RNA polymerase variants and RNA synthesis

Abstract

A variant of RNA polymerase is provided, characterized in that the amino acid corresponding to arginine at position 386 in the amino acid sequence of the wild-type T7 RNA polymerase described in SEQ ID NO. 1 is substituted with an aromatic amino acid.

Description

Technology Field

[0001] The present invention relates to an RNA polymerase useful for the production of single-strand RNA, a nucleic acid encoding said RNA polymerase, and a method for the production of single-strand RNA using said RNA polymerase. Background Technology

[0002] mRNA drugs are attracting attention as a new pharmaceutical modality. mRNA drugs are medicines that contain mRNA, which codes for artificially manufactured proteins, as an active ingredient; by inducing the production of proteins within the body upon administration, medical effects based on the function of said proteins are exerted. During the global spread of SARS-CoV-2 infection in 2020, mRNA vaccines, a type of mRNA drug, were put into practical use and administered to many people, making a significant contribution to preventing infection. Advantages compared to other drugs include the fact that they can be designed in a short period based on the genetic information of the virus, and that different types of vaccines can be mass-produced using the same manufacturing process, that is, common facilities.

[0003] In vivo, RNA is synthesized by a transcription reaction using double-strand DNA as a template, catalyzed by RNA polymerase. Although RNA transcription can be reproduced in vitro by providing the necessary elements for the reaction, many RNA polymerases present in prokaryotic and eukaryotic cells are composed of multiple subunits, so the production of each of them poses an obstacle to practical application. On the other hand, RNA polymerases derived from bacteriophages (T7 phage, T3 bacteriophage, K11 phage, etc.), chloroplasts, and mitochondria are composed of a single polypeptide (Single-subunit DNA-dependent RNA polymerase: Non-patent Literature 1).

[0004] RNA polymerase is useful as a tool for genetic engineering research. RNA polymerase derived mainly from T7 phage or SP6 phage has been commercially available as a research reagent. Representative applications include the preparation of labeled RNA used as hybridization probes and nucleic acid amplification methods combined with reverse transcription and double-strand cDNA synthesis reactions. Since its basic enzymatic properties have already been elucidated, bacteriophage-derived RNA polymerase is also used in the industrial production of RNA.

[0005] In the industrial production of RNA, it is advantageous to use RNA polymerase with high stability. Accordingly, variants of T7 RNA polymerase with increased thermal stability are being produced (Patent Document 1). In addition, attempts are being made to produce variants focusing on E42, S43, Y44, E45, M46, G47, A255, R257, A258, G259, A260, L261, and A262 of T7 RNA polymerase (Patent Document 2, Non-Patent Document 2).

[0006] On the other hand, when used in pharmaceuticals administered to humans, RNA needs to have a cap structure at its 5' end so that it is translated efficiently. In addition, it is known that double-strand RNA (dsRNA) is produced as a byproduct in transcription reactions by RNA polymerase, but, for example, double-strand RNA administered into the human body causes an undesirable immune response in therapeutic terms through Toll-like receptors or other pathways. Therefore, for the manufacture of mRNA drugs composed of single-strand RNA, a transcription reaction system capable of synthesizing RNA with a cap structure by suppressing the production of double-strand RNA is advantageous. Prior art literature

[0007] International Open Pamphlet WO2001 / 066705 International Open Pamphlet WO2019 / 036682

[0008] Biotechnology Journal International, 20(3), p1-35, 2017NATURE BIOTECHNOLOGY VOLUME 41, p560-568, 2023 The problem to be solved

[0009] The objective of the present invention is to provide a variant of RNA polymerase that is suitable for producing single-strand RNA having a cap structure, which is more suitable than conventional RNA polymerase, and at the same time has reduced ability to produce double-strand RNA, in order to stably supply high-purity single-strand RNA that can be used for pharmaceuticals, etc. means of solving the problem

[0010] As a result of diligent efforts to solve the above problem, the inventors identified a variant of RNA polymerase capable of efficiently transcribing RNA in the presence of a cap analog by introducing a new mutation at a specific location in the RNA polymerase, and further constructed a high-purity single-strand RNA synthesis system using said variant, thereby completing the present invention.

[0011] For example, the following forms are provided.

[0012] (1) A variant of RNA polymerase, characterized in that the amino acid corresponding to arginine at position 386 in the amino acid sequence of wild-type T7 RNA polymerase described in SEQ ID NO. 1 is substituted with an aromatic amino acid.

[0013] (2) The RNA polymerase variant described in (1), wherein the RNA polymerase variant is a variant in which the above substitution mutation is introduced into an RNA polymerase having an amino acid sequence having 70% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1.

[0014] (3) In (1) or (2) above, an RNA polymerase variant comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1.

[0015] (4) In any one of (1) to (3) above, additionally, an amino acid selected from the group consisting of glutamic acid at position 48, arginine at position 50, phenylalanine at position 51, arginine at position 52, and proline at position 72 in the amino acid sequence described in SEQ ID NO. 1 is substituted with an amino acid different from the original amino acid.

[0016] (5) In the above (4), an RNA polymerase variant in which the amino acid corresponding to the position selected from the group consisting of the 48th, 50th, 51st, 52nd and 72nd positions in the amino acid sequence described in SEQ ID NO. 1 is substituted with alanine.

[0017] (6) In any one of (1) to (5) above, additionally, an amino acid corresponding to an amino acid selected from the group consisting of serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 in the amino acid sequence described in SEQ ID NO. 1 is substituted with an amino acid different from the original amino acid.

[0018] (7) RNA polymerase variant in which, in (6) above, the amino acid corresponding to serine at position 430 of the amino acid sequence described in SEQ ID NO. 1 is substituted with proline, the amino acid corresponding to serine at position 633 of the amino acid sequence described in SEQ ID NO. 1 is substituted with proline, the amino acid corresponding to phenylalanine at position 849 of the amino acid sequence described in SEQ ID NO. 1 is substituted with isoleucine, or the amino acid corresponding to phenylalanine at position 880 of the amino acid sequence described in SEQ ID NO. 1 is substituted with tyrosine.

[0019] (8) A nucleic acid encoding an RNA polymerase variant described in any one of (1) to (7) above.

[0020] (9) A vector containing the nucleic acid described in (8) above.

[0021] (10) A method for producing an RNA polymerase variant comprising the process of culturing a cell into which the nucleic acid described in (8) above has been introduced.

