A T7 RNA polymerase mutant and its application

Abstract

The present invention relates to a T7 RNA polymerase mutant and its application. Specifically, the present invention discloses a T7 RNA polymerase mutant, which has at least one amino acid mutation at a site corresponding to the wild-type T7 RNA polymerase selected from the group consisting of: position 44, position 45, position 46, position 172, position 389, position 425, position 441, position 632, position 643, position 754, and position 789; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO:1, and the mutation results in a significant reduction in the by-product dsRNA produced during the process of the enzyme synthesizing mRNA, and at the same time reduces the production of RNA aggregates.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a T7 RNA polymerase mutant and its applications. Background Art

[0002] T7 RNA polymerase (T7 RNAP) was first discovered in Escherichia coli infected with bacteriophage T7. It is a single-chain enzyme encoded by the T7 phage chromosome, which has high promoter specificity and can strictly and specifically recognize the T7 promoter without the assistance of other transcription factors. The transcription of a target gene containing a T7 promoter in the cytoplasm can be efficiently induced by T7 RNAP without entering the nucleus, which can overcome the barrier effect of the nuclear membrane on the transcription of foreign genes. T7 RNA polymerase has a mature and stable heterologous expression and purification process, can normally exercise its transcriptional activity without any other auxiliary factors, and can produce full-length RNA transcripts with high fidelity. Therefore, it has become the most mainstream tool enzyme for in vitro transcription.

[0003] However, although T7 RNA polymerase has significant advantages, it also has some unavoidable disadvantages as an in vitro RNA synthesis tool. It will produce many by-products during the process of synthesizing RNA. Currently, under the process system, the most concerned is the production of double-stranded RNA (dsRNA), because it will trigger a serious immune response in mammals. Industrially, dsRNA can be effectively removed by high performance liquid chromatography (HPLC) and cellulose chromatography. However, the development of industrial purification methods for mRNA is difficult and costly, and it is difficult for companies lacking funds and relevant equipment to implement this method. Therefore, if the production of dsRNA can be reduced from the source, it will greatly reduce the difficulty of downstream purification, shorten the production cycle, and reduce the production cost.

[0004] Therefore, there is an urgent need in the art to develop a T7 RNA polymerase mutant with high efficiency and low by-products. Summary of the Invention

[0005] The object of the present invention is to provide a T7 RNA polymerase mutant with high efficiency and low by-products.

[0006] In the first aspect of the present invention, there is provided a T7 RNA polymerase mutant, wherein the polymerase mutant has at least one amino acid mutation at a site corresponding to the wild-type T7 RNA polymerase selected from the group consisting of: position 44, position 45, position 46, position 172, position 389, position 425, position 441, position 632, position 643, position 754, and position 789; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1.

[0007] In another preferred example, the content of dsRNA produced by the polymerase mutant is reduced by ≥25% compared to the wild type.

[0008] In another preferred example, the sequence of the wild-type T7 RNA polymerase is as shown in SEQ ID NO: 1.

[0009] In another preferred example, the polymerase mutant comprises one or more amino acid mutations selected from the group consisting of: Y44E, M46G, K172A, R632H, Q754A, and S789P mutations; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1; and the content of dsRNA produced by the polymerase mutant is reduced by ≥25% compared to the wild-type T7 RNA polymerase.

[0010] In another preferred example, the polymerase mutant further comprises one or more amino acid mutations selected from the group consisting of: R173H, E45M, K441F, K441G, K389M, R425H, and E643F mutations.

[0011] In another preferred example, the content of dsRNA produced by the polymerase mutant is reduced by ≥30% compared to the wild-type T7 RNA polymerase, preferably reduced by ≥50%, more preferably reduced by ≥70%.

[0012] In another preferred example, the content of dsRNA produced by the polymerase mutant is 0.01 - 0.75 ng / μg; preferably 0.01 - 0.5 ng / μg, more preferably 0.01 - 0.3 ng / μg.

