T7 RNA polymerase mutant and application thereof

Abstract

The invention relates to a T7 RNA polymerase mutant and application thereof. Specifically, the invention discloses a T7 RNA polymerase mutant, and the polymerase mutant has at least one amino acid mutation at sites corresponding to a wild type T7 RNA polymerase and selected from the following groups: the 44th site, the 45th site, the 46th site, the 172nd site, the 389th site, the 425th site, the 441st site, the 632th site, the 643th site, the 754th site and the 789th site; the serial number of the mutation site is based on the sequence shown in SEQ ID NO: 1, the mutation leads to obvious reduction of a byproduct dsRNA generated in the process of enzyme synthesis of mRNA, and meanwhile, the generation of RNA aggregates is reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a T7 RNA polymerase mutant and an application thereof. 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 chromosome of T7 bacteriophage. It has high promoter specificity and can strictly and specifically recognize T7 promoters without the assistance of other transcription factors. The transcription of target genes containing T7 promoters 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 exogenous genes. T7 RNA polymerase has a mature and stable heterologous expression purification process, and does not require any other auxiliary factors to exercise normal transcription activity. It can also produce high-fidelity full-length RNA transcripts, making it the most mainstream tool enzyme for in vitro transcription.

[0003] However, although T7 RNA polymerase has significant advantages, it also has some disadvantages as an in vitro RNA synthesis tool that cannot be ignored. It produces many by-products in the process of synthesizing RNA. Under the current process system, people are most concerned about the production of double-stranded RNA (dsRNA) because it can cause severe immune responses in mammals. In industry, dsRNA can be effectively removed by high-performance liquid chromatography (HPLC) and cellulose chromatography, but the development of industrial mRNA purification methods is difficult and costly. It is difficult for companies that lack funds and related 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 production costs.

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

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

[0006] In a first aspect of the present invention, a T7 RNA polymerase mutant is provided, wherein the polymerase mutant has at least one amino acid mutation at a position 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 dsRNA content produced by the polymerase mutant is reduced by ≥25% compared with the wild type.

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

[0009] In another preferred embodiment, 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 dsRNA content produced by the polymerase mutant is reduced by ≥25% compared with the wild-type T7 RNA polymerase.

[0010] In another preferred embodiment, 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 dsRNA content produced by the polymerase mutant is reduced by ≥30%, preferably ≥50%, and more preferably ≥70%, compared with that of the wild-type T7 RNA polymerase.

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

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

[0014] In another preferred embodiment, the polymerase mutant comprises a combination of mutation sites selected from the following group: (a) R632H, R173H and E45M; (b) R632H and Y44E; (c) R632H and Q754A.

[0015] In another preferred example, the amino acid sequence of the polymerase mutants (R632H, R173H and E45M) is shown in SEQ ID NO:2.

[0016] In another preferred embodiment, the polymerase mutant comprises a K172A mutation; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1; and the dsRNA content produced by the polymerase mutant is reduced by ≥25% compared with the wild type.

[0017] In another preferred embodiment, the polymerase mutant comprises K172A and Q754A mutations; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1.

[0018] In another preferred embodiment, the polymerase mutant comprises a mutation combination selected from the following group: (a) K172A and Q754A; (b) K172A, K441F and Q754A; (c) K172A, S789P, and R425H.

[0019] In another preferred embodiment, the polymerase mutant comprises an R173H mutation; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1; and the dsRNA content produced by the polymerase mutant is reduced by ≥25% compared with the wild type.

[0020] In another preferred embodiment, the polymerase mutant comprises a mutation combination selected from the following group: (a) R173H, R632H and E45M; (b) R173H and K389M.

[0021] In another preferred embodiment, the polymerase mutant comprises a Y44E mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0022] In another preferred embodiment, the polymerase mutant comprises a mutation combination selected from the following group: (a) Y44E and R632H; (b) Y44E, E643F, and S789P.

[0023] In another preferred embodiment, the polymerase mutant comprises a Q754A mutation; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1.

