Modified t7 RNA polymerase

Abstract

The present disclosure addresses the problem of providing a modified T7 RNA polymerase.　The aforementioned problem is solved by providing a T7 RNA polymerase selected from any of the following (i) to (iii). (i) A T7 RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 1, said amino acid sequence including at least the amino acid substitution indicated by (1): (1) a substitution in which an amino acid residue corresponding to the valine residue at position 114 in SEQ ID NO: 1 is substituted with an isoleucine residue, a threonine residue, an alanine residue, a cysteine residue, a glutamic acid residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a glutamine residue, an arginine residue, a serine residue, or a tryptophan residue; (ii) a T7 RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 1, said amino acid sequence including at least the amino acid substitution indicated by (1), and furthermore including one or more of a substitution, a deletion, an insertion, and an addition of one or several amino acid residues at one or several positions other than the amino acid substitution indicated by (1), and the T7 RNA polymerase having enzymatic activity; or (iii) a T7 RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 1, said amino acid sequence having 70% or greater identity to the entire amino acid sequence including at least the amino acid substitution indicated by (1), with the proviso that the amino acid substitution indicated by (1) is maintained, and the T7 RNA polymerase having enzymatic activity.

Description

Modified T7 RNA polymerase

[0001] The present disclosure relates to a modified T7 RNA polymerase.

[0002] T7 RNA polymerase is known as an RNA polymerase that synthesizes RNA using DNA as a template, that is, a DNA-dependent RNA polymerase. Also, T7 RNA polymerase is known to have high specificity for the T7 phage promoter and is often used in in vitro transcription reactions.

[0003] In Patent Document 1, mutations such as Q786L / M, V685A, and K179E / R / C mutations have been reported as mutations that improve the thermal stability and / or specific activity of T7 RNA polymerase.

[0004] In Patent Document 2, the M490V mutation that improves the productivity of T7 RNA polymerase has been reported.

[0005] In Patent Document 3, mutations such as S430P, F849I, and F880Y mutations have been reported as mutations that improve the thermal stability of T7 RNA polymerase.

[0006] In Patent Document 4, mutations such as Q744, F849I, F880Y, S430P, C510R, and S767G mutations have been reported as mutations that improve the thermal stability of T7 RNA polymerase.

[0007] WO2010 / 016621 JP 2012-075418 A JP 2001-54387 A JP 2014-528730 A

[0008] The object of this disclosure is to provide modified T7 RNA polymerases. Specifically, in one embodiment, the object is to provide a T7 RNA polymerase with improved thermal stability. In another embodiment, the object is to provide a T7 RNA polymerase with improved resistance to reaction inhibition by contaminants. In yet another embodiment, the object is to provide a T7 RNA polymerase with improved productivity. In yet another embodiment, the object is to provide a T7 RNA polymerase with improved thermal stability and productivity. In yet another embodiment, the object is to provide a T7 RNA polymerase with improved specific activity (RNA productivity).

[0009] The inventors have found amino acid mutations that improve the thermal stability, resistance to reaction inhibition by contaminants, productivity, and / or specific activity (RNA productivity) of T7 RNA polymerase. That is, this disclosure provides the following embodiments.

[0010] [1] A T7 RNA polymerase selected from any of (i) to (iii) below: (i) A T7 RNA polymerase having an amino acid sequence described in SEQ ID NO: 1, which includes at least one of the amino acid substitutions shown in (1) to (12) below: (1) The amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue, threonine residue, alanine residue, cysteine ​​residue, glutamic acid residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, or tryptophan residue. (2) The amino acid residue corresponding to the 835th threonine residue in SEQ ID NO: 1 is replaced with a leucine residue. (3) The amino acid residue corresponding to the 127th threonine residue in SEQ ID NO: 1 is replaced with an alanine residue. (4) The amino acid residue corresponding to the 128th serine residue in SEQ ID NO: 1 is replaced with an asparagine residue. (5) The amino acid residue corresponding to the 242nd glutamic acid residue in SEQ ID NO: 1 is replaced with an isoleucine residue. (6) The amino acid residue corresponding to the 384th valine residue in SEQ ID NO: 1 is replaced with an alanine residue. (7) The amino acid residue corresponding to the 543rd isoleucine residue in SEQ ID NO: 1 is replaced with a leucine residue. (8) The amino acid residue corresponding to the 581st isoleucine residue in SEQ ID NO: 1 is replaced with a methionine residue. (9) The amino acid residue corresponding to the 630th threonine residue in SEQ ID NO: 1 is replaced with a valine residue. (10) The amino acid residue corresponding to the 687th valine residue in SEQ ID NO: 1 is replaced with a glutamic acid residue. (11) The amino acid residue corresponding to the 861st methionine residue in SEQ ID NO: 1 is replaced with a leucine residue. (12) The amino acid residue corresponding to the 810th isoleucine residue in SEQ ID NO: 1 is replaced with a valine residue. (ii) An amino acid sequence described in Sequence ID No. 1, which includes at least one of the amino acid substitutions shown in (1) to (12) above, and further includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (12), and has enzymatic activity, and is a T7 RNA polymerase;(iii) An amino acid sequence described in Sequence ID No. 1, which has 70% or more identity with the entire amino acid sequence containing at least one of the amino acid substitutions shown in (1) to (12) above, provided that the amino acid substitution shown in (1) above is maintained, and is a T7 RNA polymerase that has enzymatic activity.

[0011] [2] The T7 RNA polymerase according to [1], having the amino acid sequence described in Sequence ID No. 1, which includes at least the amino acid substitution shown in (1'); (1') The amino acid residue corresponding to the 114th valine residue of Sequence ID No. 1 is substituted with an isoleucine residue.

[0012] [3] The T7 RNA polymerase of [1] or [2], wherein the amino acid sequence having the T7 RNA polymerase further comprises one or more amino acid substitutions selected from (a) to (q) below: (a) The amino acid residue corresponding to the 430th serine residue of SEQ ID NO: 1 is substituted with a proline residue; (b) The amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 is substituted with a valine or alanine residue; (c) The amino acid residue corresponding to the 510th cysteine ​​residue of SEQ ID NO: 1 is substituted with an arginine residue; (d) The amino acid residue corresponding to the 767th serine residue of SEQ ID NO: 1 is substituted with a glycine residue; (e) The amino acid residue corresponding to the 786th glutamine residue of SEQ ID NO: 1 is substituted with a methionine residue; (f) The amino acid residue corresponding to the 849th phenylalanine residue of SEQ ID NO: 1 is substituted with an isoleucine residue; (g) The amino acid residue corresponding to the 880th phenylalanine residue of SEQ ID NO: 1 is substituted with a tyrosine residue (h) The amino acid residue corresponding to the 108th glutamic acid residue in SEQ ID NO: 1 is replaced with a glycine residue; (i) The amino acid residue corresponding to the 441st lysine residue in SEQ ID NO: 1 is replaced with an arginine residue; (j) The amino acid residue corresponding to the 446th leucine residue in SEQ ID NO: 1 is replaced with a phenylalanine residue; (k) The amino acid residue corresponding to the 495th serine residue in SEQ ID NO: 1 is replaced with an aspartic acid residue; (l) The amino acid residue corresponding to the 505th glutamine residue in SEQ ID NO: 1 is replaced with an alanine residue; (m) The amino acid residue corresponding to the 530th cysteine ​​residue in SEQ ID NO: 1 is replaced with a glycine residue; (n) The amino acid residue corresponding to the 534th leucine residue in SEQ ID NO: 1 is replaced with a valine residue; (o) The amino acid residue corresponding to the 633rd serine residue in SEQ ID NO: 1 is replaced with a proline residue; (p) The amino acid residue corresponding to the 650th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue; (q) The amino acid residue corresponding to the 832nd methionine residue in Sequence ID No. 1 is replaced with a phenylalanine residue.

[0013] [4] Any T7 RNA polymerase from [1] to [3], wherein (i) to (iii) are (iv) to (vi) below: (iv) A T7 RNA polymerase having the amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or 105; (v) A T7 RNA polymerase having an amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, which includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and which has enzymatic activity; (vi) A T7 RNA polymerase having an amino acid sequence that has 70% or more identity with the amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, and which is enzymatically active.

[0014] [5] A polynucleotide encoding any of the T7RNA polymerases from [1] to [4]. [6] An expression vector containing the polynucleotide from [5]. [7] A transformant obtained by transforming a host with the expression vector from [6]. [8] A transformant from [7] in which the host is Escherichia coli. [9] A method for producing T7RNA polymerase, comprising the steps of culturing the transformant from [7] or [8] to express T7RNA polymerase, and recovering the polymerase expressed from the resulting culture.

[10] An amplification reagent for a target nucleic acid containing any of the T7RNA polymerases from [1] to [4].

[0015] Figure showing the relative transcriptional activity of T7RNA polymerase T7m5 with known amino acid substitutions introduced, and T7m6 with the additional amino acid substitution of V114I added to T7m5. Figure showing the purified yield (relative value) per culture medium of each T7RNA polymerase variant: T7m5 with known amino acid substitutions introduced, T7m6 with the additional amino acid substitution of V114I added to T7m5, and T7V114T with the additional amino acid substitution of V114T added to T7m5. Figure showing the results of measuring the change in transcript amount at each reaction temperature for commercially available heat-stable T7RNA polymerase and T7RNA polymerase T7m5 with known amino acid substitutions introduced, and T7m12-a, T7m12-b, and T7m12-c with the amino acid substitutions introduced in this disclosure, measured by fluorescence intensity. The figure shows the changes in transcript yield at various reaction temperatures for commercially available heat-resistant T7RNA polymerase, T7RNA polymerase T7m5 with known amino acid substitutions introduced, and T7m12-a, T7m12-b, and T7m12-c with the amino acid substitutions described herein, expressed as relative values ​​with the fluorescence intensity of each T7RNA polymerase at 46°C set to 100%. The figure also shows a comparison of the purified yield (relative value) per culture medium for T7RNA polymerase T7m5 with known amino acid substitutions introduced and for T7RNA polymerase mutants with amino acid substitutions introduced at predetermined positions.

[0016] <1> T7 RNA polymerase This disclosure provides a T7 RNA polymerase having the “specific mutations” described herein. More specifically, the present disclosure provides a T7 RNA polymerase selected from any of (i) to (iii) below: (i) a T7 RNA polymerase having the amino acid sequence described in SEQ ID NO: 1, wherein at least one of the following amino acid substitutions is included: (1) the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue, a threonine residue, an alanine residue, a cysteine ​​residue, a glutamic acid residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a glutamine residue, an arginine residue, a serine residue, or a tryptophan residue; (2) the amino acid residue corresponding to the 835th threonine residue in SEQ ID NO: 1 is replaced with a leucine residue; (3) the amino acid residue corresponding to the 127th threonine residue in SEQ ID NO: 1 is replaced with an alanine residue; (4) the amino acid residue corresponding to the 128th serine residue in SEQ ID NO: 1 is replaced with an asparagine residue (5) The amino acid residue corresponding to the 242nd glutamic acid residue in SEQ ID NO: 1 is replaced with an isoleucine residue. (6) The amino acid residue corresponding to the 384th valine residue in SEQ ID NO: 1 is replaced with an alanine residue. (7) The amino acid residue corresponding to the 543rd isoleucine residue in SEQ ID NO: 1 is replaced with a leucine residue. (8) The amino acid residue corresponding to the 581st isoleucine residue in SEQ ID NO: 1 is replaced with a methionine residue. (9) The amino acid residue corresponding to the 630th threonine residue in SEQ ID NO: 1 is replaced with a valine residue. (10) The amino acid residue corresponding to the 687th valine residue in SEQ ID NO: 1 is replaced with a glutamic acid residue. (11) The amino acid residue corresponding to the 861st methionine residue in SEQ ID NO: 1 is replaced with a leucine residue. (12) The amino acid residue corresponding to the 810th isoleucine residue in SEQ ID NO: 1 is replaced with a valine residue.(ii) A T7 RNA polymerase having an amino acid sequence as described in Sequence ID No. 1, which includes at least one of the amino acid substitutions shown in (1) to (12) above, and further includes at least one substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (12), and having enzymatic activity; (iii) A T7 RNA polymerase having an amino acid sequence as described in Sequence ID No. 1, which has 70% or more identity with the entire amino acid sequence including at least one of the amino acid substitutions shown in (1) to (12) above, provided that the amino acid substitutions shown in (1) to (12) above are maintained, and having enzymatic activity.

[0017] T7RNA polymerase may refer to RNA polymerase originating from RNA polymerase derived from T7 phage. RNA polymerase may refer to a protein that has the activity to synthesize RNA. T7RNA polymerase preferably has the activity to catalyze the transcription reaction that synthesizes RNA using DNA as a template. This activity is also called "transcription enzyme activity." It is also called "DNA-dependent RNA polymerase activity." In other words, the enzyme activity of T7RNA polymerase preferably includes DNA-dependent RNA polymerase activity. Hereinafter, unless otherwise specified, "enzyme activity" in this application means the "enzyme activity of T7RNA polymerase" described above. Wild-type T7RNA polymerase typically has DNA-dependent RNA polymerase activity. Note that T7RNA polymerase having a certain amino acid sequence may refer to T7RNA polymerase composed of a protein containing that amino acid sequence.

[0018] The gene that codes for T7 RNA polymerase is also called the "T7 RNA polymerase gene."

[0019] The T7RNA polymerase of this disclosure has a "specific mutation." A T7RNA polymerase having a "specific mutation" is also referred to as a modified T7RNA polymerase or mutant T7RNA polymerase. In other words, the T7RNA polymerase of this disclosure is a mutant T7RNA polymerase. The gene encoding the mutant T7RNA polymerase is also referred to as the "mutant T7RNA polymerase gene." As an example, the "mutant T7RNA polymerase gene" may represent a polynucleotide encoding the mutant T7RNA polymerase.

[0020] T7RNA polymerase that does not possess the "specific mutation" is also called "wild-type T7RNA polymerase." The gene encoding wild-type T7RNA polymerase is also called the "wild-type T7RNA polymerase gene." The term "wild-type" here is merely a convenient designation to distinguish wild-type T7RNA polymerase from mutant T7RNA polymerase, and is not limited to naturally occurring polymerase as long as it does not possess the "specific mutation." Wild-type T7RNA polymerase may or may not possess mutations other than the "specific mutation," as long as it does not possess the "specific mutation."

[0021] When a certain wild-type T7RNA polymerase and a certain mutant T7RNA polymerase are identical except for the presence or absence of a "specific mutation," the wild-type T7RNA polymerase may also be referred to as "the wild-type T7RNA polymerase corresponding to a certain mutant T7RNA polymerase," and the mutant T7RNA polymerase may also be referred to as "the mutant T7RNA polymerase corresponding to a certain wild-type T7RNA polymerase."

[0022] The following describes wild-type T7 RNA polymerase.

[0023] Wild-type T7 RNA polymerase may or may not have enzymatic activity, as long as its corresponding mutant T7 RNA polymerase does. Wild-type T7 RNA polymerase usually has enzymatic activity. Specifically, wild-type T7 RNA polymerase may have transcription activity, i.e., DNA-dependent RNA polymerase activity.

[0024] Wild-type T7 RNA polymerase is not limited to naturally occurring forms, as long as it does not contain the "specific mutation," and may contain mutations other than the "specific mutation." Here, mutations other than the "specific mutation" include known mutations that have been reported to be introduced into T7 RNA polymerase. Specifically, mutations other than the "specific mutation" include, for example, the mutations described in WO2010 / 016621, JP 2012-075418, JP 2001-54387, or JP 2014-528730.

[0025] More specifically, mutations other than the "specific mutations" include, for example, the substitution of the amino acid residue corresponding to the lysine residue at position 179 of SEQ ID NO: 1 (described below) with a glutamate residue, arginine residue, or cysteine ​​residue; the substitution of the amino acid residue corresponding to the serine residue at position 430 of SEQ ID NO: 1 with a proline residue; the substitution of the amino acid residue corresponding to the methionine residue at position 490 of SEQ ID NO: 1 with a valine residue or alanine residue; the substitution of the amino acid residue corresponding to the cysteine ​​residue at position 510 of SEQ ID NO: 1 with an arginine residue; the substitution of the amino acid residue corresponding to the valine residue at position 687 of SEQ ID NO: 1 with an alanine residue; the substitution of the amino acid residue corresponding to the glutamine residue at position 744 of SEQ ID NO: 1 with a leucine residue, arginine residue, or proline residue; the substitution of the amino acid residue corresponding to the serine residue at position 767 of SEQ ID NO: 1 with a glycine residue; the substitution of the amino acid residue corresponding to the glutamine residue at position 786 of SEQ ID NO: 1 with a methionine residue or leucine residue; the substitution of the amino acid residue corresponding to the phenylalanine residue at position 849 of SEQ ID NO: 1 with an isoleucine residue; and the substitution of the phenylalanine residue at position 880 of SEQ ID NO: 1 with a glycine residue. The mutation may include one or more mutations selected from the following: substitution of an amino acid residue corresponding to a nyalanine residue with a tyrosine residue; substitution of an amino acid residue corresponding to the 108th glutamic acid residue in SEQ ID NO: 1 with a glycine residue; substitution of an amino acid residue corresponding to the 441st lysine residue in SEQ ID NO: 1 with an arginine residue; substitution of an amino acid residue corresponding to the 446th leucine residue in SEQ ID NO: 1 with a phenylalanine residue; substitution of an amino acid residue corresponding to the 495th serine residue in SEQ ID NO: 1 with an aspartic acid residue; substitution of an amino acid residue corresponding to the 505th glutamine residue in SEQ ID NO: 1 with an alanine residue; substitution of an amino acid residue corresponding to the 530th cysteine ​​residue in SEQ ID NO: 1 with a glycine residue; substitution of an amino acid residue corresponding to the 534th leucine residue in SEQ ID NO: 1 with a valine residue; substitution of an amino acid residue corresponding to the 633rd serine residue in SEQ ID NO: 1 with a proline residue; substitution of an amino acid residue corresponding to the 650th valine residue in SEQ ID NO: 1 with an isoleucine residue; and substitution of an amino acid residue corresponding to the 832nd methionine residue in SEQ ID NO: 1 with a phenylalanine residue.

