T7 rna polymerase mutant with molecular tag and use thereof
By introducing a DNA-binding protein molecule tag into the T7 RNA polymerase mutant, its thermal stability and activity were improved, solving the problem of insufficient activity of wild-type T7 RNA polymerase at high temperatures and achieving efficient RNA synthesis under high-temperature conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-11-29
- Publication Date
- 2026-05-29
AI Technical Summary
Wild-type T7 RNA polymerase has poor thermal stability under high temperature conditions, which leads to the production of double-stranded RNA during RNA synthesis, affecting its application in the medical field. Furthermore, existing mutants have insufficient activity at high temperatures.
Thermostability of T7 RNA polymerase mutants is enhanced by introducing molecular tags, particularly DNA-binding proteins or their binding fragments, such as Sso7d and lacI-like proteins, and their function is optimized by modifying the amino acid sequence.
It improves the thermostability and activity of T7 RNA polymerase, enabling it to maintain high transcriptional activity at 50°C, which is superior to commercial products and suitable for RNA synthesis applications under high-temperature conditions.
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Abstract
Description
Technical Field
[0001] This invention relates to a T7 RNA polymerase mutant with a molecular tag and its application, belonging to the field of nucleic acid tool enzyme technology. Background Technology
[0002] T7 RNA polymerase (T7RNAP) recognizes specific promoter regions and synthesizes RNA using DNA as a template. Due to its high transcriptional activity and single-subunit protein nature, it has a wide range of applications, such as highly sensitive in vitro nucleic acid detection and mRNA synthesis. In these applications, the thermostable T7RNAP has certain advantages. For example, in the NASBA isothermal amplification method, high-temperature reactions may be more favorable than room-temperature reactions (reference DOI: 10.1093 / protein / gzt040). In isothermal amplification detection using DNA polymerases such as Bst and Bsu (whose optimal temperature is generally above 50℃) combined with T7RNAP (reference DOI: 10.1016 / j.synbio.2018.02.005), the thermostable T7RNAP will be more advantageous. In mRNA synthesis, R7RNAP undergoes a significant conformational change as it transitions from transcription initiation to elongation, resulting in small abortive transcripts of 2-10 nt in length. Furthermore, T7RNAP possesses RNA-dependent RNA polymerase activity, leading to the production of small amounts of double-stranded RNA during RNA synthesis. Double-stranded RNA can trigger an immune response in the human body, which is detrimental to the application of mRNA in pharmaceuticals such as vaccines and drugs. It has been reported that in vitro transcription at 50°C can prevent the production of double-stranded RNA (Reference DOI: 10.1261 / rna.073858.119).
[0003] Wild-type T7RNAP has poor thermostability. Literature reports that T7RNAP activity decreases by 50% after treatment at 43.5℃ for 10 min (reference DOI: 10.1093 / protein / gzt040). In NEB's patent literature CN108779446B, the Tm value of wild-type T7RNAP is about 42.5℃. After mutation, the highest Tm can be increased to about 56℃. However, in actual tests, the transcription yield of commercial Hi-T7RNAP at 50℃ is much lower than that of WT at 37℃.
[0004] The activity of T7RNAP under high temperature conditions such as 50℃ still needs to be further improved. Summary of the Invention
[0005] One object of the present invention is to provide a modified T7 RNA polymerase to improve its thermal stability.
[0006] Another object of the present invention is to provide relevant applications of the modified T7 RNA polymerase.
[0007] On one hand, the present invention provides a T7 RNA polymerase mutant with a molecular tag, wherein the molecular tag includes a DNA-binding protein or a binding fragment thereof, located at the N-terminus of the amino acid sequence of the T7 RNA polymerase mutant.
[0008] According to a specific embodiment of the present invention, the molecular tag helps to further improve the thermostability of the T7 RNA polymerase mutant.
[0009] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the binding fragment of the DNA-binding protein preferably has substantially the same function as the DNA-binding protein.
