vaccinia virus capping enzyme, when the two are independent proteins, the expression and purification efficiency of the two proteins is higher, the total yield of the mRNA combination capping enzyme is improved, and the production quality of the mRNA combination capping enzyme is more controllable and the production cost is further reduced. The present invention has found through a large number of experiments that in the mRNA capping reaction, the reaction mediated by the MTase domain expression protein has the greatest impact on the yield of the capping product. By increasing the molar amount of the MTase domain expression protein or using a more active MTase domain expression protein, the yield of the capping product can be improved. The experimental results show that the mRNA combination capping of the present invention retains the capping enzyme activity and has the improvement of one or more of the following characteristics: capping enzyme activity, thermal stability and capping efficiency, etc.
[0015] The TPase domain, GTase domain and MTase domain may include but are not limited to the TPase domain, GTase domain and MTase domain of the naturally occurring capping enzyme, and may also include mutant, recombinant, fused or otherwise engineered, chemically modified domains of these domains.
[0016] The TPase-GTase domain expression protein of the vaccinia virus capping enzyme in the present invention has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 1; preferably, an amino acid sequence with an identity of more than 90%, more preferably more than 95%, and even more preferably more than 98% with the amino acid sequence shown in SEQ ID NO: 1 is feasible.
[0017] The TPase-GTase domain expression protein of the amoeba virus capping enzyme in the present invention has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 2; preferably, an amino acid sequence with an identity of more than 90%, more preferably more than 95%, and more preferably more than 98% with the amino acid sequence shown in SEQ ID NO: 2 is feasible.
[0018] The TPase-GTase domain expression protein of the African swine fever virus capping enzyme in the present invention has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 3; preferably, an amino acid sequence with an identity of more than 90%, further preferably more than 95%, and further preferably more than 98% with the amino acid sequence shown in SEQ ID NO: 3 is feasible.
[0019] The MTase domain expression protein of the African swine fever virus capping enzyme in the present invention has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO:4; preferably, an amino acid sequence with an identity of more than 90%, further preferably more than 95%, and further preferably more than 98% with the amino acid sequence shown in SEQ ID NO:4 is feasible.
[0020] The MTase domain expression protein of the amoeba virus capping enzyme in the present invention has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 5. Preferably, an amino acid sequence with an identity of more than 90%, more preferably more than 95%, and more preferably more than 98% with the amino acid sequence shown in SEQ ID NO: 5 is feasible.
[0021] The present invention also provides a method for preparing the mRNA combination capping enzyme, which comprises the following steps:
[0022] a, recombining the gene sequences of the TPase-GTase domain expression protein and the MTase domain expression protein into expression vectors respectively, to obtain a recombinant expression vector of the TPase-GTase domain expression protein and a recombinant expression vector of the MTase domain expression protein;
[0023] b. Transfecting the recombinant expression vector of the TPase-GTase domain expression protein and the recombinant expression vector of the MTase domain expression protein into expression strains respectively to obtain the expression strain of the TPase-GTase domain expression protein and the expression strain of the MTase domain expression protein;
[0024] c. amplifying and induced expression of the expression strain of the TPase-GTase domain protein and the expression strain of the MTase domain protein to obtain expression bacterial solution; centrifuging the expression bacterial solution and collecting bacterial precipitates, adding lysis buffer to the bacterial precipitates to resuspend the bacteria, then ultrasonically disrupting and centrifuging, collecting supernatants, and obtaining expression supernatants of the TPase-GTase domain protein and the MTase domain protein;
[0025] d. Affinity chromatography is performed on the expression supernatant of the TPase-GTase domain expression protein and the expression supernatant of the MTase domain expression protein to obtain the TPase-GTase domain expression protein and the MTase domain expression protein; the TPase-GTase domain expression protein and the MTase domain expression protein are mixed to obtain the mRNA combination capping enzyme. Preferably, in step a, the expression vector is pET28a(+) or pETDuet-1, purchased from YEASEN and Novagen, respectively.
[0026] Preferably, in step b, the expression strain is Escherichia coli Rosetta (DE3) pLysS strain.
[0027] Preferably, in step c, the lysis buffer comprises 48-52 mM Tris, 480-520 mM NaCl, 8-12 v% Glycerol and 18-22 mM Imidazole, and the pH of the lysis buffer is 7.8-8.2. More preferably, the lysis buffer comprises 50 mM Tris, 500 mM NaCl, 10 v% Glycerol (glycerol) and 20 mM Imidazole (imidazole), and the pH of the lysis buffer is 8.0.
