JP6720632B2 - Fusion protein - Google Patents

Description

The present invention relates to a novel nucleic acid synthase. More specifically, it relates to a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity.

The synthesis of DNA from a template nucleic acid using a DNA polymerase is used and applied to various methods such as a sequencing method and a nucleic acid amplification method in the field of molecular biology. Among them, the nucleic acid amplification method has already been put to practical use not only in the field of research but also in the field of forensic medicine such as gene diagnosis and paternity testing, or in the inspection of microorganisms in food and the environment.

Various methods have been developed to date for the nucleic acid amplification method, such as a polymerase chain reaction (PCR) method, a Loop-Mediated Isomeric Amplification (LAMP) method, a Transcribed Reverse Transcribing Reconclusion Reconcription Acupuncture (TRC) method, and a Transcribed Reversed Condensed Reconcription (TRC) method. Nucleic acid amplification methods such as the (NASBA) method are relatively popular.

Among them, the PCR method used for amplifying a specific sequence of DNA is an extremely widespread technique from research fields to applied fields. Currently, the PCR method is being further developed, and there are various technologies such as the Multiplex PCR method for simultaneously amplifying multiple primers and the real-time PCR method for detecting the PCR amplification product in real time using a fluorescent dye. To do. These techniques have already been put to practical use not only in the field of research but also in the field of forensic medicine, foods, microbiological tests in the environment, and the like.

As means for improving the performance and accuracy of the above PCR method, modification of DNA polymerase and improvement of reaction buffer have been carried out.

Fusion DNA polymerase is mentioned as one of the methods of modification of DNA polymerase. The fusion polymerase can improve the performance of the DNA polymerase over the wild type by combining the protein having the desired activity with the DNA polymerase.

As one of the proteins having a desired activity in a fusion protein, a DNA binding protein that improves processability and extensibility has been used. Specifically, Sso7d derived from Sulfolobus solfataricus, Sac7d derived from Sulfolobus acidocaldarius, and Pae3192 patent derived from Pyrobaculum aerophilum have been used. 1, Patent Document 2).

Among them, as a typical fusion DNA polymerase, a sequence non-specific double-stranded nucleic acid binding protein (Sso7d) derived from Sulfolobus sofataricus and a DNA polymerase (Pfu DNA polymerase) derived from Pyrococcus furiosus are combined. Sso7d-Pfu DNA polymerase improved the elongation property and processability in PCR as compared with the wild type.

Alba has low homology to these existing DNA-binding proteins and has a new feature that it can bind not only to double-stranded DNA but also to RNA. It has been only recently that the detailed mechanism and properties of Alba have been clarified, and there has been no actual study on a fusion protein of Alba and DNA polymerase.

Japanese Patent No. 4405725 U.S. Patent Application Publication No. 2011/0086406

The Journal of Biological Chemistry Vol. 289, No. 3, pp. 1478-1490 (2014). PLOS ONE Vol.8, No.2, p.e. 58237 (2013). Genome Research Vol.15, No.3, p.352-363 (2005)

The present invention has been made against the background of the problems of the related art. That is, it is to provide a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity.

As a result of intensive studies in view of the above circumstances, the present inventors have found that a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity can be applied to various buffer compositions that could not be used conventionally. Furthermore, they have found that salt resistance is improved as compared with conventional fusion proteins, and have completed the present invention.

That is, the present invention has the following configurations.
Item 1. A fusion protein comprising a DNA/RNA binding protein fused with a protein having a polymerase activity.
Item 2. Item 2. The fusion protein according to Item 1, wherein the protein having polymerase activity is DNA polymerase.
Item 3. Item 2. The fusion protein according to Item 1, wherein the protein having a polymerase activity is a reverse transcriptase.
Item 4. Item 3. The fusion protein according to Item 2, wherein the protein having polymerase activity is a thermostable DNA polymerase having an activity optimum temperature of 50°C or higher.
Item 5. Item 3. The fusion protein according to Item 2, wherein the protein having a polymerase activity is a thermostable DNA polymerase having a residual activity of 90% or more after heat treatment at 60°C for 30 minutes.
Item 6. Item 3. The fusion protein according to Item 2, wherein the thermostable DNA polymerase is Taq DNA polymerase or Tth DNA polymerase.
Item 7. Item 7. The fusion protein according to any one of Items 1 to 6, wherein the DNA/RNA binding protein is Alba (Acetylation Lower Binding Affinity).
Item 8. Item 8. The fusion protein according to Item 7, wherein the Alba is derived from Thermococcus kodakaraensis.

