WO2017090685A1 - Dnaポリメラーゼ変異体 - Google Patents
Dnaポリメラーゼ変異体 Download PDFInfo
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- WO2017090685A1 WO2017090685A1 PCT/JP2016/084808 JP2016084808W WO2017090685A1 WO 2017090685 A1 WO2017090685 A1 WO 2017090685A1 JP 2016084808 W JP2016084808 W JP 2016084808W WO 2017090685 A1 WO2017090685 A1 WO 2017090685A1
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- WIPO (PCT)
- Prior art keywords
- pip
- dna polymerase
- pcna
- dna
- fusion polypeptide
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- 238000002741 site-directed mutagenesis Methods 0.000 description 1
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- 238000000638 solvent extraction Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
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- 210000001541 thymus gland Anatomy 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to a DNA polymerase mutant.
- the DNA polymerase mutant of the present invention is particularly useful for nucleic acid amplification in the presence of PCNA.
- DNA polymerase is an enzyme that can synthesize a new DNA strand along a template DNA strand in a test tube.
- a primer and four types of deoxynucleotides are used for the reaction. If (dATP, dGTP, dCTP, dTTP) is present, a new DNA strand is synthesized.
- DNA polymerase is used in many operations such as nucleic acid amplification methods including base sequence determination and polymerase chain reaction (PCR).
- thermophilic archaea As related factors, for example, a plurality of proteins derived from Pyrococcus furiosus have been isolated (Patent Document 1).
- Thermococcus kodakarensis KOD1 strain also has PCNA (proliferating cell nuclear antigen), RFC-S (replication factor C small subunit: replication factor C small sub-cLFlCunLuClFlCunLuClFlClClFlClFlClFlClFlClFlClFlClFlClFlClFlClClCcL Subunit: replication factor C large subunit) was isolated (Patent Document 2).
- PCNA is a homomultimer and forms a circular structure called “sliding clamp” to promote the DNA synthesis reaction.
- PCNA is highly conserved from yeast to humans.
- PCNA plays an important role in post-replication modifications such as cell division, DNA replication, repair, cell cycle regulation, DNA methylation and chromatin remodeling. I'm in charge.
- RFC replication factor C
- RFC is a protein complex consisting of five subunits, and is also called “clamp loader” because of its function of loading PCNA into DNA.
- RFC is equivalent to the ⁇ complex of Escherichia coli ( ⁇ Complex). The function of the RFC as a clamp loader is described. (1) RFC binds to a DNA strand, (2) RFC cleaves cyclic PCNA using energy generated by hydrolyzing ATP, (3) PCNA (4) Further, by hydrolyzing ATP, RFC is dissociated from DNA and PCNA binds to DNA.
- PCNA forms complexes with various proteins other than DNA polymerase and RFC, and is involved in DNA repair / replication and other gene regulatory functions. It is known that at least 12 proteins bind to PCNA in humans. Each protein is retained on the DNA strand by binding to PCNA via a PIP box (PIP box: PCNA interaction protein box).
- PIP box PCNA interaction protein box
- Non-patent Document 1 It has been clarified that a highly common amino acid sequence exists in the PIP box and that some proteins bind to PCNA through the PIP box site (Non-patent Document 1).
- DNA replication is performed in the natural world by the function of PCNA and RFC in cooperation. Of these, attempts have been made to increase the efficiency of PCR using PCNA, which plays a central role.
- the family A (pol I type) DNA polymerase also referred to as “Taq polymerase” derived from the thermophilic eubacteria Thermus aquaticus, which is widely used in PCR, does not interact with PCNA.
- a chimeric fusion protein in which 50 amino acids including the PIP box derived from PolB of Archaeoglobus fulgidus are fused to the C-terminus of the Taq polymerase so as to have a form similar to that of Family B ( ⁇ -type) DNA polymerase, A. It was reported that DNA was amplified in the presence of P. fulgidus-derived PCNA. However, when the size of the amplified DNA was 5 kb, there was still a problem in extensibility because the band derived from the synthesized product became thin (Non-patent Document 2).
- the present invention is intended to solve the problems of such a conventional family A (pol I type) DNA polymerase, which is more convenient and easy to use, and has excellent extensibility and rapidity.
- An object of the present invention is to provide a nucleic acid amplification method using the polymerase.
- the present inventors have conducted a study from the N-terminal side to the C-terminal side.
- the present inventors have found that a nucleic acid amplification reaction in the presence of PCNA is promoted by using a fusion polypeptide comprising one or more peptides that bind to PCNA and a polypeptide having DNA polymerase activity, and completed the present invention I let you.
- the present invention is outlined as follows: [1] From the N-terminal side to the C-terminal side, a) one or more peptides that bind to PCNA and b) a polypeptide having DNA polymerase activity, A fusion polypeptide comprising, [2] The fusion polypeptide according to [1], wherein the peptide that binds to PCNA is a peptide having a PIP box, [3] The fusion polypeptide according to [2], wherein the PIP box is a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 52 to 91 in the sequence listing, [4] The fusion polypeptide according to [1] or [2], wherein the peptide that binds to PCNA is a peptide having a PIP box derived from a DNA polymerase-related factor, [5] The fusion polypeptide according to any one of [1], [2] and [4], wherein the peptide that binds to PCNA is
- the present invention makes it possible to amplify long-chain DNA in a short time in nucleic acid amplification in the presence of PCNA even with Pol I type DNA polymerase.