[0024] In another preferred embodiment, the polymerase mutant comprises a mutation combination selected from the following group: (a) Q754A and K172A; (b) Q754A, K172A and K441F; (c) Q754A and R623H.

[0025] In another preferred embodiment, the polymerase mutant comprises an S789P mutation; the numbering of the mutation site is based on the sequence shown in SEQ ID NO: 1.

[0026] In another preferred embodiment, the polymerase mutant comprises a mutation combination selected from the following group: (a) S789P and K441G; (b) S789P, E643F, and Y44E; (c) S789P, K172A, and R425H.

[0027] In another preferred embodiment, the polymerase mutant comprises a mutation or a combination of mutations selected from the following group: (1) R632H, R173H and E45M; (2) R632H and Y44E; (3) R632H and Q754A; (4) M46G; (5) K441F, K172A and Q754A; (6) E643F, S789P, and Y44E; (7) K172A and Q754A; (8) S789P and K441G; (9) S789P, K172A and R425H; (10) Q754A and R632H; (11) K172A; (12) K389M and R173H.

[0028] In another preferred embodiment, the polymerase mutant comprises a mutation or a combination of mutations selected from the following group: (1) R632H, R173H and E45M; (2) K172A and Q754A; (3) K172A.

[0029] In another preferred embodiment, the T7 RNAP polymerase mutant is used for in vitro transcription (IVT) reaction, and its performance in IVT reaction is better than that of wild-type T7 RNAP, and the performance includes: (a) increasing the yield of the target product (i.e., single-stranded RNA) in an IVT reaction; and / or (b) Reducing the formation of byproducts in IVT reactions, including double-stranded RNA (dsRNA).

[0030] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the T7 RNA polymerase mutant according to the first aspect of the present invention.

[0031] In another preferred embodiment, the polynucleotide is DNA, RNA, cDNA, or a combination thereof.

[0032] In the third aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the second aspect of the present invention.

[0033] In another preferred embodiment, the vector is an expression vector.

[0034] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0035] In the fourth aspect of the present invention, a genetically engineered host cell is provided, wherein the host cell contains the vector as described in the third aspect of the present invention, or the genome of the host cell is integrated with the polynucleotide as described in the second aspect of the present invention.

[0036] In another preferred embodiment, the host cells include prokaryotic cells and eukaryotic cells.

[0037] In another preferred embodiment, the host cell is a prokaryotic cell.

[0038] In another preferred embodiment, the host cell includes cells derived from the following microorganisms: Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, and Escherichia coli.

[0039] In another preferred embodiment, the host cell is Escherichia coli.

[0040] In a fifth aspect of the present invention, there is provided a method for preparing the T7 RNA polymerase mutant as described in the first aspect of the present invention, comprising the following steps: (1) introducing the polynucleotide according to the second aspect of the present invention into a host cell to obtain a genetically engineered host cell; (2) culturing the genetically engineered host cell under conditions suitable for protein expression, thereby obtaining a culture containing the T7 RNA polymerase mutant; (3) isolating and / or purifying the T7 RNA polymerase mutant from the culture.

[0041] In the sixth aspect of the present invention, there is provided a use of the bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention for in vitro transcription reaction (IVT), or for preparing a reagent or kit for in vitro transcription (IVT) reaction.

[0042] In a seventh aspect of the present invention, a reagent for in vitro transcription (IVT) reaction is provided, wherein the reagent comprises the T7 RNA polymerase mutant as described in the first aspect of the present invention.

[0043] In an eighth aspect of the present invention, a kit is provided, wherein the kit comprises the reagent as described in the seventh aspect of the present invention.

[0044] In another preferred embodiment, the kit is used for in vitro transcription (IVT) reaction, and further comprises other reagents required for in vitro transcription (IVT) reaction, including: DNA template, NTPs, in vitro transcription reaction buffer, and optionally RNase inhibitor.

