JP5789949B2 - Improved RNA polymerase variants - Google Patents

Description

  The present invention relates to a recombinant RNA polymerase produced using a host cell, wherein the RNA polymerase whose productivity in the host cell is improved by mutating the amino sequence of the wild type (natural type) RNA polymerase, the RNA The present invention relates to a gene encoding polymerase and a method for producing RNA polymerase using the gene.

  Polymerases, which are enzymes that perform nucleic acid elongation reactions, are broadly classified into DNA polymerases and RNA polymerases depending on the nucleic acids that serve as substrates. DNA polymerases can be further divided into two types: DNA-dependent DNA polymerases that play a central role in DNA replication and DNA repair, and RNA-dependent DNA polymerases that perform reverse transcription and telomere synthesis. On the other hand, RNA polymerase is divided into three types: DNA-dependent RNA polymerase that synthesizes mRNA, rRNA, and the like, RNA-dependent RNA polymerase that performs important functions in RNA viruses, and RNA polymerase that does not require a specific template. .

  Polymerase is not only an important research object in understanding life phenomena, but also an industrially important enzyme, and is applied in various industrial fields. For example, Taq polymerase is applied in the PCR method which is a method for amplifying DNA, and AMV reverse transcriptase and T7 RNA polymerase are applied in the TRC method (a patent document 1 and non-patent document 1) which is a method for amplifying RNA.

  As described above, many of the polymerases already applied in the industrial field have known mutant enzymes with enhanced functions. For example, in T7 RNA polymerase, a promoter sequence recognized by amino acid substitution is modified (Patent Document 2), a specific activity and thermal stability at high temperature are enhanced (Patent Document 3), and amino acid deletion and substitution 3 One with enhanced '-deoxyribonucleotide uptake ability (Patent Document 4) is known.

JP 2000-014400 A US Pat. No. 5,385,834 Special table 2003-525627 gazette JP 2003-061683 A

Ishiguro T. et al. , Analytical Biochemistry, 314, 77-86 (2003).

  As for polymerases such as T7 RNA polymerase that have already been applied in industrial fields, various mutant enzymes with enhanced functions are known. However, such functional enhancements are only functional enhancements such as improving activity, improving specificity, improving some resistance, etc., and their productivity, ie, productivity in host cells (expression in host cells). It is not functional enhancement from the viewpoint of producing a larger amount of the enzyme itself, such as improvement of the property.

  Accordingly, the present invention provides a recombinant RNA polymerase produced using a host cell, the RNA polymerase having improved productivity in the host cell by mutating the amino sequence of wild-type (natural) RNA polymerase, A gene encoding RNA polymerase and a method for producing RNA polymerase using the gene are provided.

  As a result of intensive studies to solve the above problems, the present inventors have improved the productivity of T7 RNA polymerase by mutating the amino acid of wild-type T7 RNA polymerase (substitution with other amino acids) by genetic engineering techniques. The present inventors have found that this can be done and have completed the present invention.

  That is, the present invention relates to a recombinant T7 RNA polymerase produced using a host cell, wherein the amino acid residue corresponding to the 490th methionine in the amino acid sequence constituting the wild type (natural type) T7 RNA polymerase is other than It is a T7 RNA polymerase mutant in which the productivity in the host cell is improved compared to the wild type productivity by substitution with an amino acid residue. The present invention also relates to a gene encoding such a T7 RNA polymerase mutant. Further, the present invention is a cell transformed with a vector containing a gene encoding such a T7 RNA polymerase mutant. The present invention is a method for producing T7 RNA polymerase, comprising culturing cells transformed with a vector containing a gene encoding such a T7 RNA polymerase mutant. Hereinafter, the present invention will be described in detail.

  The T7 RNA polymerase mutant of the present invention is obtained by replacing the amino acid residue corresponding to the 490th methionine in the amino acid sequence (SEQ ID NO: 6) constituting the wild type T7 RNA polymerase with another amino acid. The “other amino acid residue” to be substituted is not particularly limited, but a hydrophobic amino acid such as valine, leucine, isoleucine or fanylalanine can be exemplified as a preferred amino acid residue, and valine is exemplified as a particularly preferred amino acid residue. be able to. As a result, it is considered that the substitution of the amino acid residue corresponding to the 490th methionine as described above increased the hydrophobicity of T7 RNA polymerase and improved the stability.