According to the present invention, a buffer protein that can be used can be expanded by a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity, and salt resistance can be improved as compared with a conventional protein having a polymerase activity. Therefore, it is less likely to be affected by the inhibitor and less affected by the influence of the salt contained in the sample, and therefore it is also useful for a sample having a large difference between samples such as a clinical sample.

It is the figure which evaluated Alba-Taq and Taq with respect to various buffers. It is a figure which evaluated Alba-Taq, Sso7d-Taq, and Taq using the BlendTaq (TOYOBO) attachment buffer about the resistance with respect to NaCl. It is the figure which evaluated Alba-Taq and Sso7d-Taq using the buffer attached to KOD-Plus-(TOYOBO) about the tolerance with respect to NaCl. It is a figure which evaluated Alba-Tth, Sso7d-Tth, and Tth about resistance with respect to NaCl.

Hereinafter, the present invention will be described in detail.
One of the embodiments of the present invention is a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity.

In the present invention, the DNA/RNA binding protein means a protein having a binding domain for both DNA and RNA nonspecifically in the double-stranded structure.

The polymerase activity means an activity of polymerizing a single nucleotide to synthesize a polynucleotide.

In the present invention, the polymerase means an enzyme having a polymerase activity. Specific examples include DNA polymerase and RNA polymerase.

Fusion proteins are mainly artificial (
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Made)
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And more than two proteins
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Is a protein that is integrally transcribed and expressed to form a single protein.

DNA polymerase is a single-stranded
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As a template
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Can synthesize DNA chains with
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Means

In the present invention, when the protein having polymerase activity is DNA polymerase, it is preferably thermostable DNA polymerase. The thermostable DNA polymerase is preferably a thermostable DNA polymerase having an optimum activity temperature of 50° C. or higher, more preferably 60° C. or higher, further preferably 65° C. or higher. Further, it may have a residual activity of 90% or more, more preferably 95% or more by heat treatment at 60° C. for 30 minutes.

Thermostable DNA polymerases used in the present invention include Pyrococcus furiosus, Thermococcus kodakaraensis, Thermococcus litoralis, and Bacillus stearothermophilus. Examples include separated DNA polymerases and the like. Preferred is a DNA polymerase (Taq DNA polymerase) derived from Thermus aquaticus (SEQ ID NO: 1) or a DNA polymerase derived from Thermus thermophilus (Tth DNA polymerase) (SEQ ID NO: 3), It is not limited to these.

In the present invention, when the protein having a polymerase activity is reverse transcriptase, it includes, but is not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, and Tth DNA polymerase.

The DNA/RNA binding protein in the present invention is not particularly limited, but examples thereof include Alba (Acetylation Lower Binding Affinity) and double-stranded RNA binding protein (Double Stranded RNA Binding protein). Of these, Alba is particularly preferable.

Alba is a protein that binds to the double-stranded structure of DNA or RNA in a non-sequence-specific manner (Non-Patent Documents 1 and 2). Sso10b derived from Sulfolobus sofataricas and Sac10b derived from Sulfolobus acidocaldarius have also been referred to as Alba because their acetylation of lysine residues reduces their binding to DNA.