[0045] In another preferred embodiment, the kit is used to prepare circular RNA (circRNA), which further comprises reagents required for in vitro cyclization reaction.

[0046] In another preferred example, the kit is used to prepare self-amplifying RNA (saRNA).

[0047] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to produce mRNA, circRNA or saRNA.

[0048] In a ninth aspect of the present invention, a method for performing an in vitro transcription (IVT) reaction is provided, characterized in that it comprises the step of performing a reaction using the T7 RNA polymerase mutant described in the first aspect of the present invention.

[0049] In another preferred embodiment, the method has one or more properties selected from the following group relative to the method using wild-type T7 RNA polymerase: (a) Improving the yield of the target product (i.e., single-stranded RNA) in the IVT reaction; (b) Reducing the formation of byproducts in IVT reactions, including double-stranded RNA (dsRNA).

[0050] In another preferred embodiment, the method is not for diagnostic or therapeutic purposes.

[0051] In another preferred example, the dsRNA content produced by the polymerase mutant is reduced by ≥30%, preferably ≥50%, and more preferably ≥70%, compared with that of the wild-type T7 RNA polymerase.

[0052] In another preferred embodiment, the dsRNA content produced by the polymerase mutant is 0.01-0.75 ng / μg; preferably 0.01-0.5 ng / μg, and more preferably 0.01-0.3 ng / μg.

[0053] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The gel electrophoresis results of wild-type T7 RNA polymerase and the mutants of the present invention are shown. Mut1-13 represent T7 RNA polymerase mutants numbered 1-13, respectively, and WT represents wild-type T7 RNA polymerase; RNA polymer represents T7 RNA polymerase mutant.

[0055] Figure 2 The result graph shows the dsRNA content produced in total RNA by wild-type T7 RNA polymerase and the mutant of the present invention when transcribing a 2k template, wherein the sequence of the 2k template is shown in SEQ ID NO:3. DETAILED DESCRIPTION

[0056] After extensive and in-depth research, the inventors used deep learning to predict the mutation sites of amino acids, and screened out T7 RNA polymerase mutants that can reduce dsRNA production and RNA aggregates through extensive experiments; the dsRNA content produced by the T7 RNA polymerase mutant 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 mutant of the present invention has higher transcriptional activity and product integrity, improves the purity of the product to a certain extent, and reduces the burden of the downstream purification process. The present invention was completed on this basis.

[0057] the term In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, 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 belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] As used herein, the terms "wild-type T7 RNA polymerase" or "WT T7 RNA polymerase" both comprise a protein having an amino acid sequence as shown in SEQ ID NO: 1.

[0060] As used herein, the terms "T7 RNA polymerase mutant", "mutant T7 RNA polymerase", and "T7 RNA polymerase mutant protein" are used interchangeably and refer to a mutant protein having at least two or more amino acid substitutions, deletions, or insertions compared to the amino acid sequence of wild-type T7 RNA polymerase.

[0061] As used herein, the term "corresponding to" has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means a position in one sequence that corresponds to a specified position in another sequence after the two sequences have been aligned for homology or sequence identity. Thus, for example, "corresponding to wild-type T7 RNA polymerase" means that an amino acid sequence is aligned with the amino acid sequence of wild-type T7 RNA polymerase to find the site on the amino acid sequence that corresponds to the wild-type T7 RNA polymerase.

[0062] Mutants of the present invention As used herein, the term "mutant of the present invention" refers to a bacteriophage T7 RNA polymerase mutant.

[0063] In the present invention, a preferred class of mutants is a T7 RNA polymerase mutant having R173H, R632H and E45M mutations corresponding to the wild-type T7 RNA polymerase (amino acid sequence as shown in SEQ ID NO: 1). Such mutants have significantly improved performance compared to the wild-type T7 RNA polymerase and other single-site mutants, specifically, the dsRNA content produced by the polymerase mutant is reduced by ≥95% compared to the wild-type T7 RNA polymerase.

