JP2014082936A - Variant reverse transcriptase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: a variant reverse transcriptase excellent in industrial productivity and capable of efficiently conducting reverse transcription reaction even under a temperature condition suppressing the formation of a secondary structure; a nucleic acid encoding the variant reverse transcriptase; a method for producing the variant reverse transcriptase; a method for reverse transcription; a reverse transcription reaction kit; and a detection kit.SOLUTION: There are provided: a variant reverse transcriptase in which amino acid residues corresponding to at least one residue selected from the group consisting of F303, L432, V433, and I434 are substituted by positively charged amino acid residues in an amino acid sequence of a wild-type MMLV reverse transcriptase; a nucleic acid encoding the variant reverse transcriptase; a method for producing the variant reverse transcriptase by collecting the variant reverse transcriptase from a culture obtained by cultivating cells retaining the nucleic acid; a reverse transcription method for synthesizing cDNA from RNA using the variant reverse transcriptase; a reverse transcription reaction kit containing the variant reverse transcriptase; and a detection kit containing the variant reverse transcriptase and a reagent for detecting a marker.

Description

  The present invention relates to a mutant reverse transcriptase. More specifically, the present invention relates to a mutant reverse transcriptase, a nucleic acid encoding the same, a reverse transcription method using the mutant reverse transcriptase, a reversal useful for gene analysis, disease inspection, useful gene cloning, and the like. The present invention relates to a copy reaction kit and a detection kit.

  Reverse transcriptase has an activity of synthesizing cDNA using RNA as a template (hereinafter referred to as “RNA-dependent DNA polymerase activity”). Among the reverse transcriptases, Moloney murine leukemia virus reverse transcriptase (hereinafter referred to as “MMLV reverse transcriptase”) and avian myeloblastosis virus reverse transcriptase (hereinafter referred to as “removal transcriptase”) have high catalytic activity and accuracy. "AMV reverse transcriptase") is widely used, for example, for gene analysis, disease testing, gene cloning, and the like.

  Among the reverse transcriptases, MMLV reverse transcriptase can be easily produced as a soluble protein in E. coli by genetic engineering techniques. However, MMLV reverse transcriptase has a drawback that the reaction rate during cDNA synthesis is lower than that of AMV reverse transcriptase.

  By the way, when RNA has a base sequence that can easily form a secondary structure, synthesis of cDNA by reverse transcriptase is hindered by the secondary structure, so that the formation of the secondary structure is suppressed by increasing the reaction temperature. It is desirable to synthesize cDNA. However, wild-type reverse transcriptase has low thermal stability and may be inactivated at a temperature at which formation of RNA secondary structure is suppressed. Thus, a mutant reverse transcriptase with improved heat resistance has been proposed (see, for example, Patent Document 1).

  However, the mutant reverse transcriptase has a drawback of low industrial productivity. Therefore, there is a need for a highly versatile reverse transcriptase that is excellent in industrial productivity, can be used even under temperature conditions where secondary structure formation is suppressed, can perform a reverse transcription reaction efficiently, and Yes.

Special table 2009-504162

  The present invention has been made in view of the above prior art, and has excellent industrial productivity, high thermal stability, and efficient reverse transcription reaction even under temperature conditions where secondary structure formation is suppressed. It is an object of the present invention to provide a versatile mutant reverse transcriptase. Another object of the present invention is to provide a nucleic acid and a method for producing the mutant reverse transcriptase from which the mutant reverse transcriptase can be easily obtained. Another object of the present invention is to provide a highly versatile reverse transcription reaction kit and detection kit that can efficiently perform a reverse transcription reaction even under temperature conditions where the formation of secondary structure is suppressed. To do. Furthermore, an object of the present invention is to provide a reverse transcription method capable of performing a reverse transcription reaction efficiently even under temperature conditions where formation of secondary structures is suppressed.

That is, the gist of the present invention is as follows.
[1] A mutant reverse transcriptase having reverse transcriptase activity, which has an amino acid sequence corresponding to SEQ ID NO: 2, and in the amino acid sequence, a phenylalanine residue at position 303 of SEQ ID NO: 2, and position 432 An amino acid residue corresponding to at least one residue selected from the group consisting of a leucine residue, a valine residue at position 433, and an isoleucine residue at position 434 is substituted with a positively charged amino acid residue. Characteristic mutant reverse transcriptase,
[2] (a) substitution of a residue corresponding to the phenylalanine residue at position 303 of SEQ ID NO: 2 with a lysine residue or arginine residue;
(B) substitution of a residue corresponding to the leucine residue at position 432 with a lysine residue or an arginine residue;
(C) substitution of a residue corresponding to a valine residue at position 433 in SEQ ID NO: 2 with a lysine residue or arginine residue; and (d) a residue corresponding to an isoleucine residue at position 434 in SEQ ID NO: 2. The mutant reverse transcriptase according to the above [1], having at least one selected from the group consisting of substitution of a group with a lysine residue or arginine residue,
[3] In the sequence shown in SEQ ID NO: 2, aspartic acid residue at position 108, glutamic acid residue at position 117, aspartic acid residue at position 124, glutamic acid residue at position 286, and aspartic acid residue at position 524 The mutant inversion according to [1] or [2] above, wherein an amino acid residue corresponding to at least one residue selected from the group consisting of is substituted with a positively charged amino acid residue or a nonpolar amino acid residue Photoenzyme,
[4] The amino acid sequence corresponding to SEQ ID NO: 2 corresponds to the phenylalanine residue at position 303 of SEQ ID NO: 2, the leucine residue at position 432, the valine residue at position 433, and the isoleucine residue at position 434, respectively. The mutant reverse transcriptase according to any one of the above [1] to [3], which is an amino acid sequence having a conservative substitution of amino acid residues excluding residues
[5] The amino acid sequence corresponding to SEQ ID NO: 2 is
(1) In the sequence shown in SEQ ID NO: 2, substitution, deletion, insertion or addition of one or several amino acid residues is further added to the sequence excluding the threonine residue at position 24 to the proline residue at position 474 An amino acid sequence of a polypeptide having reverse transcriptase activity, or (2) sequence identity to a sequence excluding a threonine residue at position 24 to a proline residue at position 474 in the sequence shown in SEQ ID NO: 2. The mutant reverse transcriptase according to any one of the above [1] to [4], which is an amino acid sequence of a polypeptide having a reverse transcriptase activity of at least 80%,
[6] A nucleic acid encoding the mutant reverse transcriptase according to any one of [1] to [5],
[7] A method for producing the mutant reverse transcriptase according to any one of [1] to [5],
(A) a step of culturing cells retaining the nucleic acid according to [6] to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture, and (B) in the step (A) A method for producing a mutant reverse transcriptase comprising a step of recovering the mutant reverse transcriptase from the obtained culture,
[8] A reverse transcription method comprising synthesizing cDNA from RNA using the mutant reverse transcriptase according to any one of [1] to [5],
[9] A kit for performing a reverse transcription reaction, comprising the mutant reverse transcriptase according to any one of [1] to [5] above, and [10 ] A kit for detecting a marker in a sample containing RNA obtained from a living body, comprising the mutant reverse transcriptase according to any one of [1] to [5] above and a reagent for detecting the marker The present invention relates to a detection kit comprising:

  The mutant reverse transcriptase of the present invention is excellent in industrial productivity, has high thermal stability, and can perform a reverse transcription reaction efficiently even under temperature conditions where secondary structure formation is suppressed, It is characterized by high versatility. Moreover, the method for producing the nucleic acid of the present invention and the mutant reverse transcriptase of the present invention has an excellent effect that the mutant reverse transcriptase can be easily obtained. Furthermore, the reverse transcription reaction kit and the detection kit of the present invention are characterized in that the reverse transcription reaction can be carried out efficiently even under temperature conditions where the formation of secondary structure is suppressed, and the versatility is high. In addition, the reverse transcription method of the present invention has an excellent effect that the reverse transcription reaction can be performed efficiently even under temperature conditions in which the formation of secondary structure is suppressed.

FIG. 2 is a schematic explanatory diagram showing the structure of a WT expression plasmid pET-MRT obtained in Production Example 1. In Test Example 2, it is a graph which shows the result of having investigated the relationship between a test sample and specific activity. In Experiment 2, it is a graph which shows the result of having investigated the relationship between a test sample and the residual activity after heat processing at 50 degreeC. In Experiment 3, it is a graph which shows the result of having investigated the relationship between a test sample and the residual activity after heat processing. (A) is a graph showing the results of SDS-PAGE equivalent to 2.7 μL of soluble fraction in Test Example 4, and (B) is SDS-PAGE equivalent to 0.9 μL of soluble fraction in Test Example 4. It is a graph which shows the result of having performed.

1. Mutant reverse transcriptase The mutant reverse transcriptase of the present invention is a mutant reverse transcriptase having reverse transcriptase activity, and has an amino acid sequence corresponding to SEQ ID NO: 2. In the amino acid sequence, SEQ ID NO: Amino acid residue corresponding to at least one residue selected from the group consisting of phenylalanine residue at position 303, leucine residue at position 432, valine residue at position 433, and isoleucine residue at position 434: It is characterized by being substituted with a positively charged amino acid residue.

  The mutant reverse transcriptase of the present invention comprises a phenylalanine residue at position 303, a leucine residue at position 432, a valine residue at position 433, and a position at position 434 in the amino acid sequence corresponding to SEQ ID NO: 2. Since the amino acid residue corresponding to at least one residue selected from the group consisting of isoleucine residues has an amino acid sequence substituted with a positively charged amino acid residue, formation of secondary structure is suppressed. Even under temperature conditions, it has a high residual activity compared to wild-type MMLV reverse transcriptase. Therefore, the mutant reverse transcriptase of the present invention has high thermostability, can perform a reverse transcription reaction efficiently even under temperature conditions where secondary structure formation is suppressed, and is highly versatile. Therefore, according to the mutant reverse transcriptase of the present invention, even when the template RNA has a base sequence that easily forms a secondary structure, the reaction temperature can be increased, and the secondary structure can be formed. CDNA can be synthesized while suppressing the above. In addition, the mutant reverse transcriptase of the present invention has an improved expression level in cells such as E. coli cells, for example, compared to wild-type MMLV reverse transcriptase or other mutant reverse transcriptases. Therefore, the mutant reverse transcriptase of the present invention is excellent in industrial productivity.

  In the present specification, “wild type” means a naturally occurring one in which no mutation has been artificially introduced, and “mutant type” means one in which a mutation has been artificially introduced. In the present specification, “sequence identity” refers to a value calculated by alignment under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3 by the BLAST algorithm.