[0026] The wild-type T7 RNA polymerase may specifically be, for example, a T7 RNA polymerase having the registered sequence (SEQ ID NO: 1) of GenBank No. ACO57213.1, or a T7 RNA polymerase having the amino acid sequence (SEQ ID NO: 2) in which the amino acid sequence of SEQ ID NO: 1 is modified by substituting the amino acid residue corresponding to the 430th serine residue with a proline residue, the amino acid residue corresponding to the 490th methionine residue with a valine residue, the amino acid residue corresponding to the 510th cysteine ​​residue with an arginine residue, the amino acid residue corresponding to the 767th serine residue with a glycine residue, the amino acid residue corresponding to the 786th glutamine residue with a methionine residue, the amino acid residue corresponding to the 849th phenylalanine residue with an isoleucine residue, and the amino acid residue corresponding to the 880th phenylalanine residue with a tyrosine residue. An example of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 14. Note that the nucleotide sequence shown in SEQ ID NO: 14 is registered with GenBank No. This is the nucleotide sequence encoding wild-type T7 RNA polymerase, extracted from FJ881694.1. An example of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 3. The wild-type T7 RNA polymerase gene may, for example, be a gene having the nucleotide sequence shown in SEQ ID NO: 14 or 3. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the said nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the said nucleotide sequence or amino acid sequence.

[0027] Wild-type T7RNA polymerase may be a variant of the wild-type T7RNA polymerase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 1 or 2), as long as it does not have a "specific mutation." Similarly, a wild-type T7RNA polymerase gene may be a variant of the wild-type T7RNA polymerase gene exemplified above (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 14 or 3), as long as the encoding T7RNA polymerase does not have a "specific mutation." In other words, the term "wild-type T7RNA polymerase" may include the wild-type T7RNA polymerase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 1 or 2) as well as their variants. Similarly, the term "wild-type T7RNA polymerase gene" may include the wild-type T7RNA polymerase gene exemplified above (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 14 or 3) as well as their variants. Examples of variants include artificially modified versions of the genes and proteins exemplified above.

[0028] The wild-type T7RNA polymerase gene may encode a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 1 or 2), provided that the encoding T7RNA polymerase does not have a "specific mutation". For example, the encoded protein may have an elongated or shortened N-terminus and / or C-terminus. Note that the above "one or several" or "one or several" may vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically, for example, it may be 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0029] The above substitutions, deletions, insertions, or additions of one or more amino acids are conservative mutations that maintain the original function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Glun and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered to be conservative substitutions include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include substitutions of e to Leu, Met, Val, or Phe; substitutions of Leu to Ile, Met, Val, or Phe; substitutions of Lys to Asn, Glu, Gln, His, or Arg; substitutions of Met to Ile, Leu, Val, or Phe; substitutions of Phe to Trp, Tyr, Met, Ile, or Leu; substitutions of Ser to Thr or Ala; substitutions of Thr to Ser or Ala; substitutions of Trp to Phe or Tyr; substitutions of Tyr to His, Phe, or Trp; and substitutions of Val to Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants), such as those based on individual differences in the organism from which the gene originates.

[0030] Furthermore, the wild-type T7RNA polymerase gene may also be a gene that encodes a protein having an amino acid sequence that has, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity with respect to the entire amino acid sequence, as long as the encoding T7RNA polymerase does not have a "specific mutation".

[0031] Furthermore, the wild-type T7RNA polymerase gene may also be a gene, such as DNA, that hybridizes under stringent conditions with a probe, such as a complementary sequence to all or part of the above base sequence, which can be prepared from the above base sequence (for example, the base sequence shown in SEQ ID NO: 14 or 3), provided that the encoding T7RNA polymerase does not have a "specific mutation." "Stringent conditions" may mean conditions under which a so-called specific hybrid is formed and a nonspecific hybrid is not formed. For example, one example is a condition in which DNA with high identity, for instance, DNA with 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity, hybridizes with each other, while DNA with lower identity does not hybridize. Alternatively, one example is a condition in which the DNA is washed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0032] As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When using a DNA fragment of about 300 bp in length as the probe, the conditions for washing during hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0033] Furthermore, since codon degeneracy differs depending on the host, the wild-type T7RNA polymerase gene may be modified by substituting any codon with an equivalent codon. In other words, the wild-type T7RNA polymerase gene may be a variant of the wild-type T7RNA polymerase gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type T7RNA polymerase gene may be modified to have the optimal codons depending on the codon usage frequency of the host being used.

[0034] Note that "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11, Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. Furthermore, "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated using the default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear) by blastn.

[0035] The mutant T7 RNA polymerase is described below.

[0036] The mutant T7 RNA polymerase possesses enzymatic activity.

[0037] As mentioned above, "enzyme activity" refers to "the enzymatic activity possessed by T7 RNA polymerase." As a preferred example, the enzymatic activity of a mutant T7 RNA polymerase is transcriptional activity, that is, DNA-dependent RNA polymerase activity.

[0038] Mutant T7RNA polymerase possesses "specific mutations" compared to wild-type T7RNA polymerase.

[0039] In other words, mutant T7RNA polymerase may be an enzyme having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in, for example, SEQ ID NO: 1 or 2. Alternatively, mutant T7RNA polymerase may be an enzyme having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in, for example, SEQ ID NO: 1 or 2, and further includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and is enzymatically active.

[0040] In other words, mutant T7RNA polymerase may be an enzyme having the same amino acid sequence as wild-type T7RNA polymerase, except for having a "specific mutation." That is, mutant T7RNA polymerase may be an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2, except for having a "specific mutation." Also, mutant T7RNA polymerase may be an enzyme having an amino acid sequence that includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in the amino acid sequence shown in SEQ ID NO: 1 or 2, except for having a "specific mutation," and possessing enzymatic activity. Furthermore, mutant T7RNA polymerase may be an enzyme having an amino acid sequence that has 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, except for having a "specific mutation," and possessing enzymatic activity.

[0041] More specifically, the mutant T7 RNA polymerase may be an enzyme selected from any of (i) to (iii) below: (i) an enzyme having the amino acid sequence described in SEQ ID NO: 1, which includes at least one of the amino acid substitutions shown in (1) to (12) below: (1) The amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue, threonine residue, alanine residue, cysteine ​​residue, glutamic acid residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, or tryptophan residue. (2) The amino acid residue corresponding to the 835th threonine residue in SEQ ID NO: 1 is replaced with a leucine residue. (3) The amino acid residue corresponding to the 127th threonine residue in SEQ ID NO: 1 is replaced with an alanine residue. (4) The amino acid residue corresponding to the 128th serine residue in SEQ ID NO: 1 is replaced with an asparagine residue. (5) The amino acid residue corresponding to the 242nd glutamic acid residue in SEQ ID NO: 1 is replaced with an isoleucine residue. (6) The amino acid residue corresponding to the 384th valine residue in SEQ ID NO: 1 is replaced with an alanine residue. (7) The amino acid residue corresponding to the 543rd isoleucine residue in SEQ ID NO: 1 is replaced with a leucine residue. (8) The amino acid residue corresponding to the 581st isoleucine residue in SEQ ID NO: 1 is replaced with a methionine residue. (9) The amino acid residue corresponding to the 630th threonine residue in SEQ ID NO: 1 is replaced with a valine residue. (10) The amino acid residue corresponding to the 687th valine residue in SEQ ID NO: 1 is replaced with a glutamic acid residue. (11) The amino acid residue corresponding to the 861st methionine residue in SEQ ID NO: 1 is replaced with a leucine residue. (12) The amino acid residue corresponding to the 810th isoleucine residue in SEQ ID NO: 1 is replaced with a valine residue. (ii) An enzyme having an amino acid sequence as described in Sequence ID No. 1, which includes at least one of the amino acid substitutions shown in (1) to (12) above, and further includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (12) above, and having enzymatic activity;(iii) An amino acid sequence described in Sequence ID No. 1, which has 70% or more identity with the entire amino acid sequence containing at least one of the amino acid substitutions shown in (1) to (12) above, provided that the amino acid substitutions shown in (1) to (12) above are maintained, and which has enzymatic activity.

[0042] Note that "one or several" in (ii) can vary depending on the position and type of amino acid residue in the protein's three-dimensional structure, but specifically, for example, it could be 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0043] Furthermore, the "identity" in (iii) may also refer to an amino acid sequence that has, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity with respect to the entire amino acid sequence.

[0044] The mutant T7 RNA polymerase is an enzyme selected from any of the above (i) to (iii), and the amino acid sequence of the enzyme may further have an amino acid sequence having one or more amino acid substitutions selected from the following (a) to (q): (a) the amino acid residue corresponding to the 430th serine residue of SEQ ID NO: 1 is substituted with a proline residue; (b) the amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 is substituted with a valine residue or an alanine residue; (c) the amino acid residue corresponding to the 510th cysteine residue of SEQ ID NO: 1 is substituted with an arginine residue; (d) the amino acid residue corresponding to the 767th serine residue of SEQ ID NO: 1 is substituted with a glycine residue; (e) the amino acid residue corresponding to the 786th glutamine residue of SEQ ID NO: 1 is substituted with a methionine residue; (f) the amino acid residue corresponding to the 849th phenylalanine residue of SEQ ID NO: 1 is substituted with an isoleucine residue; (g) the amino acid residue corresponding to the 880th phenylalanine residue of SEQ ID NO: 1 is substituted with a tyrosine residue; (h) the amino acid residue corresponding to the 108th glutamic acid residue of SEQ ID NO: 1 is substituted with a glycine residue; (i) the amino acid residue corresponding to the 441st lysine residue of SEQ ID NO: 1 is substituted with an arginine residue; (j) the amino acid residue corresponding to the 446th leucine residue of SEQ ID NO: 1 is substituted with a phenylalanine residue; (k) the amino acid residue corresponding to the 495th serine residue of SEQ ID NO: 1 is substituted with an aspartic acid residue; (l) the amino acid residue corresponding to the 505th glutamine residue of SEQ ID NO: 1 is substituted with an alanine residue; (m) the amino acid residue corresponding to the 530th cysteine residue of SEQ ID NO: 1 is substituted with a glycine residue; (n) the amino acid residue corresponding to the 534th leucine residue of SEQ ID NO: 1 is substituted with a valine residue; (o) the amino acid residue corresponding to the 633rd serine residue of SEQ ID NO: 1 is substituted with a proline residue; (p) the amino acid residue corresponding to the 650th valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue; (q) the amino acid residue corresponding to the 832nd methionine residue of SEQ ID NO: 1 is substituted with a phenylalanine residue.

[0045] Preferably, the mutant T7 RNA polymerase may be an enzyme selected from any of the following (i') to (iii'): (i') an enzyme having an amino acid sequence set forth in SEQ ID NO: 1 and having an amino acid sequence containing at least the amino acid substitution shown in the following (1'): (1') the amino acid residue corresponding to the 114th valine residue of SEQ ID NO: 1 is substituted with an isoleucine residue; (ii') an enzyme having an amino acid sequence set forth in SEQ ID NO: 1, being an amino acid sequence containing at least the amino acid substitution shown in the above (1'), further having an amino acid sequence containing any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or several positions other than the amino acid substitution shown in the above (1'), and having enzyme activity; (iii') an enzyme having an amino acid sequence set forth in SEQ ID NO: 1, being an amino acid sequence having 70% or more identity to the entire amino acid sequence containing at least the amino acid substitution shown in the above (1'), provided that it has an amino acid sequence in which the amino acid substitution shown in the above (1') is maintained and has enzyme activity.

[0046] Note that "one or several" in (ii') varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically, for example, it may be 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0047] Also, the "identity" in (iii') may be an amino acid sequence having 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0048] Preferably, the mutant T7RNA polymerase is an enzyme selected from any of (i') to (iii') above, wherein the amino acid sequence of the enzyme has an amino acid sequence having one or more amino acid substitutions selected from the following (a) to (q): (a) the amino acid residue corresponding to the 430th serine residue of SEQ ID NO: 1 is replaced with a proline residue; (b) the amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 is replaced with a valine or alanine residue; (c) the amino acid residue corresponding to the 510th cysteine ​​residue of SEQ ID NO: 1 is replaced with an arginine residue; (d) the amino acid residue corresponding to the 767th serine residue of SEQ ID NO: 1 is replaced with a glycine residue; (e) the amino acid residue corresponding to the 786th glutamine residue of SEQ ID NO: 1 is replaced with a methionine residue; (f) the amino acid residue corresponding to the 849th phenylalanine residue of SEQ ID NO: 1 is replaced with an isoleucine residue; (g) the amino acid residue corresponding to the 880th phenylalanine residue of SEQ ID NO: 1 is replaced with a tyrosine residue; (h) The amino acid residue corresponding to the 108th glutamic acid residue in SEQ ID NO: 1 is replaced with a glycine residue; (i) The amino acid residue corresponding to the 441st lysine residue in SEQ ID NO: 1 is replaced with an arginine residue; (j) The amino acid residue corresponding to the 446th leucine residue in SEQ ID NO: 1 is replaced with a phenylalanine residue; (k) The amino acid residue corresponding to the 495th serine residue in SEQ ID NO: 1 is replaced with an aspartic acid residue; (l) The amino acid residue corresponding to the 505th glutamine residue in SEQ ID NO: 1 is replaced with an alanine residue; (m) The amino acid residue corresponding to the 530th cysteine ​​residue in SEQ ID NO: 1 is replaced with a glycine residue; (n) The amino acid residue corresponding to the 534th leucine residue in SEQ ID NO: 1 is replaced with a valine residue; (o) The amino acid residue corresponding to the 633rd serine residue in SEQ ID NO: 1 is replaced with a proline residue; (p) The amino acid residue corresponding to the 650th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue; (q) The amino acid residue corresponding to the 832nd methionine residue in Sequence ID No. 1 is replaced with a phenylalanine residue.

[0049] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an isoleucine residue in Sequence ID 2. As described above, Sequence ID 2 is an amino acid sequence in which the amino acid sequence of Sequence ID 1 is modified by replacing the amino acid residue corresponding to the 430th serine residue with a proline residue, the amino acid residue corresponding to the 490th methionine residue with a valine residue, the amino acid residue corresponding to the 510th cysteine ​​residue with an arginine residue, the amino acid residue corresponding to the 767th serine residue with a glycine residue, the amino acid residue corresponding to the 786th glutamine residue with a methionine residue, the amino acid residue corresponding to the 849th phenylalanine residue with an isoleucine residue, and the amino acid residue corresponding to the 880th phenylalanine residue with a tyrosine residue. Therefore, the amino acid sequence of Sequence ID 6 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an isoleucine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0050] Sequence ID 16 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a threonine residue in Sequence ID 2. The amino acid sequence of Sequence ID 16 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a threonine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0051] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the glutamic acid residue at position 108 in Sequence ID 1 is replaced with a glycine residue, as shown in Sequence ID 18. The amino acid sequence of Sequence ID 18 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the glutamic acid residue at position 108 in Sequence ID 1 is replaced with a glycine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0052] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the threonic acid residue at position 127 of Sequence ID 1 is replaced with an alanine residue, as shown in Sequence ID 20. The amino acid sequence of Sequence ID 20 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonic acid residue at position 127 of Sequence ID 1 is replaced with an alanine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0053] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the serine residue at position 128 in Sequence ID 1 is replaced with an asparagine residue, as shown in Sequence ID 22. The amino acid sequence of Sequence ID 22 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the serine residue at position 128 in Sequence ID 1 is replaced with an asparagine residue, and which has any of the amino acid substitutions described in (a) to (g) above.

[0054] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the glutamic acid residue at position 242 in Sequence ID 1 is replaced with an isoleucine residue, as shown in Sequence ID 24. The amino acid sequence of Sequence ID 24 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the glutamic acid residue at position 242 in Sequence ID 1 is replaced with an isoleucine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0055] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the valine residue at position 384 in Sequence ID 1 is replaced with an alanine residue, as shown in Sequence ID 26. The amino acid sequence of Sequence ID 26 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the valine residue at position 384 in Sequence ID 1 is replaced with an alanine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0056] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the lysine residue at position 441 in Sequence ID 1 is replaced with an arginine residue, as shown in Sequence ID 28. The amino acid sequence of Sequence ID 28 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the lysine residue at position 441 in Sequence ID 1 is replaced with an arginine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0057] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the leucine residue at position 446 in Sequence ID 1 is replaced with a phenylalanine residue, as shown in Sequence ID 30. The amino acid sequence of Sequence ID 230 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the leucine residue at position 446 in Sequence ID 1 is replaced with a phenylalanine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0058] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the methionine residue at position 490 in Sequence ID 1 is replaced with an alanine residue, as shown in Sequence ID 32. The amino acid sequence of Sequence ID 32 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the methionine residue at position 490 in Sequence ID 1 is replaced with an alanine residue, and also has any of the amino acid substitutions described in (a) and (c) to (g) above.

[0059] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the serine residue at position 495 in Sequence ID 1 is replaced with an aspartic acid residue, as shown in Sequence ID 34. The amino acid sequence of Sequence ID 34 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the serine residue at position 495 in Sequence ID 1 is replaced with an aspartic acid residue, and which has any of the amino acid substitutions described in (a) to (g) above.