  The three-dimensional structure of T7 RNA polymerase has been elucidated. Substitution of the amino acid residue corresponding to the 490th methionine is essential for achieving “improving productivity”. In the present invention, in addition to the 490th mutation, the activity of a conventionally known T7 RNA polymerase is improved. Mutation etc. for doing so may be added in a superimposed manner. Such mutations include, for example, one or more amino acid residues of an amino acid residue corresponding to the 179th lysine, an amino acid residue corresponding to the 786th glutamine, and an amino acid residue corresponding to the 685th valine. The substitution to the amino acid residue can be illustrated. Here, in the T7 RNA polymerase to which a mutation for substituting the amino acid residue corresponding to the 179th lysine with glutamic acid or the like is added, it is possible to achieve further effects of improving the thermal stability and specific activity, and the 786th position. T7 RNA polymerase to which a mutation that replaces the amino acid residue corresponding to glutamine of leucine and methionine is added can achieve the effects of further improving the thermal stability and specific activity. With T7 RNA polymerase to which a mutation for substituting the corresponding amino acid residue with alanine or the like is added, it is possible to achieve the effect of further improving the thermal stability.

  The gene of the present invention encoding a mutant T7 RNA polymerase can be obtained by introducing a mutation into the wild type T7 RNA polymerase gene (SEQ ID NO: 6). Methods for introducing desired mutations into a given nucleic acid sequence are known to those skilled in the art, for example, site-directed mutagenesis, PCR using degenerate oligonucleotides, exposure of cells containing nucleic acids to mutagens or radiation. Known techniques such as these can be used as appropriate, and for example, the methods described in the embodiments can be referred to. The wild-type T7 RNA polymerase gene is, for example, an appropriate primer prepared from its genomic information from T7 phage (DSM No. 4623, ATCC 11303-B7, NCIMB10380, etc.). Can be obtained by PCR.

  The mutant T7 RNA polymerase of the present invention can be produced by transforming a cell (host cell) with a vector containing the gene of the present invention and culturing the cell.

  The vector containing the gene of the present invention is not particularly limited. For example, a self-replicating vector may be used, or a vector that is integrated into the host cell genome when introduced into the host cell. On the other hand, an expression vector in which elements necessary for the transcription (for example, a promoter and the like) are operatively linked is particularly preferred. A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and may be appropriately selected depending on the type of host. Examples of suitable promoters for E. coli include lac, trp, trc or tac promoters. In addition, useful sequences other than the promoter may be added to the T7 RNA polymerase gene. For example, a gene encoding a signal peptide for secreting the produced T7 RNA polymerase of the present invention out of the host cell is added, or a histidine hexamer is included at the end for the purpose of simplifying the purification operation after production. It can be exemplified by adding a gene that adds a tag sequence.

  For example, when using Escherichia coli as a host cell, it is convenient to incorporate the gene of interest into an appropriate plasmid to obtain the vector of the present invention. Examples of such plasmids include plasmids for expression such as pTrc99A (manufactured by GE Healthcare Bioscience) and pCDF-1b (manufactured by Takara Bio).

  In order to produce the mutant T7 RNA polymerase of the present invention, various cultured cells such as yeast, animal cell lines, plant cells, and insect cells can be used as host cells, but infection with bacteriophages such as JM109 strain and HB101 strain is also possible. Examples of suitable cells (hosts) include E. coli that is easy to handle.

  Transformation of a host cell with a vector may be performed according to a usual method. The mutant T7 RNA polymerase of the present invention can be produced by culturing the thus obtained transformant in an appropriate nutrient medium under conditions that allow expression of the introduced gene of the present invention. . Extraction of mutant T7 RNA polymerase from the culture medium can be performed by applying an ordinary protein purification method. For example, when the mutant T7 RNA polymerase of the present invention is expressed in cells, the cells are collected by centrifugation after culturing, suspended in an appropriate aqueous buffer, then treated with a surfactant, lysozyme, ultrasonic waves. Extract with a crusher or a commercially available protein extraction reagent.