[0028] Preferably, in step c, the inducing agent used for inducing expression is IPTG, and the final concentration of IPTG after addition is 0.08-0.12 mM. More preferably, the final concentration of IPTG after addition is 0.10 mM.
[0029] The present invention also provides an mRNA capping kit, which comprises the mRNA combined capping enzyme, a capping buffer, guanosine triphosphate and S-adenosylmethionine.
[0030] Preferably, the capping buffer comprises 50 mM Tris HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, and has a pH of 8.0.
[0031] The present invention also provides a method for RNA capping using the mRNA capping kit, specifically comprising:
[0032] S-adenosylmethionine and guanosine triphosphate are added to the capping buffer, and then the mRNA combination capping enzyme and the uncapped target RNA are added to obtain an RNA capping system, which is incubated at 35-55° C. to complete the capping of the target RNA.
[0033] Preferably, the concentration of S-adenosylmethionine in the RNA capping system is 1.8-2.2 mM.
[0034] Preferably, the concentration of the guanosine triphosphate in the RNA capping system is 0.3-0.7 mM.
[0035] Preferably, the added amount of the combined capping enzyme is 0.01-0.05 pmol.
[0036] Preferably, the incubation time is 50-70 min.
[0037] Compared with the prior art, the mRNA combination capping enzyme provided by the present invention combines the TPase-GTase domain expression protein and the MTase domain expression protein from a specific source for mRNA capping reaction, and its mRNA capping efficiency is significantly improved compared to the existing capping enzyme derived from vaccinia virus. At the same time, the TPase-GTase domain expression protein and the MTase domain expression protein in the mRNA combination capping enzyme provided by the present invention can be efficiently expressed by means of a recombinant vector, and the obtained expression protein is easy to purify, significantly reducing the production cost of the mRNA capping enzyme. In addition, the mRNA combination capping enzyme provided by the present invention is more conducive to controlling the ratio of TPase, GTase and MTase in the capping enzyme through the separate expression of each domain, thereby improving the activity of the capping enzyme, and the combined capping enzyme has higher thermal stability than the existing capping enzyme derived from vaccinia virus.
[0038] The mRNA capping enzyme combination provided by the present invention is used in an mRNA capping kit or other mRNA capping products, which can effectively promote the improvement and development of mRNA capping technology in biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 is the recombinant expression vector map of VCE12 in Example 1;
[0040] Figure 2 is the recombinant expression vector map of FCE12 in Example 1;
[0041] Figure 3 This is the recombinant expression vector map of ACE12 in Example 1;
[0042] Figure 4 is the recombinant expression vector map of FCE3 in Example 1;
[0043] Figure 5 This is the recombinant expression vector map of ACE3 in Example 1;
[0044] Figure 6 This is the SDS-PAGE electrophoresis detection diagram of VCE12 protein expression in Example 1;
[0045] Figure 7 This is the SDS-PAGE electrophoresis detection diagram of FCE12 protein expression in Example 1;
[0046] Figure 8 This is the SDS-PAGE electrophoresis detection diagram of ACE12 protein expression in Example 1;
[0047] Fig. 9 This is the SDS-PAGE electrophoresis detection diagram of FCE3 protein expression in Example 1;
[0048] Fig.10 This is the SDS-PAGE electrophoresis detection diagram of ACE3 protein expression in Example 1;
[0049] Fig.11 The HPLC spectrum of GTP and GDP in the TPase activity detection in Example 2;
[0050] Fig.12 This is a bar graph of TPase enzyme activity detection of VCE12, FCE12, ACE12, vaccinia virus capping enzyme, and amoeba virus capping enzyme in Example 2;
[0051] Fig.13 HPLC spectrum of GpppA, m7GpppA, SAM and SAH in the MTase activity detection in Example 2;
[0052] Fig.14 This is a bar graph of MTase enzyme activity detection of ACE3, FCE3, vaccinia virus capping enzyme and amoeba virus capping enzyme in Example 2;
[0053] Fig.15This is the LC-MS spectrum of the 23nt RNA before capping in Example 2;
[0054] Fig.16 This is the LC-MS spectrum of the 23nt RNA after capping in Example 2;
[0055] Fig.17 This is a bar graph of GTase enzyme activity detection of FCE12, VCE12, ACE12, vaccinia virus capping enzyme and amoeba virus capping enzyme in Example 2;
[0056] Fig.18 This is the LC-MS spectrum of the capped 4233nt RNA in Example 4;
[0057] Fig.19 It is a bar graph showing the capping efficiency of the capping enzymes in the 9 mRNA capping kits tested in Example 4;
[0058] Fig. 20 The capping efficiency bar graph is of the eight combined capping enzymes tested in Example 5, as well as the vaccinia virus capping enzyme and the amoeba virus capping enzyme. DETAILED DESCRIPTION
[0059] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
[0060] In the embodiments, VCE represents vaccinia virus capping enzyme, FCE represents amoeba virus capping enzyme, ACE represents African swine fever virus capping enzyme, VCE12 represents TPase-GTase domain expression protein of vaccinia virus capping enzyme, FCE12 represents TPase-GTase domain expression protein of amoeba virus capping enzyme, ACE12 represents TPase-GTase domain expression protein of African swine fever virus capping enzyme, ACE3 represents African swine fever virus capping enzyme MTase domain expression protein, and FCE3 represents amoeba virus capping enzyme MTase domain expression protein.