[0013] In another preferred example, the polymerase mutant comprises the R632H mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0014] In another preferred example, the polymerase mutant comprises a combination of mutation sites selected from the group consisting of:

[0015] (a) R632H, R173H, and E45M;

[0016] (b) R632H and Y44E;

[0017] (c) R632H and Q754A.

[0018] In another preferred example, the amino acid sequence of the polymerase mutant (R632H, R173H, and E45M) is as shown in SEQ ID NO: 2.

[0019] In another preferred example, the polymerase mutant contains the K172A mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1; and the content of dsRNA produced by the polymerase mutant is reduced by ≥25% compared to the wild type.

[0020] In another preferred example, the polymerase mutant contains the K172A and Q754A mutations; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0021] In another preferred example, the polymerase mutant contains a mutation combination selected from the group consisting of:

[0022] (a) K172A and Q754A;

[0023] (b) K172A, K441F and Q754A;

[0024] (c) K172A, S789P and R425H.

[0025] In another preferred example, the polymerase mutant contains the R173H mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1; and the content of dsRNA produced by the polymerase mutant is reduced by ≥25% compared to the wild type.

[0026] In another preferred example, the polymerase mutant contains a mutation combination selected from the group consisting of:

[0027] (a) R173H, R632H and E45M;

[0028] (b) R173H and K389M.

[0029] In another preferred example, the polymerase mutant contains the Y44E mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0030] In another preferred example, the polymerase mutant contains a mutation combination selected from the group consisting of:

[0031] (a) Y44E and R632H;

[0032] (b) Y44E, E643F and S789P.

[0033] In another preferred example, the polymerase mutant contains the Q754A mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0034] In another preferred example, the polymerase mutant contains a mutation combination selected from the group consisting of:

[0035] (a) Q754A and K172A;

[0036] (b) Q754A, K172A and K441F;

[0037] (c) Q754A and R623H.

[0038] In another preferred example, the polymerase mutant comprises an S789P mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0039] In another preferred example, the polymerase mutant comprises a mutation combination selected from the following group:

[0040] (a) S789P and K441G;

[0041] (b) S789P, E643F and Y44E;

[0042] (c) S789P, K172A and R425H.

[0043] In another preferred example, the polymerase mutant comprises a mutation or mutation combination selected from the following group:

[0044] (1) R632H, R173H and E45M;

[0045] (2) R632H and Y44E;

[0046] (3) R632H and Q754A;

[0047] (4) M46G;

[0048] (5) K441F, K172A and Q754A;

[0049] (6) E643F, S789P and Y44E;

[0050] (7) K172A and Q754A;

[0051] (8) S789P and K441G;

[0052] (9) S789P, K172A and R425H;

[0053] (10) Q754A and R632H;

[0054] (11) K172A;

[0055] (12) K389M and R173H.

[0056] In another preferred embodiment, the polymerase mutant comprises a mutation or combination of mutations selected from the group consisting of:

[0057] (1) R632H, R173H, and E45M;

[0058] (2) K172A and Q754A;

[0059] (3) K172A.

[0060] In another preferred embodiment, the T7 RNAP polymerase mutant is used in an in vitro transcription (IVT) reaction, and its efficiency in the IVT reaction is superior to that of wild-type T7 RNAP. The efficiency includes:

[0061] (a) increasing the yield of the target product (i.e., single-stranded RNA) in the IVT reaction; and / or

[0062] (b) reducing the formation of by-products in the IVT reaction, where the by-products include double-stranded RNA (dsRNA).

[0063] In a second aspect of the present invention, there is provided an isolated polynucleotide that encodes the T7 RNA polymerase mutant described in the first aspect of the present invention.

[0064] In another preferred embodiment, the polynucleotide is DNA, RNA, cDNA, or a combination thereof.

[0065] In a third aspect of the present invention, there is provided a vector that contains the polynucleotide described in the second aspect of the present invention.

[0066] In another preferred embodiment, the vector is an expression vector.

[0067] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0068] In a fourth aspect of the present invention, there is provided a genetically engineered host cell that contains the vector described in the third aspect of the present invention, or the genome of the host cell is integrated with the polynucleotide described in the second aspect of the present invention.