  The amount of RNA polymerase contained in the obtained extract can be measured by an analysis method used for ordinary proteins. For example, electrophoresis using SDS-polyacrylamide, various types of chromatography such as ion exchange, hydrophobic interaction, and gel filtration can be used. In addition, an electrophoresis method using a capillary, a microchip, or the like that can quickly measure even a small amount of sample can be used.

  The T7 RNA polymerase mutant of the present invention is a recombinant RNA polymerase produced using a host cell, and is an RNA polymerase whose productivity in the host cell is improved by mutating the amino sequence of wild-type RNA polymerase. . As shown in the examples described later, a product having an activity equivalent to that of the wild type can be produced with about twice the productivity as compared to the wild type (see Example 4).

  As a result, the T7 RNA polymerase mutant of the present invention can be produced in a larger amount at the same cost as the wild type. This makes it possible to provide T7 RNA polymerase, which is increasingly applied in the industrial field, at a lower cost, and to reduce the costs of various reagents, tests, etc. to which T7 RNA polymerase is applied.

The restriction enzyme map of plasmid pTrc99A-T7RNApol which cloned T7 RNA polymerase. Results of time-lapse analysis by wild-type and M490V mutant T7 RNA polymerase polyacrylamide electrophoresis. Lane 1 is molecular weight marker, lane 2 is before IPTG addition (wild type), lane 3 is 1 hour after IPTG addition (wild type), lane 4 is 2 hours after IPTG addition (wild type), lane 5 is 3 hours after IPTG addition After (wild type), lane 6 is molecular weight marker, lane 7 is molecular weight marker, lane 8 is before IPTG addition (M490V), lane 9 is 1 hour after IPTG addition (M490V), lane 10 is 2 hours after IPTG addition (M490V) ), Lane 11 shows the result of 3 hours after addition of IPTG (M490V), and lane 12 shows the molecular weight marker. The result of having analyzed the microbial cell extract of the wild type T7 RNA polymerase before IPTG addition with a fully automatic chip electrophoresis system. The vertical axis represents fluorescence intensity (read value from the apparatus), and the horizontal axis represents time (seconds). The result of having analyzed the microbial cell extract of the wild type T7 RNA polymerase 3 hours after addition of IPTG with the fully automatic chip electrophoresis system. The vertical axis represents fluorescence intensity (read value from the apparatus), and the horizontal axis represents time (seconds). The result of having analyzed the cell extract of M490V mutant type T7 RNA polymerase before IPTG addition with the fully automatic chip electrophoresis system. The vertical axis represents fluorescence intensity (read value from the apparatus), and the horizontal axis represents time (seconds). The result of having analyzed the microbial cell extract of M490V mutant T7 RNA polymerase 3 hours after addition of IPTG with the fully automatic chip electrophoresis system. The vertical axis represents fluorescence intensity (read value from the apparatus), and the horizontal axis represents time (seconds). The result of having analyzed the purified wild type T7 RNA polymerase with a fully automatic chip electrophoresis system. The vertical axis represents fluorescence intensity (read value from the apparatus), and the horizontal axis represents time (seconds).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.

  Example 1 Cloning of T7 RNA polymerase gene

Cloning of the T7 RNA polymerase gene was performed according to the following method.
(1) Using the T7 phage genomic DNA library (manufactured by Sigma) as a template plasmid, the T7 RNA polymerase gene was amplified using the PCR method with the following reagent composition and reaction conditions divided into the first half and the second half. In the reagent composition, the synthetic DNA primer is primer FF (SEQ ID NO: 1, 20 bases on the 3 ′ end corresponds to the 1st to 20th bases of SEQ ID NO: 5) and primer FR (SEQ ID NO: 5) for amplification of the first half 2, corresponding to the complementary strand of the 1813th to 1839th bases of SEQ ID NO: 5) and the primer RF (corresponding to the 1825th to 1853th bases of SEQ ID NO: 3 and SEQ ID NO: 5) and the primer RR SEQ ID NO: 4, 3 ′ terminal 20 bases corresponded to the complementary strand of the 2633th to 2652th bases of SEQ ID NO: 5), respectively.