[0060] Sequence ID 36 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the glutamine residue at position 505 in Sequence ID 1 is replaced with an alanine residue in Sequence ID 6. The amino acid sequence of Sequence ID 36 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the glutamine residue at position 505 in Sequence ID 1 is replaced with an alanine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0061] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the cysteine ​​residue at position 530 in Sequence ID 1 is replaced with a glycine residue, as shown in Sequence ID 38. The amino acid sequence of Sequence ID 38 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the cysteine ​​residue at position 530 in Sequence ID 1 is replaced with a glycine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0062] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the leucine residue at position 534 in Sequence ID 1 is replaced with a valine residue, as shown in Sequence ID 40. The amino acid sequence of Sequence ID 40 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the leucine residue at position 534 in Sequence ID 1 is replaced with a valine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0063] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the isoleucine residue at position 543 of Sequence ID 1 is replaced with a leucine residue, as shown in Sequence ID 42. The amino acid sequence of Sequence ID 42 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the isoleucine residue at position 543 of Sequence ID 1 is replaced with a leucine residue, and which has any of the amino acid substitutions described in (a) to (g) above.

[0064] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the isoleucine residue at position 581 in Sequence ID 1 is replaced with a methionine residue, as shown in Sequence ID 44. The amino acid sequence of Sequence ID 44 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the isoleucine residue at position 581 in Sequence ID 1 is replaced with a methionine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0065] Sequence ID 46 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the threonine residue at position 630 in Sequence ID 1 is replaced with a valine residue in Sequence ID 6. The amino acid sequence of Sequence ID 46 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 630 in Sequence ID 1 is replaced with a valine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0066] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, as shown in Sequence ID 48. The amino acid sequence of Sequence ID 48 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0067] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the valine residue at position 650 in Sequence ID 1 is replaced with an isoleucine residue, as shown in Sequence ID 50. The amino acid sequence of Sequence ID 50 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the valine residue at position 650 in Sequence ID 1 is replaced with an isoleucine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0068] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the valine residue at position 687 in Sequence ID 1 is replaced with a glutamic acid residue, as shown in Sequence ID 52. The amino acid sequence of Sequence ID 52 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the valine residue at position 687 in Sequence ID 1 is replaced with a glutamic acid residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0069] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the isoleucine residue at position 810 in Sequence ID 1 is replaced with a valine residue, as shown in Sequence ID 54. The amino acid sequence of Sequence ID 54 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the isoleucine residue at position 810 in Sequence ID 1 is replaced with a valine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0070] Sequence ID 56 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the methionine residue at position 832 in Sequence ID 1 is replaced with a phenylalanine residue in Sequence ID 6. The amino acid sequence of Sequence ID 56 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the methionine residue at position 832 in Sequence ID 1 is replaced with a phenylalanine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0071] Sequence ID 6 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the threonine residue at position 835 in Sequence ID 1 is replaced with a leucine residue, as shown in Sequence ID 58. The amino acid sequence of Sequence ID 58 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 in Sequence ID 1 is replaced with a leucine residue, and which also has any of the amino acid substitutions described in (a) to (g) above.

[0072] Sequence ID 60 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the methionine residue at position 861 in Sequence ID 1 is replaced with a leucine residue. The amino acid sequence of Sequence ID 60 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the methionine residue at position 861 in Sequence ID 1 is replaced with a leucine residue, and which has any of the amino acid substitutions described in (a) to (g) above.

[0073] Sequence ID 58 is an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the glutamic acid residue at position 242 of Sequence ID 1 is replaced with an isoleucine residue, as shown in Sequence ID 62. The amino acid sequence of Sequence ID 62 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the glutamic acid residue at position 242 of Sequence ID 1 is replaced with an isoleucine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0074] Sequence ID 58 is an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the lysine residue at position 441 of Sequence ID 1 is replaced with an arginine residue, as shown in Sequence ID 64. The amino acid sequence of Sequence ID 64 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the lysine residue at position 441 of Sequence ID 1 is replaced with an arginine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0075] Sequence ID 58 is an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the threonine residue at position 630 in Sequence ID 1 is replaced with a valine residue, as shown in Sequence ID 66. The amino acid sequence of Sequence ID 66 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 in Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 in Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the threonine residue at position 630 in Sequence ID 1 is replaced with a valine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0076] Sequence ID 58 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, as shown in Sequence ID 68. The amino acid sequence of Sequence ID 68 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0077] Sequence ID 68 is shown in Sequence ID 70 as an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the glutamic acid residue at position 242 of Sequence ID 1 is replaced with an isoleucine residue. The amino acid sequence of Sequence ID 70 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and the amino acid residue corresponding to the glutamic acid residue at position 242 of Sequence ID 1 is replaced with an isoleucine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0078] Sequence ID 68 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the lysine residue at position 441 of Sequence ID 1 is replaced with an arginine residue, as shown in Sequence ID 72. The amino acid sequence of Sequence ID 72 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and the amino acid residue corresponding to the lysine residue at position 441 of Sequence ID 1 is replaced with an arginine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0079] Sequence ID 74 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the isoleucine residue at position 543 of Sequence ID 1 is replaced with a leucine residue in Sequence ID 68. The amino acid sequence of Sequence ID 74 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and the amino acid residue corresponding to the isoleucine residue at position 543 of Sequence ID 1 is replaced with a leucine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0080] Sequence ID 76 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the threonine residue at position 630 of Sequence ID 1 is replaced with a valine residue in Sequence ID 68. The amino acid sequence of Sequence ID 76 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID 1 is replaced with a proline residue, and the amino acid residue corresponding to the threonine residue at position 630 of Sequence ID 1 is replaced with a valine residue, and also has any of the amino acid substitutions described in (a) to (g) above.

[0081] Sequence ID 78 shows an example of a mutant T7 RNA polymerase in which, in Sequence ID 68, the amino acid residue corresponding to the methionine residue at position 490 in Sequence ID 1 is replaced with an alanine residue, the amino acid residue corresponding to the cysteine ​​residue at position 530 is replaced with a glycine residue, the amino acid residue corresponding to the isoleucine residue at position 810 is replaced with a valine residue, and the amino acid residue corresponding to the methionine residue at position 861 is replaced with a leucine residue. The amino acid sequence of Sequence ID No. 78 is an amino acid sequence having any of the amino acid substitutions described in (a) and (c) to (g) above, in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID No. 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID No. 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID No. 1 is replaced with a proline residue, the amino acid residue corresponding to the methionine residue at position 490 of Sequence ID No. 1 is replaced with an alanine residue, the amino acid residue corresponding to the cysteine ​​residue at position 530 of Sequence ID No. 1 is replaced with a glycine residue, the amino acid residue corresponding to the isoleucine residue at position 810 of Sequence ID No. 1 is replaced with a valine residue, and the amino acid residue corresponding to the methionine residue at position 861 of Sequence ID No. 1 is replaced with a leucine residue.

[0082] Sequence ID 80 shows an example of a mutant T7 RNA polymerase in which, in Sequence ID 68, the amino acid residue corresponding to the methionine residue at position 490 in Sequence ID 1 is replaced with an alanine residue, the amino acid residue corresponding to the cysteine ​​residue at position 530 is replaced with a glycine residue, the amino acid residue corresponding to the isoleucine residue at position 543 is replaced with a leucine residue, and the amino acid residue corresponding to the methionine residue at position 861 is replaced with a leucine residue. The amino acid sequence of Sequence ID No. 80 is an amino acid sequence having any of the amino acid substitutions described in (a) and (c) to (g) above, in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID No. 1 is replaced with an isoleucine residue, the amino acid residue corresponding to the threonine residue at position 835 of Sequence ID No. 1 is replaced with a leucine residue, the amino acid residue corresponding to the serine residue at position 633 of Sequence ID No. 1 is replaced with a proline residue, the amino acid residue corresponding to the methionine residue at position 490 of Sequence ID No. 1 is replaced with an alanine residue, the amino acid residue corresponding to the cysteine ​​residue at position 530 of Sequence ID No. 1 is replaced with a glycine residue, the amino acid residue corresponding to the isoleucine residue at position 543 of Sequence ID No. 1 is replaced with a leucine residue, and the amino acid sequence having any of the amino acid substitutions described in (a) and (c) to (g) above.

[0083] Sequence ID 82 shows an example of a mutant T7 RNA polymerase in which, in Sequence ID 68, the amino acid residue corresponding to the glutamic acid residue at position 108 in Sequence ID 1 is replaced with a glycine residue, the amino acid residue corresponding to the isoleucine residue at position 581 is replaced with a methionine residue, the amino acid residue corresponding to the valine residue at position 650 is replaced with an isoleucine residue, and the amino acid residue corresponding to the isoleucine residue at position 810 is replaced with a valine residue. The amino acid sequence of Sequence ID No. 82 is an amino acid sequence having any of the above amino acid substitutions (a) to (g).

[0084] Sequence ID 85 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an alanine residue in Sequence ID 2. As described above, Sequence ID 2 is an amino acid sequence in which the amino acid sequence of Sequence ID 1 is modified by replacing the amino acid residue corresponding to the 430th serine residue with a proline residue, the amino acid residue corresponding to the 490th methionine residue with a valine residue, the amino acid residue corresponding to the 510th cysteine ​​residue with an arginine residue, the amino acid residue corresponding to the 767th serine residue with a glycine residue, the amino acid residue corresponding to the 786th glutamine residue with a methionine residue, the amino acid residue corresponding to the 849th phenylalanine residue with an isoleucine residue, and the amino acid residue corresponding to the 880th phenylalanine residue with a tyrosine residue. Therefore, the amino acid sequence of Sequence ID 85 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an alanine residue, and also has any of the amino acid substitutions described in (a) to (g) above. Sequence ID 87 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a cysteine ​​residue in Sequence ID 2. The amino acid sequence of Sequence ID 87 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a cysteine ​​residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 89 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a glutamic acid residue in Sequence ID 2. The amino acid sequence of Sequence ID 89 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a glutamic acid residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 91 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a lysine residue in Sequence ID 2.The amino acid sequence of SEQ ID NO: 91 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with a lysine residue, and also has any of the amino acid substitutions described in (a) to (g). SEQ ID NO: 93 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with a leucine residue in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 93 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with a leucine residue, and also has any of the amino acid substitutions described in (a) to (g). SEQ ID NO: 95 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with a methionine residue in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 95 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with a methionine residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 97 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an asparagine residue in Sequence ID 2. The amino acid sequence of Sequence ID 97 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an asparagine residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 99 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a glutamine residue in Sequence ID 2. The amino acid sequence of Sequence ID 99 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a glutamine residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 101 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with an arginine residue in Sequence ID 2. The amino acid sequence of Sequence ID No. 101 is an amino acid sequence in which the amino acid residue corresponding to the valine residue at position 114 of Sequence ID No. 1 is replaced with an arginine residue, and also has any of the amino acid substitutions described in (a) to (g) above.Sequence ID 103 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a serine residue in Sequence ID 2. The amino acid sequence of Sequence ID 103 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a serine residue, and also has any of the amino acid substitutions described in (a) to (g). Sequence ID 105 shows an example of a mutant T7 RNA polymerase in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a tryptophan residue in Sequence ID 2. The amino acid sequence of Sequence ID 105 is an amino acid sequence in which the amino acid residue corresponding to the 114th valine residue in Sequence ID 1 is replaced with a tryptophan residue, and also has any of the amino acid substitutions described in (a) to (g).

[0085] The mutant T7 RNA polymerase may specifically be an enzyme selected from any of the following (iv) to (vi): (iv) an enzyme having the amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105; (v) An amino acid sequence described in Sequence ID Nos. 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the specific mutation is maintained, and having an enzyme that possesses enzymatic activity; (vi) An enzyme having an amino acid sequence that has 70% or more identity with the amino acid sequences described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, provided that a specific mutation is maintained, and that the amino acid sequence has enzymatic activity.

[0086] Note that "one or several" in (v) can vary depending on the position and type of amino acid residue in the protein's three-dimensional structure, but specifically, for example, it could be 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0087] Furthermore, the "identity" in (vi) may also refer to an amino acid sequence that has, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity with respect to the entire amino acid sequence.

[0088] A mutant T7 RNA polymerase may specifically be an enzyme selected from any of the following (vii) to (ix): (vii) an enzyme having the amino acid sequence described in SEQ ID NOs. 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105; (viiii) an enzyme having an amino acid sequence described in SEQ ID NOs. 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, which includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the specific mutation is maintained, and which is enzymatically active; (ix) An enzyme having an amino acid sequence that has 70% or more identity with the amino acid sequence described in SEQ ID NOs. 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, provided that a specific mutation is maintained, and that has enzymatic activity.

[0089] Furthermore, in terms of improved productivity and maintained activity, the mutant T7RNA polymerase is more preferably an enzyme selected from any of the following (x) to (xi): (x) a T7RNA polymerase having the amino acid sequence described in SEQ ID NO: 87, 93, 95, or 103; (xi) a T7RNA polymerase having the amino acid sequence described in SEQ ID NO: 87, 93, 95, or 103, which includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and having enzymatic activity; (xi) a T7RNA polymerase having an amino acid sequence that is 70% or more identical to the amino acid sequence described in SEQ ID NO: 87, 93, 95, or 103, and having enzymatic activity.

[0090] Note that the "one or several" in (viiii) and (xi) can vary depending on the position and type of amino acid residue in the protein's three-dimensional structure, but specifically, for example, it could be 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0091] Furthermore, the "identity" of (ix) and (xi) may be an amino acid sequence having, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity with respect to the entire amino acid sequence.

[0092] The mutant T7RNA polymerase may contain other amino acid sequences in addition to the amino acid sequence of the mutant T7RNA polymerase exemplified above. These other amino acid sequences are also called "additional sequences." That is, the mutant T7RNA polymerase may contain a fusion protein with an additional sequence. Furthermore, the mutant T7RNA polymerase may be expressed, for example, in a form containing an additional sequence (i.e., as a fusion protein with an additional sequence), and may ultimately lose part or all of the additional sequence. Unless otherwise specified, "the mutant T7RNA polymerase contains an additional sequence" or "the mutant T7RNA polymerase is a fusion protein with an additional sequence" means that the ultimately obtained mutant T7RNA polymerase contains an additional sequence. On the other hand, "the mutant T7RNA polymerase is expressed in a form containing an additional sequence" or "the mutant T7RNA polymerase contains an additional sequence at the time of expression" means, unless otherwise specified, that the mutant T7RNA polymerase contains an additional sequence at least at the time of expression, and does not necessarily mean that the ultimately obtained mutant T7RNA polymerase contains an additional sequence. In other words, the mutant T7RNA polymerase gene may include a nucleotide sequence encoding an additional sequence in addition to the nucleotide sequence of the mutant T7RNA polymerase gene as exemplified above. The same applies to wild-type T7RNA polymerase and the wild-type T7RNA polymerase gene. The additional sequence is not particularly limited as long as the mutant T7RNA polymerase has enzymatic activity. The additional sequence can be appropriately selected according to various conditions such as its intended use. Examples of additional sequences include peptide tags, signal peptides (also called signal sequences), and protease recognition sequences. The additional sequence may be ligated to the N-terminus, C-terminus, or both of the mutant T7RNA polymerase. The additional sequence may be a single amino acid sequence, or a combination of two or more amino acid sequences.

[0093] Peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphate), and the Fc region of antibodies. A 6xHis tag is an example of a His tag. Peptide tags can be used, for example, for the detection and purification of expressed mutant T7 RNA polymerase.

[0094] The signal peptide is not particularly limited as long as it functions in a host expressing mutant T7RNA polymerase. Examples of signal peptides include those recognized by the Sec secretory pathway and those recognized by the Tat secretory pathway. Signal peptides can be used, for example, in the secretory production of mutant T7RNA polymerase. When mutant T7RNA polymerase is secreted using a signal peptide, the signal peptide is cleaved during secretion, and mutant T7RNA polymerase without the signal peptide may be secreted outside the bacterial cell. In other words, typically, the mutant T7RNA polymerase ultimately obtained does not need to have a signal peptide.

[0095] Specific examples of protease recognition sequences include those for Factor Xa protease and proTEV protease. Protease recognition sequences can be used, for example, to cleave expressed mutant T7RNA polymerase. Specifically, for example, when expressing mutant T7RNA polymerase as a fusion protein with a peptide tag, introducing a protease recognition sequence to the junction between the mutant T7RNA polymerase and the peptide tag allows the expressed mutant T7RNA polymerase to use the protease to cleave the peptide tag, thereby obtaining mutant T7RNA polymerase without the peptide tag.

[0096] In this specification, "the Xth amino acid in the amino acid sequence described in SEQ ID NO: 1" means the amino acid located at position X from the N-terminus of the amino acid sequence described in SEQ ID NO: 1. In a specific amino acid sequence, "the amino acid residue corresponding to the Xth amino acid in the amino acid sequence described in SEQ ID NO: 1" means an amino acid residue in that specific amino acid sequence that is located at the same position as the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the alignment between that specific amino acid sequence and the amino acid sequence of SEQ ID NO: 1. For example, "the amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1" means an amino acid residue in that specific amino acid sequence that is located at the same position as the 114th valine in the amino acid sequence shown in SEQ ID NO: 1 in the alignment between that specific amino acid sequence and the amino acid sequence of SEQ ID NO: 1. Furthermore, in the amino acid sequence described in SEQ ID NO: 1, "the amino acid residue corresponding to the Xth amino acid in the amino acid sequence described in SEQ ID NO: 1" means the Xth amino acid itself in the amino acid sequence described in SEQ ID NO: 1. In other words, the positions of the amino acid substitutions exemplified above (i.e., amino acid substitutions at the specific positions and optionally other amino acid substitutions) do not necessarily represent absolute positions in the protein of this disclosure, but rather relative positions based on the amino acid sequence described in SEQ ID NO: 1. That is, for example, if the protein of this disclosure includes insertions, deletions, or additions of amino acid residues on the N-terminal side of the position of the amino acid substitution exemplified above, the absolute position of the amino acid substitution may vary accordingly. The positions of the amino acid substitutions exemplified above in the protein of this disclosure can be identified, for example, by alignment of the amino acid sequence of the protein of this disclosure with the amino acid sequence described in SEQ ID NO: 1. This alignment can be performed, for example, using an alignment program such as BLAST or FASTA. The same applies to the positions of the amino acid substitutions exemplified above in any amino acid sequence, such as a variant sequence of the amino acid sequence described in SEQ ID NO: 1.Furthermore, the amino acid residues prior to the amino acid substitutions of the T7 RNA polymerase exemplified in this disclosure (for example, the amino acid substitutions shown in (1) to (12) or (a) to (q) above) indicate the type of amino acid residue prior to the substitution in the amino acid sequence described in SEQ ID NO: 1, and may or may not be conserved in unmodified amino acid sequences other than the amino acid sequence described in SEQ ID NO: 1.