Alba is known to have amino residues involved in binding to nucleic acid (Non-patent Document 1), and one or several amino acid sequence residues are substituted, deleted, inserted and/or added (hereinafter, these are summarized. It may also be referred to as “mutation”), and thus Alba whose binding to DNA is weakened may be used. For example, from Non-Patent Document 1, the 16th lysine residue, the 17th lysine residue, the 22nd tyrosine residue, or the 44th arginine residue in SEQ ID NO: 5 may be replaced with an alanine residue. However, the present invention is not limited to this.

In the present specification, a position corresponding to a certain position (order) on SEQ ID NO: 5 in an amino acid sequence which is not completely identical to the amino acid sequence represented by SEQ ID NO: 5 means that when the primary structures of the sequences are compared (aligned), The position corresponds to the position of SEQ ID NO:5.

Since Alba forms a dimer or a multimer in a concentration-dependent manner, a mutation may be introduced to facilitate formation of a monomer. For example, according to Non-Patent Document 1, the 60th phenylalanine residue in SEQ ID NO: 5 may be replaced with an alanine residue, but the present invention is not limited thereto.

The amino acid sequence of Alba is highly conserved in archaea with respect to the mutation site (Non-Patent Document 1), and its position is predicted with high probability by comparing (alignment) the primary structures of the amino acid sequences. You can check it.

The origin of Alba is Pyrococcus furiosus (Pfu), Thermococcus kodakaraensis (Kod), Sulfolobus sofatalicas (Sso), Sulfolobus cibatae (Ssh), and Sulfolobus acidocardarius (Sac), which are the archaea. However, the present invention is not limited to this. It is preferably derived from Thermococcus kodakaraensis (SEQ ID NO: 6).

The fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity preferably has a DNA/RNA binding protein at the N-terminus and a polymerase moiety at the C-terminus, but is not particularly limited to this embodiment. is not. With respect to the DNA sequence encoding the fusion protein, it is preferable, but not particularly limited, to arrange the DNA/RNA binding protein portion on the 5'side upstream of the polymerase portion. The two moieties may each be directly linked or may be separated by a peptide linker. The peptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40. Alternatively, it may be 50 or more amino acids in length. The length is preferably about 5 amino acids, but is not particularly limited.

The vector used in the present invention may be any vector as long as it enables cloning and expression of the fusion protein, and examples thereof include phage and plasmid. Examples of the plasmid include pBluescript. Examples of other plasmids include pUC19, pBR322, pSP73, pGW7, pET3A, pET21b and the like. Examples of the phage include λgt11, λDASH, λZapII and the like. Examples of host cells used in the present invention include E. coli and yeast. Examples of Escherichia coli include JM109, 101, XL1, PR1, BL21(DE3)polysS and the like. In the present invention, a gene encoding the above fusion protein is inserted into the above vector to prepare a recombinant expression vector, and a host cell is transformed with this recombinant expression vector.

As a method for producing the fusion protein in the present invention, a conventionally known method can be used. As an example, the recombinant host cell is cultured to express the fusion protein gene. For example, using Escherichia coli as a host, transformation is performed with the expression vector, and then the cells are applied to an agar medium containing a drug such as ampicillin to form colonies. The colony is inoculated into a nutrient medium such as LB medium or 2×YT medium and cultured at 30° C. to 37° C. for 12 to 20 hours, and then the cells are crushed to extract a crude enzyme solution. A vector derived from pBluescript is preferable. Any known method may be used as a method for disrupting the bacterial cells. For example, ultrasonic treatment, a physical disruption method such as French press or glass bead disruption, or a lytic enzyme such as lysozyme may be used. This crude enzyme solution is heat-treated at 70° C. for 1 hour, centrifuged to remove the host-derived protein, and subjected to SDS-PAGE to confirm the expression of the target protein.

Any method may be used as a method for obtaining the purified fusion protein from the strain selected by the above method, and for example, the following method is available. That is, the bacterial cells obtained by culturing in a nutrient medium are recovered and then crushed and extracted by an enzymatic or physical crushing method to obtain a crude enzyme solution. The obtained crude enzyme extract is heat-treated, for example, treated at 70° C. for 1 hour, and then the fusion protein fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution is desalted by a method such as dialysis, and then separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to the extent that it shows a nearly single band.

Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to the following examples.

(Example 1)
Preparation of plasmid of Alba-Taq The Alba gene (SEQ ID NO: 6) derived from Thermococcus kodakaraensis KOD1 strain and the DNA polymerase (Taq DNA polymerase) gene (SEQ ID NO: 1) derived from Thermus aquaticus were fused. A plasmid was created. The fused Alba-Taq nucleic acid sequence is shown in SEQ ID NO:10.

The Alba gene derived from Thermococcus kodakaraensis KOD1 strain was produced by artificial synthesis with reference to the information described in Non-Patent Document 3. The artificially synthesized nucleic acid was cloned into pBlueScript with the codon frame being aligned upstream of the Taq DNA polymerase gene. Gly-Gly-Gly-Val-Thr was interposed as a peptide linker between the Alba region and the Taq DNA polymerase region. The obtained plasmid was transformed into Escherichia coli JM109 competent cells (TOYOBO) by the heat shock method, and cultured on an LB medium plate containing 100 μg/ml ampicillin for 16 hours. Single colonies obtained by transformation were used for enzyme preparation.

(Example 2)
Preparation of Alba-Taq Cultivation of the bacterial cells obtained in Example 2 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. This culture medium was preliminarily cultured in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin at 30° C. for 16 hours. Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerated at 30° C. for 16 hours. The cells were collected from the culture solution by centrifugation, suspended in 15 mL of a disruption buffer solution (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells were subjected to sonication treatment to remove the cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting-out, heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Alba-Taq.

(Example 3)
Construction of Sso7d-Taq plasmid Double- stranded DNA binding protein gene (Sso7d) (Sso7d) derived from Sulfolobus solfataricus (SEQ ID NO: 8) and Thermus aquaticus were prepared for use in the evaluation of DNA polymerase in Examples described later. A plasmid in which the DNA polymerase (Taq DNA polymerase) gene (SEQ ID NO: 1) derived from the gene was fused was prepared. The fused Sso7d-Taq nucleic acid sequence is shown in SEQ ID NO:12.

The double-stranded DNA binding protein gene (Sso7d) derived from Sulfolobus solfataricus was produced by artificial synthesis with reference to the information described in Patent Document 1. The artificially synthesized nucleic acid was cloned into pBlueScript with the codon frame being aligned upstream of the Taq DNA polymerase gene. Gly-Gly-Gly-Val-Thr was interposed as a peptide linker between the Sso7d region and the Taq DNA polymerase region. Escherichia coli JM109 was transformed with the obtained plasmid in the same manner as in Example 1 and used for enzyme preparation.

(Example 4)
Preparation of Sso7d-Taq Cultivation of the bacterial cells obtained in Example 3 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin in advance in this medium at 30° C. for 16 hours. Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerobically cultured at 30° C. for 16 hours. The cells were collected from the culture solution by centrifugation, suspended in 15 mL of a disruption buffer solution (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells were subjected to sonication treatment to remove the cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting-out, heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Sso7d-Taq.

(Example 5)
Preparation of Taq plasmid A plasmid containing a DNA polymerase (Taq DNA polymerase) gene (SEQ ID NO: 1) derived from Thermus aquaticus was prepared for use in the evaluation of DNA polymerase in Examples described later. A DNA polymerase (Taq DNA polymerase) gene derived from Thermus aquaticus was cloned into pBluescript and constructed as a plasmid. Escherichia coli JM109 was transformed with the obtained plasmid in the same manner as in Example 1 and used for enzyme preparation.

(Example 6)
Production of Taq The culture of the bacterial cells obtained in Example 5 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin in advance in this medium at 30° C. for 16 hours. Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerobically cultured at 30° C. for 16 hours. The bacterial cells were collected from the culture solution by centrifugation, suspended in 50 mL of a disruption buffer (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the bacterial cells were treated with sonication to suspend the bacterial cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Taq.