[0069] In another preferred embodiment, the host cell includes a prokaryotic cell and a eukaryotic cell.

[0070] In another preferred embodiment, the host cell is a prokaryotic cell.

[0071] In another preferred example, the host cell includes cells derived from the following microorganisms: Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, Escherichia coli.

[0072] In another preferred example, the host cell is Escherichia coli.

[0073] In the fifth aspect of the present invention, a method for preparing the T7 RNA polymerase mutant as described in the first aspect of the present invention is provided, including the following steps:

[0074] (1) Introduce the polynucleotide as described in the second aspect of the present invention into a host cell to obtain a genetically engineered host cell;

[0075] (2) Cultivate the genetically engineered host cell under conditions suitable for protein expression, so as to obtain a culture containing the T7 RNA polymerase mutant;

[0076] (3) Isolate and / or purify the T7 RNA polymerase mutant from the culture.

[0077] In the sixth aspect of the present invention, a use of the phage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention is provided, for in vitro transcription reaction (IVT), or for preparing reagents or kits for in vitro transcription (IVT) reaction.

[0078] In the seventh aspect of the present invention, a reagent for in vitro transcription (IVT) reaction is provided, and the reagent contains the T7 RNA polymerase mutant as described in the first aspect of the present invention.

[0079] In the eighth aspect of the present invention, a kit is provided, which contains the reagent as described in the seventh aspect of the present invention.

[0080] In another preferred embodiment, the kit is used for in vitro transcription (IVT) reaction, and further contains other reagents required for in vitro transcription (IVT) reaction, including: DNA template, NTPs, in vitro transcription reaction buffer, and optionally RNase inhibitor.

[0081] In another preferred embodiment, the kit is used for preparing circular RNA (circRNA), and further contains reagents required for in vitro cyclization reaction.

[0082] In another preferred embodiment, the kit is used for preparing self-amplifying RNA (saRNA).

[0083] In another preferred embodiment, the kit further contains an instruction manual, which indicates that the kit is used for producing mRNA, circRNA or saRNA.

[0084] In the ninth aspect of the present invention, a method for performing in vitro transcription (IVT) reaction is provided, which is characterized by including the step of using the T7 RNA polymerase mutant as described in the first aspect of the present invention for the reaction.

[0085] In another preferred embodiment, the method has one or more performances selected from the following group compared with the method using wild-type T7 RNA polymerase:

[0086] (a) Increasing the yield of the target product (i.e., single-stranded RNA) in the IVT reaction;

[0087] (b) Reducing the formation of by-products in the IVT reaction, and the by-products include double-stranded RNA (dsRNA).

[0088] In another preferred embodiment, the method is for non-diagnostic or therapeutic purposes.

[0089] In another preferred embodiment, the content of dsRNA produced by the polymerase mutant is reduced by ≥30%, preferably by ≥50%, more preferably by ≥70% compared with wild-type T7 RNA polymerase.

[0090] In another preferred embodiment, the content of dsRNA produced by the polymerase mutant is 0.01 - 0.75 ng / μg; preferably 0.01 - 0.5 ng / μg, more preferably 0.01 - 0.3 ng / μg.

[0091] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The figure shows the gel electrophoresis results of the wild-type T7 RNA polymerase and the mutants of the present invention. Among them, Mut1-13 respectively represent the T7 RNA polymerase mutants numbered 1-13, and WT represents the wild-type T7 RNA polymerase; among them, RNA aggregates represent the T7 RNA polymerase mutants.

[0093] Figure 2 The figure shows the results of the content of dsRNA produced in total RNA during the transcription of the 2k template by the wild-type T7 RNA polymerase and the mutants of the present invention. Among them, the sequence of the 2k template is as shown in SEQ ID NO:3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0094] Through extensive and in-depth research, the inventors of the present invention used deep learning to predict the mutation sites of amino acids and screened out T7 RNA polymerase mutants with the ability to reduce the production of dsRNA and at the same time reduce RNA aggregates through extensive experiments; the content of dsRNA produced by the T7 RNA polymerase mutants of the present invention is significantly reduced to less than 95% compared with the wild-type T7 RNA polymerase. In addition, the T7 RNA polymerase mutants of the present invention have higher transcriptional activity and product integrity, improve the purity of the product to a certain extent, and reduce the burden of the downstream purification process. The present invention has been completed on this basis.