The reverse transcriptase activity is the following steps 1) to 6):
1) Reaction solution [Composition: 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, 12.5 μM poly (rA) · p (dT) ₁₅ (p (dT) ₁₅ concentration in terms), and 0.2 mM [methyl - ³ H] dTTP] steps of incubation at 37 ° C. the reverse transcriptase in,
2) collecting 20 μL of the product obtained in step 1) and spotting it on a glass filter;
3) The glass filter after step 2) is washed with a cooled 5% by mass trichloroacetic acid aqueous solution for 10 minutes, and then with a cooled 95% by volume ethanol aqueous solution, and the poly (( removing [ ³ H] dTTP not incorporated in rA) · p (dT) ₁₅ ;
4) After drying the glass filter after step 3), placing the glass filter in 2.5 mL of a liquid scintillation reagent and counting the radioactivity;
5) A step of calculating the amount of [ ³ H] dTTP taken into poly (rA) · p (dT) ₁₅ (hereinafter referred to as “dTTP uptake amount”) based on the radioactivity obtained in step 4). 6) Based on the dTTP uptake calculated in step 5), the step of determining the amount of reverse transcriptase that incorporates 1 nmol of dTTP into poly (rA) · p (dT) ₁₅ in 10 minutes is performed. Can be measured.

  Positions 1 to 671 of SEQ ID NO: 2 are the amino acid sequences of wild-type MMLV reverse transcriptase. Note that positions 672 to 677 in SEQ ID NO: 2 are His tags composed of six histidine residues. In the present specification, the “amino acid sequence corresponding to SEQ ID NO: 2” is 50% or more of the sequence identity with the amino acid sequence of wild-type MMLV reverse transcriptase or the sequence shown in SEQ ID NO: 2. The amino acid sequence of wild-type reverse transcriptase exhibiting reverse transcriptase activity under the same conditions as wild-type MMLV reverse transcriptase. The sequence identity is preferably 70% or more, more preferably 80%, particularly preferably 100%, from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity.

  In the mutant reverse transcriptase of the present invention, in the amino acid sequence corresponding to SEQ ID NO: 2, the amino acid residue corresponding to the phenylalanine residue at position 303 of SEQ ID NO: 2 and the leucine residue at position 432 of SEQ ID NO: 2 Amino acid residues corresponding to the group, amino acid residues corresponding to the valine residue at position 433 of SEQ ID NO: 2 and amino acid residues corresponding to the isoleucine residue at position 434 of SEQ ID NO: 2, of these Three amino acid residues, two of these amino acid residues, or any one amino acid residue are replaced with positively charged amino acid residues. Among these, at least one selected from the group consisting of a valine residue at position 433 and an isoleucine residue at position 434 of SEQ ID NO: 2 from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity. The amino acid residue corresponding to the residue is preferably substituted with a positively charged amino acid residue.

  The amino acid residue corresponding to the phenylalanine residue at position 303 in SEQ ID NO: 2 is obtained by converting the amino acid sequence corresponding to SEQ ID NO: 2 to GAP Costs (Existence 11) with respect to the sequence of SEQ ID NO: 2 using the BLAST algorithm. , Extension 1), Extract 10, and Word Size 3 are amino acid residues that are present at positions corresponding to the phenylalanine residue at position 303 in SEQ ID NO: 2. The “amino acid residue corresponding to the leucine residue at position 432 of SEQ ID NO: 2” means that the amino acid sequence corresponding to SEQ ID NO: 2 is compared with the sequence of SEQ ID NO: 2 by the BLAST algorithm. Is an amino acid residue present at a position corresponding to the leucine residue at position 432 of SEQ ID NO: 2. “The amino acid residue corresponding to the valine residue at position 433 of SEQ ID NO: 2” is the same as described above by using the BLAST algorithm for the amino acid sequence corresponding to SEQ ID NO: 2 with respect to the sequence of SEQ ID NO: 2. An amino acid residue present at a position corresponding to the valine residue at position 433 of SEQ ID NO: 2 when aligned under conditions. In addition, the “amino acid residue corresponding to the isoleucine residue at position 434 of SEQ ID NO: 2” refers to the amino acid sequence corresponding to SEQ ID NO: 2 by the BLAST algorithm with respect to the sequence of SEQ ID NO: 2. The amino acid residue which exists in the position corresponded to the isoleucine residue of the 434th position of sequence number: 2 when alignment is carried out on the same conditions as.

  “Positively charged amino acid residue” refers to an amino acid residue that is positively charged at a pH suitable for performing a reverse transcription reaction (eg, pH 6.0 to 9.5). Examples of the positively charged amino acid residue include an arginine residue and a lysine residue. Among these positively charged amino acid residues, they are positively charged at a pH suitable for conducting a reverse transcription reaction (for example, pH 6.0 to 9.5), and have high thermal stability under such pH conditions. Arginine residues and lysine residues are preferable because the property can be secured.

The mutant reverse transcriptase of the present invention ensures high thermostability and ensures sufficient specific activity,
(A) substitution of a residue corresponding to the phenylalanine residue at position 303 of SEQ ID NO: 2 with a lysine residue or arginine residue;
(B) substitution of a residue corresponding to the leucine residue at position 432 with a lysine residue or an arginine residue;
(C) substitution of a residue corresponding to a valine residue at position 433 in SEQ ID NO: 2 with a lysine residue or arginine residue; and (d) a residue corresponding to an isoleucine residue at position 434 in SEQ ID NO: 2. It preferably has at least one selected from the group consisting of substitution of a group with a lysine residue or arginine residue.

  From the viewpoint of ensuring high thermal stability, the mutant reverse transcriptase of the present invention has a threonine residue at position 24 to a proline residue at position 474 in SEQ ID NO: 2 in the amino acid sequence corresponding to SEQ ID NO: 2. It is preferable that at least one of the negatively charged amino acid residues among the amino acid residues localized in the corresponding region is substituted with the positively charged amino acid residue or the nonpolar amino acid residue.

The “region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2” (hereinafter also referred to as “region _T24-P474 ”) is the amino acid sequence corresponding to SEQ ID NO: 2. A region corresponding to the region of the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2 when the sequence of SEQ ID NO: 2 is aligned under the same conditions as described above by the BLAST algorithm It is.

  Examples of the negatively charged amino acid residue include an aspartic acid residue and a glutamic acid residue. The negatively charged amino acid residue has a shape as long as the polypeptide in which the negatively charged amino acid residue is substituted with the positively charged amino acid residue or the nonpolar amino acid residue can express reverse transcriptase activity. It may be a residue present at a position that causes a change in Specific examples of the negatively charged amino acid residue include an amino acid residue corresponding to the aspartic acid residue at position 108 of SEQ ID NO: 2, an amino acid residue corresponding to the glutamic acid residue at position 117 of SEQ ID NO: 2, and a sequence No. 2: aspartic acid residue at position 124, amino acid residue corresponding to glutamic acid residue at position 286 in SEQ ID NO: 2, amino acid residue corresponding to aspartic acid residue at position 524 in SEQ ID NO: 2, and the like Although mentioned, this invention is not limited only to this illustration. Among these negatively charged amino acid residues, aspartic acid at position 108 of SEQ ID NO: 2 in the sequence shown in SEQ ID NO: 2 from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity. Residue, glutamic acid residue at position 117 of SEQ ID NO: 2, aspartic acid residue at position 124 of SEQ ID NO: 2, glutamic acid residue at position 286 of SEQ ID NO: 2, and aspartic acid at position 524 of SEQ ID NO: 2. An amino acid residue corresponding to at least one residue selected from the group consisting of residues is preferred.

  Examples of the nonpolar amino acid residues include alanine residues, glycine residues, valine residues, leucine residues, isoleucine residues, methionine residues, cysteine residues, tryptophan residues, phenylalanine residues, and proline residues. Etc. Among these nonpolar amino acid residues, an alanine residue is preferred because the size of the side chain is small and the shape change caused by substitution is considered to be small.

The mutant reverse transcriptase of the present invention ensures high thermostability and ensures sufficient specific activity,
(I) an alanine residue, arginine residue, lysine residue, serine residue or threonine residue, preferably an alanine residue or arginine residue corresponding to the aspartic acid residue at position 108 of SEQ ID NO: 2 Replacement with,
(II) To the alanine residue, arginine residue, lysine residue, serine residue or threonine residue of the residue corresponding to the glutamic acid residue at position 117 of SEQ ID NO: 2, preferably an alanine residue or arginine residue Replacement,
(III) Alanine residue, arginine residue, lysine residue, serine residue or threonine residue, preferably alanine residue or arginine residue corresponding to the aspartic acid residue at position 124 of SEQ ID NO: 2 Replacement with,
(IV) Substitution of a residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 with an arginine or lysine residue, preferably arginine residue, and (V) asparagine at position 524 of SEQ ID NO: 2. Substitution of an acid residue corresponding to an alanine or asparagine residue, preferably an alanine residue,
It is preferable to have at least one selected from the group consisting of: When a reverse transcriptase having no RNase H activity is desired as the reverse transcriptase, the mutant reverse transcriptase of the present invention preferably has the substitution (V).

The amino acid sequence corresponding to SEQ ID NO: 2 is a phenylalanine residue at position 303 in SEQ ID NO: 2, a leucine residue at position 432 in SEQ ID NO: 2, It may be an amino acid sequence having a conservative substitution of amino acid residues except for a residue corresponding to a valine residue at position 433 and an isoleucine residue at position 434 of SEQ ID NO: 2. Examples of conservative substitutions, for example, a particular amino acid residue, hydrophobicity, charge, pK _a, the amino acid residues that exhibits a function similar to the specific amino acid residues in terms of features on three-dimensional structure And the like. More specific examples of the conservative substitution include substitution of one amino acid residue belonging to any of the following groups I to VI with another amino acid residue belonging to the same group.
Group I: Glycine and alanine residues Group II: Valine, isoleucine and leucine residues Group III: Aspartate, glutamate, asparagine and glutamine residues Group IV: Serine and Threonine residue Group V: Arginine residue and lysine residue Group VI: Phenylalanine residue and tyrosine residue

In the present invention, the amino acid sequence corresponding to SEQ ID NO: 2 is within the range that does not interfere with the object of the present invention.
(1) In the sequence shown in SEQ ID NO: 2, substitution, deletion, insertion or addition of one or several amino acid residues is further added to the sequence excluding the threonine residue at position 24 to the proline residue at position 474 An amino acid sequence of a polypeptide having reverse transcriptase activity, or (2) sequence identity to a sequence excluding a threonine residue at position 24 to a proline residue at position 474 in the sequence shown in SEQ ID NO: 2. May be an amino acid sequence of a polypeptide having at least 80% and having reverse transcriptase activity.