[0061] Example 1
[0062] Five proteins, VCE12, FCE12, ACE12, FCE3 and ACE3, were prepared respectively, and the preparation methods were as follows:
[0063] 1) The gene expression sequence of VCE12 protein was recombined into pETDuet-1; the gene expression sequences of FCE12, ACE12, FCE3, and ACE3 proteins were recombined into pET28a(+) vector respectively; and the recombinant expression vectors of VCE12, FCE12, ACE12, FCE3, and ACE3 were obtained;
[0064] The recombinant expression vector map of VCE12 is as follows Figure 1 As shown, the recombinant expression vector map of FCE12 is as follows Figure 2 As shown, the recombinant expression vector of ACE12 is as follows Figure 3 As shown, the recombinant expression vector of FCE3 is as follows Figure 4 As shown, the recombinant expression vector of ACE3 is as follows Figure 5 As shown;
[0065] 2) The obtained recombinant expression vectors of VCE12, FCE12 and ACE12, FCE3 and ACE3 were transfected into the expression strain E. coli Rosetta (DE3) pLysS, respectively, to obtain five expression strains expressing VCE12, FCE12, ACE12, FCE3 and ACE3, respectively;
[0066] 3) The VCE12 expression strain was inoculated into 30 mL of LB medium containing 50 μg / mL ampicillin for shaking culture, and the four expression strains of FCE12, ACE12, FCE3 and ACE3 were respectively inoculated into 30 mL of LB medium containing 50 μg / mL kanamycin sulfate for shaking culture. The culture temperature of the above five single strains was 37° C., the time was 12 h, and the shaking speed was 200 r / min to obtain seed liquids of VCE12, FCE12, ACE12, FCE3 and ACE3; wherein the formula of the LB medium is: 10 g of peptone, 5 g of yeast powder and 10 g of sodium chloride per liter of liquid culture medium;
[0067] The VCE12 seed solution was inoculated into a 2YT medium (containing 16 g of peptone, 10 g of yeast powder and 5 g of sodium chloride per liter) containing 50 μg / mL ampicillin at a volume ratio of 2:100 for shaking culture. The FCE12, ACE12, FCE3 and ACE3 seed solutions were inoculated into a 2YT medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 2:100 for shaking culture. The culture temperature was 37°C and the shaking speed was 200 r / min. When the OD of the culture solution reached 600 When the p-value reached 0.8, IPTG was added to make the final concentration of IPTG 0.1 mM, and the expression was induced at 16°C for 18 h to obtain the expression bacterial solutions of VCE12, FCE12, ACE12, FCE3 and ACE3 respectively;
[0068] 1 mL of the above-mentioned VCE12, FCE12, ACE12, FCE3 and ACE3 expression bacterial solution was taken respectively, the bacterial cells were collected by centrifugation, and the bacterial cells were ultrasonically disrupted to obtain the supernatant and bacterial precipitate. Then, the content of the target protein in the supernatant and bacterial precipitate was detected by SDS-PAGE electrophoresis. The detection results were as follows: Figure 6-10As shown, it can be seen that the above five expression strains successfully expressed VCE12, FCE12, ACE12, FCE3 and ACE3 with high expression levels, and obvious protein bands were present in the supernatant, confirming their soluble expression.