[0064] In another preferred embodiment, under 2K template conditions, the dsRNA content produced by the polymerase mutant is 0.01-0.75 ng / μg; preferably 0.01-0.5 ng / μg, and more preferably 0.01-0.3 ng / μg.

[0065] The sequence of wild-type T7 RNA polymerase is as follows (SEQ ID NO: 1): MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA 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 indicates the added His tag): MNHKVHHHHHH* In a specific embodiment, the mutant is a T7 RNA polymerase mutant with R173H, R632H and E45M mutations, which comprises the amino acid sequence shown in SEQ ID NO: 2. The term also includes variant forms and derivative polypeptides of the SEQ ID NO: 2 sequence that have the same function as the polypeptide shown. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0066] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with R173H mutation, and further comprises an amino acid residue mutation selected from the following group: K389M, R632H or E45M; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0067] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with R632H mutation, and further comprises one or more amino acid residue mutations selected from the following group: Y44E, Q754A, R173H and E45M; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO:1.

[0068] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with K172A mutation, and further comprises one or more amino acid residue mutations selected from the following group: Q754A, K441F, S789P and R425H; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0069] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with Y44E mutation, and further comprises one or more amino acid residue mutations selected from the following group: R632H, E643F and S789P; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0070] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with Q754A mutation, and further comprises one or more amino acid residue mutations selected from the following group: R623H, K172A and K441F; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0071] More preferably, the mutant of the present invention is based on the T7 RNA polymerase mutant with S789P mutation, and further comprises one or more amino acid residue mutations selected from the following group: K441G, E643F, Y44E, K172A and R425H; wherein the site is based on the wild-type T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0072] Among them, taking the T7 RNA polymerase mutant based on the R173H site mutation as an example, when there is only a single mutant at the R173H site, the dsRNA content cannot be significantly reduced; but the dsRNA content of the multi-site mutant enzyme formed after combining with the R632H and E45M mutations is significantly reduced.

[0073] 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.

[0074] The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein / tag sequence used to purify the polypeptide). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0075] As used herein, "isolated protein (or polypeptide)" means that the protein is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the protein using standard protein purification techniques. 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.

[0076] The amino 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 tag may be FLAG, HA, c-Myc, Poly-His, Poly-Arg, StrepII, etc. These tags can be used to purify the protein.

[0077] Polynucleotides, vectors and host cells of the present invention The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0078] The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0079] 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 the polynucleotides may be naturally occurring variants or non-naturally occurring variants, but will not substantially change the function of the encoded polypeptide.

[0080] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are preferably purified to homogeneity. The full-length nucleotide sequence of the T7 RNA polymerase mutant of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on 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 is used as a template to amplify and obtain the relevant sequence.

[0081] Once the relevant sequence is obtained, the recombinant protein can be prepared by bioengineering methods. This is usually done by cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, then transferring the expression vector into cells for expression, and harvesting and purifying the recombinant protein.

[0082] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing plasmids (or vectors) known in the art, transferred into cells, and the protein can be obtained after expression and purification.

[0083] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0084] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0085] Transforming host cells with recombinant DNA and recombinantly expressing 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 described in detail here.

[0086] application The present invention also provides the use of the T7 RNA polymerase mutant and its derivatives of the present invention to prepare reagents or kits for in vitro RNA synthesis, cell-free protein expression, nucleic acid detection, etc.

[0087] 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 to synthesize RNA drugs, including RNA vaccines, RNA interfering agents, RNA inhibitors, gene editing tools, etc. In one embodiment of the present invention, the in vitro RNA synthesis reaction can be used to synthesize mRNA, typically including mRNA vaccines. In one embodiment of the present invention, the in vitro RNA synthesis reaction can be used to synthesize non-coding RNA, including sgRNA, tRNA, siRNA, snoRNA, etc. Reduce the generation of by-products during the reaction, including by-products such as double-stranded RNA (dsRNA).