[0022] (11) A method for producing single-strand RNA characterized by using a reaction solution containing an RNA polymerase variant described in any one of (1) to (7) above.

[0023] (12) A method of preparation characterized in that, in the above (11), the reaction solution also includes a single-strand DNA binding protein, a single-strand RNA binding protein, or a low-temperature shock protein.

[0024] (13) A method of manufacturing in (12) above, wherein the single-strand DNA binding protein is the T4 phage gene 32 protein.

[0025] (14) A manufacturing method characterized in that, in the above (12), the reaction solution contains a low-temperature shock protein.

[0026] (15) A method for manufacturing the low-temperature shock protein in the above (14) as CspA. Effects of the invention

[0027] The present invention provides an RNA polymerase variant having RNA transcription activity suitable for producing high-purity RNA in vitro, and a method for producing the same. According to the present invention, an RNA polymerase in which a mutation is introduced at a specific amino acid can synthesize single-strand RNA more efficiently in a reaction solution containing a cap analog, while simultaneously suppressing the generation of double-strand RNA. Therefore, the RNA polymerase variant of the present invention exhibits excellent effects in supplying RNA of higher purity than conventional methods. Brief explanation of the drawing

[0028] Figure 1 shows the total amount of RNA synthesized using the RNA polymerase variant of the present invention in the presence or absence of a cap analog. Figure 2 shows the amount of protein expressed from RNA synthesized using the RNA polymerase variant of the present invention. Figure 3 shows the content of dsRNA in RNAs of different chain lengths synthesized using the RNA polymerase variant of the present invention in the presence of a cap analog. Figure 4 shows the cap retention rate of RNA synthesized using the RNA polymerase variant of the present invention. Specific details for implementing the invention

[0029] (Definition of Terms)

[0030] In this specification, nonpolar aliphatic amino acids refer to glycine, alanine, leucine, isoleucine, valine, and proline, and may be denoted as G, A, L, I, V, and P, respectively. Polar amino acids refer to serine, threonine, glutamine, and asparagine, and are denoted as S, T, Q, and N, respectively. Acidic amino acids refer to glutamic acid and aspartic acid, and are denoted as E and D, respectively. Basic amino acids refer to lysine, arginine, and histidine, and are denoted as K, R, and H, respectively. Aromatic amino acids refer to phenylalanine, tyrosine, and tryptophan, and are denoted as F, Y, and W, respectively. Sulfur-containing amino acids refer to cysteine ​​and methionine, and are denoted as C and M, respectively.

[0031] In this specification, identity of amino acid sequences refers to the ratio of the number of identical amino residues in two amino acid sequences when they are optimally aligned with each other. If a position in the first sequence is occupied by the same amino residue as that of the corresponding position in the second sequence, the sequences are said to exhibit identity with respect to that residue position. Proper alignment of the two sequences can be performed using various algorithms, for example, the BLAST algorithm.

[0032] In this specification, a mutation of an amino acid includes the deletion, addition, insertion, or substitution of one or more amino acids.

[0033] The present invention will be described in detail below.

[0034] 1. RNA polymerase variant of the present invention

[0035] The present invention provides a variant of a single subunit DNA-dependent RNA polymerase suitable for the production of single-strand RNA used in mRNA medicines, etc.

[0036] RNA polymerase refers to an enzyme that has the activity of synthesizing an RNA chain complementary to its sequence using DNA as a template, and among them, those consisting of a single polypeptide chain are called single-subunit DNA-dependent RNA polymerase. The RNA polymerase variant of the present invention is one in which a mutation is introduced into the amino acid sequence of this type of RNA polymerase. Although not specifically limited, the RNA polymerase variant provided by the present invention has improved transcription efficiency in a reaction solution containing a cap analog.

[0037] The RNA polymerase variant of the present invention (hereinafter referred to as the variant of the present invention) is suitably a variant of RNA polymerase derived from a bacteriophage, and examples include RNA polymerase of T7 phage and a variant of RNA polymerase having high amino acid sequence identity with said enzyme (e.g., RNA polymerase derived from T3 bacteriophage, K11 phage, or SP6 phage). Although the present invention is not particularly limited, an RNA polymerase variant of the present invention is exemplified as having an amino acid sequence having 70% or more sequence identity with the amino acid sequence of the wild-type T7 RNA polymerase described in SEQ ID NO. 1. The amino acid sequences of RNA polymerase of T3 bacteriophage (NCBIACCESSION_CAC86264) and K11 phage (NCBIACCESSION_P18147) have 70% or more identity with the amino acid sequence of T7 RNA polymerase. Accordingly, the amino acids of T3 polymerase and K11 polymerase corresponding to specific amino acids present in the amino acid sequence of wild-type T7 RNA polymerase described in SEQ ID NO. 1 can be easily identified (see Non-Patent Literature 1). For example, the position corresponding to R386 of T7 RNA polymerase is position 387 in T3 polymerase and position 409 in K11 polymerase. In one embodiment of the present invention, a variant polypeptide is provided having sequence identity of 90% or more, preferably 95% or more, and more preferably 98% or more with the amino acid sequence of T7 RNA polymerase described in SEQ ID NO. 1. The variant polypeptide has RNA polymerase activity, that is, activity of transcribing RNA using DNA as a template.

[0038] The present invention will be described below with reference to a variant derived from T7 RNA polymerase. A variant in which R386 in the amino acid sequence of the wild-type T7 RNA polymerase described in SEQ ID NO. 1 is substituted with an aromatic amino acid synthesizes single-strand RNA having a cap structure with higher efficiency than the wild-type in a reaction solution containing a cap analog. Suitably, in the variant of the present invention, R386 is substituted with phenylalanine, tyrosine, or tryptophan.

[0039] The RNA polymerase variant of the present invention may have one or more additional mutations, for example, amino acid substitutions, in addition to the substitution of an amino acid corresponding to position R386 in the amino acid sequence of wild-type T7 RNA polymerase. These additional mutations are not particularly limited unless they cause a significant decrease in the activity of RNA transcription of the variant of the present invention. Examples of additional substitutions include the substitution of an amino acid corresponding to an amino acid selected from E48, R50, F51, R52, or P72 in T7 RNA polymerase. By substituting one or more of these five amino acids with other amino acids, the ability to generate double-strand RNA in the variant of the present invention may be reduced. The amino acid after substitution is not particularly limited, but is preferably a nonpolar aliphatic amino acid, and more preferably alanine.