[0069] 4) The above-mentioned VCE12, FCE12, ACE12, FCE3 and ACE3 expression bacterial solutions were centrifuged at 4°C and 5500g for 20 min, the supernatant was removed, and lysis buffer was added to the bacterial pellet. The amount of lysis buffer added was 6 mL of lysis buffer per gram of bacterial pellet. The cells were ultrasonically treated at a power of 640 W for 3s and then stopped for 5s for 30 min. The cells were then centrifuged at 4°C and 12000g for 30 min. The supernatant was collected to obtain the expression supernatants of VCE12, FCE12, ACE12, FCE3 and ACE3, respectively.
[0070] The lysis buffer includes 50 mM Tris, 500 mM NaCl, 10 v% Glycerol, and 20 mM Imidazole, and the pH of the lysis buffer is 8.0;
[0071] 5) Purifying the expression supernatants of VCE12, FCE12, ACE12, FCE3 and ACE3 by nickel ion affinity chromatography to obtain VCE12, FCE12, ACE12, FCE3 and ACE3;
[0072] Among them, the amino acid sequences of the five expressed proteins VCE12, FCE12, ACE12, ACE3 and FCE3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;
[0073] The binding buffer used for nickel ion affinity chromatography is buffer A, which includes 50 mM Tris, 500 mM NaCl, 10 v% Glycerol, 20 mM Imidazole, and has a pH of 8.0;
[0074] The elution buffer used for nickel ion affinity chromatography is buffer B, which includes 50 mM Tris, 800 mM NaCl, 10 v% Glycerol, 20 mM Imidazole, and has a pH of 8.0;
[0075] The elution buffer used for nickel ion affinity chromatography is buffer C, which includes 50 mM Tris, 800 mM NaCl, 10 v% Glycerol, 500 mM Imidazole, and pH 8.0;
[0076] The chromatography column used for nickel ion affinity chromatography was SDL-100, 5 mL, purchased from YEASEN;
[0077] The specific operation method of nickel ion affinity chromatography is as follows: the nickel ion affinity chromatography column is first washed with ultrapure water, then the column is balanced with buffer A, and then the expression supernatant is loaded; after the loading is completed, the column is first rinsed with buffer B, and then a 0-100% linear elution is performed with buffer C, and the eluted components are collected to obtain the expressed protein.
[0078] Example 2
[0079] Enzyme activity detection
[0080] TPase activity assay
[0081] Take five reaction tubes, add 50 μL of 1× RNA capping buffer (50 mM Tris HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0), add 0.01 pmol of VCE12, FCE12, ACE12 obtained in Example 1, as well as vaccinia virus capping enzyme and amoeba virus capping enzyme, respectively, at 37°C, add GTP, the final concentration of GTP is 0.5 mM, and incubate for 60 min.
[0082] High performance liquid chromatography (HPLC) was used to analyze the TPase activity of VCE12, FCE12, ACE12, vaccinia virus capping enzyme, and amoeba virus capping enzyme. The peak areas corresponding to GTP and GDP were quantified and used to calculate the relative activity of TPase at the end of the 60-min capping reaction. The HPLC chromatogram obtained after the TPase reaction is shown in Figure 2. Fig.11 The TPase activity of VCE12, FCE12, ACE12, vaccinia virus capping enzyme and amoeba virus capping enzyme is shown in Fig.12 The test results showed that VCE12, FCE12, ACE12, vaccinia virus capping enzyme and amoeba virus capping enzyme all had TPase activity.
[0083] MTase activity assay
[0084] Take four reaction tubes and add 20 μL of 1× RNA capping buffer (50 mM Tris HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, pH 8.0), and SAM to a final concentration of 2 mM. 0.01 pmol of ACE3, FCE3, vaccinia virus capping enzyme and amoeba virus capping enzyme obtained in Example 1 were added at 37° C., and then 0.5 mM GpppA was added and incubated for 60 min.
[0085] High performance liquid chromatography (HPLC) was used to analyze the MTase activity of ACE3, FCE3, vaccinia virus capping enzyme, and amoeba virus capping enzyme. The peak areas corresponding to GpppA and m7GpppA were quantified and used to calculate the relative activity of MTase at the end of the 60-min capping reaction. The HPLC profile after MTase reaction is shown in Figure 2. Fig.13 The MTase activity of ACE3, FCE3, vaccinia virus capping enzyme and amoeba virus capping enzyme is shown in Fig.14 As shown, the test results showed that ACE3, FCE3, vaccinia virus capping enzyme and amoeba virus capping enzyme all had certain MTase enzyme activity.