[0010] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the binding fragment of the DNA-binding protein includes the DNA-binding domain of the DNA-binding protein.
[0011] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the DNA binding protein is selected from Sso7d and / or lacI-like proteins.
[0012] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the DNA binding protein or its binding fragment comprises the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5.
[0013] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the DNA binding protein or its binding fragment can be a derived fragment based on the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5, after shortening or adding one or two amino acid residues at the N-terminus.
[0014] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the DNA binding protein or its binding fragment can be a derived fragment that is truncated or has one or two amino acid residues added at the C-terminus based on the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5.
[0015] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the DNA binding protein or its binding fragment can be a derived fragment based on the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5, after shortening or adding 1 or 2 amino acid residues at the N-terminus and C-terminus respectively.
[0016] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the T7 RNA polymerase mutant is selected from:
[0017] (1) A T7 RNA polymerase mutant consisting of the amino acid sequence shown in SEQ ID No. 2;
[0018] (2) A T7 RNA polymerase mutant with essentially the same function as (1) obtained by replacing, deleting or inserting one or more amino acids based on the amino acid sequence shown in SEQ ID No.2.
[0019] According to a specific embodiment of the present invention, the T7 RNA polymerase mutant with molecular tag of the present invention has mutations at the following sites compared to the wild-type T7 RNA polymerase:
[0020] S430, F880, F849, Q744, H205, D388, L534, I109, V567, G618.
[0021] According to a specific embodiment of the present invention, the T7 RNA polymerase mutant with molecular tag of the present invention has one or more of the following mutations:
[0022] S430P, F880Y, F849I, Q744R / Q744L / Q744P, H205S, D388E, L534V, I109L, V567P, G618Q.
[0023] According to a specific embodiment of the present invention, the T7 RNA polymerase mutant with molecular tag of the present invention has the following mutations:
[0024] S430P, F880Y, F849I, Q744R or Q744L or Q744P, H205S, D388E, L534V, I109L, V567P and G618Q.
[0025] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the amino acid sequence of the T7 RNA polymerase mutant is a truncated sequence obtained by shortening one, two or three amino acid residues at the N end of the amino acid sequence shown in SEQ ID No. 2.
[0026] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the amino acid sequence of the T7 RNA polymerase mutant is a truncated sequence obtained by shortening the C-terminus of the amino acid sequence shown in SEQ ID No. 2 by 1, 2 or 3 amino acid residues.
[0027] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the amino acid sequence of the T7 RNA polymerase mutant is a truncated sequence obtained by shortening one or two amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID No. 2 and by shortening one or two amino acid residues at the C-terminus of the amino acid sequence shown in SEQ ID No. 2.
[0028] According to a specific embodiment of the present invention, in the T7 RNA polymerase mutant with molecular tag of the present invention, the linker can be a linker commonly used in CRISPR gene editing to construct Cas9 fusion proteins, such as Linker: (SGGS)2-XTEN-(SGGS)2: SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or other commonly used linkers: (G m S) n m≥1, n≥1, for example GGGGS or GGGSGGGS(GSG) n (n≤5) etc.
[0029] According to specific embodiments of the present invention, the T7 RNA polymerase mutant with molecular tags of the present invention may optionally be further labeled with tags for enhancing protein expression, tags for protein purification, and / or tags for protein detection, such as His tags.
[0030] On the other hand, the present invention also provides a nucleic acid molecule that encodes the T7 RNA polymerase mutant with molecular tag described in the present invention.
[0031] According to a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule of the present invention, which encodes the amino acid sequence portion of the T7 RNA polymerase mutant, is shown in SEQ ID No. 3.
[0032] According to a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule of the present invention, which encodes the amino acid sequence portion of the T7 RNA polymerase mutant, is shown as positions 298-2943 of SEQ ID No. 10.
[0033] According to a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule of the present invention, which encodes the amino acid sequence portion of the T7 RNA polymerase mutant, is shown as positions 298-2943 of SEQ ID No. 10.