  The above-mentioned “substitution, deletion, insertion or addition of one or several amino acid residues” means substitution, deletion, insertion or addition of a number of amino acid residues within a range showing reverse transcriptase activity. The “one or several” refers to 1 to 30 pieces. The “one or several” is preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 3, particularly preferably 1 or 2.

  The sequence identity is at least 80%, preferably 90% or more, more preferably 95% or more, particularly preferably 100% from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity. is there.

  As described above, the mutant reverse transcriptase of the present invention has excellent industrial productivity, high thermal stability, and efficient reverse transcription reaction even under temperature conditions where secondary structure formation is suppressed. Since it is highly versatile, it is useful for gene analysis, examination of diseases, cloning of useful genes, and the like.

2. Nucleic acid encoding mutant reverse transcriptase The nucleic acid of the present invention is a nucleic acid encoding the mutant reverse transcriptase of the present invention. Since the nucleic acid of the present invention encodes the mutant reverse transcriptase, the mutant reverse transcriptase can be easily obtained by expressing the mutant reverse transcriptase encoded by the nucleic acid.

  Examples of the nucleic acid include DNA and mRNA, but the present invention is not limited to such examples.

The nucleic acid of the present invention is, for example, a nucleic acid consisting of a base sequence corresponding to SEQ ID NO: 1,
A positive amino acid residue corresponding to at least one residue selected from the group consisting of a valine residue at position 433 and an isoleucine residue at position 434 in the amino acid sequence corresponding to SEQ ID NO: 2; Substitution to a charged amino acid residue,
_-In the amino acid sequence corresponding to SEQ ID NO: 2, substitution of at least any positively charged amino acid residue or nonpolar amino acid residue of a negatively charged amino acid residue among amino acid residues localized in region _T24-P474 Can be obtained by introducing site-specific mutations so as to cause such mutations.

  The 1st to 2013th positions of SEQ ID NO: 1 are the nucleotide sequences of nucleic acids encoding wild type MMLV reverse transcriptase. Note that positions 2014 to 2034 in SEQ ID NO: 1 are the base sequence of a nucleic acid encoding a His tag consisting of six histidine residues. The “base sequence corresponding to SEQ ID NO: 1” has a nucleotide sequence of 50% or more of the nucleotide sequence of a nucleic acid encoding wild-type MMLV reverse transcriptase or the sequence shown in SEQ ID NO: 2, and is wild A nucleotide sequence of a nucleic acid encoding a reverse transcriptase exhibiting reverse transcriptase activity under the same conditions as type MMLV reverse transcriptase.

  The introduction of site-specific mutation into the nucleic acid is, for example, a non-PCR method using a non-PCR primer designed to cause the mutation, a PCR method using a PCR primer designed to cause the mutation Etc.

  As described above, since the nucleic acid of the present invention encodes the mutant reverse transcriptase of the present invention, the mutant reverse transcriptase of the present invention can be easily obtained. It is useful for industrial production.

3. Method for Producing Mutant Reverse Transcriptase The mutant reverse transcriptase of the present invention can be obtained by expressing the mutant reverse transcriptase encoded by the nucleic acid using the nucleic acid of the present invention. The present invention also includes a method for producing such a mutant reverse transcriptase.

The production method of the present invention is a method for producing the aforementioned mutant reverse transcriptase,
(A) a step of culturing cells retaining the nucleic acid of the present invention to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture, and (B) a mutation from the culture obtained in the step It is a method characterized by including the process of collect | recovering type | mold reverse transcriptase.

  First, a cell retaining the nucleic acid of the present invention is cultured to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture [“Step (A)”].

  The cell holding the nucleic acid can be obtained, for example, by transforming a host cell with a gene introduction carrier containing the nucleic acid.

  Examples of the host cell include bacterial cells such as Escherichia coli, insect cells, yeast cells, plant cells, animal cells and the like, but the present invention is not limited to such examples. Among these, bacterial cells are preferable, and Escherichia coli cells are more preferable because the mutant reverse transcriptase can be easily purified and the mutant reverse transcriptase can be produced in large quantities. Examples of the E. coli cells include BL21 (DE3), but the present invention is not limited to such examples.

  The carrier for gene introduction may be a biological carrier or a non-biological carrier. Examples of biological carriers include vectors such as plasmid vectors, phage vectors, and viral vectors, but the present invention is not limited to such examples. Examples of the non-biological carrier include gold particles, dextran, and liposomes, but the present invention is not limited to such examples. Such a carrier for gene transfer can be appropriately selected depending on the host cell to be used. For example, when a cell of BL21 (DE3) which is Escherichia coli is used as a host cell, a pET plasmid vector can be used. In this case, the mutant reverse transcriptase can be expressed in a large amount, and the mutant reverse transcriptase can be easily purified.

  The vector may be a secretion vector that secretes the protein outside the cell, or an intracellular expression vector that accumulates the protein inside the cell. In addition, the vector may contain elements for facilitating purification of the mutant reverse transcriptase, such as a His tag and an extracellular secretion signal.

  When the gene introduction carrier is a vector that is the biological carrier, the gene introduction carrier is inserted into the vector cloning site by inserting the nucleic acid and operably linked under the control of a promoter. Can be produced. In the present specification, “operably linked” means that the expression of a polypeptide encoded by a nucleic acid is linked so that it is expressed in a state exhibiting biological activity under the control of an element such as a promoter. Means that

  On the other hand, when the carrier for gene introduction is the non-biological carrier, the carrier for gene introduction is a nucleic acid construct obtained by operably linking the nucleic acid under the control of a promoter, if necessary. , And can be prepared by supporting the non-biological carrier. Such a nucleic acid construct may appropriately contain elements necessary for expression of a gene such as a replication origin and a terminator.

  The introduction of the carrier for gene introduction into the host cell can be carried out by a method according to the type of carrier used for gene introduction, the type of host cell, and the like. Examples of such a transformation method include a transfection method, an electroporation method, a calcium phosphate method, a DEAE-dextran method, and a particle gun method, but the present invention is not limited to such examples.

  In the step (A), the culture condition of the cell holding the nucleic acid varies depending on the type of the host cell used, and therefore cannot be determined unconditionally. Accordingly, it is preferable to set appropriately. When the nucleic acid is operably linked under the control of an inducible promoter, cells that retain the nucleic acid may be cultured under expression-inducing conditions according to the type of the promoter.

  Next, the mutant reverse transcriptase is recovered from the culture obtained in the step (A) [“Step (B)”].

  If the mutant reverse transcriptase is secreted extracellularly in the culture, the culture is subjected to centrifugation, filtration, etc. to recover the culture supernatant, and the mutant reverse transcriptase in the culture supernatant is purified. Can be isolated. Examples of methods for performing such purification include centrifugation, ultracentrifugation, ultrafiltration, salting out, dialysis, ion exchange column chromatography, adsorption column chromatography, affinity chromatography, and gel filtration column chromatography. The present invention is not limited to such examples.

  On the other hand, when the mutant reverse transcriptase is accumulated in the cells in the culture, in step (B), the culture is subjected to centrifugation or the like to recover the cells, and the mutant reversal is obtained from the cell disruption. The transcriptase can be isolated. In this case, for example, the cells are disrupted by ultrasonic disruption, lysis, freeze disruption, etc., and then the mutant reverse transcriptase in the cell-free extract obtained from the obtained disrupted product is purified to obtain a single cell. Can be separated.

  For example, when a BL21 (DE3) cell, which is Escherichia coli, is used as a host cell and a pET-type plasmid vector is used as a carrier for gene transfer, as in the method described in the Examples below, mutant reverse transcription is carried out in the cell. The enzyme can be expressed in large quantities, and after the collected cells are crushed, the extract obtained from the crushed material is subjected to anion exchange column chromatography and nickel affinity chromatography. Can be easily purified.

  As described above, since the nucleic acid of the present invention is used in the method for producing a mutant reverse transcriptase of the present invention, the mutant reverse transcriptase can be easily obtained. Useful for industrial production of reverse transcriptase.

4). Reverse transcription method The reverse transcription method of the present invention is characterized by synthesizing cDNA from RNA using the mutant reverse transcriptase of the present invention. The mutant reverse transcriptase of the present invention has a higher thermal stability than the wild type reverse transcriptase. Therefore, according to the reverse transcription method of the present invention, the reverse transcription reaction can be performed in a wide temperature range including the temperature at which the formation of RNA secondary structure is suppressed. Therefore, the reverse transcription method of the present invention can use various RNAs as templates, and can perform a reverse transcription reaction efficiently.

  In the reverse transcription method of the present invention, the mutant reverse transcriptase, RNA, an oligonucleotide primer complementary to the RNA, and four types of deoxyribonucleoside triphosphates are incubated in a reverse transcription reaction buffer. Thus, a reverse transcription reaction can be performed.

  Since the reaction temperature in the reverse transcription reaction varies depending on the type of RNA used, the type of mutant reverse transcriptase used, etc., it cannot be determined unconditionally, so the type of RNA used, the type of mutant reversal used It is preferable to set appropriately according to the type of the coenzyme. The reaction temperature can be set to the optimum reaction temperature, for example, when the RNA used is an RNA that does not readily form a secondary structure at the optimum reaction temperature of wild-type reverse transcriptase. The reaction temperature is higher than the optimum reaction temperature of the wild-type reverse transcriptase, for example, when the RNA used is an RNA that easily forms a secondary structure at the optimum reaction temperature of the wild-type reverse transcriptase. For example, it can set so that it may become 45-65 degreeC.

  Since the concentration of the reverse transcriptase in the reaction system during the reverse transcription reaction varies depending on the use of the reverse transcription method of the present invention and the like, it cannot be determined unconditionally, so it is appropriately set according to the use etc. It is preferable. The concentration of the reverse transcriptase is usually 0.001 to 0.1 μM.

  The concentration of the oligonucleotide primer in the reaction system during the reverse transcription reaction is usually 0.1 to 10 μM.

  Since the concentration of the four types of deoxyribonucleoside triphosphates in the reaction system during the reverse transcription reaction varies depending on the concentration and length of the target RNA, it cannot be determined in general. It is preferable to set appropriately depending on the concentration and length of RNA to be obtained. The concentration of the four deoxyribonucleoside triphosphates is usually 0.01 to 1 μM.

  The reverse transcription reaction buffer can be appropriately selected depending on the type of mutant reverse transcriptase used. The reverse transcription reaction buffer may contain a divalent cation, such as magnesium ion or manganese ion. In addition, the reverse transcription reaction buffer is not limited to the purpose of the present invention, and if necessary, a reducing agent (for example, dithiothreitol), a stabilizer (for example, glycerol, trehalose, etc.), organic Components such as a solvent (for example, dimethyl sulfoxide, formamide, etc.) may be contained.