(Example 7)
Preparation of plasmid of Alba-Tth The Alba gene (SEQ ID NO: 6) derived from Thermococcus kodakaraensis KOD1 strain was fused with the DNA polymerase (Tth DNA polymerase) gene (SEQ ID NO: 3) derived from Thermus thermophilus. A plasmid was prepared. The fused Alba-Tth nucleic acid sequence is shown in SEQ ID NO:14.

The Alba gene derived from Thermococcus kodakaraensis KOD1 strain was produced by artificial synthesis with reference to the information described in Non-Patent Document 3. The artificially synthesized nucleic acid was cloned into pBlueScript with the codon frame aligned upstream of the Tth DNA polymerase gene. Gly-Gly-Gly-Val-Thr was interposed as a peptide linker between the Alba region and the Tth DNA polymerase region. Escherichia coli JM109 was transformed with the obtained plasmid in the same manner as in Example 1 and used for enzyme preparation.

(Example 8)
Preparation of Alba-Tth The culture of the bacterial cells obtained in Example 7 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin in advance at 37° C. for 16 hours Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerobically cultured at 37° C. for 16 hours. The cells were recovered from the culture solution by centrifugation, suspended in 15 mL of a disruption buffer (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells were sonicated to suspend the cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Alba-Tth.

(Example 9)
Construction of Sso7d-Tth plasmid For use in the evaluation of DNA polymerase in Examples described later, a double-stranded DNA binding protein gene (Sso7d) derived from Sulfolobus solfataricus (Sso7d) (SEQ ID NO: 8) and Thermus thermo A plasmid in which the DNA polymerase (Tth DNA polymerase) gene derived from Fils (SEQ ID NO: 3) was fused was prepared. The fused Sso7d-Tth nucleic acid sequence is shown in SEQ ID NO:16.

The double-stranded DNA binding protein gene (Sso7d) derived from Sulfolobus solfataricus was produced by artificial synthesis with reference to the information described in Patent Document 1. The artificially synthesized nucleic acid was cloned into pBlueScript with the codon frame aligned upstream of the Tth DNA polymerase gene. Gly-Gly-Gly-Val-Thr was interposed as a peptide linker between the Sso7d region and the Tth DNA polymerase region. Escherichia coli JM109 was transformed with the obtained plasmid in the same manner as in Example 1 and used for enzyme preparation.

(Example 10)
Production of Sso7d-Tth The culture of the bacterial cells obtained in Example 9 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin in advance at 37° C. for 16 hours Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerobically cultured at 37° C. for 16 hours. The cells were recovered from the culture solution by centrifugation, suspended in 15 mL of a disruption buffer (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells were sonicated to suspend the cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Sso7d-Tth.

(Example 11)
Preparation of Tth plasmid A plasmid containing a DNA polymerase (Tth DNA polymerase) gene (SEQ ID NO: 3) derived from Thermus thermophilus was prepared for use in the evaluation of DNA polymerase in Examples described later. A DNA polymerase (Tth DNA polymerase) (SEQ ID NO:) derived from Thermus thermophilus was cloned into pBluescript and constructed as a plasmid. Escherichia coli JM109 was transformed with the obtained plasmid in the same manner as in Example 1 and used for enzyme preparation.

(Example 12)
Preparation of Tth The culture of the bacterial cells obtained in Example 11 was carried out as follows. First, 80 mL of TB medium (Molecular Cloning 2nd Edition, p.A.2) containing 100 μg/mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/mL ampicillin in advance at 37° C. for 16 hours Escherichia coli JM109 (plasmid transformant) (using a test tube) was inoculated and aerobically cultured at 37° C. for 16 hours. The bacterial cells were collected from the culture solution by centrifugation, suspended in 50 mL of a disruption buffer (30 mM Tris-HCl buffer solution (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the bacterial cells were treated with sonication to suspend the bacterial cells. The cells were crushed to obtain a cell lysate. Next, the cell lysate was treated at 70° C. for 1 hour, and the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were performed, and finally storage buffer (20 mM Tris-HCl buffer (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM). Dithiothreitol, 0.5% Tween 20, 0.5% nonidet P40, 50% glycerin) was substituted to obtain Tth.