[0095] TERMS

[0096] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0098] As used herein, the terms "wild-type T7 RNA polymerase" or "WT T7 RNA polymerase" both encompass a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0099] As used herein, the terms "T7 RNA polymerase mutant", "mutant T7 RNA polymerase", and "T7 RNA polymerase mutant protein" are used interchangeably and all refer to a mutant protein having at least two or more amino acid substitutions, deletions, or insertions as compared to the amino acid sequence of wild-type T7 RNA polymerase.

[0100] The term "corresponding to" as used herein has the meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means the position in one sequence that corresponds to a specified position in another sequence after a homology or sequence identity alignment of the two sequences. Thus, for example, "corresponding to wild-type T7 RNA polymerase" means aligning a given amino acid sequence with the amino acid sequence of wild-type T7 RNA polymerase to find the sites on the amino acid sequence that correspond to wild-type T7 RNA polymerase.

[0101] The mutant of the present invention

[0102] As used herein, the term "the mutant of the present invention" refers to a phage T7 RNA polymerase mutant.

[0103] In the present invention, a preferred class of mutants is a T7 RNA polymerase mutant corresponding to wild-type T7 RNA polymerase (amino acid sequence shown in SEQ ID NO: 1) having R173H, R632H, and E45M mutations. Such mutants have significantly improved efficacy as compared to wild-type T7 RNA polymerase and other single-site mutants, specifically manifested as a ≥95% decrease in the content of dsRNA produced by the polymerase mutant as compared to wild-type T7 RNA polymerase.

[0104] In another preferred embodiment, under 2K template conditions, the content of dsRNA produced by the polymerase mutant is 0.01 - 0.75 ng / μg; preferably 0.01 - 0.5 ng / μg, more preferably 0.01 - 0.3 ng / μg.

[0105] The sequence of wild-type T7 RNA polymerase is as follows (SEQ ID NO: 1):

[0106] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0107] The sequences of the T7 RNA polymerase mutants (Mut-4) with R173H, R632H, and E45M mutations are as follows (SEQ ID NO:2) (where the underlined part represents the added His tag):

[0108] MNHKVHHHHHH MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEYYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKHVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKHSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA*

[0109] In a specific embodiment, the mutant is a T7 RNA polymerase mutant with R173H, R632H, and E45M mutations, which contains the amino acid sequence shown in SEQ ID NO:2. This term also includes variant forms and derivative polypeptides of the SEQ ID NO:2 sequence that have the same function as the shown polypeptide. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0110] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with R173H mutation and further contains an amino acid residue mutation selected from the group consisting of: K389M, R632H, or E45M; wherein, the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0111] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with R632H mutation and further contains one or more amino acid residue mutations selected from the group consisting of: Y44E, Q754A, R173H, and E45M; wherein, the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0112] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with K172A mutation and further contains one or more amino acid residue mutations selected from the group consisting of: Q754A, K441F, S789P, and R425H; wherein, the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0113] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with Y44E mutation and further contains one or more amino acid residue mutations selected from the group consisting of: R632H, E643F, and S789P; wherein, the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0114] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with Q754A mutation and further contains one or more amino acid residue mutations selected from the group consisting of: R623H, K172A, and K441F; wherein, the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0115] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with the S789P mutation and further comprises one or more amino acid residue mutations selected from the group consisting of K441G, E643F, Y44E, K172A, and R425H; wherein, the sites are based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0116] Among them, taking the T7 RNA polymerase mutant with the R173H site mutation as an example, when there is only the single mutant at the R173H site, the dsRNA content cannot be significantly reduced; but when combined with the R632H and E45M mutations to form a multi-site mutant enzyme, the dsRNA content is significantly reduced.