[0040] Additionally, the RNA polymerase variant of the present invention may be one in which a known mutation has been introduced. Patent Document 1 discloses a heat-resistant RNA polymerase variant in which four amino acid substitutions (serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 are each substituted with proline, proline, isoleucine, and tyrosine, respectively) have been introduced into T7 RNA polymerase, a single subunit DNA-dependent RNA polymerase (the amino acid sequence of wild-type T7 RNA polymerase is shown in SEQ ID NO. 1). In the RNA polymerase variant of the present invention, a substitution may be introduced to any one or more amino acids corresponding to the amino acids selected from S430, S633, F849, or F880 above, and preferably, one or more mutations selected from a substitution in proline of the amino acid corresponding to S430, a substitution in proline of the amino acid corresponding to S633, a substitution in isoleucine of the amino acid corresponding to F849, or a substitution in tyrosine of the amino acid corresponding to F880 may be introduced.

[0041] A variant in which R386 of T7 RNA polymerase is substituted with tryptophan, and additionally S430, S633, F849, and F880 are substituted with proline, proline, isoleucine, and tyrosine, respectively, is named T7-a2-a27. The amino acid sequence of T7-a2-a27 is shown in SEQ ID NO. 6. That is, a polypeptide containing the amino acid sequence described in SEQ ID NO. 6 is an example of a variant of the present invention. Compared to the wild-type T7 RNA polymerase, T7-a2-a27 exhibits improved RNA transcription efficiency, particularly in the presence of a cap analog. Additionally, variants in which R50 or F51 of T7-a2-a27 is substituted with alanine are named T7-b7 and T7-b8, respectively. These two variants are examples of variants of the present invention in which the ability to produce double-strand RNA is significantly reduced compared to wild-type T7 RNA polymerase.

[0042] As one embodiment of the present invention, the variant of the present invention may have other peptides added. Examples of peptides other than those mentioned above include signal peptides necessary for the secretory expression of the variant of the present invention and affinity tags useful for the purification of said variant, but are not limited thereto. The above signal peptide may be selected as a suitable one derived from a known secretory protein or a modified one thereof corresponding to the host used for the production of the variant of the present invention, and is usually added to the N-terminus of the variant. In addition, the above affinity tag may be one for which a ligand capable of being used for capture is known, and may be selected from known ones. Examples of affinity tags include the histidine (His) tag, HN tag, HAT tag, glutathione S-transferase (GST) tag, maltose binding protein (MBP) tag, C-Myc tag, and Strep(II) tag consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). These tags may be attached to either the N-terminus or the C-terminus of the variant. Additionally, the affinity tags may be removed from the variant of the present invention after purification is complete. For this purpose, an affinity tag is used that can insert a protease recognition sequence, such as Facto rXa, PreScission Protease, Thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protease), between the RNA polymerase variant of the present invention and the affinity tag.

[0043] The RNA polymerase variant of the present invention can be prepared using known recombinant protein production methods. Based on the gene sequence information of a wild-type single-subunit DNA-dependent RNA polymerase, the amino acid codon corresponding to R386 in the amino acid sequence of T7 RNA polymerase can be identified and changed to an aromatic amino acid codon. A nucleic acid having the sequence designed in this way, i.e., a nucleic acid encoding the variant of the present invention, can be prepared, and by using a suitable expression vector and host, a variant as described below can be obtained. If it is desired to introduce a mutation other than at the position corresponding to R386, a sequence encoding the desired variant can be designed by the same operation. The nucleic acid encoding the variant of the present invention may be chemically synthesized or may be produced by introducing a site-specific mutation into a nucleic acid encoding a wild-type RNA polymerase.

[0044] The RNA polymerase variant of the present invention can be used not only for the production of RNA but also for nucleic acid amplification methods that include RNA polymerase as a component (e.g., NASBA method described in U.S. Patent No. 5130238: Nucleic Acid Sequence-based Amplification; 3SR method described in WO90 / 06995: Self-sustained sequence replication reaction; SMART method described in WO99 / 37806: Signal mediated amplification of RNA technology; TMA method described in WO91 / 01384: Transcription-mediated amplification, etc.). These are methods capable of efficiently amplifying target nucleic acids under isothermal conditions.

[0045] 2. Nucleic acid encoding the RNA polymerase variant of the present invention

[0046] The present invention provides a nucleic acid encoding the RNA polymerase variant described in 1. above (hereinafter referred to as the nucleic acid of the present invention).

[0047] The nucleic acid of the present invention may encode the RNA polymerase variant of the present invention and is not limited to a nucleic acid of a specific nucleotide sequence. As described above, the nucleotide sequence of the nucleic acid of the present invention is designed by specifying a codon corresponding to an amino acid to be substituted based on the gene nucleotide sequence information of the wild-type RNA polymerase, and changing it to a codon of another amino acid. Furthermore, in order to increase the expression level of the RNA polymerase variant of the present invention in a host, the codons of the designed nucleotide sequence may be changed to be suitable for the host without changing the amino acid sequence coded in the sequence (optimization of codons).

[0048] As described above, the RNA polymerase variant of the present invention may have a signal peptide or an affinity tag added to it. Accordingly, the nucleic acid of the present invention comprises a nucleic acid encoding the variant of the present invention to which a signal peptide or an affinity tag is added. The base sequence of the nucleic acid encoding the signal peptide or the affinity tag can be designed according to their amino acid sequences, and in that case, the selection of codons may be performed considering the host being used.

[0049] The nucleic acid encoding the RNA polymerase variant of the present invention may be chemically synthesized or produced by introducing site-specific mutations into a nucleic acid encoding a wild-type RNA polymerase. The method of introducing site-specific mutations into a nucleic acid is well known to those skilled in the art, and kits that can be used for the above method are also commercially available.

[0050] A PET system, a recombinant protein expression system using E. coli as a host, is a method that utilizes RNA polymerase derived from bacteriophages. By placing a nucleic acid encoding a desired protein downstream of a promoter recognized by RNA polymerase and introducing it into E. coli, and then expressing RNA polymerase within the same E. coli, the expression of the desired protein is strongly induced. In this system, RNA polymerase is supplied from an RNA polymerase gene incorporated into the E. coli chromosome or from an expression vector introduced into E. coli. The nucleic acid of the present invention can be used as a source of RNA polymerase in a PET system or a similar expression system.