[0086] GTase activity assay
[0087] Take five reaction tubes, add 20 μL of 1× RNA capping buffer (50 mM Tris HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0), SAM and GTP, so that the final concentration of SAM is 2 mM, and the final concentration of GTP is 0.5 mM, add 0.01 pmol of FCE12, VCE12, ACE12 obtained in Example 1, as well as vaccinia virus capping enzyme and amoeba virus capping enzyme at 37°C, and then add 10 μg of 23nt uncapped RNA obtained by in vitro transcription (sequence as shown in SEQ ID NO: 6) and incubate for 60 min.
[0088] LC-MS was used to analyze the GTase activity of FCE12, VCE12, ACE12, vaccinia virus capping enzymes, and amoeba virus capping enzymes. The peak areas corresponding to capped RNA and uncapped RNA were quantified and used to calculate the relative activity of GTase at the end of the 60-min capping reaction. The LC-MS spectra of 23nt RNA before and after capping are shown in Figure 2. Figure 15-16 The GTase activity of FCE12, VCE12, ACE12, vaccinia virus capping enzyme and amoeba virus capping enzyme is shown in Fig.17 As shown, FCE12, VCE12, ACE12, vaccinia virus capping enzyme and amoeba virus capping enzyme all have certain GTase activity.
[0089] Example 3
[0090] Preparation of mRNA combination capping enzymes
[0091] VCE12, FCE12 or ACE12 obtained in Example 1 and FCE3 or ACE3 obtained in Example 1 were mixed in a specific ratio to obtain 8 mRNA combination capping enzymes, and the specific ratios are shown in Table 1.
[0092] Table 1 mRNA combination capping enzyme and protein composition
[0093] mRNA combinatorial capping enzyme Protein composition and molar ratio mRNA combinatorial capping enzyme-1 VCE12:ACE3=1:1 mRNA combinatorial capping enzyme-2 FCE12:ACE3=1:1 mRNA combined capping enzyme-3 VCE12:FCE3=1:1 mRNA combinatorial capping enzyme-4 VCE12:FCE3=1:1.5 mRNA combined capping enzyme-5 VCE12:FCE3=1:2 mRNA combined capping enzyme-6 FCE12:FCE3=1:1 mRNA combinatorial capping enzyme-7 FCE12:FCE3=1:1.5 mRNA combined capping enzyme-8 FCE12:FCE3=1:2
[0094] Example 4
[0095] The vaccinia virus capping enzyme (VCE) and the 8 mRNA combination capping enzymes obtained in Example 3 were used to prepare mRNA capping kits, respectively, to obtain 9 mRNA capping kits.
[0096] The mRNA capping kit includes: one of the above 9 capping enzymes, a capping buffer (composition: 50mM Tris-HCl, 5mM KCl, 1mM MgCl 2 and 1 mM DTT, pH 8.0), guanosine triphosphate (GTP), and S-adenosylmethionine (SAM).
[0097] The RNA capping efficiency of the capping enzymes in the above 9 mRNA capping kits was determined as follows:
[0098] 1) RNA capping
[0099] Take 9 reaction tubes, add 20 μL of the above-mentioned capping buffer to each tube, then add SAM and GTP to make the final concentration of SAM 2 mM and the final concentration of GTP 0.5 mM, place at 37°C, and at the same time take the 9 capping enzymes in the above-mentioned 9 mRNA capping kits respectively, the total enzyme molar amount in each reaction tube is 0.02 pmol, and then add 10 μg of 4233nt uncapped RNA obtained by in vitro transcription (sequence is SEQ ID NO: 7), incubate for 60 minutes, and obtain capped RNA.
[0100] 2) DNAzyme treatment of capped RNA
[0101] DNAzyme (sequence as SEQ ID NO: 8) was added to the capped RNA to obtain a 27 μL reaction system, which contained 0.8 μM capped RNA and 10 μM specific DNAzyme, which was pre-denatured at an annealing temperature of 85°C for 5 min, then maintained at 85°C for 10 sec, and then gradually cooled by -0.1°C per cycle, and after 600 cycles, the temperature was reduced to 25°C and maintained for 5 min, and then an additional 3 μL of 10xDNAzyme Reaction Buffer (containing 10 mM Tris and 50 mM MgCl 2 ), react at 37°C for 2h to obtain the RNA fragments after enzyme digestion.