[0117] The present invention also includes fragments, derivatives, and analogs of the protein (or polypeptide). As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity of the polypeptide.

[0118] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides having substituent groups in one or more amino acid residues, or (ii) polypeptides formed by fusing additional amino acid sequences to this polypeptide sequence (such as fusion protein / tag sequences used to purify this polypeptide). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well-known to those skilled in the art.

[0119] As used herein, an "isolated protein (or polypeptide)" means that the protein is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it. Those skilled in the art can purify the protein using standard protein purification techniques. The substantially pure protein can produce a single major band on a non-reducing polyacrylamide gel. The purity of the protein can also be further analyzed using techniques such as amino acid sequence determination.

[0120] The amino terminus or carboxyl terminus of the protein of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. For example, the tags may be FLAG, HA, c-Myc, Poly–His, Poly-Arg, StrepII, etc. These tags can be used for purifying the protein.

[0121] The polynucleotides, vectors, and host cells of the present invention

[0122] The polynucleotides of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

[0123] The polynucleotides encoding the mature polypeptide of SEQ ID NO:2 include: coding sequences encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0124] The present invention also relates to variants of the above polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring variants or non-naturally occurring variants, but will not substantially change the function of the polypeptide encoded thereby.

[0125] The polypeptides and polynucleotides in the present invention are preferably provided in isolated form, and more preferably purified to homogeneity. The full-length nucleotide sequence of the T7 RNA polymerase mutant of the present invention or its fragment can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences.

[0126] Once the relevant sequences are obtained, recombinant protein preparation can be carried out by bioengineering methods. This is usually to clone the polynucleotide sequence encoding the recombinant protein into an expression vector, then transfer the expression vector into cells for expression, and harvest and purify to obtain the recombinant protein.

[0127] Currently, the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention can be completely obtained by chemical synthesis. Then this DNA sequence can be introduced into various existing plasmids (or vectors) known in the art, and the protein can be obtained after being transferred into cells for expression and purification.

[0128] The present invention also relates to vectors containing the above appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0129] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0130] Transforming host cells with recombinant DNA and recombinant expression of proteins are conventional techniques well known to those skilled in the art. The recombinantly expressed proteins can be separated and purified by conventional techniques well known to those skilled in the art, which will not be elaborated here.

[0131] Application

[0132] The present invention also provides the use of the T7 RNA polymerase mutants and their derivatives of the present invention for preparing reagents or kits for in vitro RNA synthesis, cell-free protein expression, nucleic acid detection, etc.

[0133] In one embodiment of the present invention, the use of the mutant of the present invention in in vitro RNA synthesis is provided. The mutant of the present invention can be used for synthesizing RNA drugs, including RNA vaccines, RNA interference agents, RNA inhibitors, gene editing tools, etc. In one embodiment of the present invention, the in vitro RNA synthesis reaction can be used for synthesizing mRNA, typically including mRNA vaccines. In one embodiment of the present invention, the in vitro RNA synthesis reaction can be used for synthesizing non-coding RNA, including sgRNA, tRNA, siRNA, snoRNA, etc. The generation of by-products during the reaction process is reduced, including by-products such as double-stranded RNA (dsRNA).

[0134] The main advantages of the present invention include:

[0135] (1) The T7 RNA polymerase mutant screened in the present invention significantly reduces the content of dsRNA by-products and RNA aggregates in in vitro transcription. Among them, compared with the wild type, the content of dsRNA produced by the T7 RNA polymerase mutant of the present invention is significantly reduced by less than 95%.

[0136] (2) The T7 RNA polymerase mutant of the present invention has higher transcriptional activity.

[0137] (3) The T7 RNA polymerase mutant of the present invention has higher product integrity.

[0138] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0139] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent companies unless otherwise specified. In the following embodiments, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0140] Example 1: Construction, Expression and Purification of T7 RNA Polymerase Mutants

[0141] 1. Search for the amino acid sequence of the wild-type T7 RNA polymerase in the NCBI database. The wild-type (WT) amino acid sequence of the T7 RNA polymerase involved in the present invention is shown in SEQ ID NO: 1.