[0051] 3. A vector comprising a nucleic acid encoding the RNA polymerase variant of the present invention

[0052] The present invention provides a vector having a nucleic acid encoding a variant of the RNA polymerase of the present invention. The vector of the present invention is useful for introducing the nucleic acid encoding the variant of the RNA polymerase of the present invention into a suitable host for the production of the variant of the RNA polymerase of the present invention or for other purposes.

[0053] There are no particular limitations on the vector into which the nucleic acid encoding the RNA polymerase variant of the present invention is inserted. Vectors capable of self-replication in host cells or vectors capable of being incorporated into the host chromosome may be used. For example, plasmid vectors, phage vectors, viral vectors, artificial chromosomes, etc., may be used. The vector is selected to be suitable for the host being used. Vectors suitable for various types of hosts (E. coli, Bacillus species, yeast, filamentous fungi, insect cells, mammalian cells, etc.) are well known to those skilled in the art, and many are also commercially available. In the present invention, these known vectors or their variants may be used. Numerous expression vectors for expressing recombinant proteins in a host have also been established.

[0054] Expression vectors possess a suitable promoter capable of functioning within the host or other factors involved in transcription and translation (operators, terminators, enhancers, ribosome binding sites, etc.), making them suitable for producing the product of the loaded gene in the host. For example, in expression vectors for E. coli, promoters such as the trp promoter, lac promoter, PL promoter, and PR promoter, or their variants, are used, but are not limited to the above. If the promoter is inducible, the expression of the gene product can be induced by appropriate manipulation.

[0055] The expression vector used in the present invention may additionally contain a nucleic acid encoding a signal peptide or an affinity tag. The nucleic acid encoding these peptides is arranged to be expressed as a fusion protein of the RNA polymerase variant of the present invention and the affinity tag. Since a vector into which a nucleic acid encoding an affinity tag is inserted is also known, such a vector may be made into a vector of the present invention by inserting the nucleic acid of the present invention into such a vector. The expression vector loaded with the nucleic acid of the present invention constructed in this way is useful for the production of the RNA polymerase variant of the present invention.

[0056] 4. Method for preparing the RNA polymerase variant of the present invention

[0057] The present invention provides a method for preparing an RNA polymerase variant of the present invention. The method comprises a process of culturing cells into which a nucleic acid encoding the variant of the present invention has been introduced, and is carried out by collecting the RNA polymerase variant from the obtained culture.

[0058] There are no specific limitations on the cell (host) used for the production of the variant of the present invention, as long as it is a host used for the production of recombinant proteins. For example, bacteria (E. coli, Bacillus subtilis, etc.), yeast, filamentous fungi, insect cells, eukaryotic cells, and animal cells (including mammalian cells such as human cells) may be used.

[0059] Representative hosts used for the industrial production of recombinant proteins, such as bacteria of the genus Bacillus (e.g. Escherichia coli and Bacillus subtilis), are well known to those skilled in the art, and many strains are commercially available. Among these, a suitable host strain can be selected and used by considering the production scale and other conditions. In addition, methods for producing recombinant proteins using yeast (genus Saccharomyces or genus Schizosaccharomyces) or insect cells as hosts are also known. By introducing a nucleic acid encoding the RNA polymerase variant of the present invention into these host cells, or by producing a host cell in which the said nucleic acid is incorporated on a chromosome, and by culturing said host, the RNA polymerase variant can be expressed and produced.

[0060] The nucleic acid encoding the RNA polymerase variant of the present invention described above can be loaded into a suitable vector and introduced into a host. As for the vector, for example, one that can be used as an expression vector can be selected. When using a plasmid vector, the method of introduction can be appropriately selected according to the host, for example, a method using calcium ions, the lipoporosis method, the electroporation method, the spheroplast method, the lithium acetate method, etc. Phage vectors or viral vectors can be used to infect host cells using a method corresponding to these vectors, and cells into which the nucleic acid encoding the RNA polymerase variant has been introduced can be obtained. In addition, the above PET system can be used for the production of the variant of the present invention.

[0061] Cells possessing the nucleic acid encoding the RNA polymerase variant obtained in this way can be cultured, and the variant of the present invention can be obtained from the culture. The culture conditions are not particularly limited as long as they are suitable for the host, expression vector, etc. used. In addition, known protein purification methods may be used for the isolation or purification of the RNA polymerase variant. The case in which Escherichia coli is used as a host is described below. Escherichia coli into which the nucleic acid encoding the variant of the present invention has been introduced is cultured at a temperature suitable for its growth, for example, 37°C. If the nucleic acid encoding the RNA polymerase variant is positioned downstream of an inducible promoter, the expression of the RNA polymerase variant can be induced by performing operations suitable for said promoter during culture. After recovering and washing the E. coli cells from the culture, an E. coli lysate containing the variant of the present invention can be obtained by ultrasonic disruption, lysozyme treatment, or other lysis treatments. When the variant of the present invention is secreted and expressed, the supernatant of the culture is recovered. Purification of the RNA polymerase variant of the present invention is carried out by appropriately combining purification methods used in the field, such as ammonium sulfate precipitation, anion exchange column, cation exchange column, gel filtration column, affinity chromatography column, filtration, dialysis, etc., using this lysate or culture supernatant as a starting material. A variant with an affinity tag attached can be easily purified by using an affinity carrier corresponding to the properties of the affinity tag. For example, a variant of the present invention having a plurality of histidines as an affinity tag (histidine tag, HN tag, HAT tag, etc.) can be purified by using a carrier bound with a metal such as nickel.

[0062] 5. Method for preparing single-strand RNA of the present invention

[0063] The present invention provides a method for producing single-strand RNA using a variant of the RNA polymerase of the present invention (in vitro transcription method). In the presence of a double-strand DNA template and four types of ribonucleotide triphosphate, the variant of the present invention synthesizes single-strand RNA having a sequence complementary to the template DNA. The RNA produced by the method of the present invention is not limited to mRNA, RNA probes, ribozymes, guide RNA (used for genome editing by Cas9, etc.), etc. are examples.