[0088] The main advantages of the present invention include: (1) The T7 RNA polymerase mutant screened by the present invention significantly reduces the content of dsRNA byproducts and RNA polymers in in vitro transcription, wherein the dsRNA content produced by the T7 RNA polymerase mutant of the present invention is significantly reduced by less than 95% compared with the wild type.

[0089] (2) The T7 RNA polymerase mutant of the present invention has higher transcription activity.

[0090] (3) The T7 RNA polymerase mutant of the present invention has a higher product integrity.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0092] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0093] Example 1: Construction, expression and purification of T7 RNA polymerase mutants 1. The wild-type amino acid sequence of T7 RNA polymerase was searched in the NCBI database. The wild-type (WT) amino acid sequence of T7 RNA polymerase involved in the present invention is shown in SEQ ID NO: 1.

[0094] The T7 RNA polymerase mutant was constructed by site-directed mutagenesis, wherein the site-directed mutagenesis process comprises the following steps: obtaining the following mutants by directed evolution, constructing the DNA sequence of the T7 RNA polymerase mutant and the N-terminal 6*His tag and the thrombin cleavage site into the pColdII vector by homologous recombination, enzyme ligation, sequence synthesis, etc., and transforming into Escherichia coli BL21, and the mutation sites of the T7 RNA polymerase mutant obtained by screening relative to the wild type are shown in Table 1: Table 1

[0095] 2. The prokaryotic expression vector pColdII containing the above mutation site was transformed into the E. coli BL21 expression strain, and cultured in LB medium containing kanamycin (Kan) at 180 rpm and 37°C until OD600 reached 0.6 to 1.2, then 0.5 mM isopropylthiogalactoside was added and protein expression was induced at 180 rpm and 20°C for 12 h; 3. Centrifuge at 4°C, 6000rpm for 20min to collect bacterial precipitates. Thaw the bacteria at room temperature in advance and pour the prepared buffer (20mM Tris-HCl, 250mM NaCl, pH8.0) into the corresponding container; 4. Put the thawed bacteria into the solution, use a stirring rod to break up the lumps of bacteria and add them to the rotor, put the bacterial solution on a magnetic stirrer to accelerate the dissolution (>500rpm), resuspend the bacteria at a ratio of about 1:10 (for example, 10g of bacteria is resuspended in 100ml of buffer), turn on and check whether the cell disruptor is normal, and use a No. 10 probe 400W ultrasonic disruption (4S working, 6S intermittent) 100 times for 100ml of bacterial solution in an ice bath; 5. The bacterial solution was balanced with a 50ml centrifuge cup, and then placed in a centrifuge for centrifugation at a speed of 12000rpm*15min. The supernatant after centrifugation was poured into a sample container, and the supernatant was sampled and run on a gel, and the precipitate was stored separately for later use. The AKTA Pure-150 equipment was debugged for protein purification, and the purification process was a two-step chromatography, affinity chromatography followed by ion exchange chromatography.

[0096] 5.1 The column and solution information used in affinity chromatography are as follows: Chelating SFF (Ni) column enrichment (affinity chromatography); Column volume: 6ml; Column diameter: 16mm; Volume flow rate: 5ml / min (loading); 5ml / min (elution); Affinity chromatography buffer A: 20 mM Tris-HCl, 250 mM NaCl, pH 8.0; Affinity chromatography buffer B: 20 ​​mM Tris-HCl, 250 mM NaCl, 500 mM imidazole, pH 8.0; 5.1.1 Pretreatment: Use NaOH to rinse the chromatography column and then use caustic soda. After that, rinse with 0.2M NiSO4, pyrogen-free water, buffer B, and pyrogen-free water in sequence.

[0097] 5.1.2 Balance: Rinse the cleaned affinity chromatography column with buffer A until the UV280 peak value does not change.

[0098] 5.1.3 Sampling: Take 40 μl of sample solution for detection. After A280 reading is >100 mAu, collect the sample effluent.

[0099] 5.1.4 Balance after loading: After loading, continue to rinse the affinity chromatography column with buffer A until the A280 reading stabilizes and no longer decreases. When the A280 reading is <100mAu, stop collecting the loading effluent.