  The concentration of the divalent cation in the reverse transcription reaction buffer varies depending on the type of reverse transcriptase and other components contained in the reverse transcription reaction buffer. Therefore, it is preferable to set appropriately depending on the type of reverse transcriptase and other components contained in the buffer for reverse transcription reaction. The concentration of the divalent cation is usually 1 to 30 mM.

  Since the pH of the reverse transcription reaction buffer solution varies depending on the type of reverse transcriptase and other components contained in the reverse transcription reaction buffer solution, it cannot be generally determined. It is preferable to set appropriately depending on the type of the above and other components contained in the reverse transcription reaction buffer. The pH of the reverse transcription reaction buffer is generally 6.0 to 9.5.

  As described above, since the reverse transcription enzyme of the present invention is used in the reverse transcription method of the present invention, the reverse transcription reaction is efficiently performed even under temperature conditions in which the formation of secondary structure is suppressed. Since it is versatile, it is useful for gene analysis, examination of diseases, cloning of useful genes, and the like.

5. Reverse Transcription Reaction Kit The reverse transcription reaction kit of the present invention is a kit for carrying out a reverse transcription reaction, and is characterized by containing the mutant reverse transcriptase of the present invention. Since the reverse transcription reaction kit of the present invention contains the mutant reverse transcriptase of the present invention having high thermal stability, the reverse transcription in a wide temperature range including the temperature at which the formation of RNA secondary structure is suppressed. Suitable for photoreaction. Therefore, the reverse transcription reaction kit of the present invention is highly versatile because the reverse transcription reaction can be efficiently performed regardless of the type of RNA. Since the reverse transcription reaction kit of the present invention contains the mutant reverse transcriptase of the present invention, it is excellent in industrial productivity.

  The reverse transcription reaction kit of the present invention may contain a reagent necessary for carrying out a reverse transcription reaction in addition to the mutant reverse transcriptase. Examples of such a reagent include RNA used as a template for a reverse transcription reaction, oligonucleotide primers complementary to the RNA, four types of deoxyribonucleoside triphosphates, a buffer for reverse transcription reaction, an organic solvent, and the like. The reverse transcription reaction buffer is the same as the reverse transcription reaction buffer used in the reverse transcription method.

  In the reverse transcription reaction kit of the present invention, the mutant reverse transcriptase may be enclosed in a container containing a storage buffer containing a stabilizer such as glycerol or trehalose. Examples of such a storage buffer include a buffer having a pH corresponding to the pH stability of the mutant reverse transcriptase.

  The reagent necessary for performing the reverse transcription reaction may be enclosed in a container different from the container containing the mutant reverse transcriptase, and the progress of the reverse transcription reaction during storage of the reagent. May be enclosed in the same container as the mutant reverse transcriptase. The reagent may be enclosed in a container so as to have an amount suitable for performing a reverse transcription reaction. This eliminates the need for the user to mix each reagent in an amount suitable for the reverse transcription reaction, and is easy to handle.

  As described above, the reverse transcription reaction kit of the present invention uses the mutant reverse transcriptase of the present invention, so that the reverse transcription reaction can be performed efficiently even under temperature conditions in which the formation of secondary structure is suppressed. Since it can be carried out and has high versatility, it is suitable for conducting a reverse transcription reaction in gene analysis, examination of diseases, cloning of useful genes, and the like.

6). Detection Kit The detection kit of the present invention is a kit for detecting a marker in a sample containing RNA obtained from a living body, and contains the mutant reverse transcriptase and the reagent for detecting the marker. It is a feature. Since the detection kit of the present invention contains the mutant reverse transcriptase having high thermostability, it is suitable for a reverse transcription reaction in a wide temperature range including a temperature at which formation of RNA secondary structure is suppressed. It is. Therefore, the detection kit of the present invention can be used for various samples and is highly versatile. Moreover, since the detection kit of the present invention contains the mutant reverse transcriptase of the present invention, it is excellent in industrial productivity.

  Examples of the marker include base sequences peculiar to viruses or bacteria contained in living bodies, RNA having base sequences peculiar to diseases, and the like. In the present specification, the “base sequence peculiar to a virus or a bacterium” refers to a base sequence that exists in a virus or bacterium but does not exist in a living body. Further, the “base sequence peculiar to a disease” refers to a base sequence that exists in a living body affected with a disease but does not exist in a normal living body that does not suffer from the disease.

  Although it does not specifically limit as said virus, For example, HPV, HIV, influenza virus, HCV, Norovirus, West Nile virus etc. are mentioned. Examples of the bacterium include Bacillus cereus, Salmonella, enterohemorrhagic Escherichia coli, Vibrio, Campylobacter, and methicillin-resistant Staphylococcus aureus that cause food poisoning. Examples of the disease include cancer, diabetes, heart disease, high blood pressure, and infectious diseases.

  Examples of the reagent for detecting the marker include a probe that is complementary to the RNA serving as the marker and bound with a fluorescent substance or a radioactive substance, or a fluorescent substance that specifically intercalates with a double-stranded nucleic acid (for example, Ethidium bromide).

  In addition to the mutant reverse transcriptase and the marker detection reagent, the detection kit of the present invention includes, for example, four types of deoxyribonucleoside triphosphates, a reverse transcription reaction buffer, an organic solvent, RNA serving as a positive control, It may contain RNA that serves as a negative control. The reverse transcription reaction buffer is the same as the reverse transcription reaction buffer used in the reverse transcription method.

  In the detection kit of the present invention, the mutant reverse transcriptase may be enclosed in a container containing a storage buffer containing a stabilizer such as glycerol or trehalose. Such a storage buffer solution is the same as the storage buffer solution in the reverse transcription reaction kit.

  The four types of deoxyribonucleoside triphosphates, reverse transcription reaction buffer, and the like may be sealed in a container different from the container containing the mutant reverse transcriptase, and the reagent may be stored. As long as the progress of the reverse transcription reaction therein is stopped, it may be enclosed in the same container as the mutant reverse transcriptase. From the same viewpoint as in the case of the reverse transcription reaction kit, the reagent may be enclosed in a container so as to have an amount suitable for performing the reverse transcription reaction.

  As described above, since the detection kit of the present invention uses the mutant reverse transcriptase of the present invention, the detection kit can efficiently perform a reverse transcription reaction even under temperature conditions where secondary structure formation is suppressed. Therefore, it is highly versatile and can detect a base sequence peculiar to a virus or a bacterium or a base sequence peculiar to a disease with high accuracy regardless of the kind of RNA used as a test sample.

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

Production Example 1
DNA (SEQ ID NO: 1) encoding wild-type MMLV reverse transcriptase (hereinafter also simply referred to as “WT”) was inserted into the pET-22b (+) plasmid to obtain a WT expression plasmid pET-MRT. The WT expression plasmid expresses WT as a His tag polypeptide with (His) ₆ added to the C-terminus. The structure of the WT expression plasmid pET-MRT obtained in Production Example 1 is shown in FIG.

Example 1
Using the pET-MRT obtained in Production Example 1, a V433R primer pair, and a site-directed mutagenesis kit (Stratagene, trade name: Quikchange ^™ site-directed mutagenesis kit), pET- A point expression mutation causing substitution of a valine residue at position 433 of SEQ ID NO: 2 to an arginine residue was introduced into DNA encoding WT contained in MRT to obtain a mutant expression plasmid. Whether or not a predetermined point mutation was introduced into the DNA contained in the obtained plasmid for expressing a mutant was confirmed by a DNA sequencer [manufactured by Shimadzu Corporation, trade name: DSQ-2000]. “V433R” indicates substitution of an amino acid residue corresponding to the valine residue at position 433 in the sequence shown in SEQ ID NO: 2 with an arginine residue. The primers constituting the V433R primer pair are as shown in Table 1. In Table 1, the underlined portion indicates a sequence mismatched with DNA encoding WT contained in pET-MRT.

  The obtained mutant expression plasmid was used to transform E. coli BL21 (DE3), which is a host cell. The obtained cells were cultured at 37 ° C. in L broth [composition: 1% by mass tryptone, 0.5% by mass yeast extract, 1% by mass sodium chloride and the remaining water] containing 50 μg / mL ampicillin for transformation. Cells were obtained.

  Next, the transformed cells were seeded in 3 mL of L broth containing 50 μg / mL ampicillin and incubated at 37 ° C. for 16 hours with shaking to obtain a culture solution. 2 mL of the culture solution was added to 3 mL of L broth containing 50 μg / mL ampicillin, and the transformed cells were incubated at 37 ° C. for 2 hours. Thereafter, 200 μL of 0.5 M isopropyl-β-thiogalactopyranoside (IPTG) aqueous solution was added, and the transformed cells were incubated at 30 ° C. for 4 hours to express the protein.

  The obtained culture was subjected to centrifugation at 5840 × g for 10 minutes to collect cells. The collected cells were suspended in 20 mL of buffer A [composition: 20 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 10 volume% glycerol, pH 7.2] and disrupted by sonication. The obtained crushed material was subjected to centrifugation at 20000 × g for 20 minutes, and the supernatant was recovered. The collected supernatant was subjected to anion using a column (2.5 × 12 cm) packed with an anion exchange resin [trade name: Toyopearl DEAE-650M, manufactured by Tosoh Corp.] equilibrated with the buffer A. The sample was subjected to ion exchange column chromatography. After the column was washed with the buffer A to remove non-adsorbed protein from the column, the buffer B [composition: 300 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 10 vol% glycerol, pH 7. 2] was used to elute the protein adsorbed on the column to obtain a fraction showing reverse transcriptase activity.

  The obtained fraction was purified with a histidine tag binding protein parallelized with buffer C [composition: 20 mM imidazole, 20 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 10 vol% glycerol, pH 7.2]. The sample was applied to a prepacked column (manufactured by GE Healthcare, trade name: HisTrap HP 1 mL). After removing the non-adsorbed protein from the column by washing the column with buffer D [composition: 80 mM imidazole, 20 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 10 vol% glycerol, pH 7.2]. The protein adsorbed on the column was eluted with buffer E [composition: 500 mM imidazole, 20 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 10 volume% glycerol, pH 7.2], and reverse transcriptase activity An affinity-purified fraction showing was obtained. The obtained fraction was dialyzed against buffer F [composition: 20 mM potassium phosphate buffer, 2.0 mM dithiothreitol and 50 vol% glycerol, pH 7.2], and in the sequence shown in SEQ ID NO: 2. A mutant reverse transcriptase (V433R) having an amino acid sequence in which the valine residue at position 433 was substituted with an arginine residue was obtained.