[0064] The double-strand DNA that can be used as a template in the method for producing single-strand RNA of the present invention has a promoter sequence recognized by the variant of the present invention. Promoter sequences recognized by bacteriophage-derived RNA polymerase are known (see, for example, FEBS Letters, 4, p264-267, 1998). In addition, it may contain a sequence encoding a poly(A) tail at the 3' end in the region encoding the RNA to be transcribed. There is no particular limitation on the origin of the template double-strand DNA, and it may be plasmid DNA or a DNA fragment obtained by nucleic acid amplification. For example, a double-strand DNA that can be used as a template can be prepared by inserting DNA with a nucleotide sequence encoding the desired RNA downstream of the promoter in a plasmid loaded with a promoter recognized by RNA polymerase. In addition, it is preferable to use circular DNA identical to the plasmid after linearization (e.g., digestion by restriction enzymes).

[0065] Ribonucleotide triphosphate is typically used as one of four types (ATP, CTP, GTP, UTP) that serve as substrates for natural RNA, but one or more of the above four types may be replaced with an analog of ribonucleotide triphosphate. There are no specific limitations on the analogs of ribonucleotide triphosphate. Known analogs known as substrates for RNA polymerase may be used in the method of the present invention. For example, in the preparation of RNA administered to a living organism, pseudouridine triphosphate, 1-methylpseudouridine triphosphate, or other UTP analogs may be used instead of UTP. The concentration of ribonucleotide triphosphate or its analog may be appropriately set according to the purpose, and is typically in the range of 0.2 to 15 mM.

[0066] The reaction solution used for in vitro transfer contains magnesium ions and a reducing agent (such as dithiothreitol) in addition to a buffer component to maintain the pH at an appropriate value. It may also contain various other components, such as salts (NaCl, KCl, etc.), polyamines (such as spermidine), ribonuclease inhibitors, proteins (such as bovine serum albumin), surfactants, etc. The concentrations of these components can be set by referring to known reaction solution compositions. In addition, the pH of the reaction solution is typically prepared in the range of 7.0 to 9.0.

[0067] A modification called a cap structure exists at the 5' end of eukaryotic mRNA. This modification consists of 7-methylguanosine attached to the 5' end via a 5'-5' triphosphate structure and methylation at the 2'-O position on an adjacent nucleotide. The cap structure contributes to the stabilization of mRNA while improving translation efficiency by being recognized by translation initiation factors in eukaryotic cells.

[0068] A cap structure can be added to a single-strand RNA prepared by the method of the present invention. By treating the single-strand RNA with a capping enzyme, for example, a capping enzyme derived from vaccinia virus, 7-methylguanosine is added to the 5' end of the single-strand RNA (Cap0). Subsequently, by acting on mRNA Cap 2'-O-methyltransferase, the 2'-O position of the nucleotide adjacent to the 7-methylguanosine is methylated (Cap1).

[0069] In the method of the present invention, a cap structure can be added in parallel with the transcription of single-strand RNA. In this case, transcription of single-strand RNA using a variant of the present invention is carried out in a reaction solution containing a compound called a cap analog. There are no particular limitations on the cap analog, and a cap analog composed of a dinucleotide, a trinucleotide, or more nucleotides may be used. For example, commercially available cap analogs such as m7G(5')ppp(5')G (standard cap), ARCA (Anti-Reverse Cap Analog), and the CleanCap (registered trademark) series cap analogs (TriLink) may also be used in the present invention.

[0070] The reaction solution of the present invention may contain inorganic pyrophosphatase. When RNA is synthesized by RNA polymerase, pyrophosphate accumulates as a byproduct, but pyrophosphate inhibits the transcription reaction. Since inorganic pyrophosphatase has the activity of catalyzing the hydrolysis of pyrophosphate to produce orthophosphate, the RNA synthesis reaction is promoted by the breakdown of pyrophosphate by said enzyme.

[0071] Furthermore, the present invention provides a method for reducing the production of double-strand RNA in an RNA transcription reaction, characterized by using a reaction solution containing a protein having an affinity for nucleic acids. Examples of proteins having an affinity for nucleic acids may include single-strand DNA binding proteins (SSBs), single-strand RNA binding proteins, or cold shock proteins. When a reaction solution for RNA transcription containing proteins having an affinity for nucleic acids is used, the production of double-strand RNA is suppressed compared to cases where these proteins are not contained. Examples of single-strand DNA binding proteins include T4 phage gene 32 protein (T4 gp32), T7 phage single-strand DNA binding protein, and single-strand DNA binding protein of E. coli. Cold shock proteins are a general term for proteins that are transiently expressed at high levels when the growth temperature is lowered in bacteria, etc. In E. coli, a cold shock protein named CspA is known. In addition, eight types of proteins, CspB to CspI, which have high amino acid sequence identity with CspA, are known, and among them, CspB, CspG, and CspI are cold shock proteins [J. Bacteriol., 181, p1603-1609, 1999). Furthermore, homologues of these cold shock proteins exist in microorganisms such as Bacillus subtilis (CspB), Bacillus caldolticus (CspB), Thermotegamaritima (CspB, CspL), and Lactobacillus plantarum (CspL). Although the present invention is not specifically limited to the RNA transcription reaction, CspA of Escherichia coli is suitable. In the method for reducing the production of double-strand RNA in the above RNA transcription reaction, any RNA polymerase may be used in the above RNA transcription reaction, and wild-type RNA polymerase or mutant-type RNA polymerase may be used. The above variant RNA polymerase is also not limited to the RNA polymerase variant of the present invention.In addition, in the single-strand RNA preparation method of the present invention, a reaction solution containing a protein having affinity for nucleic acid may be used.

[0072] 6. Key of the present invention crack

[0073] The present invention provides a kit containing an RNA polymerase variant of the present invention for use in the synthesis of a desired RNA.

[0074] In addition to the variant of the present invention, the kit of the present invention may contain components necessary for preparing a reaction solution for RNA transcription, such as buffer components, magnesium salts, four types of NTPs or NTP analogs, polyamines, reducing agents, surfactants, or other components. These components may be packaged as individual components so that they can be mixed to prepare a reaction solution at use, or they may be packaged as a mixture of multiple components to facilitate the preparation of the reaction solution. Examples include a kit containing a premix solution prepared so that the reaction solution is completed by adding only template DNA or water (sterile water, etc.) at use, and a kit containing the said premix in a dry state. In addition, components that may be contained in the kit include ribonuclease inhibitors, cap analogs, inorganic pyrophosphatases, bovine serum albumin, or other proteins (single-strand DNA binding proteins, single-strand RNA binding proteins, cryo-shock proteins, etc.).