[0100] 5.1.5 Elution: Use different concentration gradients of imidazole (50mM-500mM) to elute T7 RNA polymerase from the column.

[0101] 5.1.6 Sample testing: The load, outflow and elution samples shall be tested for purity, concentration, turbidity, conductivity and pH.

[0102] 5.2 The chromatographic columns and solutions used in ion exchange chromatography are as follows: Q-HP column purification (ion exchange chromatography) Column volume: 15ml; Column diameter: 26mm; Volume flow rate: 8ml / min (loading); 8ml / min (elution) Ion exchange chromatography buffer A: 20mM Tris-HCl, pH8.0 Ion exchange chromatography buffer B: 20mM Tris-HCl, 1M NaCl, pH8.0 5.2.1 Dilute the sample 4 times with ion exchange chromatography buffer A, mix well, and purify it on a Q-HP column.

[0103] 5.2.2 Pretreatment: Use NaOH to rinse the chromatography column and soak it in caustic soda. After completion, rinse it with 0.2M NiSO4, depyrogenated water, buffer B, and depyrogenated water in sequence.

[0104] 5.2.3 Equilibration: Rinse the treated Q-HP column with ion exchange chromatography buffer A until the UV280 peak value no longer changes.

[0105] 5.2.4 Sampling: Take 40 μl of sample solution for detection. When A280 reading is >100 mAu, collect the sample effluent.

[0106] 5.2.5 Balance after loading: Continue to rinse Q-HP with buffer A until the A280 reading is less than 100mAu, and then stop collecting the loading solution.

[0107] 5.2.6 Elution: Use different concentration gradients of NaCl (200mM-300mM) to elute T7 RNA polymerase from the column.

[0108] 5.2.7 Sample testing: The loaded and desalted samples shall be tested for purity, concentration, turbidity, conductivity and pH.

[0109] Dialysis and filtration: Prepare the dialysis bag washing solution according to the sample volume: 50mM Tris-HCl, 100mM NaCl, 1mMEDTA, 50% glycerol, pH7.9, dialyze 56.8 times for 16 hours, change the solution once and dialyze for another 3 hours. After dialysis, collect the protein and store it at -20℃.

[0110] Example 2: In vitro transcription test of T7 RNA polymerase mutants and wild-type T7 RNA polymerase 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 mutant to 200 U / μL, add the same volume of enzyme into the reaction, and react at 37°C for 3 hours; Table 2

[0111] The in vitro transcription test template was a 2k template (sequence shown in SEQ ID NO: 3), and 10× transcription buffer, ATP / GTP / CTP / UTP, inorganic pyrophosphatase, and RNase inhibitor were all biological products purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0112] 2. Add 30 μl of lithium chloride precipitation solution (7.5 M lithium chloride, 50 mM EDTA) to 20 μl of reaction solution, precipitate at -20°C for 30 min, and centrifuge to remove the supernatant.

[0113] 3. Wash twice with pre-cooled 75% ethanol.

[0114] 4. Dissolve the RNA precipitate in 100 μL RNase-free water.

[0115] 5. Dilute the wild-type and mutant transcripts of T7 RNA polymerase 50 and 100 times, respectively, to ensure that the RNA concentration does not exceed the detection range of Nanodrop. Test the RNA concentration of the mutant under two dilution conditions. When the numerical deviation is not large after multiplying the 5-fold and 10-fold sample concentrations by the dilution factor, take the average as the average value of the mutant transcripts. If the difference between the two dilution factors is large, the RNA concentration needs to be retested.

[0116] The RNA yield results of T7 RNA polymerase wild type (WT) and each mutant (Mut) are shown in Table 3 below: Table 3

[0117] Nanodrop was used to detect the sample concentrations. The wild-type and mutant transcription products were diluted 50 times and 100 times, respectively. When the numerical deviation of the sample detection concentration multiplied by the dilution multiple was not large, the mean value was taken as the average value of the mutant transcription products. If the deviation was large (>10%), the sample was re-diluted and tested to improve the accuracy of the test results.