[0097] The mutant T7RNA polymerase gene is not particularly limited as long as it encodes the mutant T7RNA polymerase described above. In this specification, the term "gene" is not limited to DNA, but may include any polynucleotide as long as it encodes the protein of interest. That is, "mutant T7RNA polymerase gene" may mean any polynucleotide that encodes mutant T7RNA polymerase. The mutant T7RNA polymerase gene may be DNA, RNA, or a combination thereof. The mutant T7RNA polymerase gene may be single-stranded or double-stranded. The mutant T7RNA polymerase gene may be single-stranded DNA or single-stranded RNA. The mutant T7RNA polymerase gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The mutant T7RNA polymerase gene may contain both DNA residues and RNA residues in a single polynucleotide chain. If the mutant T7 RNA polymerase gene contains RNA, the descriptions of DNA such as the nucleotide sequence exemplified above may be appropriately modified to reflect the RNA. The form of the mutant T7 RNA polymerase gene can be appropriately selected according to various conditions such as its intended use.

[0098] Examples of mutant T7RNA polymerase genes include the nucleotide sequences shown in SEQ ID NOs: 7, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106.

[0099] A mutant T7RNA polymerase gene may be a variant of the mutant T7RNA polymerase gene exemplified above, as long as the encoded T7RNA polymerase has a "specific mutation." In other words, the term "mutant T7RNA polymerase gene" may include the mutant T7RNA polymerase genes exemplified above, as well as their variants. Examples of variants include artificially modified versions of the genes exemplified above.

[0100] A mutant T7RNA polymerase gene may encode a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105) insofar as the encoding T7RNA polymerase has a "specific mutation". For example, the encoded protein may have an elongated or shortened N-terminus and / or C-terminus. The above-mentioned "one or several" or "one or several" may vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically, for example, it may be 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0101] Furthermore, the mutant T7RNA polymerase gene may also be a gene that encodes a protein having an amino acid sequence that has, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity with respect to the entire amino acid sequence, as long as the encoding T7RNA polymerase has a "specific mutation".

[0102] Furthermore, the mutant T7RNA polymerase gene may also be a gene, such as DNA, that hybridizes under stringent conditions with a probe, such as a complementary sequence to all or part of the above base sequence, which can be prepared from the above base sequence, as long as the encoding T7RNA polymerase has a "specific mutation." "Stringent conditions" may mean conditions under which a so-called specific hybrid is formed and a nonspecific hybrid is not formed. For example, one example is a condition in which DNA with high identity, for instance, DNA with 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity, hybridizes with each other, while DNA with lower identity does not hybridize. Alternatively, one example is a condition in which the DNA is washed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0103] As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When using a DNA fragment of about 300 bp in length as the probe, the conditions for washing during hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0104] Furthermore, since codon degeneracy differs depending on the host, the mutant T7RNA polymerase gene may be one in which any codon is replaced with an equivalent codon. In other words, the mutant T7RNA polymerase gene may be a variant of the mutant T7RNA polymerase gene exemplified above due to the degeneracy of the genetic code. For example, the mutant T7RNA polymerase gene may be modified to have the optimal codons depending on the codon usage frequency of the host being used.

[0105] The following describes "specific mutations."

[0106] "Specific mutations" refer to mutations useful for transcription reactions, and may particularly refer to mutations useful for thermal stability and / or resistance to reaction inhibition by contaminants. Specifically, "specific mutations" refer to mutations that, when introduced into wild-type T7 RNA polymerase, confer improved thermal stability and / or resistance to reaction inhibition by contaminants to wild-type T7 RNA polymerase.

[0107] "Specific mutations" may refer to mutations that are further useful for improving productivity. Specifically, "specific mutations" may refer to mutations that, when introduced into wild-type T7RNA polymerase, confer productivity-enhancing effects to wild-type T7RNA polymerase.

[0108] "Specific mutations" include one or more amino acid substitutions listed in (1) to (12) below: (1) The amino acid residue corresponding to the valine residue at position 114 of SEQ ID NO: 1 is replaced with an isoleucine residue, threonine residue, alanine residue, cysteine ​​residue, glutamic acid residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, or tryptophan residue. (2) The amino acid residue corresponding to the threonine residue at position 835 of SEQ ID NO: 1 is replaced with a leucine residue. (3) The amino acid residue corresponding to the threonine residue at position 127 of SEQ ID NO: 1 is replaced with an alanine residue. (4) The amino acid residue corresponding to the serine residue at position 128 of SEQ ID NO: 1 is replaced with an asparagine residue. (5) The amino acid residue corresponding to the glutamic acid residue at position 242 of SEQ ID NO: 1 is replaced with an isoleucine residue. (6) The amino acid residue corresponding to the valine residue at position 384 of SEQ ID NO: 1 is replaced with an alanine residue. (7) The amino acid residue corresponding to the 543rd isoleucine residue in SEQ ID NO: 1 is replaced with a leucine residue. (8) The amino acid residue corresponding to the 581st isoleucine residue in SEQ ID NO: 1 is replaced with a methionine residue. (9) The amino acid residue corresponding to the 630th threonine residue in SEQ ID NO: 1 is replaced with a valine residue. (10) The amino acid residue corresponding to the 687th valine residue in SEQ ID NO: 1 is replaced with a glutamic acid residue. (11) The amino acid residue corresponding to the 861st methionine residue in SEQ ID NO: 1 is replaced with a leucine residue. (12) The amino acid residue corresponding to the 810th isoleucine residue in SEQ ID NO: 1 is replaced with a valine residue.

[0109] By possessing a "specific mutation," mutant T7RNA polymerase exhibits improved thermal stability, resistance to reaction inhibition by contaminants, and / or productivity compared to wild-type T7RNA polymerase. In other words, mutant T7RNA polymerase may exhibit improved thermal stability, resistance to reaction inhibition by contaminants, and / or productivity compared to wild-type T7RNA polymerase by having one or more of the amino acid substitutions described in (1) to (12) above.

[0110] Improved thermal stability may be demonstrated, for example, as improved residual activity after heat treatment, as an increase in the amount of residual enzyme after heat treatment, or as reactivity at high temperatures. Specifically, improved thermal stability may be demonstrated by a reduction in the time it takes to detect the amplification product after performing an isothermal gene amplification reaction with transcription at a high reaction temperature using mutant T7RNA polymerase, compared to wild-type T7RNA polymerase, or by the detection of the amplification product without thermal inactivation at a reaction temperature higher than that of wild-type T7RNA polymerase. Here, "high reaction temperature" may specifically mean, for example, 45-60°C, 51-55°C, or 53-54°C. As another example, improved thermal stability may be demonstrated by a reduction in the time it takes to detect the amplification product after heating mutant T7RNA polymerase at 45-65°C for 1-60 minutes and then performing an isothermal gene amplification reaction with transcription, compared to wild-type T7RNA polymerase. As yet another example, improved thermal stability may be demonstrated by heating mutant T7RNA polymerase at 45-65°C for 1-60 minutes, followed by a transcription reaction, and observing an increase in the amount of DNA produced compared to wild-type T7RNA polymerase. As yet another example, improved thermal stability may be demonstrated by heating mutant T7RNA polymerase at 45-65°C for 1-60 minutes, and observing an increase in the amount of enzyme remaining without thermal denaturation compared to wild-type T7RNA polymerase.

[0111] Improved resistance to reaction inhibition by contaminants may be demonstrated, for example, by showing superior activity in the presence of contaminants compared to T7 RNA polymerase without the "specific mutation." While improved resistance to reaction inhibition by contaminants may be demonstrated under high-temperature conditions as an example, it is not limited to this and may be demonstrated regardless of temperature conditions. Examples of contaminants are not particularly limited, but include biological contaminants such as saliva, urine, feces, nasal secretions, sputum, blood, puncture fluid (pleural fluid, ascites, etc.), and nasal / pharyngeal mucus, as well as contaminants derived from natural components such as soil and environmental water (river water, seawater, lake water), and components of specimens or measurement samples containing the target nucleic acid. Another example of contaminants is components of reagents used for collecting, storing, and transporting the aforementioned specimens or measurement samples (virus transport solutions, transport media, blood preservation solutions, fecal preservation solutions, buffers, physiological saline, etc.). Further examples of impurities include components of reagents (such as alcohols like ethanol, surfactants, guanidine salts, buffers, and organic solvents) used to extract, separate, and purify the target nucleic acid from the specimen, measurement sample, and viruses, bacteria, and cells containing the target nucleic acid. The presence of impurities may refer to conditions in which, for example, the aforementioned impurities, or purified versions thereof, are present in the reaction solution. Improved resistance to reaction inhibition by impurities may be specifically demonstrated, for example, by shortening the time it takes to detect the amplification product produced when an isothermal gene amplification reaction involving reverse transcription is performed using mutant T7RNA polymerase in a reaction solution containing saliva or urine purification, compared to wild-type T7RNA polymerase. The saliva or urine purification may include components of the aforementioned reagents, such as reagents for the purification of saliva or urine.

[0112] The improvement in productivity may be demonstrated, for example, as an improvement in the purified yield per culture medium. Specifically, first, E. coli is transformed with a plasmid capable of expressing the mutant T7RNA polymerase to produce the mutant T7RNA polymerase, and the culture is cultured and purified. Then, the purified mutant T7RNA polymerase is quantified using a NanoDrop micro-spectrophotometer to calculate the purified yield per culture medium. It may then be shown that the mutant T7RNA polymerase yields a higher purified yield per culture medium than wild-type T7RNA polymerase.

[0113] <2> Production of mutant T7RNA polymerase Mutant T7RNA polymerase can be produced, for example, by expressing the mutant T7RNA polymerase gene in a host that possesses the gene.

[0114] The following describes in detail the production of mutant T7RNA polymerase using a host possessing the mutant T7RNA polymerase gene.

[0115] <2-1> A host possessing a host-mutant T7RNA polymerase gene can be obtained by introducing the mutant T7RNA polymerase gene into a suitable host. "Introducing the mutant T7RNA polymerase gene into a host" may also include modifying the wild-type T7RNA polymerase gene in a host that has been introduced with the wild-type T7RNA polymerase gene, etc., so that it encodes the mutant T7RNA polymerase. "Possessing the mutant T7RNA polymerase gene" is also referred to as "possessing the mutant T7RNA polymerase."

[0116] The host is not particularly limited as long as it can express a functional mutant T7 RNA polymerase. Examples of hosts include microorganisms, plant cells, insect cells, and animal cells. Microorganisms are particularly noteworthy as hosts. Examples of microorganisms include bacteria and yeast. Bacteria are particularly noteworthy as microorganisms.

[0117] Examples of bacteria include those belonging to the family Enterobacteriaceae, Corynebacteria, and Bacillus.

[0118] Bacteria belonging to the Enterobacteriaceae family include those belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified under the Enterobacteriaceae family according to the classification method used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. While there are no particular restrictions on Escherichia bacteria, those classified under the Escherichia genus according to classifications known to microbiology experts are generally considered. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p.2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli, also known as Escherichia coli.Examples of Escherichia coli include Escherichia coli B strains such as BL21 (DE3); Escherichia coli K-12 strains such as JM109 (ATCC 53323), HB101 (ATCC 33694), W3110 (ATCC 27325), and MG1655 (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); and their derivative strains. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Examples of Klebsiella bacteria include Klebsiella planticola.

[0119] Examples of coryne-type bacteria include those belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0120] Furthermore, the genus Corynebacterium includes bacteria that were previously classified under the genus Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Additionally, the genus Corynebacterium statyonis includes bacteria that were previously classified under Corynebacterium ammoniagenes but have been reclassified as Corynebacterium statyonis based on 16S rRNA sequencing analysis (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0121] Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis strain 168 Marburg (ATCC 6051) and Bacillus subtilis strain PY79 (Plasmid, 1984, 12, 1-9). Examples of Bacillus amyloricephasiens include Bacillus amyloricephasiens strain T (ATCC 23842) and Bacillus amyloricephasiens strain N (ATCC 23845).

[0122] Examples of yeasts include those belonging to genera such as Saccharomyces cerevisiae, Candida utilis, Pichia pastoris, Hansenula polymorpha, and Schizosaccharomyces pombe.

[0123] These strains can be obtained, for example, from the American Type Culture Collection (PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a registration number, which can be used to obtain them (see http: / / www.atcc.org / ). The registration numbers for each strain are listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the depositary institutions where each strain is deposited.

[0124] A mutant T7RNA polymerase gene can be obtained, for example, by modifying a wild-type T7RNA polymerase gene so that the encoded T7RNA polymerase has a "specific mutation." The wild-type T7RNA polymerase gene used as the basis for modification can be obtained, for example, by cloning from a virus containing the wild-type T7RNA polymerase gene, or by chemical synthesis. Alternatively, the wild-type T7RNA polymerase gene used as the basis for modification may be obtained, for example, by artificially modifying a wild-type T7RNA polymerase gene obtained by cloning from a virus. Furthermore, a mutant T7RNA polymerase gene can also be obtained without going through the wild-type T7RNA polymerase gene. The mutant T7RNA polymerase gene may be obtained directly, for example, by chemical synthesis. The obtained mutant T7RNA polymerase gene may be used as is or after further modification. For example, a mutant T7RNA polymerase gene of a different nature may be obtained by modifying a mutant T7RNA polymerase gene of a certain nature.

[0125] Gene modification can be performed using known methods. For example, site-directed mutagenesis (SMU) can be used to introduce a desired mutation at a target site in DNA. That is, for example, SMU can be used to modify the coding region of a gene so that the encoded protein includes substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Examples of SMU methods include methods using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0126] There are no particular restrictions on the method for introducing the mutant T7RNA polymerase gene into a host. The mutant T7RNA polymerase gene only needs to be held in the host in an expressible state. The mutant T7RNA polymerase gene can be introduced into the host in the same manner as described in detail in the "Methods for Introducing the Gene" section below.

[0127] Furthermore, if a host already possesses a T7RNA polymerase gene, such as a wild-type T7RNA polymerase gene, on its chromosomes, the host can be modified to possess the mutant T7RNA polymerase gene by modifying the T7RNA polymerase gene to encode a mutant T7RNA polymerase. Modification of the T7RNA polymerase gene present on chromosomes can be carried out, for example, by spontaneous mutation, mutagenesis, or genetic engineering. Note that a host possessing a T7RNA polymerase gene on its chromosomes may be obtained, for example, by introducing a T7RNA polymerase gene, such as a wild-type T7RNA polymerase gene, into the host's chromosomes beforehand.

[0128] The host may have any properties as long as it can produce mutant T7 RNA polymerase.

[0129] <2-2> Methods for introducing genes The following describes methods for introducing genes into a host.

[0130] Gene introduction into a host can be achieved by introducing the gene into the host's chromosomes. Gene introduction into chromosomes can be performed, for example, using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include methods using linear DNA such as Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl.Acad. Sci. US A. 97:6640-6645 (2000)), methods using plasmids containing temperature-sensitive origins of replication, methods using conjugate-transferable plasmids, methods using suicide vectors that do not have origins of replication that function in the host, and phage-based transduction methods. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of the gene can be introduced into the chromosome by performing homologous recombination targeting sequences that have many copies on the chromosome. Sequences that have multiple copies on a chromosome include repetitive DNA sequences and inverted repeats located at both ends of transposons. Homologous recombination may also be performed targeting appropriate sequences on the chromosome, such as genes that are unnecessary for the production of mutant T7 RNA polymerase. Genes can also be randomly introduced onto the chromosome using transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1).

[0131] The introduction of a target gene onto a chromosome can be confirmed by Southern hybridization using a probe with a sequence complementary to all or part of the gene, or by PCR using primers created based on the gene's sequence.

[0132] Gene introduction into a host can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the target gene can be linked to a vector that functions in the host to construct an expression vector for that gene, and the gene can be introduced into the host by transforming the host with this expression vector. A host transformed with an expression vector is also called a transformant. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism possessing the target gene as a template. As the vector, a vector capable of autonomous replication within the host cell can be used. The vector may be a multicopy vector. In addition, the vector may have markers such as antibiotic resistance genes in order to select transformants. The vector may also have a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, pCold vectors (all available from Takara Bio), pACYC177, pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pET vectors (Merck), and pQE vectors (Qiagen).

[0133] When introducing a gene, it is sufficient that the gene is expressible by the host. Specifically, the gene should be maintained in a state where it is expressed under the control of a promoter that functions in the host. "Promoter that functions in the host" may mean a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a different species. The promoter may be the specific promoter of the gene being introduced or a promoter of another gene. Specific examples of promoters include, for example, the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Bifidobacterium-derived Pm1 promoter, PR promoter, PL promoter, P4 promoter, and P8 promoter.