(Example 13)
Application to Various Buffers Various buffer compositions described below were used to compare the amounts of Ala-Taq, Taq amplified. For comparison, 100 ng of Alba-Taq and Taq were used per 50 μl reaction system.

As one of PCR reaction compositions, a buffer attached to KOD-Plus- (manufactured by Toyobo) was used, 1×PCR Buffer, 1.5 mM MgSO ₄ , 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NOS: 18, 19), A 50 μl reaction solution containing 50 ng of human genomic DNA (Roche) and 100 ng of each enzyme was prepared.

As one of PCR reaction compositions, KOD-Plus-ver. 2 (manufactured by Toyobo) using 1x PCR Buffer, 1.5 mM MgSO ₄ , 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NO: 18, 19), 50 ng of human genomic DNA (Roche) and 100 ng of each enzyme. 50 μl of reaction solution was prepared.

As one of PCR reaction compositions, a buffer attached to KOD-Plus-Neo (manufactured by Toyobo) was used, 1×PCR Buffer, 1.5 mM MgSO ₄ , 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NOS: 18, 19). A 50 μl reaction solution containing 50 ng of human genomic DNA (Roche) and 100 ng of each enzyme was prepared.

As one of the PCR reaction compositions, a buffer attached to KOD FX (manufactured by Toyobo) was used, and 1×PCR Buffer, 0.4 mM dNTPs, 15 pmol of each primer (SEQ ID NO: 18, 19), human genomic DNA (Roche) 50 ng and A 50 μl reaction solution containing 100 ng of each enzyme was prepared.

As one of the PCR reaction compositions, Buffer attached to KOD FX Neo (manufactured by Toyobo) was used, and 1×PCR Buffer, 0.4 mM dNTPs, 15 pmol of each primer (SEQ ID NO: 18, 19), human genomic DNA (Roche) 50 ng. And 50 μl of reaction solution containing 100 ng of each enzyme was prepared.

As one of the PCR reaction compositions, a buffer attached to KOD-Multi & Epi-(registered trademark) (manufactured by Toyobo) was used, and 1×PCR Buffer, 15 pmol of each primer (SEQ ID NO: 18, 19), human genomic DNA (Roche) 50 ng. And 50 μl of reaction solution containing 100 ng of each enzyme was prepared.

The reaction with the above reaction solution was performed using PCR system GeneAmp (registered trademark) 9700 (Applied Biosystem) on a schedule in which 94° C., 30 seconds→60° C., 30 seconds→68° C., and 1 minute were repeated 40 cycles. After completion of the reaction, 5 μl of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under UV irradiation.

The results are shown in Figure 1. In Taq, amplification was confirmed only in one of the eight kinds of buffers, whereas in Alba-Taq, amplification was confirmed only in eight kinds of the eight kinds of buffers. It is considered that this is because it has excellent resistance to the salt of the buffer component.

(Example 14)
Salt resistance-1
Amplification amount comparison was performed for salt resistance (NaCl: 50 mM to 200 mM) of Alba-Taq, Sso7d-Taq, and Taq. For comparison, 25 ng of Alba-Taq, Sso7d-Taq, Taq were used per 50 μl reaction system.

PCR was performed using Buffer attached to Blend Taq (manufactured by Toyobo), 1×PCR Buffer, 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NO: 20, 21), 50 ng of human genomic DNA (Roche), and 50 μl of each enzyme 25 ng. The reaction solution of was prepared. The reaction was carried out using a PCR system GeneAmp (registered trademark) 9700 (Applied Biosystem) in a schedule in which 30 cycles of 94° C., 30 seconds→65° C., 30 seconds→68° C., and 1 minute 30 seconds were repeated. After completion of the reaction, 5 μl of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under UV irradiation.