Examples 2-4
In Example 1, instead of using the V433R primer pair as a primer pair, a V433K primer pair (Example 2), an I434R primer pair (Example 3) or an I434K primer pair (Example 4) was used. The mutant reverse transcriptase (V433K) having the amino acid sequence in which the valine residue at position 433 in the sequence shown in SEQ ID NO: 2 was replaced with a lysine residue was performed in the same manner as in Example 1 except for Example 2), mutant reverse transcriptase (I434R) having an amino acid sequence in which the isoleucine residue at position 434 in the sequence shown in SEQ ID NO: 2 is substituted with an arginine residue (Example 3) or SEQ ID NO: Mutant reverse transcriptase (I434K) having an amino acid sequence in which the isoleucine residue at position 434 in the sequence shown in FIG.施例 4) was obtained. “V433K” is a substitution of an amino acid residue corresponding to the valine residue at position 433 in the sequence shown in SEQ ID NO: 2 with a lysine residue, and “I434R” is in the sequence shown in SEQ ID NO: 2. Substitution of amino acid residue corresponding to isoleucine residue at position 434 to arginine residue and “I434K” is a lysine residue of amino acid residue corresponding to isoleucine residue at position 434 in the sequence shown in SEQ ID NO: 2 Indicates a replacement for. The primers constituting each primer pair are as shown in Table 1.

Comparative Examples 1 and 2
In Example 1, instead of using the V433R primer pair as the primer pair, the same operation as in Example 1 was used, except that the L304R primer pair (Comparative Example 1) or the L304K primer pair (Comparative Example 2) was used. To the mutant reverse transcriptase (L304R) (Comparative Example 1) or SEQ ID NO: 2 having an amino acid sequence in which the leucine residue at position 304 in the sequence shown in SEQ ID NO: 2 is substituted with an arginine residue. A mutant reverse transcriptase (L304K) (Comparative Example 2) having an amino acid sequence in which the leucine residue at position 304 in the sequence shown was substituted with a lysine residue was obtained. “L304R” is a substitution of an amino acid residue corresponding to the leucine residue at position 304 in the sequence shown in SEQ ID NO: 2 with an arginine residue, and “L304K” is in the sequence shown in SEQ ID NO: 2. The substitution of an amino acid residue corresponding to the leucine residue at position 304 to a lysine residue is shown. The primers constituting each primer pair are as shown in Table 1.

Comparative Example 3
In Example 1, instead of transforming E. coli BL21 (DE3) with the mutant expression plasmid, E. coli BL21 (DE3) was transformed with the WT expression plasmid obtained in Production Example 1, The same operation as in Example 1 was performed to obtain WT.

Test example 1
As a test sample, a dialysis fraction containing the mutant reverse transcriptase obtained in Example 1, 2, 3 or 4 or Comparative Example 1 or 2 was used as sodium dodecyl sulfate-denatured polyacrylamide gel electrophoresis (hereinafter referred to as “SDS”). -PAGE "). As a result, the dialysis fractions containing the mutant reverse transcriptase obtained in Examples 1 to 4 and Comparative Examples 1 and 2 all showed a single band of 75 kDa.

Test example 2
(1) Measurement of reverse transcriptase activity and specific activity As a test sample, the mutant reverse transcriptase obtained in Example 1, 2, 3 or 4, Comparative Example 1 or 2 or WT obtained in Comparative Example 3 was used. Reverse transcription reaction was performed using the contained dialysis fraction. The composition of the reaction solution used in the reverse transcription reaction was 30 nM mutant reverse transcriptase or WT, 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, 12.5 μM poly a - ^[3 H-methyl] dTTP (1.85Bq / pmol) [GE Healthcare (GE Healthcare) Co. Ltd.] (rA) · p (dT) 15 [p (dT) ₁₅ in terms of concentration] and 0.2 mM. The volume of the reaction solution used for the reverse transcription reaction is 72 μL. The reverse transcription reaction was performed by incubating the reaction solution at 37 ° C.

Collect 20 μL of the product after 2.5 minutes, 5.0 minutes or 7.5 minutes from the start of the incubation, and immediately take a glass filter (manufactured by Whatman, trade name: GF / C, diameter 2.5 cm). ] Spotted. Next, the glass filter was washed with a cooled 5% by mass trichloroacetic acid aqueous solution for 10 minutes. Thereafter, the glass filter was washed with a cooled 95% by volume aqueous ethanol solution. This removed [ ³ H] dTTP that was not incorporated into poly (rA) · p (dT) ₁₅ . Such washing with an aqueous trichloroacetic acid solution was repeated three times. Moreover, the washing | cleaning by ethanol aqueous solution was performed once.

Thereafter, the glass filter was dried. The glass filter was placed in 2.5 mL of a liquid scintillation reagent (trade name: Ecoscint H, manufactured by National Diagnostics), and the radioactivity was counted. Based on the radioactivity, the amount of [ ³ H] dTTP incorporated into poly (rA) · p (dT) ₁₅ (referred to as “dTTP incorporation”) was calculated. Next, the initial reaction rate was calculated based on the change over time in the amount of dTTP uptake, and the reverse transcriptase activity was calculated. One unit in reverse transcriptase activity was defined as the amount of reverse transcriptase that incorporated 1 nmol of dTTP into poly (rA) · p (dT) ₁₅ in 10 minutes.

  Also, based on a calibration curve prepared according to the Bradford method using a known amount of bovine serum albumin preparation and a protein quantification reagent (trade name: Protein Assay CBB Solution, manufactured by Nacalai Tesque) The amount of mutant reverse transcriptase or WT contained in the dialysis fraction was examined.

  Thereafter, the reverse transcriptase activity of the dialysis fraction containing the mutant reverse transcriptase obtained in Example 1, 2, 3 or 4, Comparative Example 1 or 2 or WT obtained in Comparative Example 3, and Comparative Example Specific activity was calculated from the mutant reverse transcriptase obtained in 1 or 2 or the amount of WT obtained in Comparative Example 3.

  FIG. 2 shows the results of examining the relationship between the test sample and the specific activity in Test Example 2. In the figure, lane 1 is the mutant reverse transcriptase (L433R) obtained in Example 1, lane 2 is the mutant reverse transcriptase (L433K) obtained in Example 2, and lane 3 is obtained in Example 3. Mutant reverse transcriptase (I434R), Lane 4 is the mutant reverse transcriptase (I434R) obtained in Example 4, Lane 5 is the mutant reverse transcriptase (L304R) obtained in Comparative Example 1, Lane 6 Represents the mutant reverse transcriptase (L304K) obtained in Comparative Example 2 and Lane 7 represents the WT obtained in Comparative Example 3.

  From the results shown in FIG. 2, each of the mutant reverse transcriptases obtained in Examples 1 to 4 has a specific activity (40000 units / mg) that is generally considered to be practically sufficient. I understand that. In contrast, the specific activities of the mutant reverse transcriptases obtained in Comparative Examples 1 and 2 are both extremely low, indicating that they are unsuitable for practical use.

(2) Measurement of residual activity The mutant reverse transcriptase obtained in Example 1, 2, 3 or 4 or the WT obtained in Comparative Example 3 has a concentration of the mutant reverse transcriptase or WT of 30 nM. Thus, 200 μL of the incubation solution [composition: 10 mM potassium phosphate buffer (pH 7.6), 2 mM dithiothreitol, 0.2 vol% Triton ^™ X-100 and 10 vol% glycerol] was added. By incubating the obtained mixture at 48 ° C. or 50 ° C. for 10 minutes, the mutant reverse transcriptase obtained in Example 1, 2, 3 or 4 or the WT obtained in Comparative Example 3 was subjected to heat treatment. did. Next, the mixture containing mutant reverse transcriptase after heat treatment or WT after heat treatment was incubated on ice for 30 to 60 minutes.

  Next, the mutant reverse transcriptase after the heat treatment or the WT after the heat treatment is added to the reaction solution so that the concentration of the mutant reverse transcriptase after the heat treatment or the WT after the heat treatment is 30 nM. The resulting mixture was incubated at 37 ° C. to perform a reverse transcriptase reaction.

Collect 20 μL of the product after 2.5 minutes, 5.0 minutes or 7.5 minutes from the start of the incubation, and immediately take a glass filter (manufactured by Whatman, trade name: GF / C, diameter 2.5 cm). ] Spotted. Next, the glass filter is washed with a 5% by mass trichloroacetic acid aqueous solution cooled on ice for 10 minutes, and then washed with a 95% by volume aqueous ethanol solution cooled on ice to obtain poly (rA) · p (dT ) [ ³ H] dTTP that was not incorporated into ₁₅ was removed. The washing with the aqueous trichloroacetic acid solution was performed three times, and the washing with the aqueous ethanol solution was performed once.

  Thereafter, the glass filter was dried. The glass filter was placed in 2.5 mL of a liquid scintillation reagent (trade name: Ecoscint H, manufactured by National Diagnostics), and the radioactivity was counted with a liquid scintillation counter. The dTTP uptake amount was calculated based on the radioactivity.

The initial reaction rate was calculated based on the change in dTTP uptake over time. Next, the residual activity was calculated from the initial reaction rate when the heat treatment was not performed (referred to as “initial reaction rate A”) and the initial reaction rate when the heat treatment was performed (referred to as “initial reaction rate B”). . The residual activity is represented by the formula (I):
[Residual activity (%)] = initial reaction rate B / initial reaction rate A × 100 (I)
It calculated based on the formula represented by.

  FIG. 3 shows the result of examining the relationship between the test sample and the residual activity after heat treatment at 50 ° C. in Test Example 2. In the figure, lane 1 is the mutant reverse transcriptase (L433R) obtained in Example 1, lane 2 is the mutant reverse transcriptase (L433K) obtained in Example 2, and lane 3 is obtained in Example 3. Mutant reverse transcriptase (I434R), lane 4 shows the mutant reverse transcriptase (I434R) obtained in Example 4, and lane 5 shows the WT obtained in Comparative Example 3.

  From the results shown in FIG. 3, the mutant reverse transcriptase (L433R) obtained in Example 1, the mutant reverse transcriptase (L433K) obtained in Example 2, and the mutant type obtained in Example 3 were used. The residual activity of the reverse transcriptase (I434R) and the mutant reverse transcriptase (I434R) obtained in Example 4 after the heat treatment at 50 ° C. (lanes 1 to 4) is the residual activity of the WT after the heat treatment at 50 ° C. It can be seen that it is higher than the activity (lane 5). Further, the initial reaction rate after heat treatment at 50 ° C. of each of the mutant reverse transcriptases obtained in Examples 1 to 4 and the initial reaction rate after heat treatment at 50 ° C. of WT obtained in Comparative Example 3 were determined. The comparison results are shown in Table 2.