[0134] A terminator for transcription termination can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be of host origin or of heterologous origin. The terminator may be specific to the gene being introduced or may be a terminator of another gene. Specific examples of terminators include, for example, the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0135] <2-3> Mutant T7RNA polymerase can be expressed by culturing a host that possesses the mutant T7RNA polymerase gene. For example, mutant T7RNA polymerase may be expressed by culturing a transformant obtained by transforming a host with an expression vector containing mutant T7RNA polymerase.

[0136] The culture medium used is not particularly limited, as long as it allows the host to grow and expresses a functional mutant T7 RNA polymerase. For example, a standard culture medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain, as needed, a carbon source, nitrogen source, phosphate source, sulfur source, and other various organic and inorganic components. The types and concentrations of the culture medium components may be appropriately set according to various conditions, such as the type of host.

[0137] Specific carbon sources include, for example, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, hydrolyzed starch, and hydrolyzed biomass; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived raw materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds; plant bodies containing these organs; and decomposition products of these plant organs. The form of use of plant-derived raw materials is not particularly limited and can be used in any form, such as unprocessed products, juices, pulverized products, or refined products. In addition, pentose sugars such as xylose, hexose sugars such as glucose, or mixtures thereof can be obtained and used, for example, from plant biomass. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis by enzymes such as cellulase, and alkaline treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed beforehand to release pentose sugars, and then cellulose may be hydrolyzed to produce hexose sugars. Xylose may also be supplied by converting hexose sugars, such as glucose, into xylose, for example, by providing the host with a conversion pathway from hexose sugars. As a carbon source, one type of carbon source may be used, or two or more carbon sources may be used in combination.

[0138] The concentration of the carbon source in the culture medium is not particularly limited, as long as the host can grow and express a functional mutant T7RNA polymerase. The concentration of the carbon source in the culture medium may be as high as possible, for example, without inhibiting the production of mutant T7RNA polymerase. The initial concentration of the carbon source in the culture medium may be, for example, usually 5 to 30 w / v%, preferably 10 to 20 w / v%. In addition, the carbon source may be supplied to the culture medium as appropriate. For example, the carbon source may be supplied to the culture medium in response to the decrease or depletion of the carbon source as the culture progresses. The carbon source may be temporarily depleted as long as mutant T7RNA polymerase is eventually produced, but it is preferable in some cases to carry out the culture in a way that prevents the carbon source from being depleted or to prevent a state of carbon source depletion from continuing.

[0139] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, peptone, organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may also be used as a nitrogen source. One type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0140] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One phosphate source may be used, or two or more phosphate sources may be used in combination.

[0141] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. One sulfur source may be used, or two or more sulfur sources may be used in combination.

[0142] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other organic and inorganic components may be used individually, or in combination of two or more components.

[0143] Furthermore, when using a nutrient-dependent mutant strain that requires nutrients such as amino acids for growth, it is preferable to supplement the culture medium with such required nutrients.

[0144] The culture conditions are not particularly limited, as long as the host can grow and a functional mutant T7RNA polymerase is expressed. Culturing can be carried out under the usual conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions may be set appropriately depending on various factors, such as the type of host. Furthermore, if necessary, the expression of the mutant T7RNA polymerase gene can be induced.

[0145] Culturing can be carried out using a liquid medium. During culturing, for example, the host may be cultured on a solid medium such as agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured on a liquid medium and then inoculated into the liquid medium for the main culture. In other words, culturing may be carried out separately as seed culture and main culture. In this case, the culture conditions for seed culture and main culture may be the same or different. Mutant T7 RNA polymerase should be expressed at least in the main culture. The amount of host contained in the medium at the start of culturing is not particularly limited. For example, a seed culture solution with OD660 = 4 to 100 may be added to the medium for the main culture at the start of culturing in an amount of 0.1% to 100% by mass, preferably 1% to 50% by mass.

[0146] Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium used at the start of culture is also called the "initial culture medium." The culture medium supplied to the culture system (e.g., a fermenter) in fed-batch or continuous culture is also called the "fed-batch medium." The act of supplying fed-batch medium to the culture system in fed-batch or continuous culture is also called "fed-batch." When culture is carried out separately as a seed culture and a main culture, the culture forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out as batch cultures, or the seed culture may be carried out as a batch culture and the main culture as a fed-batch or continuous culture.

[0147] Various components, such as carbon sources, may be contained in the initial culture medium, the fed-batch medium, or both. That is, during the culture process, various components, such as carbon sources, may be added to the medium individually or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial culture medium may or may not be the same as the types of components contained in the fed-batch medium. Furthermore, the concentration of each component contained in the initial culture medium may or may not be the same as the concentration of each component contained in the fed-batch medium. In addition, two or more types of fed-batch mediums with different types and / or concentrations of components may be used. For example, if multiple feedings are performed intermittently, the types and / or concentrations of components contained in each fed-batch medium may or may not be the same.

[0148] Culturing can be carried out, for example, under aerobic conditions. "Aerobic conditions" may mean that the dissolved oxygen concentration in the culture medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 100% of the saturated oxygen concentration, preferably 20 to 100%. Culturing can be carried out, for example, by aeration culture or shaking culture. The pH of the culture medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During cultivation, the pH of the culture medium can be adjusted as needed. The pH of the culture medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and phosphoric acid aqueous solution. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. The culture may be continued, for example, until the carbon source in the culture medium is consumed or until the host becomes inactive.

[0149] By culturing the host in this manner, a culture containing mutant T7RNA polymerase can be obtained. Mutant T7RNA polymerase can accumulate, for example, within the host's cells. "Bacterial cells" may be replaced with "cells" as appropriate, depending on the type of host. Depending on the host used and / or the design of the mutant T7RNA polymerase gene, it may also be possible to accumulate mutant T7RNA polymerase in the periplasm or to secrete and produce mutant T7RNA polymerase outside the cell.

[0150] The mutant T7RNA polymerase may be recovered as it is contained in the culture (specifically, the culture medium or bacterial cells) or recovered from the culture (specifically, the culture medium or bacterial cells). The mutant T7RNA polymerase may be purified during the recovery process from the culture. Purification can be carried out to any desired degree. That is, examples of mutant T7RNA polymerase include purified mutant T7RNA polymerase and fractions containing mutant T7RNA polymerase. In other words, the mutant T7RNA polymerase may be recovered in the form of purified enzyme, in the form of such fractions (i.e., in the form contained in such fractions), or in combination thereof. Such fractions are not particularly limited, as long as they contain mutant T7RNA polymerase in a way that allows it to act on its substrate. Such fractions include cultures of hosts possessing the mutant T7RNA polymerase gene (i.e., hosts possessing mutant T7RNA polymerase), bacterial cells recovered from the culture, culture supernatant recovered from the culture, processed products thereof (e.g., cell lysates, cell lysates, cell extracts, and other processed products of the cells described later), partially purified products thereof (i.e., crude products), and combinations thereof. Note that "purified mutant T7RNA polymerase" may include crude products. These fractions can be recovered individually or in appropriate combinations. Mutant T7RNA polymerase recovered in any manner including these can be used for any application, including "amplification of target nucleic acids" described later, and may be used in "reagents used for amplification of target nucleic acids" described later. Furthermore, the manner in which mutant T7RNA polymerase is recovered may be appropriately determined according to the application.

[0151] For example, mutant T7 RNA polymerase may be recovered in a form contained within the bacterial cells. The method for recovering the bacterial cells from the culture medium is not particularly limited, and known methods can be used, for example. Such methods include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be used. These methods can be used individually or in appropriate combinations. The recovered bacterial cells can be washed as appropriate using a suitable medium. The recovered bacterial cells can also be resuspended as appropriate using a suitable medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

[0152] As another example, the mutant T7RNA polymerase may be recovered from its form contained within the bacterial cells by subjecting the bacterial cells to appropriate processing. Examples of bacterial cell processing include immobilization on a carrier such as acrylamide or carrageenan, freeze-thaw processing, processing to increase membrane permeability, and physical disruption by sonication or pressure homogenization. Membrane permeability can be increased, for example, by using a surfactant or organic solvent. These processing methods can be used individually or in appropriate combinations. Furthermore, the recovered mutant T7RNA polymerase may be purified to a higher purity, desalted, and / or concentrated by applying various chromatography methods such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and / or ultrafiltration using a membrane, either individually or in combination.

[0153] The mutant T7 RNA polymerase may be produced alone or together with other proteins.

[0154] <2-4> Production of mutant T7RNA polymerase Mutant T7RNA polymerase may be produced by the method described above.

[0155] For example, mutant T7RNA polymerase may be produced by a method that includes the steps of expressing the mutant T7RNA polymerase gene in a host having the gene, and recovering the expressed enzyme. Such a method is also called a "method for producing T7RNA polymerase."

[0156] A method for producing T7RNA polymerase may preferably include the steps of: expressing T7RNA polymerase by culturing a transformant obtained by transforming a host with an expression vector having a mutant T7RNA polymerase gene; and recovering the polymerase expressed from the resulting culture.

[0157] The expression vector is as described above. An expression vector containing the mutant T7RNA polymerase gene may be an expression vector containing the nucleotide encoding the mutant T7RNA polymerase.

[0158] The host and its culture are as described above.

[0159] The recovery of the expressed mutant T7RNA polymerase is as described above. The mutant T7RNA polymerase may be purified during the recovery process from the culture. The purification of the enzyme is also as described above.

[0160] <3> Use of Mutant T7 RNA Polymerase The uses of mutant T7 RNA polymerase are not particularly limited. Mutant T7 RNA polymerase can be used, for example, for the synthesis of RNA from a target nucleic acid or for the amplification of a target nucleic acid. Specifically, mutant T7 RNA polymerase can be used, for example, for the synthesis of RNA from a target nucleic acid by a transcription reaction or for the amplification of a target nucleic acid accompanied by a transcription reaction. Both the synthesis of RNA from a target nucleic acid and the amplification of a target nucleic acid may specifically include a step of carrying out a transcription reaction in which RNA is synthesized using DNA as a template using mutant T7 RNA polymerase. Both the synthesis of RNA from a target nucleic acid and the amplification of a target nucleic acid may include any known steps as other steps, typically including, for example, a step of carrying out a polymerase chain reaction by DNA-dependent DNA polymerase that synthesizes DNA using DNA as a template, and / or a step of carrying out a reverse transcription reaction that synthesizes DNA using RNA as a template.

[0161] In RNA synthesis from a target nucleic acid, the target nucleic acid may be DNA. Specifically, RNA synthesis from a target nucleic acid may be carried out, for example, by synthesizing RNA using DNA, which is the target nucleic acid, as a template via a T7 RNA polymerase transcription reaction. The RNA synthesized from the target nucleic acid may be, for example, messenger RNA (mRNA). As an example, the target nucleic acid, DNA, may be amplified beforehand or during the process of RNA synthesis from the target nucleic acid by carrying out a polymerase chain reaction using DNA-dependent DNA polymerase, which synthesizes DNA using DNA as a template. In one embodiment, since mutant T7 RNA polymerase has excellent thermal stability, it is expected that DNA and / or RNA can be processed at higher temperatures during or before / after the transcription reaction, thereby suppressing the generation of by-products such as double-stranded RNA, and is therefore considered suitable for RNA synthesis by transcription reaction (Monica Z Wu et al., RNA, 26:345-360, 2020). Therefore, the synthesis of RNA from a target nucleic acid in one aspect of this disclosure may be carried out at a high temperature. Specifically, for example, the step of carrying out a reverse transcription reaction to synthesize RNA using DNA as a template, using a mutant T7 RNA polymerase, may be carried out at a high temperature. Herein, "high temperature" specifically means, for example, heating at 45 to 60°C for 1 to 60 minutes.

[0162] In the amplification of target nucleic acids, the target nucleic acid may be RNA. Specifically, the amplification of target nucleic acids may be carried out, for example, by using RNA, which is the target nucleic acid, as a template and amplifying it as RNA through a reverse transcription reaction using a reverse transcriptase such as AMV reverse transcriptase and a transcription reaction using T7 RNA polymerase. The amplification of target nucleic acids may be carried out, for example, by the NASBA (Nucleic Acid Sequence Based Amplification) method described in Japanese Patent Publication No. 2650159, the TMA (Transcription-Mediated Amplification) method described in Japanese Patent Publication No. 4-500759, or the TRC (Transcription Reverse-transcription Concerted) method described in Japanese Patent Publication No. 2000-14400. In one embodiment, since mutant RNA polymerase has excellent thermal stability, it is expected that RNA can be treated at a higher temperature during or before / after the transcription reaction, and that the formation of by-products due to RNA secondary structure formation and misannealing can be suppressed, making it suitable for amplification of target nucleic acids involving transcription reactions. Therefore, the amplification of the target nucleic acid in one aspect of this disclosure may be carried out at a high temperature. Specifically, for example, the step of carrying out a reverse transcription reaction to synthesize RNA using DNA as a template with mutant T7 RNA polymerase may be carried out at a high temperature, or the RNA treatment in the preceding and / or subsequent steps may be carried out at a high temperature, or a combination thereof. Herein, "high temperature" specifically means, for example, heating at 45 to 60°C for 1 to 60 minutes.

[0163] The reagent used for the synthesis of RNA from a target nucleic acid and / or the amplification of the target nucleic acid includes mutant T7 RNA polymerase. The reagent used for the synthesis of RNA from a target nucleic acid is also called the RNA synthesis reagent from the target nucleic acid, or simply the RNA synthesis reagent. The reagent used for the amplification of a target nucleic acid is also called the amplification reagent for the target nucleic acid. The mutant T7 RNA polymerase contained in the RNA synthesis reagent or the amplification reagent for the target nucleic acid may be mutant T7 RNA polymerase recovered in any manner, including the manner described above. The RNA synthesis reagent or the amplification reagent for the target nucleic acid may contain any known components other than mutant T7 RNA polymerase that are necessary for the transcription reaction. Furthermore, since the amplification of the target nucleic acid may be carried out by a reverse transcription reaction using RNA-dependent DNA polymerase, the reagent used for the amplification of the target nucleic acid may contain RNA-dependent DNA polymerase and any known components necessary for the reverse transcription reaction.

[0164] Furthermore, the target nucleic acid amplification reagent may also include a primer set consisting of a first oligonucleotide having a sequence complementary to a specific base sequence of the target nucleic acid and a second oligonucleotide having a sequence homologous to a specific base sequence (provided that at least one of the first and second oligonucleotides has a promoter sequence that can initiate RNA polymerase transcription added to its 5' end), an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having RNase H activity, and RNA polymerase. Such a reagent can be used, for example, in the TRC method described above. In other words, the target nucleic acid amplification reagent also includes, for example, a target nucleic acid amplification reagent using the TRC method.

[0165] In one embodiment, the mutant T7RNA polymerase may have improved resistance to reaction inhibition by contaminants. Therefore, in one embodiment, the mutant T7RNA polymerase may be used for RNA synthesis from a target nucleic acid or amplification of a target nucleic acid in the presence of contaminants. The presence of contaminants is as described above. In one embodiment, amplification of the target nucleic acid may be carried out in the presence of contaminants. Specifically, for example, amplification of the target nucleic acid may include the step of adding a substance containing contaminants to the reaction solution. Examples of substances containing contaminants include saliva purified or urine purified. Therefore, amplification of the target nucleic acid may include the step of adding saliva purified or urine purified to the reaction solution. Furthermore, amplification of the target nucleic acid may be carried out to amplify the target nucleic acid in a substance containing contaminants, for example, to amplify the target nucleic acid in saliva purified or urine purified. In one embodiment of this disclosure, for example, the mutant T7RNA polymerase may be used for such amplification of a target nucleic acid. In other words, one aspect of the present disclosure may relate to a mutant T7 RNA polymerase for amplification of a target nucleic acid in a substance containing impurities, and more specifically, for example, a mutant T7 RNA polymerase for amplification of a target nucleic acid in a saliva or urine product.

[0166] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0167] Example 1 Mutation introduction into the T7RNA polymerase gene (1) A polynucleotide (SEQ ID NO: 3) encoding a T7RNA polymerase (named T7m5) containing the amino acid sequence described in SEQ ID NO: 2 was synthesized. In T7m5, the amino acid residue corresponding to the 430th serine residue of wild-type T7RNA polymerase (GenBank: ACO57213.11, SEQ ID NO: 1) was replaced with a proline residue (hereinafter also referred to as S430P), the amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 was replaced with a valine residue (hereinafter also referred to as M490V), the amino acid residue corresponding to the 510th cysteine ​​residue of SEQ ID NO: 1 was replaced with an arginine residue (hereinafter also referred to as C510R), and the 767th serine residue of SEQ ID NO: 1 was replaced The amino acid residue corresponding to the phosphorus residue is substituted with a glycine residue (hereinafter also referred to as S767G), the amino acid residue corresponding to the glutamine residue at position 786 of SEQ ID NO: 1 is substituted with a methionine residue (hereinafter also referred to as Q786M), the amino acid residue corresponding to the phenylalanine residue at position 849 of SEQ ID NO: 1 is substituted with an isoleucine residue (hereinafter also referred to as F849I), and the amino acid residue corresponding to the phenylalanine residue at position 880 of SEQ ID NO: 1 is substituted with a tyrosine residue (hereinafter also referred to as F880Y). During synthesis, an oligonucleotide encoding a histidine tag consisting of the amino acid sequence described in SEQ ID NO: 4 (SEQ ID NO: 5) is added to the 5' end, and a stop codon (TAA) is added to the 3' end. (2) A plasmid capable of expressing T7m5 was created by inserting the synthesized polynucleotide between the restriction enzyme NcoI / HindIII cleavage sites of plasmid pTrc99A. (3) From the T7m5-expressing plasmids obtained in (2), a nucleotide substitution was introduced at a predetermined position in the polynucleotide encoding T7m5 (SEQ ID NO: 2) (SEQ ID NO: 3) to create a plasmid capable of expressing a T7RNA polymerase mutant (named T7m6) having the amino acid substitution described in (A) below. (A) Substitution of the valine residue at position 114 of SEQ ID NO: 1 with an isoleucine residue (hereinafter also referred to as V114I). The amino acid sequence of the T7RNA polymerase having the amino acid substitution described in (A) above is shown as SEQ ID NO: 6, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 7.