[0102] 3) LC-MS analysis of RNA capping efficiency
[0103] The RNA capping efficiency was analyzed using an Agilent time-of-flight LC-MS system 6230B, with mobile phase A (10 mM HFIP + 20 mM HA in water) and mobile phase B (80% ACN). The peak areas corresponding to the capped RNA and the uncapped RNA were quantified and used to calculate the capping efficiency at the end of the 60-min capping reaction in step 1). The LC-MS spectrum of the 4233nt RNA capping is shown in Figure 2. Fig.18 shown.
[0104] The capping efficiency of the capping enzymes in the above 9 mRNA capping kits was analyzed and compared by this method. The results are as follows: Fig.19 shown.
[0105] The results show that the eight combined capping enzymes tested in this example all have improved capping efficiency compared to the vaccinia virus capping enzyme (VCE), especially the combined capping enzyme composed of FCE12 and FCE3 (the molar ratio of FCE12:FCE3 is 1:1-2) has a significantly higher capping efficiency than the complete vaccinia virus capping enzyme (VCE). The capping efficiency of the combined capping enzyme of FCE12 and FCE3 with a molar ratio of FCE12:FCE3 of 1:1.5-2 is higher.
[0106] Example 5
[0107] Thermal stability test
[0108] 1) RNA capping
[0109] The mRNA capping kits were prepared using vaccinia virus capping enzyme (VCE), amoeba virus capping enzyme (FCE) and the 8 mRNA combination capping enzymes obtained in Example 3, respectively, to obtain 10 mRNA capping kits.
[0110] Take 10 reaction tubes and add 20 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl 2 and 1 mM DTT, pH 8.0), then SAM and GTP were added respectively to make the final concentration of SAM 2 mM, the final concentration of GTP 0.5 mM, 10 capping enzymes in 10 mRNA capping kits were added to 10 reaction tubes at 50°C, the total enzyme molar amount in each reaction tube was 0.05 pmol, and then 10 μg of 4233 nt uncapped RNA obtained by in vitro transcription (sequence is SEQ ID NO: 7) was added respectively, and incubated for 60 min to obtain capped RNA.
[0111] 2) DNAzyme treatment of capped RNA
[0112] DNAzyme (sequence such as SEQ ID NO: 8) was added to the capped RNA to obtain a 27 μL reaction system, which contained 0.8 μM capped RNA and 10 μM specific DNAzyme, which was pre-denatured at an annealing temperature of 85°C for 5 min, then maintained at 85°C for 10 sec, followed by gradient cooling, with each cycle cooling by -0.1°C. After 600 cycles, the temperature was reduced to 25°C and maintained for 5 min, and then 3 μL of 10xDNAzyme Reaction Buffer (containing 10 mM Tris and 50 mM MgCl2) was additionally added, and the reaction was carried out at 37°C for 2 h to obtain RNA fragments after enzyme cleavage.
[0113] 3) LC-MS analysis of RNA capping efficiency
[0114] The RNA capping efficiency was analyzed using an Agilent time-of-flight LC / MS system 6230B, with mobile phase A (10 mM HFIP + 20 mM HA in water) and mobile phase B (80% ACN). The peak areas corresponding to the capped RNA and the uncapped RNA were quantified and used to calculate the capping efficiency at the end of the 60 min capping reaction in step 1). The calculation results are shown in Fig. 20 shown.
[0115] The higher the capping efficiency, the better its thermal stability, indicating that the thermal stability of the above eight combined capping enzymes is higher than that of the complete vaccinia virus capping enzyme and the amoeba virus capping enzyme.
[0116] It can be seen that the combined capping enzyme of the present invention is more conducive to controlling the ratio of TPase, GTase and MTase compared with the vaccinia virus capping enzyme, can achieve higher enzyme activity, and has both higher capping efficiency and thermal stability. At the same time, the combined capping enzyme of the present invention can be highly expressed, easy to purify, and has low production cost, which is of great significance to the development of the mRNA capping field.
Claims
1. An mRNA combined capping enzyme, characterized in that: The protein comprises a TPase-GTase domain expression protein and a MTase domain expression protein, wherein the molar ratio of the MTase domain expression protein to the TPase-GTase domain expression protein is ≥1.