  From the results shown in Table 2, the initial reaction rate after heat treatment at 50 ° C. of each of the mutant reverse transcriptases obtained in Examples 1 to 4 is the same as that of WT obtained in Comparative Example 3 at 50 ° C. It can be seen that the initial reaction rate after the heat treatment tends to be large. In addition, when the heat treatment at 48 ° C. was performed, the initial reaction rate after the heat treatment at 48 ° C. of each of the mutant reverse transcriptases obtained in the examples was that of the WT obtained in Comparative Example 3 at 48 ° C. There was a tendency to be larger than the initial reaction rate after the heat treatment (see, for example, Table 3).

  From the above results, the amino acid residue corresponding to at least one residue selected from the group consisting of the valine residue at position 433 and the isoleucine residue at position 434 in the sequence shown in SEQ ID NO: 2 It can be seen that substitution with a positively charged amino acid residue such as a group or a lysine residue can improve the thermal stability compared to WT.

Example 5
In Example 1, instead of using the V433R primer pair as the primer pair, the same operation as in Example 1 was performed except that an E286R primer pair and a V433R primer pair were used, and a mutant reverse transcriptase ( E286R / V433R). “E286R” represents substitution of an amino acid residue corresponding to the glutamic acid residue at position 286 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “E286R / V433R” means a multiple mutant having E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for E286R is primer E286R [5'-ctgaggccagaaaaacgtactgtgtgggggca-3 '(sequence number: 15)] and primer E286R_CP [5'-tgcccccatcagttttctgtggtcctcgtggcctcag'] sequence.

Example 6
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was performed, except that the D108A primer pair, the E286R primer pair, and the V433R primer pair were used. Type reverse transcriptase (D108A / E286R / V433R) was obtained. “D108A” indicates substitution of an amino acid residue corresponding to the aspartic acid residue at position 108 in the sequence shown in SEQ ID NO: 2 with an alanine residue. “E286R / V433R” means a multiple mutant having D108A, E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D108A / E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for D108A is primer D108A [5'-acaccagggactaatgcttataggcctgtcca-3 '(sequence number: 17)] and primer D108A_CP [5'-tggacaggccattatagcccgtgtgtgtgtggt18' (sequence number: number 18).

Example 7
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was performed, except that a D108R primer pair, an E286R primer pair, and a V433R primer pair were used. Type reverse transcriptase (D108R / E286R / V433R) was obtained. “D108R” represents substitution of an amino acid residue corresponding to the aspartic acid residue at position 108 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “D108R / E286R / V433R” means a multiple mutant having D108R, E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D108R / E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for D108R is primer D108R [5'-acaccgggactataattcgttatagggcctgtcca-3 '(sequence number: 19)] and primer D108R_CP [5'-tggacaggcctatatacctgtccgtgtgtgt-3' (sequence number: 20).

Example 8
In Example 1, instead of using the V433R primer pair as the primer pair, the same operation as in Example 1 was performed except that the E117A primer pair, the E286R primer pair, and the V433R primer pair were used. Type reverse transcriptase (E117A / E286R / V433R) was obtained. “E117A” indicates substitution of an amino acid residue corresponding to the glutamic acid residue at position 117 in the sequence shown in SEQ ID NO: 2 with an alanine residue. “E117A / E286R / V433R” means a multiple mutant having E117A, E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E117A / E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for E117A is primer E117A [5'-tccagagtctgagaggcacacaag-3 '(sequence number: 21)] and primer E117A_CP [5'-ctttgtactgctctaccatcctggga-3' (sequence number: 22).

Example 9
In Example 1, instead of using the V433R primer pair as a primer pair, the same operation as in Example 1 was performed except that an E117R primer pair, an E286R primer pair, and a V433R primer pair were used. Type reverse transcriptase (E117R / E286R / V433R) was obtained. “E117R” represents substitution of an amino acid residue corresponding to the glutamic acid residue at position 117 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “E117R / E286R / V433R” means a multiple mutant having E117R, E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E117R / E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for E117R is primer E117R [5'-tccagagtctgagagtgtcaacaag-3 '(sequence number: 23)] and primer E117R_CP [5'-ctttgtacacgtctcacatcctggga-3' (sequence number: 24).

Example 10
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was performed except that the D124A primer pair, the E286R primer pair, and the V433R primer pair were used. Type reverse transcriptase (D124A / E286R / V433R) was obtained. “D124A” represents substitution of an amino acid residue corresponding to the aspartic acid residue at position 124 in the sequence shown in SEQ ID NO: 2 with an alanine residue. “D124A / E286R / V433R” means a multiple mutant having D124A, E286R and V433R. The obtained mutant reverse transcriptase (D124A / E286R / V433R) was confirmed to show a single band of 75 kDa as a result of SDS-PAGE. In addition, the primer which comprises the primer pair for D124A is primer D124A [5'-acagaggcgggtggaagcccatccaccccaccgt-3 '(sequence number: 25)] and primer D124A_CP [5'-acgggtggggtgggtgtccccccgtgt-3' (sequence number: 26).

Example 11
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was performed except that a D124R primer pair, an E286R primer pair, and a V433R primer pair were used. Type reverse transcriptase (D124R / E286R / V433R) was obtained. “D124R” represents substitution of an amino acid residue corresponding to the aspartic acid residue at position 124 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “D124R / E286R / V433R” means a multiple mutant having D124R, E286R and V433R. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D124R / E286R / V433R) was confirmed to show a single band of 75 kDa. In addition, the primer which comprises the primer pair for D124R is primer D124R [5'-acaagcgggtggaacggcatccaccccaccgt-3 '(sequence number: 27)] and primer D124R_CP [5'-acgggtgggggtgtgaccgtgccgtgt-3' (sequence number: 28).

Example 12
In Example 1, instead of using the V433R primer pair as the primer pair, the same operation as in Example 1 was performed except that an E286R primer pair, a V433R primer pair, and a D524A primer pair were used. Type reverse transcriptase (E286R / V433R / D524A) was obtained. “D524A” indicates substitution of an amino acid residue corresponding to the aspartic acid residue at position 524 in the sequence shown in SEQ ID NO: 2 with an alanine residue. “E286R / V433R / D524A” means a multiple mutant having E286R, V433R and D524A. The obtained mutant reverse transcriptase (E286R / V433R / D524A) was confirmed to show a single band of 75 kDa as a result of SDS-PAGE. In addition, the primer which comprises the primer pair for D524A is primer D524A [5'-caccactgggtacacagctggaagcagtctc-3 '(sequence number: 29)] and primer D524A_CP [5'-gagactgccctcgtgtgtgtgtgtggtgtggtgtggtgtgtggtgtgtggtgtggtgtggtgtgtggtgtgtggtgtgtggtggtgtgtggtgtgtggtgtgtggtgtgtggtgtgtggtgtgt

Example 13
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was used except that a D108A primer pair, an E286R primer pair, a V433R primer pair, and a D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (D108A / E286R / V433R / D524A). “D108A / E286R / V433R / D524A” means a multiple mutant having D108A, E286R, V433R and D524A. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D108A / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa.

Example 14
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was used except that the D108R primer pair, the E286R primer pair, the V433R primer pair, and the D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (D108R / E286R / V433R / D524A). “D108R / E286R / V433R / D524A” means a multiple mutant having D108R, E286R, V433R and D524A. The obtained mutant reverse transcriptase (D108R / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa as a result of SDS-PAGE.

Example 15
In Example 1, instead of using the V433R primer pair as the primer pair, the same as Example 1 except that the E117A primer pair, the E286R primer pair, the V433R primer pair, and the D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (E117A / E286R / V433R / D524A). “E117A / E286R / V433R / D524A” means multiple mutants having E117A, E286R, V433R and D524A. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E117A / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E117A / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa.

Example 16
In Example 1, instead of using the V433R primer pair as the primer pair, the same as Example 1 except that the E117 primer pair, the E286R primer pair, the V433R primer pair, and the D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (E117R / E286R / V433R / D524A). “E117R / E286R / V433R / D524A” means multiple mutants having E117R, E286R, V433R and D524A. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (E117R / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa.

Example 17
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was used except that the D124A primer pair, the E286R primer pair, the V433R primer pair, and the D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (D124A / E286R / V433R / D524A). “D124A / E286R / V433R / D524A” means a multiple mutant having D124A, E286R, V433R and D524A. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D124A / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa.

Example 18
In Example 1, instead of using the V433R primer pair as the primer pair, the same procedure as in Example 1 was used except that the D124R primer pair, the E286R primer pair, the V433R primer pair, and the D524A primer pair were used. The operation was performed to obtain a mutant reverse transcriptase (D124R / E286R / V433R / D524A). “D124R / E286R / V433R / D524A” means a multiple mutant having D124R, E286R, V433R and D524A. As a result of SDS-PAGE, the obtained mutant reverse transcriptase (D124R / E286R / V433R / D524A) was confirmed to show a single band of 75 kDa.

Test example 3
200 μL of the incubation solution 200 μL of the mutant reverse transcriptase obtained in Examples 1, 2, and 5 to 18 or the WT obtained in Comparative Example 3 so that the concentration of the mutant reverse transcriptase or WT is 30 nM. Added to. The obtained mixture was incubated at 46 ° C., 48 ° C., 50 ° C. or 52 ° C. for 10 minutes to obtain the mutant reverse transcriptase obtained in Examples 1, 2, 5 to 18 or Comparative Example 3. The WT was heat treated. Next, the mixture containing mutant reverse transcriptase after heat treatment or WT after heat treatment was incubated on ice for 30 to 60 minutes.

  Next, the mutant reverse transcriptase after the heat treatment or the WT after the heat treatment is added to the reaction solution so that the concentration of the mutant reverse transcriptase after the heat treatment or the WT after the heat treatment is 30 nM. The resulting mixture was incubated at 37 ° C. to perform a reverse transcriptase reaction.

Thereafter, 20 μL of the product after 2.5 minutes, 5.0 minutes, or 7.5 minutes from the start of incubation was collected, and immediately, a glass filter [manufactured by Whatman, trade name: GF / C, diameter 2 .5 cm]. Next, the glass filter is washed with a 5% by mass trichloroacetic acid aqueous solution cooled on ice for 10 minutes, and then washed with a 95% by volume aqueous ethanol solution cooled on ice to obtain poly (rA) · p (dT ) [ ³ H] dTTP that was not incorporated into ₁₅ was removed. The washing with the aqueous trichloroacetic acid solution was performed three times, and the washing with the aqueous ethanol solution was performed once.