[0168] Example 2 Preparation of T7RNA polymerase (1) Using the plasmid capable of expressing T7m5 prepared in Example 1 (2), or the plasmid capable of expressing T7m6 prepared in Example 1 (3), Escherichia coli strain JM109 (Takara Bio Inc.) was transformed to produce a T7RNA polymerase-producing strain (transformed organism). (2) The T7RNA polymerase-producing strain prepared in (1) was inoculated into 20 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing an appropriate amount of antibiotic, and cultured overnight with shaking at 37°C and 180 rpm. Then, IPTG (Isopropyl β-D-thiogalactopyranoside) was added to a final concentration of 2 mmol / L, and cultured for another day with shaking at 25°C and 180 rpm. (3) The culture medium from (2) was centrifuged at 4,000 × G for 10 minutes, and the supernatant was discarded to collect the bacterial cells. The collected bacterial cells were stored at -20°C to -80°C. (4) The bacterial cells collected in (3) were suspended in 2 mL of extraction buffer containing xTractor Buffer (Takara Bio Inc.) and left to stand on ice for at least 10 minutes. Then, the mixture was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected. (5) The supernatant collected in (4) was sterilized using a filter with a pore size of 0.22 μm, and then added to 1 mL of TALON Metal Affinity Resin (Takara Bio Inc.) that had been pre-equilibriumized with phosphate buffer (5.4 g / L sodium dihydrogen phosphate dihydrate, 11.7 g / L sodium chloride, pH 7.4). Next, the solution was washed with 15 mL of the phosphate buffer containing 10 mmol / L imidazole, and then eluted with 2 mL of the phosphate buffer containing 150 mmol / L imidazole. (6) The eluate from (5) was desalted and concentrated using an Amicon Ultra ultrafiltration filter (Merck) and the phosphate buffer without imidazole to obtain a purified T7 RNA polymerase solution. The solution was diluted to a concentration of 5 mg / mL.

[0169] Example 3 Evaluation of the heat resistance of T7 RNA polymerase (1) Polynucleotides having a T7 promoter were amplified according to the method described in Japanese Patent Application Publication No. 2022-185222. Specifically, an oligonucleotide consisting of the base sequence described in SEQ ID NO: 9, with the T7 promoter sequence described in SEQ ID NO: 8 added to the 5' end, was used as the first primer, and an oligonucleotide consisting of the base sequence described in SEQ ID NO: 10 was used as the second primer. Standard RNA based on 23S rRNA of Mycobacterium abscessus subspecies masiliense was used as the RNA sample, and polynucleotides having a T7 promoter were amplified by the TRC method. (2) After separating the amplified product from (1) by agarose gel electrophoresis, the band around 200 bp was excised and purified using Wizard SV Gel and PCR Clean-Up System (Promega) to prepare polynucleotides having a T7 promoter. (3) Prepare 15 μL of a transfer reaction solution containing the components listed in Table 1, and carry out the transfer reaction at 50°C or 54°C for 30 minutes.

[0170]

[0171] (4) The obtained transcripts were quantified by fluorescence measurement using a QuantiFluor RNA System (Promega) (excitation wavelength: 492 nm, fluorescence wavelength: 540 nm). The relative transcriptional activity was calculated by dividing the fluorescence intensity of the transcript obtained from the reaction at 54°C by the fluorescence intensity of the transcript obtained from the reaction at 50°C.

[0172] The results are shown in Figure 1. In T7m5 (SEQ ID NO: 2), which was created by introducing the amino acid substitutions S430P, M490V, C510R, S767G, Q786M, F849I, and F880Y into wild-type T7RNA polymerase (SEQ ID NO: 1), the activity decreased to approximately 65% ​​when the transcription reaction temperature was raised to 54°C. In contrast, T7m6 (SEQ ID NO: 6), which had the amino acid substitution V114I added to T7m5, maintained approximately 80% activity. These results indicate that introducing the amino acid substitution V114I into T7RNA polymerase improves the heat resistance of T7RNA polymerase.

[0173] Example 4 Evaluation of heat resistance of T7 RNA polymerase (1) Escherichia coli W3110 strain was transformed according to a standard method with a vector capable of expressing AMV reverse transcriptase consisting of the amino acid sequence described in SEQ ID NO: 11 (AMV-RT m4-2), a vector capable of expressing AMV reverse transcriptase consisting of the amino acid sequence described in SEQ ID NO: 12 (AMV-RT m11), or a vector capable of expressing AMV reverse transcriptase consisting of the amino acid sequence described in SEQ ID NO: 13 (AMV-RT m12). (2) Each of the resulting transformants was cultured overnight at 37°C in LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L purified agar) containing an appropriate amount of antibiotic to obtain each AMV reverse transcriptase producing strain. (3) Using each production strain obtained in (2), purified AMV reverse transcriptase was obtained by culturing in a fermenter and purifying by column chromatography according to the method described in Japanese Patent Publication No. 2014-209898. (4) The concentration of the obtained purified AMV enzyme was quantified by size exclusion chromatography using a TSKgel UP-SW Aggregate column (manufactured by Tosoh Corporation). (5) Among the components of the Mycobacterium tuberculosis rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation), the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase obtained in (3) (AMV-RT m4-2, AMV-RT m11, or AMV-RT m12). Furthermore, the T7RNA polymerase, which is also a component, was replaced with either the T7m6 prepared in Example 2 or the wild-type T7RNA polymerase (SEQ ID NO: 1) with the amino acid substitutions M490V and Q786M (named T7control). T7control was prepared in the same manner as in Example 2, except that a plasmid capable of expressing T7control as described in SEQ ID NO: 15 was used. (6) The positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at various reaction temperatures. The measurement was performed by monitoring the change in fluorescence intensity using the automated gene testing device TRCReady-80 (manufactured by Tosoh Corporation), and the time when the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0174] The results are shown in Table 2. Substituting T7RNA polymerase with T7m6 shortened the detection time at each reaction temperature compared to the case where it was substituted with T7control. In other words, it was found that introducing the V114I amino acid substitution into T7RNA polymerase improved the heat resistance of T7RNA polymerase and enhanced its enzyme activity. Furthermore, when AMV-RT m11 (sequence number 12) or AMV-RT m12 (sequence number 13) were used instead of AMV-RT m4-2 (sequence number 11) as the AMV reverse transcriptase, the detection time of approximately 4 minutes was maintained even when the reaction temperature was raised to 53°C, and positive standard RNA could be detected even when the temperature was raised to 54°C.

[0175]

[0176] Example 5 Evaluation of T7 RNA polymerase inhibition resistance (1) A saliva purified product was prepared by the following procedure. (1-1) Saliva from a healthy person was suspended in four times the volume of PBS buffer (Nacalai Tesque) and centrifuged at 10,000 x G for 1 minute. (1-2) 100 μL of the supernatant from (1-1) was added to the denaturation reagent of the TRCR nucleic acid purification kit (Tosoh Corporation) and purified according to the standard method of the purification kit to prepare a saliva purified product. (2) A urine purified product was prepared by the following procedure. (2-1) 500 μL of urine from a healthy person was added to the denaturation reagent of the TRCR nucleic acid purification kit (Tosoh Corporation) and centrifuged at 10,000 x G for 3 minutes. (2-2) The entire amount of the supernatant from centrifugation in (2-1) was transferred to a TRCR transfer tube (manufactured by Tosoh Corporation) and purified according to the standard method of the purification kit to prepare a urine product. (3) Positive standard RNA (detectable RNA with known concentration) included with the Mycobacterium tuberculosis rRNA detection reagent TRCRReady MTB (manufactured by Tosoh Corporation) was added to the saliva product prepared in (1) or the urine product prepared in (2) to prepare a measurement sample containing various impurities. (4) Of the components of the TRCRReady MTB reagent, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase obtained in Example 4 (3) (AMV-RT m4-2, AMV-RT m11, or AMV-RT m12), and the T7 RNA polymerase was replaced with T7m6 or T7control prepared in Example 2. The measurement samples containing the various impurities prepared in (3) were then measured at reaction temperatures of 46°C or 51°C, respectively.

[0177] The results are shown in Table 3. Substituting T7RNA polymerase with T7m6 shortened the detection time in samples containing contaminants compared to substitution with T7control. This suggests that the substitution of T7RNA polymerase with T7m6 mitigated the inhibition of the reaction in the sample caused by contaminants. Along with improving the thermal stability of T7RNA polymerase, resistance to reaction inhibition in the sample caused by contaminants also improved. Furthermore, when AMV-RT m11 (SEQ ID NO: 12) or AMV-RT m12 (SEQ ID NO: 13) were used instead of AMV-RT m4-2 (SEQ ID NO: 11), the detection time was shortened even further. By using these enzymes, reaction inhibition by various contaminants can be mitigated, and improvements in the performance of infectious disease testing for various clinical specimens using nucleic acid amplification methods can be expected.

[0178]

[0179] Example 6 Mutation Introduction into the T7RNA Polymerase Gene (Part 2) Similar to Example 1, a plasmid capable of expressing a T7RNA polymerase mutant was prepared by introducing a nucleotide substitution at a predetermined position in the polynucleotide (SEQ ID NO: 3) encoding T7m5 (SEQ ID NO: 2) from a plasmid capable of expressing T7m5. Specifically, a nucleotide substitution was introduced to replace the valine residue at position 114 of SEQ ID NO: 2 with a threonine residue. The amino acid sequence of the T7RNA polymerase (hereinafter also referred to as V114T) in which the valine residue at position 114 of SEQ ID NO: 2 is replaced with a threonine residue is shown as SEQ ID NO: 16, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 17.

[0180] Example 7 Preparation of T7RNA polymerase (Part 2) (1) Using a plasmid capable of expressing T7m5 (SEQ ID NO: 2) prepared in Example 1 (2), a plasmid capable of expressing T7m6 prepared in Example 1 (3), or a plasmid capable of expressing T7RNA polymerase having V114T, Escherichia coli BL21 strain (manufactured by Takara Bio Inc.) was transformed to produce a T7RNA polymerase producing strain (transformed organism). (2) The T7RNA polymerase producing strain prepared in (1) was inoculated into 3 mL of 2×YT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) containing an appropriate amount of antibiotic, and pre-cultured by shaking overnight at 37°C and 180 rpm. (3) The pre-culture solution from (2) was inoculated into 100 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing an appropriate amount of antibiotic, dispensed into a 500 mL baffled flask, and cultured with shaking at 37°C and 130 rpm for 5 hours. (4) IPTG was added to a final concentration of 2 mmol / L, and cultured further overnight at 25°C and 130 rpm. (5) The culture solution from (4) was centrifuged at 4°C and 15000 × G for 20 minutes, and the wet bacterial cells were collected by discarding the supernatant. The collected bacterial cells were stored at -20°C to -80°C. (6) The wet bacterial cells recovered in (5) were mixed with 50 mmol / L Tris-HCl buffer (pH 7.2) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3000 Unit / L Benzonase (Merck), 0.006% (w / v) lysozyme, 2 mmol / L phenylmethylsulfonyl fluoride, and 0.6% (w / v) Triton X-100 (trade name). After stirring at room temperature for 1 hour, the mixture was centrifuged at 4°C and 15000 rpm for 60 minutes to obtain an extract containing expressed T7 RNA polymerase.

[0181] (7) After sterilizing the supernatant collected in (4) using a filter with a pore size of 0.22 μm, it was added to 1 mL of TALON Metal Affinity Resin (Takara Bio Inc.) which had been pre-equilibrated with phosphate buffer (5.4 g / L sodium dihydrogen phosphate dihydrate, 11.7 g / L sodium chloride, pH 7.4). Subsequently, it was washed with 10 mL of the phosphate buffer containing 10 mmol / L imidazole, and then eluted with 2.5 mL of the phosphate buffer containing 150 mmol / L imidazole. (8) The productivity (purified yield per culture medium) was evaluated by quantifying the purified T7 RNA polymerase contained in the eluate obtained in (7) using a NanoDrop micro-spectrophotometer (Thermo Fisher Scientific Inc.). (9) Using a PD-10 column (Cytiva), imidazole was removed from the eluate of (7) to obtain a purified T7 RNA polymerase solution. This solution was diluted to a concentration of 5 mg / mL.

[0182] The results are shown in Figure 2. In Figure 2, the purified yield per culture medium for each T7RNA polymerase mutant is shown as a relative value obtained by dividing the purified yield per culture medium for T7m5 (SEQ ID NO: 2). From Figure 2, it was found that T7m6 and T7RNA polymerases containing V114T showed significantly higher purified yields per culture medium than T7m5. In particular, the purified yield per culture medium for T7RNA polymerase containing V114T was more than four times that of T7m5.

[0183] Example 8 Evaluation of heat resistance of T7RNA polymerase (Part 2) (1) Each purified T7RNA polymerase prepared in Example 7 (9) was heated at 58.5°C for 5 minutes using a thermal cycler, and then held at 4°C. (2) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). Similarly, the T7RNA polymerase, another component, was replaced with each T7RNA polymerase that had been heat-treated in (1), and the positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 50°C. The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time at which the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0184] The results are shown in Table 4. From Table 4, it can be seen that both the T7 RNA polymerase with the isoleucine residue (V114I) mutant (T7m6) and the T7 RNA polymerase with V114T, as shown in Example 4, showed shorter detection times than T7m5 (SEQ ID NO: 2). From these results, it can be seen that introducing the amino acid mutation V114I or V114T into T7m5 improves the thermal stability of T7 RNA polymerase.

[0185]

[0186] Example 9 Mutation introduction into the T7RNA polymerase gene (part 3) Similar to Example 1, a plasmid capable of expressing T7m6 was prepared by introducing a nucleotide substitution at a predetermined position in the polynucleotide (sequence number 7) encoding T7m6 (sequence number 6) from a plasmid capable of expressing T7m6, thereby creating a plasmid capable of expressing a T7RNA polymerase mutant. Specifically, polynucleotides were prepared to incorporate the amino acid substitutions shown below from to <W>: Substitution of the glutamic acid residue at position 108 of SEQ ID NO: 6 with a glycine residue (hereinafter also referred to as E108G). <C> Substitution of the threonine residue at position 127 of SEQ ID NO: 6 with an alanine residue (hereinafter also referred to as T127A). <D> Substitution of the serine residue at position 128 of SEQ ID NO: 6 with an asparagine residue (hereinafter also referred to as S128N). <E> Substitution of the glutamic acid residue at position 242 of SEQ ID NO: 6 with an isoleucine residue (hereinafter also referred to as E242I). <F> Substitution of the valine residue at position 384 of SEQ ID NO: 6 with an alanine residue (hereinafter also referred to as V384A). <G> Substitution of the lysine residue at position 441 of SEQ ID NO: 6 with an arginine residue (hereinafter also referred to as K441R). <H> Substitution of the leucine residue at position 446 of SEQ ID NO: 6 with a phenylalanine residue (hereinafter also referred to as L446F). Substitution of the valine residue at position 490 of SEQ ID NO: 6 with an alanine residue (hereinafter also referred to as V490A). <J> Substitution of the serine residue at position 495 of SEQ ID NO: 6 with an aspartic acid residue (hereinafter also referred to as S495D). <K> Substitution of the glutamine residue at position 505 of SEQ ID NO: 6 with an alanine residue (hereinafter also referred to as Q505A). <L> Substitution of the cysteine ​​residue at position 530 of SEQ ID NO: 6 with a glycine residue (hereinafter also referred to as C530G). <M> Substitution of the leucine residue at position 534 of SEQ ID NO: 6 with a valine residue (hereinafter also referred to as L534V). <N> Substitution of the isoleucine residue at position 543 of SEQ ID NO: 6 with a leucine residue (hereinafter also referred to as I543L). <O> Substitution of the isoleucine residue at position 581 of SEQ ID NO: 6 with a methionine residue (hereinafter also referred to as I581M). Substitution of the threonine residue at position 630 of SEQ ID NO: 6 with a valine residue (hereinafter also referred to as T630V).) <Q> Substitution of the serine residue at position 633 of SEQ ID NO: 6 with a proline residue (hereinafter also referred to as S633P). <R> Substitution of the valine residue at position 650 of SEQ ID NO: 6 with an isoleucine residue (hereinafter also referred to as V650I). <S> Substitution of the valine residue at position 687 of SEQ ID NO: 6 with a glutamic acid residue (hereinafter also referred to as V687E). <T> Substitution of the isoleucine residue at position 810 of SEQ ID NO: 6 with a valine residue (hereinafter also referred to as I810V). Substitution of the methionine residue at position 832 of SEQ ID NO: 6 with a phenylalanine residue (hereinafter also referred to as M832F). <V> Substitution of the threonine residue at position 835 of SEQ ID NO: 6 with a leucine residue (hereinafter also referred to as T835L). <W> Substitution of the methionine residue at position 861 of SEQ ID NO: 6 with a leucine residue (hereinafter also referred to as M861L).