[0142] Construct T7 RNA polymerase mutants by site-directed mutagenesis. The process of site-directed mutagenesis includes the steps of obtaining the following mutants by directed evolution, and constructing the DNA sequences of the T7 RNA polymerase mutants, the N-terminal 6*His tag, and the thrombin cleavage site into the pColdII vector by homologous recombination, enzymatic digestion and ligation, sequence synthesis, etc., and transforming it into Escherichia coli BL21. The mutation sites of the obtained T7 RNA polymerase mutants relative to the wild type are shown in Table 1:

[0143] Table 1

[0144]

[0145] 2. Transform the prokaryotic expression vector pColdII containing the above mutation sites into the E. coli BL21 expression strain, and culture it in LB medium containing kanamycin (Kan) at 180 rpm and 37 °C until the OD600 reaches 0.6 to 1.2, then add 0.5 mM isopropyl β-D-thiogalactoside, and induce protein expression at 180 rpm and 20 °C for 12 h;

[0146] 3. Centrifuge at 4 °C and 6000 rpm for 20 min to collect the cell pellet. Thaw the cells at room temperature in advance, and pour the prepared buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0) into the corresponding container.

[0147] 4. Then put the thawed cells into the solution, use a stirring rod to crush the lumpy cells and add them to the rotor, place the cell suspension on a magnetic stirrer for accelerated dissolution (>500 rpm), resuspend the cells at a ratio of about 1:10 (for example, 10 g of cells are resuspended in 100 ml of buffer), turn on and check whether the cell disruptor is normal, and ultrasonically disrupt 100 ml of cell suspension with a No. 10 probe at 400 W in an ice bath (working for 4 s, intermittent for 6 s) 100 times.

[0148] 5. Balance the bacterial solution in a 50 ml centrifuge tube. After balancing, place it in a centrifuge for centrifugation at 12,000 rpm for 15 minutes. Pour the supernatant after centrifugation into the sample loading container, take a sample of the supernatant for gel electrophoresis, and save the precipitate separately for future use. Debug the AKTA Pure-150 equipment for protein purification. The purification process is two-step chromatography, first affinity chromatography and then ion exchange chromatography.

[0149] 5.1 The chromatography column and solutions used in affinity chromatography are as follows:

[0150] Chelating SFF (Ni) column for enrichment (affinity chromatography);

[0151] Column volume: 6 ml; column diameter: 16 mm;

[0152] Volume flow rate: 5 ml / min (sample loading); 5 ml / min (elution);

[0153] Affinity chromatography buffer A: 20 mM Tris-HCl, 250 mM NaCl, pH 8.0;

[0154] Affinity chromatography buffer B: 20 mM Tris-HCl, 250 mM NaCl, 500 mM imidazole, pH 8.0;

[0155] 5.1.1 Pretreatment: Rinse and soak the chromatography column with NaOH. After completion, perform rinsing operations with 0.2 M NiSO4, pyrogen-free water, buffer B, and pyrogen-free water in sequence.

[0156] 5.1.2 Equilibration: Rinse the affinity chromatography column that has been cleaned with buffer A until the UV280 peak no longer changes.

[0157] 5.1.3 Sample loading: Take 40 μl of the sample loading solution for detection. After the A280 reading > 100 mAu, collect the sample loading effluent.

[0158] 5.1.4 Equilibration after sample loading: After sample loading, continue to rinse the affinity chromatography column with buffer A until the A280 reading is stable and no longer decreases. After the A280 reading < 100 mAu, stop collecting the sample loading effluent.

[0159] 5.1.5 Elution: Use solutions containing imidazole at different concentration gradients (50 mM - 500 mM) to elute T7 RNA polymerase from the column.

[0160] 5.1.6 Sample detection: Detect the purity, concentration, turbidity, conductivity, and pH of the sample loading, effluent, and elution samples.