[0034] In some specific embodiments of the present invention, the nucleic acid molecule of the present invention has a nucleotide sequence of the amino acid sequence portion encoding the molecular tag as shown in positions 22-297 of SEQ ID No. 10.
[0035] In some specific embodiments of the present invention, the nucleic acid molecule of the present invention has the nucleotide sequence shown in SEQ ID No. 10.
[0036] On the other hand, the present invention also provides a carrier containing the nucleotide sequence of the nucleic acid molecule described in the present invention.
[0037] On the other hand, the present invention also provides a host cell comprising the nucleic acid molecules or the vectors described herein.
[0038] On the other hand, the present invention also provides the application of the T7 RNA polymerase mutant with the molecular tag in in vitro transcription.
[0039] On the other hand, the present invention also provides an in vitro transcription system comprising the T7 RNA polymerase mutant with molecular tag described in the present invention.
[0040] According to a specific embodiment of the present invention, the in vitro transcription system of the present invention further comprises one or more of the conventional transcription reagents such as Tris-HCl, MgCl2, spermidine, DTT, NTPs, DNA template, RNA inhibitor, IPP, and enzyme-free water.
[0041] The T7 RNA polymerase mutant with molecular tag described in this invention has good stability and can still retain activity under high temperature conditions such as 50°C. Attached Figure Description
[0042] Figure 1 SDS-PAGE images of mutant M2 purified (nickel column and Q column).
[0043] Figure 2 SDS-PAGE images of purified mutant M15 (nickel column and Q column).
[0044] Figure 3 The temperature stability of each mutant was shown, with activity at 37°C as 100%.
[0045] Figure 4 Agarose gel electrophoresis images of IVT products of mutant M2 at different temperatures.
[0046] Figure 5 Agarose gel electrophoresis images of IVT products from mutants M15 and M16 under high temperature conditions.
[0047] Figure 6 Agarose gel electrophoresis image of IVT products of mutants M16 and NEB Hi-T7 RNApolymerase at 50℃.
[0048] Figure 7 Agarose gel electrophoresis images of the products of wild-type WT and mutant M16 used for IVT synthesis of FLUC and lacZ.
[0049] Figure 8 Agarose gel electrophoresis images of IVT products of wild-type WT and mutant M16 for self-replicating RNA.
[0050] Figure 9 SDS-PAGE images of the fusion proteins M2-1, M2-2, M17, and M18.
[0051] Figure 10 Electrophoretic images of mRNAs of different lengths prepared by IVT at 50℃ for wild-type WT and mutants M16, M17, and M18. In each lane, 1: GFP; 2: FLUC; 3: lacZ; 4: self-replicating RNA. Detailed Implementation
[0052] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.
[0053] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints may be selected.
[0054] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.
[0055] In this invention, the term "substantially equivalent" means that two molecules are substantially the same in terms of the function or effect they exhibit (such as thermal stability).
[0056] In this invention, the term "substantially" used in the context of combined or exhibited functions or effects is intended to mean that if the observed functional or effect representation is greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97%, then one molecule is said to have substantially equivalent functions to another molecule.
[0057] In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, equipment, and materials in the embodiments of this invention can be used to implement this invention.
[0058] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.
[0059] Example 1: Construction and purification of T7RNAP mutant
[0060] The T7RNAP expression gene (N-terminal 6×His tag, SEQ ID No. 6) was constructed into a modified pET28a plasmid (Novagen, with the lac promoter replacing the T7 promoter) for expressing wild-type (WT) T7RNAP (amino acid sequence shown in SEQ ID No. 1). Using conventional molecular biology methods, mutants M1, M2, M5, M9, M15, and M16 were constructed based on the above WT expression plasmid. The mutation points of each mutant compared to WT are shown in Table 1. The amino acid sequence of mutant M16 is shown in SEQ ID No. 2, and its encoding nucleic acid sequence is shown in SEQ ID No. 3.