[0075] Examples

[0076] The present invention is described in more detail below in the embodiments, but the scope of the present invention is not limited to these embodiments.

[0077] Experimental method

[0078] (1) Method for preparing T7 RNA polymerase variant

[0079] The nucleotide sequence of the gene encoding wild-type RNA polymerase derived from the Escherichia phage T7 (Bacteriophage T7) strain is disclosed in NCBI Reference Sequence No. [NC_001604 GeneID: 1261050]. The amino acid sequence of the wild-type T7 RNA polymerase encoded in this nucleotide sequence is shown in Sequence No. 1 of the sequence list. An artificial gene having a nucleotide sequence encoding an amino acid sequence in which a desired mutation was introduced into the above amino acid sequence was chemically synthesized. The obtained artificial gene was introduced into the plasmid pET6xHN-N (Takara Bio Inc. USA) using the In-Fusion (Trademark) HD ​​Cloning Kit (Takara Bio Inc. USA). The obtained plasmid has a nucleotide sequence encoding a T7 RNA polymerase variant with a histidine tag added to the N-terminus.

[0080] Next, Escherichia coli strain BL21 DE3 (TAKARA BIO INC.) was transformed with the above plasmid and cultured overnight at 37°C on a 1.5% agarose LB plate containing ampicillin at a concentration of 100 µg / ml. A single colony from this plate was inoculated into LB medium containing ampicillin at a concentration of 100 µg / ml (hereinafter referred to as 'LB-AP medium') and cultured overnight with shaking at 37°C. 300 µl of this culture medium was inoculated into 25 ml of LB-AP medium and cultured overnight with shaking at 37°C. When the OD600 value reached 0.6, IPTG at a final concentration of 1 mM was added to the culture medium, and induction culture was further performed at 30°C for 4 hours, after which the cells were collected.

[0081] The bacterial cells obtained above were suspended in 2 ml of a solution containing 50 mM Tris·HCl pH 8.0, 100 mM NaCl, 1 mM EDTA (pH 8.0), and 5% glycerol; lysozyme (Sinopharm Chemical Reagent Co., Ltd) was added to achieve a final concentration of 0.1 mg / ml, and the mixture was shaken at 4°C for 1 hour. After shaking, the mixture was centrifuged at 15,000 × g at 4°C for 30 minutes, and the supernatant was collected. This supernatant was applied to a Ni-NTA Agarose (Qiagen) column to adsorb RNA polymerase variants. After washing the column, the adsorbent was eluted with a buffer containing imidazole. Next, the obtained eluent was applied to a POROS HQ (Thermo Fisher Scientific Inc.) column to adsorb RNA polymerase variants. After washing the column, the adsorbate was eluted with a buffer containing sodium chloride. The obtained eluent was dialyzed, and the buffer was substituted with 20 mM potassium phosphate pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol. The obtained buffer substituted solution was used as the RNA polymerase variant solution for each test.

[0082] The activity of the polymerase contained in the above RNA polymerase variant solution was measured using a reaction solution with the composition shown below. In addition, the RNA polymerase activity (number of units) contained in the solution was 1 nmol at 37°C for 1 hour [ 3 The amount of enzyme that incorporates [H]GMP into the acid-insoluble precipitate was calculated as 1U.

[0083] [Composition of reaction solution (50 µl) for activity measurement]

[0084] 40mM Tris-HCl(pH8.0)

[0085] 8mM MgCl2

[0086] 2mM spermidine

[0087] 5mM DTT

[0088] 0.4mM ATP·UTP·CTP

[0089] 0.4mM [ 3 [H]GTP

[0090] 1㎍ / 50㎍ pT7-2DNA (USB)

[0091] (2) Method for evaluating transcriptional RNA amount and dsRNA production amount

[0092] For the prepared T7 RNA polymerase variant, the RNA transcription reaction was tested using the following method. A reaction solution with a final volume of 20 µl containing 10X T7 RNA Polymerase Buffer attached to T7 RNA Polymerase ver2.0 (Product No. 2541, TAKARA BIO INC.), a final concentration of 10 mM NTP, 1 µg of Positive Control Template (FLuc) (a linearized DNA with a sequence encoding firefly luciferase located downstream of the T7 promoter) attached to Takara IVTpro (trademark) mRNA Synthesis System (Product No. 6141, TAKARA BIO INC.) as template DNA (a linearized DNA), 0.1 U of Pyrophosphatase (inorganic) (Product No. 2450, TAKARA BIO INC.), 20 U of RNase Inhibitor ver.2.0 (Product No. 2315, TAKARA BIO INC.), and 200 U of the T7 RNA polymerase variant prepared in Experiment Method (1) was used. A reaction solution containing wild-type T7 RNA polymerase was also prepared.

[0093] RNA transcription was carried out using a Thermalcycler-TP-990 ThermalCycler Dice (registered trademark) Real Time System III (TAKARA BIO INC.) at 37°C for 120 minutes. Afterward, an amount equivalent to 10 U of DNase I (product number 2270, TAKARA BIO INC.) was added, and the reaction was treated at 37°C for 15 minutes to degrade the template DNA. After treatment, the reaction solution was stopped on ice.

[0094] The reaction solutions treated with DNase I were each purified by precipitation using lithium chloride at a final concentration of 3 M, and finally, their precipitates were dissolved in RNase-free water. The RNA concentration in the RNA solution obtained in this way was measured using a spectrophotometer (1 OD at 260 nM = 40 μg / ml RNA). The amount of RNA in the reaction solution calculated from the RNA concentration obtained in this way represents the sum of the single-strand RNA amount and the double-strand RNA amount (total RNA amount). The measurement of the double-strand RNA amount was performed using the EasyAna dsRNA (Modified) Quantitative Detection Kit (ELISA) 2.0 (product number DD3509EN, Vazyme), in accordance with the instructions.

[0095] Example 1: Preparation of T7 RNA polymerase variant

[0096] By the method of the prior art, an artificial gene encoding a variant protein in which the amino acid substitutions listed in Table 1 were introduced into the amino acid sequence of wild-type T7 RNA polymerase was designed and chemically synthesized. A recombinant plasmid containing the artificial gene obtained according to experimental method (1) was constructed, and the expression and purification of the variant were performed. The names of the T7 RNA polymerase variants in which each mutation was introduced and the sequence numbers of the amino acid sequences (sequence numbers 2 to 17) obtained in this way are shown in Table 1.