(Reagent composition) (total reaction volume: 100 μL)
Each 200 pM synthetic DNA primer 100 ng template plasmid 0.2 mM dNTPs
0.025 unit / μL Taq DNA polymerase (TaKaRa Ex
Taq (trade name), manufactured by Takara Bio)
Buffer attached to the enzyme (reaction conditions)
Using a thermal cycler (manufactured by Perkin-Elmer), after heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 58 ° C. for 30 seconds, 72 ° C. for 1 minute was repeated 25 times.
(2) After the PCR reaction solution was electrophoresed with 1% agarose electrophoresis, ethidium bromide staining was performed, and the band of the target product was cut out from the stained gel to purify the PCR product.
(3) Among the purified PCR products, the first half of the PCR product was digested with restriction enzymes BspHI (New England Biolabs) and HindIII (Takara Bio) and digested with restriction enzymes NcoI (Takara Bio) and HindIII The vector (manufactured by GE Healthcare Bioscience) was reacted at 4 ° C. for 30 minutes using T4 ligase.
(4) The reaction solution of (3) is transformed into E. coli JM109 strain, and LBG / Crb agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg) / ML Carbenicillin (pH 7.4)), and the plasmid retained by the colony grown after overnight culture at 37 ° C. was designated as pTrc99A-T7F.
(5) After preparing pTrc99A-T7F according to a conventional method, the PCR product in the latter half was digested with restriction enzyme HindIII, and reacted with pTrc99A-T7F digested with restriction enzyme HindIII at 4 ° C. for 30 minutes using T4 ligase.
(6) The reaction solution of (5) is transformed into E. coli strain JM109, and LBG / Crb agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg) / ML carbenicillin (pH 7.4)), and the plasmid retained by the colonies grown after overnight culture at 37 ° C. was designated as pTrc99A-T7RNApol. FIG. 1 shows a restriction map of pTrc99A-T7RNApol. For the T7 RNA polymerase gene, it was confirmed that no unintended mutation was introduced by determining the nucleotide sequence using the method shown in Example 2. The gene sequence of the obtained T7 RNA polymerase is shown in SEQ ID NO: 5 (corresponding to bases 1 to 2652 of GenBank No. FJ881694), and the amino acid sequence is shown in SEQ ID NO: 6 (same sequence as GenBank No. NP — 041960).

Example 2 Base Sequence Confirmation Method Recombinant E. coli producing T7 RNA polymerase prepared in Example 1 was transformed into a 37 ° C. LBG / Crb liquid medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5 % Glucose, 50 μg / mL carbenicillin (pH 7.4)), and the plasmid was extracted by a conventional method. The nucleotide sequence of the T7 RNA polymerase gene contained in the extracted plasmid was determined by the following method.
(1) Using BigDye Terminator v3.1 cycle sequencing kit (product name) (manufactured by Applied Biosystems), the attached buffer 2.0 μL, premix 4.0 μL, synthetic DNA primer 3.2 pmol, template plasmid 500 ng is sterilized water Prepare 20 μL with a thermal cycler (manufactured by Perkin-Elmer), heat at 96 ° C. for 1 minute, then repeat the temperature cycle of 96 ° C. · 10 seconds, 50 ° C. · 5 seconds, 60 ° C. · 4 minutes 25 times It was.
(2) Using the BigDye XTerminator purification kit (trade name) Centri-Sep spin column (trade name) (manufactured by Applied Biosystems) (manufactured by Applied Biosystems) using the sample for determining the base sequence prepared in (1), the method shown below Purified.
(2-1) Add 20 μL of BigDye XT terminator solution and 90 μL of SAM solution, and vigorously stir and shake with a shaker for 30 minutes.
(2-2) Centrifuge at 1000 G for 2 minutes.
(3) The base sequence was determined by analyzing the base sequence determination sample prepared in (2) with an ABI PRISM 3130 Genetic Analyzer (trade name) (manufactured by Applied Biosystems). The synthetic DNA primer used for the base sequence determination is (4) the determined base sequence is GENETYX ver. Analysis was performed using 8.0 (trade name) (manufactured by Genetics).