[0061] SEQ ID No. 1:
[0062] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA
[0063] SEQ ID No.2:
[0064] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQELKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWSKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKEKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPVAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETPQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAQQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQIADQLHESQLDKMPALPAKGNLNLRDILESDYAFA
[0065] SEQ ID No.3:
[0066]
[0067] The gene expressing T7RNAP (SEQ ID No. 6):
[0068]
[0069] Table 1
[0070]
[0071] The constructed plasmid was transformed into *E. coli* BL21 and cultured overnight at 37°C. The culture was then transferred to fresh LB medium, and once the OD600 reached 0.5-0.6, IPTG (0.5 mM) was added for induction for 4 h. The bacterial cells were collected by centrifugation, sonicated, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and purified using Ni and Q columns, respectively. The Ni column was eluted with T7 buffer containing different concentrations of imidazole (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 25 / 50 / 100 / 250 / 500 mM imidazole, 5% glycerol), and the elution was analyzed using G250. The eluent was collected, and samples of each component were analyzed by SDS-PAGE. The Q column was eluted using an elution buffer (20 mM Tris-HCl pH 8.0, 5 mM β-mercaptoethanol, 10% glycerol, 100 mM, 200 mM, 400 mM, 600 mM, 1000 mM NaCl). Each fraction was collected, and after SDS-PAGE analysis, the target protein was dialyzed at 4°C into a storage buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 50% glycerol, 0.1% Triton X-100, 2 mM DTT).
[0072] SDS-PAGE gel images of the M2 purification process are shown below. Figure 1 As shown. The purification method for WT was consistent with that for other mutations. SDS-PAGE gel images of the purified M1, M2, M5, M9, M15, and M16 are shown below. Figure 2 As shown.
[0073] Example 2: Activity assay and temperature stability comparison of T7RNAP mutants
[0074] DNA templates containing the T7 promoter were synthesized using primer complementary extension. The primer sequences are as follows:
[0075] F(SEQ ID No. 7):
[0076] GTCAGATCCATAATACGACTCACTATAGGGGCGACTACGGTGAGGGTCGGG TCCAGTAGC
[0077] R (SEQ ID No. 8):
[0078] GCGACTACGGAGCCCACACTCTACTCAACAGTAGCCGAAGCTACTGGACCCGACCCTCA
[0079] Prepare the samples and reagents according to the groups listed in Table 2.
[0080] Table 2
[0081] reagents 100 μL reaction system sterile water 40.0μL Upstream primer (10 μM) 5.0μL Downstream primer (10 μM) 5.0μL Primestar Mix 50.0μL
[0082] PCR was performed under the following conditions: 98℃ for 5 min; 98℃ for 10 s, 45℃ for 5 s, 72℃ for 5 s, 30 cycles; 72℃ for 5 min. After the reaction, the sample was purified by gel extraction, and the concentration was determined and used as a template for subsequent in vitro transcription reactions.
[0083] Prepare a 50 μL reaction system: 80 mM Tris-HCl pH 7.0, 40 mM MgCl2, 3 mM spermidine, 5 mM four NTPs, 20 mM DTT, 100 μM DFHBI, add 120 nM of the recovered DNA template and T7 RNA polymerase, and incubate at 37℃-50℃ for 30 min in a microplate reader. Read the fluorescence value in real time (excitation / emission wavelengths are 440 nm and 500 nm, respectively), and use the fluorescence value to represent the activity level.