[0118] The results are shown in Table 3: the yields of different mutant enzymes were not much different; compared with the wild type, the differences were all within 5%.

[0119] Example 3: Analysis of the residual dsRNA in the transcription product Based on the mRNA of the transcription product in Example 2, the integrity and RNA polymer content of the transcription product were analyzed by gel electrophoresis, and the residual dsRNA of the transcription product was analyzed by a dsRNA quantification kit.

[0120] 1. Take 1 μL of the mRNA of the transcription product in Example 2 above, add 8 μL of DEPC water, and then add 1 μL of 10*RNA loading buffer to mix. Prepare 1% agarose gel, run the gel at 190 V for 20 min, and perform EB detection.

[0121] The results are as follows Figure 1As shown in the figure, the target band produced by the T7 RNA polymerase mutant Mut4 is clear and single, and the RNA polymers produced are significantly lower than those of the wild-type T7 RNA polymerase. The gray value analysis of the result graph was performed using Tanon Image software, and the results showed that compared with the wild-type T7 RNA polymerase (WT), Mut1, Mut2, and Mut4-8 can all reduce the content of RNA polymers; among them, Mut2 only decreased by about 16% compared with the wild type; Mut4 can significantly reduce the content of polymers, and the content of polymers decreased by about 59% compared with the wild type.

[0122] 2. Use a dsRNA quantification kit to quantitatively analyze the IVT product: The operation steps are performed according to the dsRNA quantification kit (Cat. No. RD017) of Suzhou Nearshore Protein Technology Co., Ltd.

[0123] The dsRNA content produced in the total RNA when transcribed with 2k template is as follows Figure 2 As shown, wherein the sequence of the 2k template is shown in SEQ ID NO:3:

[0124] Figure 2 The results showed that compared with the wild-type T7 RNA polymerase (WT), both Mut1~11 and Mut13 mutants were able to reduce the dsRNA content; among them, the dsRNA content of the T7 RNA polymerase mutant (Mut-13) with the R173H single-point mutation was only reduced by about 10% compared with the wild-type T7 RNA polymerase; but the dsRNA content of the double-site mutation (Mut-2) composed of the K389M mutation was reduced by about 30%; and the dsRNA content of the multi-site mutation (Mut-4) formed by the combination of the R632H and E45M mutations was significantly reduced by more than 95%, and the dsRNA content of Mut-4 was about 0.01ng / μg.

[0125] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims

1. A T7 RNA polymerase mutant, characterized in that: The polymerase mutant comprises a combination of mutation sites selected from the group consisting of: (a) R632H, R173H and E45M; (b) R632H and Y44E; (c) R632H and Q754A; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO:

1.

2. The polymerase mutant according to claim 1, characterized in that The polymerase mutant is selected from a mutation combination of R632H, R173H and E45M, and the amino acid sequence of the polymerase mutant is shown in SEQ ID NO:

2.

3. The polymerase mutant according to claim 1, characterized in that The polymerase mutant is a combination of R632H and Y44E mutations; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO:

1.

4. The polymerase mutant according to claim 1, characterized in that The polymerase mutant is a combination of R632H and Q754A mutations; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO:

1.

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 genome of the host cell is integrated with the polynucleotide according to claim 5.

8. A method for preparing the T7 RNA polymerase mutant according to claim 1, characterized in that The following steps are involved: (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, thereby obtaining a culture containing the T7 RNA polymerase mutant; (3) isolating and / or purifying the T7 RNA polymerase mutant from the culture.

9. Use of the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1, characterized in that: Reagents or kits for use in in vitro transcription reactions (IVT), or for preparing in vitro transcription reactions (IVT).

10. A reagent for in vitro transcription reaction (IVT), characterized in that: The reagent comprises the T7 RNA polymerase mutant according to claim 1.

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