  Thereafter, the glass filter was dried. The glass filter was placed in 2.5 mL of a liquid scintillation reagent (trade name: Ecoscint H, manufactured by National Diagnostics), and the radioactivity was counted with a liquid scintillation counter. The dTTP uptake amount was calculated based on the radioactivity.

  The initial reaction rate was calculated based on the change in dTTP uptake over time. Next, the residual activity was calculated based on the formula represented by the formula (I).

  Table 4 shows the results of examining the relationship between the test sample and the initial reaction rate after heat treatment at 46 ° C, 48 ° C, 50 ° C or 52 ° C in Test Example 3. Moreover, the result of investigating the relationship between the test sample and the residual activity after heat treatment in Test Example 3 is shown in FIG. In the figure, lane 1 is the mutant reverse transcriptase (L433R) obtained in Example 1, lane 2 is the mutant reverse transcriptase (L433K) obtained in Example 2, and lane 3 is obtained in Example 5. Mutant reverse transcriptase (E286R / V433R), lane 4 is the mutant reverse transcriptase obtained in Example 6 (D108A / E286R / V433R), and lane 5 is the mutant reverse transcriptase obtained in Example 7. (D108R / E286R / V433R), lane 6 is the mutant reverse transcriptase obtained in Example 8 (E117A / E286R / V433R), and lane 7 is the mutant reverse transcriptase obtained in Example 9 (E117R / E286R). / V433R), lane 8 is the mutant reverse transcriptase obtained in Example 10 (D124A / E286R / V433R), and lane 9 is the mutant reverse transcriptase obtained in Example 11. D124R / E286R / V433R), Lane 10 is the mutant reverse transcriptase obtained in Example 12 (E286R / V433R / D524A), and Lane 11 is the mutant reverse transcriptase obtained in Example 13 (D108A / E286R / V433R / D524A), Lane 12 is the mutant reverse transcriptase obtained in Example 14 (D108R / E286R / V433R / D524A), and Lane 13 is the mutant reverse transcriptase obtained in Example 15 (E117A / E286R /). V433R / D524A), lane 14 is the mutant reverse transcriptase obtained in Example 16 (E117R / E286R / V433R / D524A), and lane 15 is the mutant reverse transcriptase obtained in Example 17 (D124A / E286R / V433R / D524A), lane 16 is the mutant type obtained in Example 18. Transcriptase (D124R / E286R / V433R / D524A) and lane 17 shows a WT obtained in Comparative Example 3. In the figure, the shaded bar indicates the remaining activity after heat treatment at 46 ° C., the lattice bar indicates the remaining activity after heat treatment at 48 ° C., the black bar indicates the remaining activity after heat treatment at 50 ° C., and the white bar indicates 52 ° C. The residual activity after heat treatment is shown.

  From the results shown in Table 4, the initial reaction rate after heat treatment at 46 ° C. of the mutant reverse transcriptase obtained in Examples 1, 2, and 5 to 18, the initial reaction rate after heat treatment at 48 ° C., It can be seen that both the initial reaction rate after the heat treatment at 50 ° C. and the initial reaction rate after the heat treatment at 52 ° C. are significantly higher than those of the WT obtained in Comparative Example 3. Further, from the results shown in FIG. 4, the residual activity after heat treatment at 46 ° C. of the mutant reverse transcriptase obtained in Examples 1, 2, and 5-18, the residual activity after heat treatment at 48 ° C., It can be seen that both the residual activity after the heat treatment at 50 ° C. and the residual activity after the heat treatment at 52 ° C. are significantly higher than those of the WT obtained in Comparative Example 3. In particular, the WT obtained in Comparative Example 3 was completely inactivated by the heat treatment at 52 ° C., whereas the mutant reverse transcriptase obtained in Examples 1, 2, and 5-18 was 52 It can be seen that sufficient reverse transcriptase activity is obtained even by heat treatment at ° C.

  Therefore, the amino acid residue corresponding to the valine residue at position 433 in the sequence shown in SEQ ID NO: 2 is substituted with a positively charged amino acid residue such as an arginine residue or a lysine residue, and SEQ ID NO: 2 At least selected from the group consisting of aspartic acid residue at position 108, glutamic acid residue at position 117, aspartic acid residue at position 124, glutamic acid residue at position 286 and aspartic acid residue at position 524 in the sequence shown. By replacing the amino acid residue corresponding to one residue with a positively charged amino acid residue such as an arginine residue or a nonpolar amino acid residue such as an alanine residue, the thermal stability is greatly improved compared to WT. You can see that

  Instead of substituting the amino acid residue corresponding to the valine residue at position 433 with a positively charged amino acid residue, the amino acid residue corresponding to the isoleucine residue at position 434 is also replaced with a positively charged amino acid residue. A similar trend is seen.

  From the above results, an amino acid residue corresponding to at least one residue selected from the group consisting of a valine residue at position 433 and an isoleucine residue at position 434 in the sequence shown in SEQ ID NO: 2 is a positively charged amino acid. Aspartic acid residue at position 108, glutamic acid residue at position 117, aspartic acid residue at position 124, glutamic acid residue at position 286, and position 524 at position 524 in the sequence shown in SEQ ID NO: 2 By substituting an amino acid residue corresponding to at least one residue selected from the group consisting of aspartic acid residues with a positively charged amino acid residue or a nonpolar amino acid residue, the thermal stability is significantly higher than that of WT. It can be seen that the mutated reverse transcriptase is obtained.

Test example 4
In Examples 1-18, when the mutant reverse transcriptase of the present invention (Examples 1-18) is expressed in a host cell, fractionation is easier than when WT is expressed in a host cell. There was a trend. Therefore, the expression pattern of the target protein and the contaminating protein produced when the mutant reverse transcriptase of the present invention (Examples 1 to 18) was expressed in the host cell was examined.

  By performing the same operations as in Examples 1 and 2, a V433R mutant expression plasmid and a V433K mutant expression plasmid were obtained. Further, in Example 1, instead of using the V433R primer pair as the primer pair, an E302K mutant expression plasmid was obtained except that an E302K primer pair was used. Next, V433R mutant expression plasmid (Example 19), V433K mutant expression plasmid (Example 20), WT expression plasmid obtained in Production Example 1 (Comparative Example 4) or E302K mutant expression plasmid (Comparative Example 5) ) Was used to transform E. coli BL21 (DE3), which is a host cell. The obtained cells were shaken at 30 ° C. while shaking in 3 mL of L broth containing 50 μg / mL ampicillin using an automatic expression induction system (manufactured by Novagen, trade name: Overnight Express) that does not require IPTG induction. Protein was expressed by incubation for 16 hours. The obtained culture was subjected to centrifugation at 5840 × g for 10 minutes to collect cells. The collected cells were suspended in 20 mL of Buffer A and sonicated to disrupt. The obtained crushed material was subjected to centrifugation at 20000 × g for 20 minutes, and the supernatant was collected to obtain a soluble fraction containing the mutant reverse transcriptase (V433R) (Example 19), mutant reverse transcription. A soluble fraction containing an enzyme (V433K) (Example 20), a soluble fraction containing WT (Comparative Example 4) or a soluble fraction containing a mutant reverse transcriptase (E302K) (Comparative Example 5) was obtained. It was.

  Each obtained soluble fraction was subjected to SDS-PAGE. In Test Example 4, the results of SDS-PAGE equivalent to a soluble fraction equivalent to 2.7 μL are shown in FIG. 5A, and the results of SDS-PAGE equivalent to a soluble fraction equivalent to 0.9 μL are shown in FIG. 5B. Shown in In the figure, lane 1 is a soluble fraction containing WT (Comparative Example 4), lane 2 is a soluble fraction containing a mutant reverse transcriptase (V433R) (Example 19), and lane 3 is a mutant reverse transcriptase. A soluble fraction containing (V433K) (Example 20), lane 4 shows a soluble fraction containing mutant reverse transcriptase (E302K) (Comparative Example 5).

  As shown in FIGS. 5 (A) and (B), each mutant reverse transcript in the soluble fraction containing mutant reverse transcriptase (V433R) and the soluble fraction containing mutant reverse transcriptase (V433K). The intensity of the 75 kDa band corresponding to the enzyme was shown to be higher than the intensity of the 75 kDa band in the soluble fraction containing WT and the soluble fraction containing the mutant reverse transcriptase (E302K). In contrast, the intensity of the contaminating protein band in the soluble fraction containing the mutant reverse transcriptase (V433R) and the soluble fraction containing the mutant reverse transcriptase (V433K) It was shown to be comparable to the intensity of the contaminating protein band in the soluble fraction containing mutant reverse transcriptase (E302K). The difference in intensity of the 75 kDa band is the same regardless of the type of vector used for the production of the expression plasmid. Moreover, the same tendency is seen also about each variant reverse transcriptase obtained in Examples 3-18.

  Therefore, it is suggested that the mutant reverse transcriptase of the present invention can be easily separated from other contaminating proteins and is excellent in industrial productivity.

Examples 21-24
In Example 1, instead of using the V433R primer pair as the primer pair, the F303R primer pair (Example 21), the F303K primer pair (Example 22), the L432R primer pair (Example 23) or the L432K primer Except for using the pair (Example 24), the same operation as in Example 1 was performed, and mutant reverse transcriptase (F303R) (Example 21), mutant reverse transcriptase (F303K) (Example 22). Mutant reverse transcriptase (L432R) (Example 23) or mutant reverse transcriptase (L432K) (Example 24) was obtained. “F303R” represents substitution of an amino acid residue corresponding to the phenylalanine residue at position 303 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “F303K” represents substitution of an amino acid residue corresponding to the phenylalanine residue at position 303 in the sequence shown in SEQ ID NO: 2 with a lysine residue. “L432R” indicates substitution of an amino acid residue corresponding to the leucine residue at position 432 in the sequence shown in SEQ ID NO: 2 with an arginine residue. “L432K” indicates substitution of an amino acid residue corresponding to the leucine residue at position 432 in the sequence shown in SEQ ID NO: 2 with a lysine residue. The primers constituting the F303R primer pair are the primer F303R (5′-gacaactaagggggcgcctagggacggcag-3 ′) (SEQ ID NO: 31) and the primer F303R_CP (5′-ctgccgtccctagcccctctgtttgttc-3 ′) (sequence No. 31). Primers constituting the primer pair for F303K are the primer F303K (5′-gacaactagggagaaaaactggggacggcag-3 ′) (SEQ ID NO: 33) and the primer F303K_CP (5′-ctgccgtcccttttcccttgttgtttgttcttgtc-3 ′) (sequence No. 33). The primers constituting the L432R primer pair are the primer L432R (5′-ccatgggacaccccacgtgtcattctggccc-3 ′) (SEQ ID NO: 35) and the primer L432R_CP (5′-ggcccagaatgacgtggtgtcccatgg-3 ′) (SEQ ID NO: 36). The primers constituting the L432K primer pair are the primer L432K (5′-ccatgggacaccccaaaagtattctggccc-3 ′) (SEQ ID NO: 37) and the primer L432K_CP (5′-ggcccagatttggtgtcgtgtccctgg-3 ′) (sequence number: 38).