[0161] 5.2 The chromatography column and solutions used in ion exchange chromatography are as follows:

[0162] Purification by Q-HP column (ion exchange chromatography)

[0163] Column volume: 15 ml; Column diameter: 26 mm

[0164] Volume flow rate: 8 ml / min (loading); 8 ml / min (elution)

[0165] Ion exchange chromatography buffer A: 20 mM Tris-HCl, pH 8.0

[0166] Ion exchange chromatography buffer B: 20 mM Tris-HCl, 1 M NaCl, pH 8.0

[0167] 5.2.1 Dilute the sample 4-fold with ion exchange chromatography buffer A, mix well, and purify through the Q-HP column.

[0168] 5.2.2 Pretreatment: Rinse the chromatography column with NaOH and soak it in alkali. After completion, perform rinsing operations with 0.2 M NiSO4, pyrogen-free water, buffer B, and pyrogen-free water in sequence.

[0169] 5.2.3 Equilibration: Rinse the processed Q-HP chromatography column with ion exchange chromatography buffer A until the UV280 peak value no longer changes.

[0170] 5.2.4 Loading: Take 40 μl of the loading solution for detection. After the A280 reading > 100 mAu, collect the loading effluent.

[0171] 5.2.5 Post-loading equilibration: Continue to rinse the Q-HP with buffer A until the A280 reading < 100 mAu, and then end the collection of the loading solution.

[0172] 5.2.6 Elution: Use NaCl-containing solutions with different concentration gradients (200 mM - 300 mM) to elute T7 RNA polymerase from the column.

[0173] 5.2.7 Sample detection: Detect the purity, concentration, turbidity, conductivity, and pH of the sample after loading and desalting.

[0174] Dialysis and filtration: Prepare dialysis bag washing solution according to the sample volume: 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 50% glycerol, pH 7.9. Dialyze for 16 h with a 56.8-fold volume change, change the solution once, and then dialyze for another 3 h. Collect the protein after dialysis and store it at -20 °C.

[0175] Example 2: In vitro transcription test of T7 RNA polymerase mutant and wild-type T7 RNA polymerase

[0176] 1. Prepare the in vitro transcription system of T7 RNA polymerase according to Table 2 below. Dilute the wild-type T7 RNA polymerase and the mutants to 200 U / μL, add the enzyme in the same volume to the reaction, and react at 37 °C for 3 h.

[0177] Table 2

[0178]

[0179] The in vitro transcription test template is a 2k template (the sequence is shown in SEQ ID NO:3). The 10× transcription buffer, ATP / GTP / CTP / UTP, inorganic pyrophosphatase, and RNase inhibitor are all biological products purchased from Novoprotein Scientific Inc., Suzhou.

[0180] 2. Add 30 μl of lithium chloride precipitation solution (7.5 M lithium chloride, 50 mM EDTA) to the 20 μl reaction solution, precipitate at -20 °C for 30 min, and centrifuge to remove the supernatant.

[0181] 3. Wash twice with pre-cooled 75% ethanol.

[0182] 4. Dissolve the RNA precipitate with 100 μL of RNase-free water.

[0183] 5. Dilute the transcription products of the wild-type and mutant T7 RNA polymerases 50-fold and 100-fold respectively to ensure that the RNA concentration does not exceed the detection range of Nanodrop. Test the RNA concentrations under the two dilution conditions of the mutants respectively. When the numerical deviation is not significant after multiplying the sample concentration by the dilution factor at 5-fold and 10-fold, take the average value as the average value of the mutant transcription products. If the difference between the two dilution factors is large, the RNA concentration needs to be retested.

[0184] The RNA yield results of the wild-type T7 RNA polymerase (WT) and each mutant (Mut) are shown in Table 3 below:

[0185] Table 3

[0186]

[0187] Use Nanodrop to detect the sample concentration respectively. Dilute the transcription products of the wild-type and each mutant 50-fold and 100-fold respectively. When the numerical deviation is not significant after multiplying the detected sample concentration by the dilution factor, take the average value as the average value of the mutant transcription products. If the deviation is large (>10%), dilute the sample again and detect it to improve the accuracy of the detection results.

[0188] The results are shown in Table 3: The yield differences of different mutant enzymes are not significant; compared with the wild-type, the differences are all within 5%.