[0084] The results are as follows Figure 3 As shown, M1 and M5 were constructed based on references US20160032260A1 and WO2010016621A1, respectively, but their thermal stability was poor, both lower than WT and lower than those reported in the literature. M2 was constructed based on reference WO2013050609A1, and its thermal stability was better than WT, with its activity at 50℃ being 51% of that at 37℃. M15 was constructed based on reference CN108779446B, and its thermal stability at 43℃-47℃ was better than M2, but its activity at 50℃ rapidly decreased to 38% of that at 37℃, lower than M2. M9 was constructed by superimposing the mutant Q786L / C510R / S767G reported in reference WO2013050609A1, and the results showed a significant decrease in activity at 50℃, which differed greatly from the reported results. The mutant M16, constructed from the combination of M2 and M15 mutations, exhibits good activity at temperatures ranging from 41℃ to 50℃. Its activity at 50℃ is 77% of its activity at 37℃, superior to both M2 and M15. At 37℃, the activities of M2, M15, and M16 are 1.15 times, 1.35 times, and 1.35 times that of WT, respectively. This demonstrates that the T7RNAP mutant M16 of this invention improves both thermal stability and activity, with its activity at 50℃ exceeding that of WT at 37℃.
[0085] Example 3: In vitro transcription of T7RNAP mutant
[0086] Prepare a 20 μl in vitro transcription system: 40 mM Tris-HCl pH 8.0, 19 mM MgCl2, 1 mM spermidine, 5 mM MTT, 5 mM NTPs, 500 ng DNA template (GFP, CDS length 720 bp, full-length mRNA 984 nt after adding UTR and polyA), 20 U RNA inhibitor, 0.004 U IPP, 0.4 μg T7 RNA polymerase, and make up the remaining volume with enzyme-free water. Incubate the prepared reaction system in a PCR instrument at different temperatures for 4 h. After the reaction, add 2 μl of DNase I to every 20 μl of system and digest at 37 °C for 30 min. After transcription, take an appropriate amount of sample, add 2x RNA loading, denature at 70 °C for 5 min, and perform agarose gel electrophoresis for detection.
[0087] from Figure 4 As can be seen, for M2, the IVT production is highest at 37℃. As the temperature increases, the IVT production gradually decreases, indicating that M2 has poor thermal stability.
[0088] from Figure 5 It can be seen that for M15 and M16, their IVT yield is higher than WT at 48℃. The IVT yield of M16 is significantly higher than that of M15. M15 has abnormal IVT products at 50℃, while M16 still maintains a high IVT yield at 50℃.
[0089] like Figure 6 As shown, at 50°C, the IVT yield of M16 was significantly higher than that of the commercially available NEB thermostable T7 RNA polymerase (Hi-T7 RNA polymerase).
[0090] The M16 mutant was used for IVT synthesis with other templates, and two templates of different lengths were tried: firefly luciferase FLUC (full-length mRNA 1917nt) and lacZ (full-length RNA 3918nt after adding IRES). The results are as follows. Figure 7 As shown, for both templates, M16 produced comparable IVT yields at 37℃ and 50℃, both higher than WT at 37℃, while WT produced no IVT product at 50℃. The results of using M16 for IVT of self-replicating RNA (VEEV-based, expressing GFP, mRNA length 8356 nt) are as follows... Figure 8 As shown, M16 can be transcribed normally at 50℃, and the IVT yield and purity are much higher than those of WT at 37℃.
[0091] Example 4: Effects of DNA-binding proteins on T7RNAP activity and thermal stability
[0092] DNA-binding domains of DNA-binding proteins were fused to the N-terminus of M2. The widely used Sso7d protein (SEQ ID No. 4: ATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRG AVSEKDAPKELLQMLEKQKK) and the Thermotoga-derived lacI-like protein (SEQ ID No. 5: KRRPTINDVAKLAGVSISTVSRYLKDPSQVSEKLGER IREAIKKLGYKPNKIAQGLRTGD) reported in literature CN108779446B were named M2-1 and M2-2, respectively. The expression and purification methods were the same as for WT. The SDS-PAGE images of the purified proteins are shown below. Figure 9 As shown. Using the fluorescence test in Example 2, its activity at 50°C was 27% and 19% of its activity at 37°C, respectively, and its thermal stability was actually lower than that of M2.