2. The mRNA capping enzyme combination according to claim 1, characterized in that: The molar ratio of the TPase-GTase domain expression protein to the MTase domain expression protein is 1:(1-2).
3. The mRNA capping enzyme combination according to claim 2, characterized in that: The molar ratio of the TPase-GTase domain expression protein to the MTase domain expression protein is 1:(1.5-2).
4. The mRNA capping enzyme combination according to any one of claims 1 to 3, characterized in that: The TPase-GTase domain expression protein includes at least one of a TPase-GTase domain expression protein of a vaccinia virus capping enzyme, a TPase-GTase domain expression protein of an amoeba virus capping enzyme, and a TPase-GTase domain expression protein of an African swine fever virus capping enzyme; The MTase domain expression protein includes at least one of an African swine fever virus capping enzyme MTase domain expression protein and an amoeba virus capping enzyme MTase domain expression protein.
5. The mRNA capping enzyme combination according to any one of claims 1 to 3, characterized in that: The TPase-GTase domain expression protein of the vaccinia virus capping enzyme has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 1; or The TPase-GTase domain expression protein of the amoeba virus capping enzyme has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 2; or The TPase-GTase domain expression protein of the African swine fever virus capping enzyme has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 3; or The MTase domain expression protein of the African swine fever virus capping enzyme has an amino acid sequence with an identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 4; or The MTase domain expression protein of the amoeba virus capping enzyme has an amino acid sequence with a consistency of more than 80% with the amino acid sequence shown in SEQ ID NO:
5.
6. The method for preparing the mRNA combination capping enzyme according to any one of claims 1 to 5, characterized in that: The following steps are involved: a, recombining the gene sequences of the TPase-GTase domain expression protein and the MTase domain expression protein into expression vectors respectively, to obtain a recombinant expression vector of the TPase-GTase domain expression protein and a recombinant expression vector of the MTase domain expression protein; b. Transfecting the recombinant expression vector of the TPase-GTase domain expression protein and the recombinant expression vector of the MTase domain expression protein into expression strains respectively to obtain the expression strain of the TPase-GTase domain expression protein and the expression strain of the MTase domain expression protein; c. amplifying and induced expression of the expression strain of the TPase-GTase domain protein and the expression strain of the MTase domain protein to obtain expression bacterial solution; centrifuging the expression bacterial solution and collecting bacterial precipitates, adding lysis buffer to the bacterial precipitates to resuspend the bacteria, then ultrasonically disrupting and centrifuging, collecting supernatants, and obtaining expression supernatants of the TPase-GTase domain protein and the MTase domain protein; d. Subjecting the expression supernatant of the TPase-GTase domain expression protein and the expression supernatant of the MTase domain expression protein to affinity chromatography respectively to obtain the TPase-GTase domain expression protein and the MTase domain expression protein; mixing the TPase-GTase domain expression protein and the MTase domain expression protein to obtain the mRNA combination capping enzyme.
7. The method for preparing the mRNA combination capping enzyme according to claim 6, characterized in that: In step c, the lysis buffer comprises 48-52 mM Tris, 480-520 mM NaCl, 8-12 v% Glycerol and 18-22 mM Imidazole, and the pH of the lysis buffer is 7.8-8.2; And / or in step c, the inducing agent used to induce expression is IPTG, and the final concentration of IPTG after addition is 0.08-0.12 mM.
8. An mRNA capping kit, characterized in that: The method comprises the mRNA combination capping enzyme according to any one of claims 1 to 5, a capping buffer, guanosine triphosphate and S-adenosylmethionine.
9. A method for RNA capping using the mRNA combined capping enzyme according to claim 1 or the mRNA capping kit according to claim 8, characterized in that: S-adenosylmethionine and guanosine triphosphate are added to the capping buffer, and then the mRNA combination capping enzyme and the uncapped target RNA are added to obtain an RNA capping system, which is incubated at 35-55° C. to complete the capping of the target RNA.
10. The method for RNA capping according to claim 9, characterized in that: The concentration of S-adenosylmethionine in the RNA capping system is 1.8-2.2 mM; and / or the concentration of the guanosine triphosphate in the RNA capping system is 0.3-0.7 mM; and / or the concentration of the mRNA combination capping enzyme in the RNA capping system is 0.01-0.05 pmol; And / or the incubation time is 50-70 min.