[0097]

[0098] Also, “SY43-44AA” in Table 1 represents a combination of two amino acid substitutions of S43A and Y44A.

[0099] Example 2: Effect of substitution at position R386

[0100] mRNA of luciferase encoded by the Positive Control Template was synthesized using variants a27, a28, and a29, which are substituted at position R386 of the wild-type T7 RNA polymerase prepared in Example 1. The reaction solution was prepared according to the composition described in Experimental Method (2), and three types were used: one containing a cap analog [CleanCap (registered trademark) Reagent AG (3' OMe) (Trilink)] at a final concentration of 2 mM or 8 mM, and one not containing a cap analog. Additionally, mRNA synthesis using wild-type T7 RNA polymerase was performed as a control. After incubating the reaction solution at 37°C for 120 minutes, the RNA was purified by DNase I treatment, and the RNA concentration was measured. The measured total RNA amount is shown in FIG. 1.

[0101] As can be seen in Figure 1, the total RNA amount of the three variants was reduced compared to the wild-type RNA polymerase. In addition, while the total RNA amount of each variant was correlated with the concentration of the cap analog in the reaction solution, the total RNA amount of the wild-type was not affected by the cap analog.

[0102] The expression of proteins from the RNA obtained in each reaction mixture was verified. RNA was introduced into HEK293T cells grown on a 24-well plate (seed at 1×10^5 cells / well and cultured for 18-24 hours) by adding a mixture of the obtained RNA (500 ng / well) and a transfection reagent (TransIT-mRNA Transfection Reagent). After introduction, luciferase activity in cells incubated at 37°C for 24 hours was measured using the ONE-Glo (trademark) Luciferase Assay System (Promega Corporation) following the procedure described in its instructions. The results (measurements for each test) are shown in Figure 2.

[0103] As shown in Figure 2, RNA obtained using variants a27, a28, and a29 in a reaction mixture containing cap analogs showed higher luciferase expression levels in cells compared to that obtained using wild-type T7 RNA polymerase. That is, by substituting the R386 position of T7 RNA polymerase with W, F, or Y, mRNA with a cap structure added was synthesized more efficiently.

[0104] Example 3: Evaluation of Transcribed RNA by T7 RNA Polymerase Variant

[0105] For the T7 RNA polymerase variant and wild-type T7 RNA polymerase prepared in Example 1, an evaluation test of transcribed RNA was performed. The test was conducted using the reaction solution, reaction conditions, and measurement method described in Experiment Method (2), but regarding the reaction solution, two types were used: one containing CleanCap (registered trademark) Reagent AG (3' OMe) at a final concentration of 8 mM and one not. The total RNA amount and double-strand RNA content obtained from each reaction solution were measured, and the variant and wild-type were compared. The results are shown in Table 2. Also, "T7-WT" in the table refers to the wild-type T7 RNA polymerase.

[0106]

[0107] As shown in Table 2, the amount of double-strand RNA in the presence of a cap analog was reduced in the variant of sequence number 6 (T7-a2_a27) compared to wild-type polymerase. In multiple variants T7-b6, b7, b8, b9, and T7-a2_a7_a27, which were produced by introducing additional mutations into this variant, the amount of dsRNA produced was further suppressed than in T7-a2_a27.

[0108] Example 5: Inhibitory effect of dsRNA production by cold shock protein (CspA)

[0109] CspA protein from E. coli was prepared according to the method described in Chatterjee et al. (Journal of Biochemistry. 114, p663-669, 1993) and used in the following experiments. A reaction solution was prepared by adding CspA of various concentrations and T7 RNA polymerase included in the Takara IVTpro (trademark) T7 mRNA Synthesis Kit (product number 6144, TAKARA BIO INC.) to the reaction solution described in experimental method (2), and the reaction was carried out at 37°C for 120 minutes. The results of measuring the total RNA amount and the double-strand RNA content after the reaction are shown in Table 3.

[0110]

[0111] As shown in Table 3, it was found that adding CspA at a final concentration of 0.8–4.8 μg / µl to the transcription reaction system inhibited the production of dsRNA. In addition, the total RNA amount was not significantly affected by CspA.

[0112] Example 6: Evaluation of dsRNA generation inhibition by T4 Gene 32 Protein

[0113] The effect of T4 gp32 on T7-b7 as a variant was investigated. To the reaction solution described in experimental method (2), T4 Gene 32 Protein (NEB) at various concentrations and T7 RNA polymerase included in the Takara IVTpro (trademark) T7 mRNA Synthesis Kit (6144, TAKARA BIO INC.) were added. The reaction was carried out for 120 minutes at temperatures of 37°C and 42°C, respectively. The results of measuring the total RNA amount and double-strand RNA content after the reaction are shown in Table 4.

[0114]

[0115] As shown in Table 4, the amount of double-strand RNA produced was suppressed by adding 0.2 to 0.8 μg / µl of T4 Gene 32 Protein to the reaction system.

[0116] Example 7: Evaluation of dsRNA generated by RNA transcription of different chain lengths

[0117] For the T7 RNA polymerase variant T7-b7 and wild-type T7 RNA polymerase prepared in Example 1, template DNA encoding RNA of different chain lengths was prepared, and an evaluation test of the transcribed RNA was performed.

[0118] As template DNA encoding RNA of different chain lengths, seven types of DNA with base sequences were prepared to generate RNA of 0.7kb (ZsGreen), 1.2kb (B-actin), 1.7kb (FLuc), 4.2kb (Cas9), 7.5kb (FASN), 10kb (Lambda DNA), and 12kb (Lambda DNA), respectively, upon transcription. Next, plasmid DNA in which each DNA was inserted downstream of the T7 promoter was constructed, and each plasmid was further linearized using restriction enzymes to prepare seven types of template DNA. Next, an RNA transcription test was performed using the reaction solution, reaction conditions, and measurement method of experimental method (2), but the above-mentioned linearized plasmids were used for the template DNA, and CleanCap (registered trademark) Reagent AG (3' OMe) with a final concentration of 4mM was added to the reaction solution. The dsRNA content (ng) per 1 μg of total RNA was calculated from the total RNA amount and double-strand RNA amount obtained from each reaction solution, and the variant T7-b7 and wild-type RNA polymerase were compared. The results are shown in Figure 3. Also, "T7-WT" in the figure refers to wild-type T7 RNA polymerase.