[0093] Based on M16, the aforementioned Sso7d and lacI-like proteins were fused together, resulting in M17 and M18, respectively. M18 was constructed by removing the first amino acid residue from the N-terminus of M16 and then fusing it to the C-terminus of the lacI-like protein's amino acid sequence via a linker (SEQ ID No. 9: SGGSSGGSSGSETPGTSESATPESSGGSSGGS). The encoding nucleic acid sequence of M18 is shown in SEQ ID No. 10.
[0094] SEQ ID No. 10:
[0095]
[0096] WT, M16 (two batches of shake-flask expression and purification, named M16-1 and M16-2 respectively), M17, and M18 were simultaneously expressed and purified in shake flasks (the purified proteins were analyzed by SDS-PAGE as shown in the image). Figure 9 As shown in Table 3, the activity of M16 at 37°C and 50°C was detected using the fluorescence method in Example 2. The results show that the activity and thermal stability of M16 were significantly improved after being fused with DNA-binding protein.
[0097] Table 3. Comparison of the activities of mutant M16 and fusion proteins M17 and M18 at 37℃ and 50℃.
[0098] mutant Average fluorescence reading of IVT products Multiples relative to WT WT (37℃) 1756722 1 M16-1(37℃) 2269097 1.29 M16-1(50℃) 2208292 1.26 M16-2(37℃) 2896659 1.65 M16-2(50℃) 2654754 1.51 M17(37℃) 3334270 1.90 M17(50℃) 4426346 2.52 M18(37℃) 3206830 1.83 M18(50℃) 4364697 2.48
[0099] IVT testing was performed using mutants, testing the four templates used in Example 3. The results are shown in [link to example]. Figure 10 The results showed that, at 50°C, the molecularly tagged M17 and M18 of this invention produced higher IVT yields of GFP and FLUC than M16.
[0100] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. A molecularly tagged T7 RNA polymerase mutant, wherein, The molecular tag includes a DNA-binding protein or a binding fragment thereof, which is directly or via a linker attached to the N-terminus of the amino acid sequence of the T7 RNA polymerase mutant.
2. The T7 RNA polymerase mutant with a molecular tag according to claim 1, wherein, The binding fragment of a DNA-binding protein includes the DNA-binding domain of the protein.
3. The T7 RNA polymerase mutant with a molecular tag according to claim 1 or 2, wherein, The DNA-binding protein is selected from Sso7d and / or lacI-like proteins; Preferably, the DNA-binding protein or its binding fragment comprises the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No.
5.
4. The T7 RNA polymerase mutant with a molecular tag according to any one of claims 1-3, wherein, The T7 RNA polymerase mutant was selected from: (1) A T7 RNA polymerase mutant consisting of the amino acid sequence shown in SEQ ID No. 2; (2) A T7 RNA polymerase mutant with essentially the same function as (1) obtained by replacing, deleting or inserting one or more amino acids based on the amino acid sequence shown in SEQ ID No.2; Preferably, the T7 RNA polymerase mutant has mutations at the following sites compared to the wild-type T7 RNA polymerase: S430, F880, F849, Q744, H205, D388, L534, I109, V567, G618; More preferably, the T7 RNA polymerase mutant has one or more of the following mutations: S430P, F880Y, F849I, Q744R / Q744L / Q744P, H205S, D388E, L534V, I109L, V567P, G618Q.
5. A nucleic acid molecule encoding a T7 RNA polymerase mutant with a molecular tag as described in any one of claims 1-4.
6. The nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding the amino acid sequence portion of the T7 RNA polymerase mutant is as shown in SEQ ID No. 3, or as shown in positions 298-2943 of SEQ ID No.
10.
7. A vector comprising the nucleotide sequence of the nucleic acid molecule of claim 5 or 6.
8. A host cell comprising the nucleic acid molecule of claim 5 or 6 or the vector of claim 7.
9. The use of the molecularly tagged T7 RNA polymerase mutant according to any one of claims 1-4 in in vitro transcription.
10. An in vitro transcription system comprising the T7 RNA polymerase mutant with a molecular tag as described in any one of claims 1-4.