[0187] The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in has been introduced is designated as SEQ ID NO: 18, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 19; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <C> has been introduced is designated as SEQ ID NO: 20, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 21; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <D> has been introduced is designated as SEQ ID NO: 22, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 23; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <E> has been introduced is designated as SEQ ID NO: 24, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 25; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <F> has been introduced is designated as SEQ ID NO: 26, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 27; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <G> has been introduced is designated as SEQ ID NO: 28, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 29; The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <H> has been introduced is designated as SEQ ID NO: 30, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 31, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in has been introduced is designated as SEQ ID NO: 32, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 33, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <J> has been introduced is designated as SEQ ID NO: 34, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 35, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <K> has been introduced is designated as SEQ ID NO: 36, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 37, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <L> has been introduced is designated as SEQ ID NO: 38, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 39, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <M> has been introduced is designated as SEQ ID NO: 40, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 41The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <N> has been introduced is designated as SEQ ID NO: 42, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 43, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <O> has been introduced is designated as SEQ ID NO: 44, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 45, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in has been introduced is designated as SEQ ID NO: 46, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 47, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <Q> has been introduced is designated as SEQ ID NO: 48, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 49, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <R> has been introduced is designated as SEQ ID NO: 50, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 51, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <S> has been introduced is designated as SEQ ID NO: 52, and the base sequence encoding the T7RNA polymerase is designated as SEQ ID NO: 53, The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <T> has been introduced is shown as SEQ ID NO: 54, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 55. The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in has been introduced is shown as SEQ ID NO: 56, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 57. The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <V> has been introduced is shown as SEQ ID NO: 58, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 59. The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <W> has been introduced is shown as SEQ ID NO: 60, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 61.

[0188] Example 10 Preparation of T7RNA polymerase (Part 3) (1) Using a plasmid capable of expressing T7m6 prepared in Example 1 (3), or a plasmid capable of expressing a T7RNA polymerase mutant containing the polynucleotide described in SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 prepared in Example 9, Escherichia coli BL21 strain (Takara Bio Inc.) was transformed to produce a T7RNA polymerase producing strain (transformed organism). (2) The T7RNA polymerase producing strain prepared in (1) was inoculated into 3 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing an appropriate amount of antibiotic, and pre-cultured by shaking overnight at 37°C and 180 rpm. (3) The pre-culture solution from (2) was inoculated into 100 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing an appropriate amount of antibiotic, dispensed into a 500 mL baffled flask, and cultured with shaking at 37°C and 130 rpm for 5 hours. (4) IPTG was added to a final concentration of 2 mmol / L, and cultured further overnight at 25°C and 130 rpm. (5) The culture solution from (4) was centrifuged at 4°C and 15000 × G for 20 minutes, and the wet bacterial cells were collected by discarding the supernatant. The collected bacterial cells were stored at -80°C to -20°C. (6) The wet bacterial cells recovered in (5) were mixed with 20 mmol / L Tris-HCl buffer (pH 7.2) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3000 Unit / L Benzonase (Merck), 0.006% (w / v) lysozyme, 2 mmol / L phenylmethylsulfonyl fluoride, 0.02% (w / v) sodium deoxycholate, and 0.6% (w / v) Triton X-100 (trade name). After stirring at room temperature for 1 hour, the mixture was centrifuged at 4°C and 15000 rpm for 60 minutes to obtain an extract containing expressed T7 RNA polymerase.(7) After sterilizing the supernatant collected in (4) using a 0.22 μm pore size filter, it was added to 1 mL of TALON Metal Affinity Resin (Takara Bio Inc.) which had been pre-equilibrated with phosphate buffer (5.4 g / L sodium dihydrogen phosphate dihydrate, 11.7 g / L sodium chloride, pH 7.4). Subsequently, it was washed with 10 mL of the phosphate buffer containing 10 mmol / L imidazole, and then eluted with 2.5 mL of the phosphate buffer containing 150 mmol / L imidazole. (8) Imidazole was removed from the eluate in (7) using a PD-10 column (Cytiva Inc.) to obtain a purified T7 RNA polymerase solution. The solution was diluted to a concentration of 5 mg / mL.

[0189] Example 11 Evaluation of heat resistance of T7RNA polymerase (Part 3) (1) Each purified T7RNA polymerase prepared in Example 10 (8) was heated at 59°C or 59.5°C for 5 minutes using a thermal cycler, and then held at 4°C. (2) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). In addition, the T7RNA polymerase, which is also a component of the reagent, was replaced with each T7RNA polymerase that had been heat-treated in (1), and the positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 50°C. Measurement was performed by monitoring changes in fluorescence intensity using the automated gene testing device TRCReady-80 (manufactured by Tosoh Corporation), and the detection time was defined as the time when the fluorescence measurement value became 1.2 times the initial fluorescence value.

[0190] The results are shown in Table 5. From Table 5, it can be seen that T7RNA polymerases containing any one amino acid substitution among E108G, T127A, S128N, E242I, V384A, K441R, L446F, V490A, S495D, Q505A, C530G, L534V, I543L, I581M, T630V, S633P, V650I, V687E, I810V, M832F, T835L, and M861L all showed a shorter detection time after heat treatment at 59°C compared to T7m6. In particular, T7 RNA polymerases containing any one amino acid substitution from among E108G, T127A, S128N, E242I, V384A, K441R, L446F, V490A, C530G, L534V, I543L, I581M, T630V, S633P, V650I, V687E, I810V, M832F, and M861L were detectable after heat treatment at 59.5°C for T7m6, whereas they were undetectable for T7m6. These results indicate that introducing any one amino acid mutation from among E108G, T127A, S128N, E242I, V384A, K441R, L446F, V490A, S495D, Q505A, C530G, L534V, I543L, I581M, T630V, S633P, V650I, V687E, I810V, M832F, T835L, and M861L into T7m6 improves the thermal stability of T7RNA polymerase.

[0191]

[0192] Example 12 Accumulation of Amino Acid Mutations Further improvement in thermal stability was attempted by accumulating the amino acid substitutions that were found to be involved in improving the thermal stability of T7 RNA polymerase in Example 11. Specifically, the T7 RNA polymerase (SEQ ID NO: 58) prepared in Example 10 by introducing the T835L amino acid substitution was introduced into T7m6, and the amino acid substitutions shown below from <X> to <AA> were introduced: <X> Substitution of the glutamic acid residue at position 242 of SEQ ID NO: 58 with an isoleucine residue <Y> Substitution of the lysine residue at position 441 of SEQ ID NO: 58 with an arginine residue <Z> Substitution of the threonine residue at position 630 of SEQ ID NO: 58 with a valine residue <AA> Substitution of the serine residue at position 633 of SEQ ID NO: 58 with a proline residue

[0193] The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <X> has been introduced is shown as SEQ ID NO: 62, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 63, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <Y> has been introduced is shown as SEQ ID NO: 64, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 65, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <Z> has been introduced is shown as SEQ ID NO: 66, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 67, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AA> has been introduced is shown as SEQ ID NO: 68, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 69.

[0194] Example 13 Evaluation of heat resistance of T7RNA polymerase (Part 4) (1) Using a plasmid capable of expressing T7RNA polymerase containing the polynucleotide described in SEQ ID NO: 59 prepared in Example 9, or a plasmid capable of expressing T7RNA polymerase in which multiple amino acid mutations have been introduced into T7m6, containing the polynucleotide described in SEQ ID NO: 63, 65, 67, or 69 prepared in Example 12, Escherichia coli BL21 strain (Takara Bio Inc.) was transformed to produce a T7RNA polymerase-producing strain (transformed organism). (2) Purified T7RNA polymerase was prepared in the same manner as in Examples 10(2) to (8). (3) Each purified T7RNA polymerase prepared in (2) was heated in a thermal cycler at 59.5°C, 60°C, or 61°C for 5 minutes, and then kept at 4°C. (4) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent for Mycobacterium tuberculosis, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). Similarly, the T7 RNA polymerase, another component, was replaced with the T7 RNA polymerase heat-treated in (3), and the positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 50°C. The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time at which the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0195] The results are shown in Table 6. From Table 6, it can be seen that T7RNA polymerases in which one of the amino acid substitutions E242I, K441R, T630V, or S633P was introduced in addition to T835L into T7m6 (SEQ ID NO: 6) showed a shorter detection time after heat treatment at 59.5°C compared to T7RNA polymerases in which only the T835L amino acid substitution was introduced. Furthermore, while T7RNA polymerases in which only the T835L amino acid substitution was introduced were undetectable after heat treatment at 60°C, T7RNA polymerases in which one of the amino acid substitutions E242I, K441R, T630V, or S633P was introduced in addition to T835L into T7m6 were detectable. In particular, T7RNA polymerases in which the amino acid substitutions S633P and T835L were introduced into T7m6 were detectable even after heat treatment at 61°C. These results show that introducing amino acid mutations E242I, K441R, T630V, or S633P in addition to T835L into T7m6 (SEQ ID NO: 6) improves thermal stability compared to T7RNA polymerase in which only the T835L amino acid substitution is introduced.

[0196]

[0197] Example 14 Accumulation of Amino Acid Mutations (Part 2) The T7 RNA polymerase (SEQ ID NO: 68, named T7m8) prepared in Example 12 by introducing the amino acid substitutions S633P and T835L into T7m6 (SEQ ID NO: 6) was subjected to the amino acid substitutions shown below from <AB> to <AE>: <AB> Substitution of the glutamic acid residue at position 242 of SEQ ID NO: 68 with an isoleucine residue <AC> Substitution of the lysine residue at position 441 of SEQ ID NO: 68 with an arginine residue <AD> Substitution of the isoleucine residue at position 543 of SEQ ID NO: 68 with a leucine residue <AE> Substitution of the threonine residue at position 630 of SEQ ID NO: 68 with a valine residue

[0198] The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AB> has been introduced is shown as SEQ ID NO: 70, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 71, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AC> has been introduced is shown as SEQ ID NO: 72, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 73, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AD> has been introduced is shown as SEQ ID NO: 74, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 75, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AE> has been introduced is shown as SEQ ID NO: 76, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 77.

[0199] Example 15 Evaluation of heat resistance of T7RNA polymerase (Part 5) (1) Using a plasmid capable of expressing T7m8 (SEQ ID NO: 68) prepared in Example 12, or a plasmid capable of expressing a T7RNA polymerase containing polynucleotides described in SEQ ID NO: 71, 73, 75, or 77 prepared in Example 14, Escherichia coli BL21 strain (Takara Bio Inc.) was transformed to produce a T7RNA polymerase-producing strain (transformed organism). (2) Purified T7RNA polymerase was prepared in the same manner as in Example 10 (2) to (8). (3) Each purified T7RNA polymerase prepared in (2) was heated in a thermal cycler at 61.5°C or 62°C for 5 minutes, and then kept at 4°C. (4) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent for Mycobacterium tuberculosis, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). Similarly, the T7 RNA polymerase, another component, was replaced with the T7 RNA polymerase heat-treated in (3), and the positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 50°C. The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time at which the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0200] The results are shown in Table 7. From Table 7, T7m8 (SEQ ID NO: 68) was undetectable after heat treatment at 61.5°C, while T7RNA polymerases with any of the amino acid substitutions E242I, K441R, I543L, or T630V introduced into T7m8 were detectable. In particular, T7RNA polymerase with the amino acid substitution E242I introduced into T7m8 was detectable even after heat treatment at 62°C. From these results, it can be seen that introducing any of the amino acid mutations E242I, K441R, I543L, or T630V into T7m8 improves its thermal stability compared to T7m8.

[0201] Example 16 Accumulation of Amino Acid Mutations (Part 3) The amino acid substitutions shown below from <AF> to <AH> were introduced into T7m8 (SEQ ID NO: 68) prepared in Example 12; <AF> Substitution of the valine residue at position 490 of SEQ ID NO: 68 with an alanine residue, substitution of the cysteine ​​residue at position 530 with a glycine residue, substitution of the isoleucine residue at position 810 with a valine residue, and substitution of the methionine residue at position 861 with a leucine residue (named T7m12-a) <AG> Substitution of the valine residue at position 490 of SEQ ID NO: 68 with an alanine residue, substitution of the cysteine ​​residue at position 530 with a glycine residue, substitution of the isoleucine residue at position 543 with a leucine residue, and substitution of the methionine residue at position 861 with a leucine residue (named T7m12-b) <AH> Substitution of the glutamic acid residue at position 108 of sequence number 68 with a glycine residue, substitution of the isoleucine residue at position 581 with a methionine residue, substitution of the valine residue at position 650 with an isoleucine residue, and substitution of the isoleucine residue at position 810 with a valine residue (named T7m12-c).

[0202] The amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AF> has been introduced is shown as SEQ ID NO: 78, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 79, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AG> has been introduced is shown as SEQ ID NO: 80, the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 81, the amino acid sequence of the T7RNA polymerase into which the amino acid substitution shown in <AH> has been introduced is shown as SEQ ID NO: 82, and the base sequence encoding the T7RNA polymerase is shown as SEQ ID NO: 83.

[0203] Example 17 Evaluation of heat resistance of T7RNA polymerase (Part 6) (1) Using a plasmid capable of expressing T7m8 (SEQ ID NO: 68) prepared in Example 12, or a plasmid capable of expressing a T7RNA polymerase containing the polynucleotide described in SEQ ID NO: 79, 81, or 83 prepared in Example 16, Escherichia coli BL21 strain (Takara Bio Inc.) was transformed to produce a T7RNA polymerase-producing strain (transformed organism). (2) Purified T7RNA polymerase was prepared in the same manner as in Example 10 (2) to (8). (3) Each purified T7RNA polymerase prepared in (2) was heated at 61°C or 62°C for 5 minutes using a thermal cycler, and then kept at 4°C. (4) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent for Mycobacterium tuberculosis, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). Similarly, the T7 RNA polymerase, another component, was replaced with the T7 RNA polymerase heat-treated in (3), and the positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 50°C. The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time at which the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0204] The results are shown in Table 8. From Table 8, T7m8 (SEQ ID NO: 68) was undetectable after heat treatment at 61°C, while T7m12-a, T7m12-b, and T7m12-c remained detectable even after heat treatment at 62°C. From these results, it can be seen that introducing amino acid mutations into T7m8 as shown in Example 16 improves the thermal stability compared to T7m8.

[0205]

[0206] Example 18 Evaluation of heat resistance of T7RNA polymerase (Part 7) (1) In the TRCReady MTB (Tosoh Corporation) reagent for detecting Mycobacterium tuberculosis rRNA, the AMV reverse transcriptase was replaced with purified AMV reverse transcriptase (AMV-RT m11) obtained in Example 4 (3). In addition, the T7RNA polymerase, which is also a component of the reagent, was replaced with T7m5 (SEQ ID NO: 2) prepared in Example 7 (9) and the T7RNA polymerases prepared in Examples 17 (1) and (2). Positive standard RNA (detection target RNA with known concentration) included with the reagent was measured at a reaction temperature of 54.5°C or 55°C (measurement at 55°C was performed with N=2). The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time when the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0207] The results are shown in Table 9. From Table 9, it can be seen that T7m8 (SEQ ID NO: 68) prepared in Example 12, and T7m12-a, T7m12-b, and T7m12-c prepared in Example 17, showed a shorter detection time at a reaction temperature of 54.5°C compared to the known mutant T7m5 (SEQ ID NO: 2). Furthermore, while T7m5 was undetectable at a reaction temperature of 55°C, T7m8, T7m12-a, T7m12-b, and T7m12-c were detectable. From these results, it can be said that the modified T7RNA polymerase of this disclosure has improved reactivity at high temperatures compared to the known T7RNA polymerase (T7m5), and is useful in fields such as genetic testing.

[0208]

[0209] Example 19 Evaluation of heat resistance of T7 RNA polymerase (Part 8) (1) 20 μL of the reaction solution described in Table 10 was prepared in a PCR tube, and the transcription reaction (RNA synthesis) was carried out by incubation at 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, or 58°C for 90 minutes, after which it was kept at 4°C. The T7 RNA polymerases used were T7m5 (SEQ ID NO: 2) prepared in Example 7 (9), T7m12-a, T7m12-b, T7m12-c prepared in Examples 17 (1) and (2), and a commercially available heat-resistant T7 RNA polymerase (Toyobo Co., Ltd., Thermo T7 RNA Polymerase <<TT7>>), and the standard DNA used was double-stranded DNA containing the T7 promoter sequence (SEQ ID NO: 84).

[0210]

[0211] (2) The obtained transcript was mixed with Quant-iT RNA Assay Kit (Invitrogen) on a microplate, and the amount of transcript was measured by detecting fluorescence with a plate reader. The results of the fluorescence intensity measurement are shown in Figure 3, and the relative values ​​with the fluorescence intensity at 46°C for each T7RNA polymerase set to 100% are shown in Figure 4. From Figure 3, it can be seen that the T7RNA polymerases of this disclosure (T7m12-a, T7m12-b, and T7m12-c) show higher specific activity (RNA productivity) at all reaction temperatures compared with known T7RNA polymerases (T7m5 and commercially available heat-resistant T7RNA polymerases). Furthermore, from Figure 4, it can be seen that while known T7RNA polymerases are inactivated by heat at reaction temperatures of 52°C to 54°C, T7m12-a, T7m12-b, and T7m12-c maintain their activity up to approximately 56°C, indicating high heat resistance. These results indicate that the modified T7RNA polymerase described herein exhibits improved specific activity and heat resistance in in vitro transcription (IVT) compared to known T7RNA polymerases (T7m5 and commercially available heat-stable T7RNA polymerase), making it useful in the field of RNA production, such as mRNA pharmaceuticals.