  Primer pTrcFs (SEQ ID NO: 7), primer pTrcRs (SEQ ID NO: 8), primer T7F0 (SEQ ID NO: 9), primer T7F1 (equivalent to the 258th to 287th bases of SEQ ID NO: 10 and SEQ ID NO: 5), primer T7F2 (SEQ ID NO: 5) 11, corresponding to bases 711 to 740 of SEQ ID NO: 5), primer T7F3 (corresponding to bases 1163 to 1192 of SEQ ID NO: 12, SEQ ID NO: 5), primer T7F4 (from SEQ ID NO: 13, 1615 of SEQ ID NO: 5) Equivalent to the 1644th base), primer T7F5 (corresponding to the 2066th to 2095th bases of SEQ ID NO: 14, SEQ ID NO: 5), primer T7F6 (corresponding to the 2520th to 2549th bases of SEQ ID NO: 15, SEQ ID NO: 5), Primer T7R0 (SEQ ID NO: 16), Primer T R1 (corresponding to the complementary strand of nucleotides 2390 to 2419 of SEQ ID NO: 17, SEQ ID NO: 5), primer T7R2 (corresponding to the complementary strand of nucleotides 1932 to 1961 of SEQ ID NO: 5), primer T7R3 ( SEQ ID NO: 19, corresponding to the complementary strand of the 1476th to 1504th bases of SEQ ID NO: 5), primer T7R4 (corresponding to the complementary strand of the 1025th to 1054th bases of SEQ ID NO: 5, SEQ ID NO: 5), primer T7R5 (SEQ ID NO: 21, corresponding to the complementary strand of nucleotides 561 to 590 of SEQ ID NO: 5), primer T7R6 (corresponding to the complementary strand of nucleotides 111 to 140 of SEQ ID NO: 5) is selected and used as necessary did.

Example 3 Production of M490V Mutant T7 RNA Polymerase T7 RNA polymerase mutant in which the methionine at the 490th amino acid sequence was mutated to valine from the T7 RNA polymerase gene of the pTrc99A-T7RNApol plasmid (FIG. 1) produced in Example 1 (M490V mutant) was prepared.
(1) A PCR reaction was performed using the pTrc99A-T7RNApol plasmid (FIG. 1) as a template plasmid under the following reagent composition and reaction conditions. The synthetic primer used was a combination of primer M490VF (SEQ ID NO: 23) and primer FR (SEQ ID NO: 2), and a combination of primer FF (SEQ ID NO: 1) and primer M490VR (SEQ ID NO: 24).

(Reagent composition) (total reaction volume: 50 μL)
Each 100 pM synthetic DNA primer 50 ng template plasmid 0.1 mM dNTPs
0.025 unit / μL DNA polymerase (PrimeSTAR HS
DNA polymerase (trade name, manufactured by Takara Bio Inc.)
Buffer attached to the enzyme (reaction conditions)
Use a thermal cycler (Perkin-Elmer) for 30 seconds at 96 ° C.
After heating, a temperature cycle of 96 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 3 minutes was repeated 30 times.
(2) The reaction solution was separated by 1% agarose electrophoresis, then ethidium bromide staining was performed, and the target product band was cut out from the stained gel and purified.
(3) Using the two types of purified PCR products obtained in (2) as templates, a PCR reaction was further performed to prepare an M490V mutant gene. The reagent composition, reaction conditions, and purification operation in the PCR reaction are the same as (1) to (2) except that a combination of primer FF (SEQ ID NO: 1) and primer FR (SEQ ID NO: 2) was used as a synthetic DNA primer. Same conditions.
(4) The pTrc99A vector digested with restriction enzymes HindIII (manufactured by Takara Bio) and BspHI (manufactured by New England Biolabs) and then digested with restriction enzymes NcoI and HindIII (manufactured by Takara Bio) (4) GE Healthcare Bioscience) was reacted at 4 ° C. for 30 minutes using T4 ligase.
(5) The reaction solution of (4) is transformed into E. coli strain JM109, and LBG / Crb agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg) / ML Carbenicillin (pH 7.4)), and the plasmid retained by the colonies grown after overnight culture at 37 ° C. was designated as pTrc99A-T7F-M490V.
(6) After preparing pTrc99A-T7F-M490V according to a conventional method, the purified PCR product of the latter half of the T7 RNA polymerase gene digested with the restriction enzyme HindIII prepared in Example 1 was converted into pTrc99A-T7F-M490V digested with the restriction enzyme HindIII. The reaction was performed at 4 ° C. for 30 minutes using T4 ligase.
(7) The reaction solution of (6) is transformed into E. coli strain JM109, and LBG / Crb agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg) / ML carbenicillin (pH 7.4)), and a plasmid carrying a colony grown after overnight culture at 37 ° C. was designated as M490V mutant. For the M490V mutation, the introduction of the mutation was confirmed by using the nucleotide sequence determination method shown in Example 2. The gene sequence is shown in SEQ ID NO: 25, and the amino acid sequence is shown in SEQ ID NO: 26.