[0189] Example 3. Analysis of the residual amount of transcribed product dsRNA

[0190] Based on the transcribed product mRNA in Example 2, the integrity and RNA polymer content of the transcribed product were analyzed by gel electrophoresis, and the residual amount of the transcribed product dsRNA was analyzed using a dsRNA quantification kit.

[0191] 1. Take 1 μL of the transcribed product mRNA in Example 2 above, add 8 μL of DEPC water, and then add 1 μL of 10×RNA loading buffer and mix. Prepare a 1% agarose gel and run the gel at 190 V for 20 min for EB detection.

[0192] The results are as Figure 1 shown. The target band produced by the T7 RNA polymerase mutant Mut4 is clear and single, and the RNA polymer produced is significantly lower than that of the wild-type T7 RNA polymerase. The results map was analyzed for gray value using Tanon Image software. The results show that compared with the wild-type T7 RNA polymerase (WT), Mut1, Mut2, and Mut4-8 can all reduce the RNA polymer content; among them, Mut2 only decreased by about 16% compared with the wild-type; Mut4 can significantly reduce the polymer content, and the polymer content decreased by about 59% compared with the wild-type.

[0193] 2. Quantitative analysis of the IVT product using a dsRNA quantification kit: The operation steps were carried out according to the dsRNA quantification kit (product number RD017) of Novoprotein Scientific Inc., Suzhou.

[0194] The results of the dsRNA content produced in the total RNA during 2k template transcription are as Figure 2 shown, where the sequence of the 2k template is as shown in SEQ ID NO:3:

[0195]

[0196] Figure 2 The results showed that compared with the wild-type T7 RNA polymerase (WT), both the Mut1-11 and Mut13 mutants were able to reduce the dsRNA content; among them, the dsRNA content of the single-point mutant T7 RNA polymerase (Mut-13) with R173H mutation was only reduced by about 10% compared with the wild-type T7 RNA polymerase; however, the dsRNA content of the double-site mutant (Mut-2) composed of the K389M mutation was reduced by about 30%; while the dsRNA content of the multi-site mutant (Mut-4) formed by the combination of the R632H and E45M mutations was significantly reduced by more than about 95%, and the dsRNA content of Mut-4 was about 0.01 ng / μg.

[0197] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A T7 RNA polymerase mutant, characterized in that, The polymerase mutant is mutated at R632H, R173H, and E45M on the basis of the sequence shown in SEQ ID NO:

1.

2. The polymerase mutant according to claim 1, wherein The amino acid sequence of the polymerase mutant is as shown in SEQ ID NO:

2.

3. The polymerase mutant according to claim 1, wherein The content of dsRNA produced by the polymerase mutant is reduced by ≥30% compared with that of the wild-type T7 RNA polymerase.

4. The polymerase mutant according to claim 1, wherein The content of dsRNA produced by the polymerase mutant is 0.01 - 0.75 ng / μg.

5. An isolated polynucleotide, characterized in that, The polynucleotide encodes the T7 RNA polymerase mutant according to claim 1.

6. A carrier, characterized in that, The vector contains the polynucleotide according to claim 5.

7. A genetically engineered host cell, characterized in that, The host cell contains the vector according to claim 6, or the polynucleotide according to claim 5 is integrated into the genome of the host cell.

8. A method for preparing the T7 RNA polymerase mutant as claimed in claim 1, characterized in that Comprising the following steps: (1) Introducing the polynucleotide according to claim 5 into a host cell to obtain a genetically engineered host cell; (2) Culturing the genetically engineered host cell under conditions suitable for protein expression to obtain a culture containing the T7 RNA polymerase mutant; (3) Isolating and / or purifying the T7 RNA polymerase mutant from the culture.

9. Use of a T7 RNA polymerase mutant as described in claim 1, characterized in that, For in vitro transcription reactions, or for preparing reagents or kits for in vitro transcription reactions.

10. A reagent for in vitro transcription reaction, characterized in that, The reagent contains the T7 RNA polymerase mutant according to claim 1.

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