[0212] Example 20 Evaluation of Transcription Byproducts (dsRNA) of T7RNA Polymerase (1) Transcription reactions (RNA synthesis) were carried out using modified T7RNA polymerase prepared in Examples 2, 10, 12, and 17 and wild-type T7RNA polymerase (TaKaRa). The reaction was carried out in the same manner as in Example 19 (1), except that the reaction temperature was set to 50°C for the modified T7RNA polymerase and 37°C for the wild-type T7RNA polymerase. (2) The transcripts from (1) were used in NucleoSpin Ｒ RNA solutions were prepared by purification using RNA (TaKaRa), and the total amount of RNA was quantified using a NanoDrop micro-spectrophotometer (Thermo Fisher Scientific). (3) The RNA solutions from (2) were diluted to the same concentration with TE buffer (10 mmol / L Tris, 0.1 mmol / LEDTA, pH 8.0), and the amount of dsRNA contained in the RNA solutions was measured using a Double-stranded RNA ELISA kit (Exalpha).

[0213] The results are shown in Table 11. In Table 11, the amount of dsRNA is shown as a relative value with the amount of dsRNA after the transcription reaction using wild-type T7RNA polymerase set to 1. From Table 11, it can be seen that when RNA synthesis is performed at high temperature using modified T7RNA polymerase, the amount of dsRNA by-products that causes adverse reactions (immunogenicity) in mRNA pharmaceuticals can be suppressed to less than 1 / 4 compared to when using wild-type T7RNA polymerase.

[0214] Furthermore, in reactions using the modified T7 RNA polymerase of this disclosure, the total amount of RNA is greater than when using the known modified T7 RNA polymerase (T7m5), and as described in Example 19, the specific activity (RNA productivity) is higher. From these results, it can be said that the modified T7 RNA polymerase of this disclosure is also suitable for RNA synthesis, such as mRNA pharmaceuticals.

[0215]

[0216] Example 21 Mutation introduction into the T7RNA polymerase gene (1) A polynucleotide (SEQ ID NO: 3) encoding a T7RNA polymerase (named T7m5) containing the amino acid sequence described in SEQ ID NO: 2 was synthesized. In T7m5, the amino acid residue corresponding to the 430th serine residue of wild-type T7RNA polymerase (GenBank: ACO57213.11, SEQ ID NO: 1) was replaced with a proline residue (hereinafter also referred to as S430P), the amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 was replaced with a valine residue (hereinafter also referred to as M490V), the amino acid residue corresponding to the 510th cysteine ​​residue of SEQ ID NO: 1 was replaced with an arginine residue (hereinafter also referred to as C510R), and the 767th serine residue of SEQ ID NO: 1 was replaced with a proline residue. The amino acid residue corresponding to the phosphorus residue is substituted with a glycine residue (hereinafter also referred to as S767G), the amino acid residue corresponding to the glutamine residue at position 786 of SEQ ID NO: 1 is substituted with a methionine residue (hereinafter also referred to as Q786M), the amino acid residue corresponding to the phenylalanine residue at position 849 of SEQ ID NO: 1 is substituted with an isoleucine residue (hereinafter also referred to as F849I), and the amino acid residue corresponding to the phenylalanine residue at position 880 of SEQ ID NO: 1 is substituted with a tyrosine residue (hereinafter also referred to as F880Y). During synthesis, an oligonucleotide encoding a histidine tag consisting of the amino acid sequence described in SEQ ID NO: 4 (SEQ ID NO: 5) is added to the 5' end, and a stop codon (TAA) is added to the 3' end. (2) A plasmid capable of expressing T7m5 was created by inserting the synthesized polynucleotide between the restriction enzyme NcoI / HindIII cleavage sites of plasmid pTrc99A. (3) From the T7m5-expressing plasmids obtained in (2), nucleotide substitutions were introduced at predetermined positions in the polynucleotide encoding T7m5 (SEQ ID NO: 2) (SEQ ID NO: 3) to create plasmids capable of expressing the T7RNA polymerase mutant. Specifically, nucleotide substitutions were introduced to replace the valine residue at position 114 of SEQ ID NO: 2 with an alanine residue, cysteine ​​residue, glutamic acid residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, or tryptophan residue, respectively.

[0217] The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with an alanine residue (also known as V114A) is shown in SEQ ID NO: 85, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 86. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a cysteine ​​residue (also known as V114C) is shown in SEQ ID NO: 87, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 88. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a glutamic acid residue (also known as V114E) is shown in SEQ ID NO: 89, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 90. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a lysine residue (also known as V114K) is shown in SEQ ID NO: 91, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 92. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a leucine residue (also denoted as V114L) is shown in SEQ ID NO: 93, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 94. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a methionine residue (also denoted as V114M) is shown in SEQ ID NO: 95, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 96. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with an asparagine residue (also denoted as V114N) is shown in SEQ ID NO: 97, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 98. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with a glutamine residue (also denoted as V114Q) is shown in SEQ ID NO: 99, and the nucleotide sequence encoding this T7RNA polymerase is shown in SEQ ID NO: 100. The amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of SEQ ID NO: 2 with an arginine residue (also denoted as V114R) is shown as SEQ ID NO: 101, and the nucleotide sequence encoding said T7RNA polymerase is shown as SEQ ID NO: 102.Sequence ID 103 shows the amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of Sequence ID 2 with a serine residue (also denoted as V114S), and Sequence ID 104 shows the nucleotide sequence encoding this T7RNA polymerase. Sequence ID 105 shows the amino acid sequence of T7RNA polymerase obtained by substituting the valine residue at position 114 of Sequence ID 2 with a tryptophan residue (also denoted as V114W), and Sequence ID 106 shows the nucleotide sequence encoding this T7RNA polymerase.

[0218] Example 22 Preparation of T7RNA polymerase (1) Using the plasmid capable of expressing T7m5 prepared in Example 21(2), or the plasmid capable of expressing a T7RNA polymerase mutant by introducing nucleotide substitutions at predetermined positions, prepared in Example 21(3), Escherichia coli BL21 strain (Takara Bio Inc.) was transformed to produce T7RNA polymerase-producing strains (transformers). (2) Each T7RNA polymerase-producing strain prepared in (1) was inoculated into 3 mL of 2×YT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) containing an appropriate amount of antibiotic, and pre-cultured by shaking overnight at 37°C and 180 rpm. (3) The pre-culture solution from (2) was inoculated into 100 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing an appropriate amount of antibiotic, dispensed into a 500 mL baffled flask, and cultured with shaking at 37°C and 130 rpm for 5 hours. (4) IPTG was added to a final concentration of 2 mmol / L, and cultured further overnight at 25°C and 130 rpm. (5) The culture solution from (4) was centrifuged at 4°C and 15000 × G for 20 minutes, and the wet bacterial cells were collected by discarding the supernatant. The collected bacterial cells were stored at -20°C to -80°C.

[0219] (6) The wet bacterial cells recovered in (5) were mixed with 50 mmol / L Tris-HCl buffer (pH 7.2) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3000 Unit / L Benzonase (Merck), 0.006% (w / v) lysozyme, 2 mmol / L phenylmethylsulfonyl fluoride, and 0.6% (w / v) Triton X-100 (trade name). After stirring at room temperature for 1 hour, the mixture was centrifuged at 4°C and 15000 rpm for 60 minutes to obtain an extract containing expressed T7 RNA polymerase. (7) After sterilizing the supernatant collected in (4) using a filter with a pore size of 0.22 μm, it was added to 1 mL of TALON Metal Affinity Resin (Takara Bio Inc.) which had been pre-equilibrated with phosphate buffer (5.4 g / L sodium dihydrogen phosphate dihydrate, 11.7 g / L sodium chloride, pH 7.4). Subsequently, it was washed with 10 mL of the phosphate buffer containing 10 mmol / L imidazole, and then eluted with 2.5 mL of the phosphate buffer containing 150 mmol / L imidazole. (8) The productivity (purified yield per culture medium) was evaluated by quantifying the purified T7 RNA polymerase contained in the eluate obtained in (7) using a NanoDrop micro-spectrophotometer (Thermo Fisher Scientific Inc.). (9) Using a PD-10 column (Cytiva), imidazole was removed from the eluate of (7) to obtain a purified T7 RNA polymerase solution. This solution was diluted to a concentration of 5 mg / mL.

[0220] The results are shown in Figure 5. In Figure 5, the purified yield per culture medium for each T7RNA polymerase variant is shown as a relative value obtained by dividing the purified yield per culture medium for T7m5 (SEQ ID NO: 2). From Figure 5, it was found that T7RNA polymerases containing V114A, V114C, V114E, V114L, V114M, V114N, V114Q, V114R, V114S, and V114W each had a higher purified yield per culture medium compared to T7m5. In particular, it was found that T7RNA polymerases containing V114A, V114C, V114K, V114M, V114N, V114Q, V114R, V114S, or V114W each had a purified yield per culture medium that was more than twice as high compared to T7m5. Furthermore, it was found that T7RNA polymerases containing V114A, V114C, V114M, V114N, V114Q, or V114W yielded more than 2.5 times higher purified yield per culture medium compared to T7m5. In addition, it was found that T7RNA polymerases containing V114A, V114N, V114Q, or V114W yielded more than 3 times higher purified yield per culture medium compared to T7m5.

[0221] Example 23 Evaluation of T7 RNA polymerase activity (1) Escherichia coli W3110 strain was transformed according to a standard method with a vector capable of expressing AMV reverse transcriptase consisting of the amino acid sequence described in SEQ ID NO: 12 (AMV-RT m11). (2) Each of the resulting transformants was cultured overnight at 37°C on LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L purified agar) containing an appropriate amount of antibiotic to obtain production strains of each AMV reverse transcriptase. (3) Using each production strain obtained in (2), purified AMV reverse transcriptase was obtained by culturing in a fermenter and purifying by column chromatography according to the method described in Japanese Patent Application Publication No. 2014-209898. (4) The concentration of the obtained purified AMV enzyme was quantified by size exclusion chromatography using a TSKgel UP-SW Aggregate column (manufactured by Tosoh Corporation). (5) In the TRCReady MTB (Tosoh Corporation) rRNA detection reagent for Mycobacterium tuberculosis, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11) obtained in (3). Similarly, the T7 RNA polymerase, another component, was replaced with either T7m5 prepared in Example 22 (9) or a T7 RNA polymerase mutant in which an amino acid mutation was introduced at a predetermined position. (6) The positive standard RNA (detection target RNA with known concentration) included with the reagent was measured. The measurement was performed by monitoring the change in fluorescence intensity using the TRCReady-80 automated gene testing device (Tosoh Corporation), and the time when the fluorescence measurement value became 1.2 times the initial fluorescence value was defined as the detection time.

[0222] The results are shown in Table 12. All T7RNA polymerase mutants were detectable in the same way as T7m5, and it was found that their activity was retained even after amino acid substitution. In particular, T7RNA polymerases containing V114A, V114C, V114K, V114L, V114M, V114S, or V114W were shown to have activity equivalent to that of T7m5. Furthermore, T7RNA polymerases containing V114C, V114L, V114M, or V114S were shown to have activity even more equivalent to that of T7m5.

[0223]

[0224] According to one aspect of this disclosure, a T7 RNA polymerase with improved thermal stability can be provided. Surprisingly, the amino acid mutations found in this disclosure can also be introduced into T7 RNA polymerase variants in combination with previously reported mutations that improve thermal stability, thereby providing a T7 RNA polymerase with even greater thermal stability. Furthermore, according to one aspect of this disclosure, a T7 RNA polymerase with improved resistance to reaction inhibition by contaminants can be provided. Furthermore, according to one aspect of this disclosure, a T7 RNA polymerase with improved productivity can be provided. Furthermore, according to one aspect of this disclosure, a T7 RNA polymerase with improved specific activity (RNA productivity) can be provided. According to this aspect, it is expected to be useful in infectious disease testing of various clinical specimens using nucleic acid amplification methods.

[0225] The modified T7RNA polymerase described herein exhibits improved thermal stability, resistance to reaction inhibition by contaminants, and / or productivity compared to conventional T7RNA polymerases. Therefore, replacing the T7RNA polymerase in reagents for RNA synthesis from target nucleic acids or amplification reagents for target nucleic acids with the enzyme described herein is expected to improve the performance of these reagents. Furthermore, the T7RNA polymerase described herein is useful in the production of mRNA pharmaceuticals such as vaccines via in vitro transcription reactions, as well as in research reagents.

Claims

1. A T7 RNA polymerase selected from any of (i) to (iii) below: (i) A T7 RNA polymerase having an amino acid sequence described in SEQ ID NO: 1, which includes at least one of the amino acid substitutions shown in (1) to (12) below: (1) The amino acid residue corresponding to the 114th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue, threonine residue, alanine residue, cysteine ​​residue, glutamic acid residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, or tryptophan residue. (2) The amino acid residue corresponding to the 835th threonine residue in SEQ ID NO: 1 is replaced with a leucine residue. (3) The amino acid residue corresponding to the 127th threonine residue in SEQ ID NO: 1 is replaced with an alanine residue. (4) The amino acid residue corresponding to the 128th serine residue in SEQ ID NO: 1 is replaced with an asparagine residue. (5) The amino acid residue corresponding to the 242nd glutamic acid residue in SEQ ID NO: 1 is replaced with an isoleucine residue. (6) The amino acid residue corresponding to the 384th valine residue in SEQ ID NO: 1 is replaced with an alanine residue. (7) The amino acid residue corresponding to the 543rd isoleucine residue in SEQ ID NO: 1 is replaced with a leucine residue. (8) The amino acid residue corresponding to the 581st isoleucine residue in SEQ ID NO: 1 is replaced with a methionine residue. (9) The amino acid residue corresponding to the 630th threonine residue in SEQ ID NO: 1 is replaced with a valine residue. (10) The amino acid residue corresponding to the 687th valine residue in SEQ ID NO: 1 is replaced with a glutamic acid residue. (11) The amino acid residue corresponding to the 861st methionine residue in SEQ ID NO: 1 is replaced with a leucine residue. (12) The amino acid residue corresponding to the 810th isoleucine residue in SEQ ID NO: 1 is replaced with a valine residue. (ii) An amino acid sequence described in Sequence ID No. 1, which includes at least one of the amino acid substitutions shown in (1) to (12) above, and further includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (12), and has enzymatic activity, and is a T7 RNA polymerase;(iii) An amino acid sequence described in Sequence ID No. 1, which has 70% or more identity with the entire amino acid sequence containing at least one of the amino acid substitutions shown in (1) to (12) above, provided that the amino acid substitutions shown in (1) to (12) above are maintained, and is a T7 RNA polymerase that has enzymatic activity.

2. The T7 RNA polymerase according to claim 1, having an amino acid sequence described in Sequence ID No. 1, which includes at least the amino acid substitution shown in (1'); (1') The amino acid residue corresponding to the 114th valine residue of Sequence ID No. 1 is substituted with an isoleucine residue.

3. The T7 RNA polymerase according to claim 1, wherein the amino acid sequence of the T7 RNA polymerase is further an amino acid sequence having one or more amino acid substitutions selected from (a) to (q) below: (a) The amino acid residue corresponding to the 430th serine residue of SEQ ID NO: 1 is substituted with a proline residue; (b) The amino acid residue corresponding to the 490th methionine residue of SEQ ID NO: 1 is substituted with a valine or alanine residue; (c) The amino acid residue corresponding to the 510th cysteine ​​residue of SEQ ID NO: 1 is substituted with an arginine residue; (d) The amino acid residue corresponding to the 767th serine residue of SEQ ID NO: 1 is substituted with a glycine residue; (e) The amino acid residue corresponding to the 786th glutamine residue of SEQ ID NO: 1 is substituted with a methionine residue; (f) The amino acid residue corresponding to the 849th phenylalanine residue of SEQ ID NO: 1 is substituted with an isoleucine residue; (g) The amino acid residue corresponding to the 880th phenylalanine residue of SEQ ID NO: 1 is substituted with a tyrosine residue (h) The amino acid residue corresponding to the 108th glutamic acid residue in SEQ ID NO: 1 is replaced with a glycine residue; (i) The amino acid residue corresponding to the 441st lysine residue in SEQ ID NO: 1 is replaced with an arginine residue; (j) The amino acid residue corresponding to the 446th leucine residue in SEQ ID NO: 1 is replaced with a phenylalanine residue; (k) The amino acid residue corresponding to the 495th serine residue in SEQ ID NO: 1 is replaced with an aspartic acid residue; (l) The amino acid residue corresponding to the 505th glutamine residue in SEQ ID NO: 1 is replaced with an alanine residue; (m) The amino acid residue corresponding to the 530th cysteine ​​residue in SEQ ID NO: 1 is replaced with a glycine residue; (n) The amino acid residue corresponding to the 534th leucine residue in SEQ ID NO: 1 is replaced with a valine residue; (o) The amino acid residue corresponding to the 633rd serine residue in SEQ ID NO: 1 is replaced with a proline residue; (p) The amino acid residue corresponding to the 650th valine residue in SEQ ID NO: 1 is replaced with an isoleucine residue; (q) The amino acid residue corresponding to the 832nd methionine residue in Sequence ID No. 1 is replaced with a phenylalanine residue.

4. The T7 RNA polymerase according to claim 1, wherein (i) to (iii) are (iv) to (vi), respectively: (iv) A T7 RNA polymerase having the amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105; (v) A T7 RNA polymerase having an amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, which includes one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and which has enzymatic activity; (vi) A T7 RNA polymerase having an amino acid sequence that has 70% or more identity with the amino acid sequence described in SEQ ID NOs: 6, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, and which is enzymatically active.

5. A polynucleotide encoding the T7 RNA polymerase according to any one of claims 1 to 4.

6. An expression vector comprising the polynucleotide described in claim 5.

7. A transformant obtained by transforming a host with the expression vector described in claim 6.

8. The transformant according to claim 7, wherein the host is Escherichia coli.

9. A method for producing T7 RNA polymerase, comprising the steps of culturing the transformant described in claim 7 to express T7 RNA polymerase, and recovering the polymerase expressed from the obtained culture.

10. A target nucleic acid amplification reagent comprising the T7 RNA polymerase described in any one of claims 1 to 4.

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