The results are shown in Figure 2. Amplification was confirmed up to 150 mM for Alba-Taq, 125 mM for Sso7d-Taq, and 50 mM for Taq. Alba-Taq showed the highest salt tolerance. Since Alba-Taq is not easily influenced by the salt contained in the sample, it is effective for a sample having a large difference between samples such as a clinical sample.

(Example 15)
Salt resistance-2
The comparison of the amplification amount was carried out for the salt resistance (NaCl: 50 mM to 200 mM) of Alba-Taq and Sso7d-Taq. For comparison, 25 ng of Alba-Taq and Sso7d-Taq were used per 50 μl reaction system.

PCR uses KOD-Plus- (manufactured by Toyobo) attached Buffer and includes 1×PCR Buffer, 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NOs: 20 and 21), human genomic DNA (Roche) 50 ng and each enzyme 25 ng. 50 μl of reaction solution was prepared. The reaction was carried out using PCR system GeneAmp (registered trademark) 9700 (Applied Biosystem) on a schedule in which 30 cycles of 94° C., 30 seconds→65° C., 30 seconds→68° C., and 1 minute 30 seconds were repeated. After completion of the reaction, 5 μl of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under UV irradiation.

Results are shown in FIG. Amplification was confirmed up to 100 mM for Alba-Taq and 50 mM for Sso7d-Taq. Alba-Taq showed high salt tolerance like the buffer attached to BlendTaq.

(Example 16)
Salt resistance-3
The amounts of amplification were compared for salt resistance (NaCl: 50 mM to 200 mM) of Alba-Tth, Sso7d-Tth, and Tth. For comparison, 100 ng of Alba-Tth, Sso7d-Tth, and Tth were used per 50 μl reaction system.

PCR was performed using a buffer attached to KOD FX (manufactured by Toyobo), 1×PCR Buffer, 0.2 mM dNTPs, 15 pmol of each primer (SEQ ID NOs: 20 and 21), 50 ng of human genomic DNA (Roche), and 50 μl of each enzyme 100 ng. A reaction solution was prepared. The reaction was carried out using PCR system GeneAmp (registered trademark) 9700 (Applied Biosystem) on a schedule in which 30 cycles of 94° C., 30 seconds→65° C., 30 seconds→68° C., and 1 minute 30 seconds were repeated. After completion of the reaction, 5 μl of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under UV irradiation.

The results are shown in Fig. 4. Amplification was confirmed up to 125 mM for Alba-Tth, 0 mM for Sso7d-Tth, and 50 mM for Tth. Alba-Tth showed high salt resistance like Alba-Taq. Fusing Alba with Taq or Tth showed higher salt tolerance than Taq and Sso7d-Taq.

INDUSTRIAL APPLICABILITY According to the present invention, a fusion protein of a DNA/RNA binding protein and a protein having a polymerase activity can be used to extend the buffer composition that can be used, and salt resistance can be improved over conventional proteins having a polymerase activity. Therefore, it is not easily affected by the influence of the salt contained in the sample, and can be widely used in the field of gene diagnosis and the like in which there is a large difference between samples such as clinical samples.

Claims (6)

A fusion protein comprising an Aba (Acetylation Lower Binding Affinity) protein derived from Thermococcus kodakaraensis and a protein having a polymerase activity fused with each other. The fusion protein according to claim 1, wherein the protein having polymerase activity is DNA polymerase. The fusion protein according to claim 1, wherein the protein having a polymerase activity is a reverse transcriptase. The fusion protein according to claim 2, wherein the protein having polymerase activity is a thermostable DNA polymerase having an activity optimum temperature of 50° C. or higher. The fusion protein according to claim 2, wherein the protein having a polymerase activity is a thermostable DNA polymerase having a residual activity of 90% or more after heat treatment at 60° C. for 30 minutes. The fusion protein according to claim 2, wherein the thermostable DNA polymerase is Taq DNA polymerase or Tth DNA polymerase.