  Mutant reverse transcriptase (F303R) (Example 21), mutant reverse transcriptase (F303K) (Example 22), mutant reverse transcriptase (L432R) (Example 23) and mutant reverse transcriptase (L432K) As a result of SDS-PAGE, (Example 24) was confirmed to show a single band of 75 kDa.

  Also, mutant reverse transcriptase (F303R) (Example 21), mutant reverse transcriptase (F303K) (Example 22), mutant reverse transcriptase (L432R) (Example 23) and mutant reverse transcriptase ( L432K) (Example 24) was measured for the initial reaction rate and residual activity after the heat treatment at 50 ° C. by performing the same operations as in Test Example 2 (2). As a result, mutant reverse transcriptase (F303R) (Example 21), mutant reverse transcriptase (F303K) (Example 22), mutant reverse transcriptase (L432R) (Example 23), and mutant reverse transcriptase Since the initial reaction rate after heat treatment at 50 ° C. of (L432K) (Example 24) was an average of 1.9 nM / s, it tends to be higher than the initial reaction rate after heat treatment at 50 ° C. of WT. I understood it.

  From the above results, in the amino acid sequence corresponding to SEQ ID NO: 2, the phenylalanine residue at position 303, the leucine residue at position 432, the valine residue at position 433, and the 434 position in the sequence shown in SEQ ID NO: 2 Heat stable compared to WT by substituting an amino acid residue corresponding to at least one residue selected from the group consisting of isoleucine residues with positively charged amino acid residues such as arginine residues and lysine residues It can be seen that the property can be improved.

  Further, in the amino acid sequence corresponding to SEQ ID NO: 2, the phenylalanine residue at position 303, the leucine residue at position 432, the valine residue at position 433, and the isoleucine residue at position 434 in the sequence shown in SEQ ID NO: 2 An amino acid residue corresponding to at least one residue selected from the group consisting of: a positively charged amino acid residue such as an arginine residue or a lysine residue; and 108 in the sequence shown in SEQ ID NO: 2 Corresponding to at least one residue selected from the group consisting of aspartic acid residue at position 117, glutamic acid residue at position 117, aspartic acid residue at position 124, glutamic acid residue at position 286 and aspartic acid residue at position 524 Obtained by substituting amino acid residues to be positively charged amino acid residues such as arginine residues or nonpolar amino acid residues such as alanine residues. Thermostability of mutant reverse transcriptase, is greatly improved as compared with the thermal stability of the WT.

  As described above, since the mutant reverse transcriptase of the present invention has high thermal stability, it exhibits high reverse transcriptase activity even when used for reactions at high reaction temperatures. To do. Therefore, according to the mutant reverse transcriptase of the present invention, even when the RNA used as the template contains a sequence that easily forms a secondary structure, the reaction temperature during the reverse transcription reaction is set to a high temperature. Thereby, formation of a secondary structure can be suppressed and a reverse transcription reaction can be performed. Therefore, the mutant reverse transcriptase of the present invention is not limited to the RNA-containing sample to be used and is a highly versatile analytical reagent (for example, reverse transcription reaction kit), a reagent for detecting viruses, bacteria, diseases, etc. It is suggested to be useful as (for example, a detection kit). In addition, the mutant reverse transcriptase of the present invention is excellent in industrial productivity.

Formulation Example 1
Examples of reverse transcription reaction kits are shown below.
(Reverse transcription reaction kit)
-Mutant reverse transcriptase obtained in Example 1-10x reverse transcriptase buffer [Composition: 250 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride,
20 mM dithiothreitol]
-2.0 mM dNTP mixture-10 μM (for standard RNA amplification) primer aqueous solution-Standard RNA solution (1.6 pg / μL)

Formulation Examples 2-24
In Formulation Example 1, instead of using the mutant reverse transcriptase obtained in Example 1, by using each mutant reverse transcriptase obtained in Examples 2-24, the reverse transcription of Formulation Examples 2-24 A reaction kit is obtained.

Formulation Example 25
Examples of detection kits are shown below.
(Detection kit)
-Mutant reverse transcriptase obtained in Example 1-10x reverse transcriptase buffer [Composition: 250 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride,
20 mM dithiothreitol]
-2.0 mM dNTP mixture-10 [mu] M RV-R26 primer aqueous solution-Standard RNA solution (1.6 pg / [mu] L)
-E. coli RNA solution (1.0 μg / μL)

Formulation Examples 26-48
In Prescription Example 25, instead of using the mutant reverse transcriptase obtained in Example 1, each of the mutant reverse transcriptases obtained in Examples 2 to 24 is used, whereby the detection kits of Prescription Examples 26 to 48 are used. Is obtained.

SEQ ID NO: 3 is the sequence of primer V433R.
SEQ ID NO: 4 is the sequence of primer V433R_CP.
SEQ ID NO: 5 is the sequence of primer V433K.
SEQ ID NO: 6 is the sequence of primer V433K_CP.
SEQ ID NO: 7 is the sequence of primer I434R.
SEQ ID NO: 8 is the sequence of primer I434R_CP.
SEQ ID NO: 9 is the sequence of primer I434K.
SEQ ID NO: 10 is the sequence of primer I434K_CP.
SEQ ID NO: 11 is the sequence of primer L304R.
SEQ ID NO: 12 is the sequence of primer L304R_CP.
SEQ ID NO: 13 is the sequence of primer L304K.
SEQ ID NO: 14 is the sequence of primer L304K_CP.
SEQ ID NO: 15 is the sequence of primer E286R.
SEQ ID NO: 16 is the sequence of primer E286R_CP.
SEQ ID NO: 17 is the sequence of primer D108A.
SEQ ID NO: 18 is the sequence of primer D108A_CP.
SEQ ID NO: 19 is the sequence of primer D108R.
SEQ ID NO: 20 is the sequence of primer D108R_CP.
SEQ ID NO: 21 is the sequence of primer E117A.
SEQ ID NO: 22 is the sequence of primer E117A_CP.
SEQ ID NO: 23 is the sequence of primer E117R.
SEQ ID NO: 24 is the sequence of primer E117R_CP.
SEQ ID NO: 25 is the sequence of primer D124A.
SEQ ID NO: 26 is the sequence of primer D124A_CP.
SEQ ID NO: 27 is the sequence of primer D124R.
SEQ ID NO: 28 is the sequence of primer D124R_CP.
SEQ ID NO: 29 is the sequence of primer D524A.
SEQ ID NO: 30 is the sequence of primer D524A_CP.
SEQ ID NO: 31 is the sequence of primer F303R.
SEQ ID NO: 32 is the sequence of primer F303R_CP.
SEQ ID NO: 33 is the sequence of primer F303K.
SEQ ID NO: 34 is the sequence of primer F303K_CP.
SEQ ID NO: 35 is the sequence of primer L432R.
SEQ ID NO: 36 is the sequence of primer L432R_CP.
SEQ ID NO: 37 is the sequence of primer L432K.
SEQ ID NO: 38 is the sequence of primer L432K_CP.

Claims (10)

  A mutant reverse transcriptase having reverse transcriptase activity, having an amino acid sequence corresponding to SEQ ID NO: 2, wherein in the amino acid sequence, a phenylalanine residue at position 303 of SEQ ID NO: 2 and a leucine residue at position 432 An amino acid residue corresponding to at least one residue selected from the group consisting of a group, a valine residue at position 433, and an isoleucine residue at position 434 is substituted with a positively charged amino acid residue Mutant reverse transcriptase. (A) substitution of a residue corresponding to the phenylalanine residue at position 303 of SEQ ID NO: 2 with a lysine residue or arginine residue;
(B) substitution of a residue corresponding to the leucine residue at position 432 with a lysine residue or an arginine residue;
(C) substitution of a residue corresponding to a valine residue at position 433 in SEQ ID NO: 2 with a lysine residue or arginine residue; and (d) a residue corresponding to an isoleucine residue at position 434 in SEQ ID NO: 2. The mutant reverse transcriptase according to claim 1, having at least one selected from the group consisting of substitution of a group with a lysine residue or arginine residue.   A group consisting of the aspartic acid residue at position 108, the glutamic acid residue at position 117, the aspartic acid residue at position 124, the glutamic acid residue at position 286, and the aspartic acid residue at position 524 in the sequence shown in SEQ ID NO: 2. The mutant reverse transcriptase according to claim 1 or 2, wherein an amino acid residue corresponding to at least one selected residue is substituted with a positively charged amino acid residue or a nonpolar amino acid residue.   The amino acid sequence corresponding to SEQ ID NO: 2 is a residue corresponding to the phenylalanine residue at position 303, the leucine residue at position 432, the valine residue at position 433, and the isoleucine residue at position 434 in SEQ ID NO: 2. The mutant reverse transcriptase according to any one of claims 1 to 3, which is an amino acid sequence having a conservative substitution of amino acid residues excluding. The amino acid sequence corresponding to SEQ ID NO: 2 is
(1) In the sequence shown in SEQ ID NO: 2, substitution, deletion, insertion or addition of one or several amino acid residues is further added to the sequence excluding the threonine residue at position 24 to the proline residue at position 474 An amino acid sequence of a polypeptide having reverse transcriptase activity, or (2) sequence identity to a sequence excluding a threonine residue at position 24 to a proline residue at position 474 in the sequence shown in SEQ ID NO: 2. 5 is an amino acid sequence of a polypeptide having at least 80% and having reverse transcriptase activity, The mutant reverse transcriptase according to any one of claims 1 to 4.   A nucleic acid encoding the mutant reverse transcriptase according to any one of claims 1 to 5. A method for producing the mutant reverse transcriptase according to any one of claims 1 to 5,
(A) a step of culturing cells retaining the nucleic acid according to claim 6 to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture; and (B) obtained in the step (A). A method for producing a mutant reverse transcriptase, comprising a step of recovering the mutant reverse transcriptase from the obtained culture.   A reverse transcription method comprising synthesizing cDNA from RNA using the mutant reverse transcriptase according to any one of claims 1 to 5.   A kit for performing a reverse transcription reaction, comprising the mutant reverse transcriptase according to any one of claims 1 to 5, wherein the kit is a reverse transcription reaction kit.   A kit for detecting a marker in a sample containing RNA obtained from a living body, comprising the mutant reverse transcriptase according to any one of claims 1 to 5 and a reagent for detecting the marker Detection kit characterized by.