[0119] As shown in Figure 3, in the presence of CleanCap (trademark) Reagent AG (3' OMe) at a final concentration of 4 mM, variant T7-b7 was RNA that was not affected by the chain length of the template DNA and had a lower dsRNA content than wild-type T7 RNA polymerase.

[0121] Example 8: Confirmation of retention rate of cap analogue by LC-MS

[0122] For RNA prepared with the T7 RNA polymerase variant T7-b7 and wild-type T7 RNA polymerase prepared in Example 1, the cap retention rate was verified using LC-MS (liquid chromatography-mass spectrometry). RNA of luciferase encoded therein was synthesized using a Positive Control Template included in the Takara IVTpro (trademark) mRNA Synthesis System as a template. The reaction solution was prepared according to the composition described in experimental method (2), and five types were used, including those containing a cap analog [CleanCap (registered trademark) Reagent AG (3' OMe) (Trilink)] at final concentrations of 2 mM, 4 mM, 6 mM, or 8 mM, and those not containing a cap analog. The reaction solution, incubated at 37°C for 120 minutes, was treated with DNase I to purify the RNA. The Cap1 retention rate of purified RNA was measured by the method of Michael Beverly et al. using analysis by LC-MS (Anal Bioana lChem. 2016 Jul; 408(18): 5021-30). The results are shown in Fig. 4. Also, "T7-WT" in the figure refers to wild-type T7 RNA polymerase.

[0123] As can be seen in Figure 4, under all cap analog concentration conditions used in the test, the cap retention rate of RNA obtained using variant T7-b7 was higher than that of wild-type polymerase. In T7-b7, even when the cap analog concentration was reduced to 2 mM, the cap retention rate was high at 95.3%, which was equivalent to 8 mM for the wild type. In addition, at each cap analog concentration, there was no significant difference in the total amount of RNA produced using both enzymes. On the other hand, when no cap analog was added, the total amount of RNA in wild-type T7 RNA polymerase exceeded that of T7-b7. This was consistent with the results shown in Figure 1.

[0124] (Industrial Applicability)

[0125] The present invention, which exhibits excellent effects when manufacturing high-purity mRNA, is useful in a wide range of fields such as genetic engineering, biology, and medicine.

[0126] Sequence List Free Text

[0127] SEQ ID NO1: T7-WT

[0128] SEQ ID NO2: a27

[0129] SEQ ID NO3: a28

[0130] SEQ ID NO4: a29

[0131] SEQ ID NO5: T7-a2

[0132] SEQ ID NO6: T7-a2_a27

[0133] SEQ ID NO7: T7-b2

[0134] SEQ ID NO8: T7-b3

[0135] SEQ ID NO9: T7-b4

[0136] SEQ ID NO10: T7-b5

[0137] SEQ ID NO11: T7-a2_a5_a27

[0138] SEQ ID NO12: T7-b1

[0139] SEQ ID NO13: T7-b6

[0140] SEQ ID NO14: T7-b7

[0141] SEQ ID NO15: T7-b8

[0142] SEQ ID NO16: T7-b9

[0143] SEQ ID NO17: T7-a2_a7_a27

Claims

Claim 1 A variant of RNA polymerase characterized in that the amino acid corresponding to arginine at position 386 in the amino acid sequence of the wild-type T7 RNA polymerase described in SEQ ID NO. 1 is substituted with an aromatic amino acid. Claim 2 In claim 1, the RNA polymerase variant is a variant in which the substitution mutation is introduced into an RNA polymerase comprising an amino acid sequence having 70% or more sequence identity with the amino acid sequence described in SEQ ID NO.

1. Claim 3 An RNA polymerase variant according to claim 1 or 2, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO.

1. Claim 4 An RNA polymerase variant according to any one of claims 1 to 3, wherein, additionally, the amino acid corresponding to the amino acid selected from the group consisting of glutamic acid at position 48, arginine at position 50, phenylalanine at position 51, arginine at position 52, and proline at position 72 in the amino acid sequence described in SEQ ID NO. 1 is substituted with an amino acid different from the original amino acid. Claim 5 In claim 4, an RNA polymerase variant in which an amino acid corresponding to a position selected from the group consisting of positions 48, 50, 51, 52, and 72 in the amino acid sequence described in SEQ ID NO. 1 is substituted with alanine. Claim 6 An RNA polymerase variant according to any one of claims 1 to 5, wherein, additionally, the amino acid corresponding to the amino acid selected from the group consisting of serine at position 430, serine at position 633, phenylalanine at position 849, and phenylalanine at position 880 in the amino acid sequence described in SEQ ID NO. 1 is substituted with an amino acid different from the original amino acid. Claim 7 In claim 6, an RNA polymerase variant in which the amino acid corresponding to serine at position 430 in the amino acid sequence described in SEQ ID NO. 1 is substituted with proline, the amino acid corresponding to serine at position 633 in the amino acid sequence described in SEQ ID NO. 1 is substituted with proline, the amino acid corresponding to phenylalanine at position 849 in the amino acid sequence described in SEQ ID NO. 1 is substituted with isoleucine, or the amino acid corresponding to phenylalanine at position 880 in the amino acid sequence described in SEQ ID NO. 1 is substituted with tyrosine. Claim 8 A nucleic acid encoding an RNA polymerase variant described in any one of claims 1 to 7. Claim 9 A vector containing the nucleic acid described in claim 8. Claim 10 A method for producing an RNA polymerase variant comprising a process of culturing cells into which the nucleic acid described in claim 8 has been introduced. Claim 11 A method for producing single-strand RNA characterized by using a reaction solution containing an RNA polymerase variant described in any one of claims 1 to 7. Claim 12 A method of preparation according to claim 11, characterized in that the reaction solution further comprises a single-strand DNA binding protein, a single-strand RNA binding protein, or a low-temperature shock protein. Claim 13 A method of preparation according to claim 12, wherein the single-strand DNA binding protein is the T4 phage gene 32 protein. Claim 14 A method of manufacturing characterized in that, in claim 12, the reaction solution contains a low-temperature shock protein. Claim 15 In claim 14, a method for manufacturing a low-temperature shock protein in which CspA is used.

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