Example 4 Confirmation of production amount of T7 RNA polymerase Wild-type and M490V mutant T7 RNA polymerase were cultured according to the following procedure.
(1) LBG / Crb liquid medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 50 μg / mL carbenicillin (pH 7.4)) obtained in Examples 1 and 3 in 3 mL Recombinant E. coli producing wild-type and M490V mutant T7 RNA polymerase was inoculated and cultured overnight at 37 ° C. in an 18 mL test tube.
(2) 2 × YTG / Crb medium (1.6% bactotryptone, 1% bacto yeast extract, 0.5% NaCl, 0.5% glucose, 50 μg / mL carbenicillin (pH 7.4)) to 100 mL (1 ) Was inoculated and cultured at 37 ° C., 150 rpm (rotary type, manufactured by Taitec Co., Ltd.) in a 500 mL pleated Erlenmeyer flask.
(3) About 3 to 4 hours after culturing (2) (OD value of about ₆₀₀ to about 3.0 to 3.0), 100 μL of 500 mM IPTG (isopropyl-β-thiogalactopyranoside) was added. The mixture was added, the temperature was lowered to 30 ° C., and the mixture was further cultured with shaking for 3 hours.
(4) After adding IPTG, sample an appropriate amount of the culture solution every 1 hour, 900 μL of that was collected by centrifugation at 14000 rpm for 5 minutes, and the collected cells were stored at −30. did.
(5) 180 μL of a protein extract BugBuster (trade name, manufactured by Novagen) containing the nucleolytic enzyme Benzonae (trade name, manufactured by Novagen) was added to the collected cells and well suspended, followed by treatment at room temperature for 1 hour. .
(6) The obtained extract was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was collected. A portion of the collected supernatant was analyzed by SDS polyacrylamide electrophoresis, and the amount of protein produced was analyzed over time. It was confirmed that T7 RNA polymerase was remarkably produced by addition of IPTG in both the wild type and M490V mutant types. From the darkness of the band corresponding to T7 RNA polymerase near 98 KDa, it can be seen that the M490V mutant is produced more than the wild type, and the mutation of M490V increases the production of T7 RNA polymerase (FIG. 2).
(7) In addition, quantitative analysis was performed using a fully automatic chip electrophoresis system, Experion (trade name, manufactured by Bio-Rad). The content of T7 RNA polymerase 3 hours after the addition of IPTG increased from 9.1% in the wild type to 16.5% in the M490V mutant, indicating that the production efficiency was also improved by the mutation of M490V (FIG. 4, FIG. 6, Table 1).

  Each sequence used in the above examples is shown in Table 2 in addition to the sequence list.

Claims (5)

A recombinant T7 RNA polymerase produced using a host cell, wherein the amino acid residue corresponding to the 490th methionine is valine in the amino acid sequence constituting the wild type (natural type) T7 RNA polymerase described in SEQ ID NO: 6. A T7 RNA polymerase mutant whose productivity in the host cell is improved as compared with wild type productivity. Of the amino acid sequences constituting the wild-type (native) T7 RNA polymerase shown in SEQ ID NO: 6, further substitution of any one or more amino acid residues from the following (1) (3) occurs, according to claim 1 The T7 RNA polymerase variant described in 1.
(1) The amino acid residue corresponding to the 179th lysine is replaced with glutamic acid.
(2) The amino acid residue corresponding to the 786th glutamine is replaced with leucine or methionine.
(3) The amino acid residue corresponding to the 685th valine is replaced with alanine. A gene encoding the T7 RNA polymerase variant according to claim 1 or 2 . A cell transformed with a vector comprising a gene encoding the T7 RNA polymerase variant according to claim 1 or 2 . A method for producing T7 RNA polymerase, comprising culturing a cell transformed with a vector comprising a gene encoding the T7 RNA polymerase variant according to claim 1 or 2 .

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