- FIG. 1 is a photograph of an SDS-PAGE gel relating to the purification of Taq DNA polymerase mutant in Example 1. The purity of Taq81 to Taq85 was confirmed by analysis by SDS-PAGE.
- FIG. 2 is a photograph of an SDS-PAGE gel relating to the purification of Taq DNA polymerase mutant in Example 1. The purity of Taq92 to Taq94 was confirmed by analysis by SDS-PAGE.
- FIG. 3 is a physical interaction analysis using a surface plasmon resonance (SPR) method in Example 2. The physical interaction between Taq DNA polymerase mutants (Taq81 to Taq85 and Taq94) and PfuPCNA was measured by SPR analysis.
- FIG. 4 shows the results of amplification of 1 kb DNA in Example 3 (1).
- FIG. 5 is a diagram showing the results of amplification of 8 kb DNA in Example 3 (2).
- the DNA amplification ability of Taq81 to Taq85 in the presence of PCNA was confirmed by PCR.
- FIG. 6 is a diagram showing the results of amplification of 8 kb DNA in Example 3 (2).
- the DNA amplification ability of Taq92 to Taq94 in the presence of PCNA was confirmed by PCR.
- FIG. 7 is a diagram showing the results of amplification of 12 kb DNA in Example 3 (3). The DNA amplification ability of Taq81 to Taq85 in the presence of PCNA was confirmed by PCR.
- FIG. 5 is a diagram showing the results of amplification of 8 kb DNA in Example 3 (2).
- the DNA amplification ability of Taq81 to Taq85 in the presence of PCNA was confirmed by PCR.
- FIG. 6 is a diagram showing the results of amplification of 8 kb DNA in Example 3 (2).
- FIG. 8 shows the results of amplification of 15 kb DNA in Example 3 (3).
- the DNA amplification ability of Taq81 to Taq85 in the presence of PCNA was confirmed by PCR.
- FIG. 9 is a diagram showing the results of amplification of 12 kb DNA by PCR in Example 4.
- PCR confirmed the DNA amplification ability of Taq95 to Taq98 in the presence of PCNA.
- FIG. 10 is a diagram showing the relative values of amplification of 12 kb DNA by PCR in Example 4.
- peptide refers to a compound in which two or more amino acid molecules are bonded by removing one molecule of water from one amino group and the other carboxyl group.
- oligopeptides those consisting of about 10 or less amino acids are called oligopeptides, and those consisting of more than 10 amino acids are called polypeptides, but there are no strict boundaries.
- fusion polypeptide refers to a polypeptide comprising two or more polypeptides that are not fused in the natural state, and a polypeptide comprising a peptide and a polypeptide that are not fused in the natural state.
- PCNA is an abbreviation for proliferating cell nuclear antigen, and is a component of a protein molecule called “sliding clamp” because of its unique shape and function.
- PCNA is a replication cofactor that promotes the DNA strand synthesis reaction by forming a circular structure with homomultimers, sandwiching the DNA strand in the center hole, binding to the DNA polymerase on the surface and tying the enzyme onto the DNA. is there.
- PCNA is highly conserved from yeast to humans. In eukaryotic cells, PCNA plays an important role in post-replication modifications such as cell division, DNA replication, repair, cell cycle regulation, DNA methylation and chromatin remodeling. I'm in charge.
- all protein molecules having the above functions belong to the category of PCNA even if their names are different.
- polypeptide having DNA polymerase activity refers to a polypeptide having an activity of synthesizing a DNA strand complementary to a template nucleic acid (DNA or RNA) using deoxyribonucleotide triphosphate as a substrate.
- a DNA polymerase known as “polypeptide having DNA polymerase activity” or a variant thereof can be used.
- the term “activity” includes DNA synthesis activity and primer extension activity unless otherwise specified.
- the DNA synthesis activity includes an activity of synthesizing a complementary DNA using DNA as a template, and an activity of synthesizing a complementary DNA using RNA as a template.
- the DNA synthesis activity can be measured as the uptake activity of the substrate deoxyribonucleotide triphosphate (dNTP).
- dNTP substrate deoxyribonucleotide triphosphate
- a radioactive isotope incorporated into the complementary strand
- the amount of DNA polymerase activity is measured as a quantity.
- This method is called a nucleotide uptake assay and is also a standard method for measuring DNA polymerase activity.
- the activity of the DNA polymerase can be evaluated by measuring the chain length of the primer extension product synthesized by the DNA polymerase using the template DNA hybridized with the primer as a substrate.
- the fusion polypeptide of the present invention comprises a) one or more peptides that bind to PCNA in the direction from the N-terminal side to the C-terminal side, and b) a polypeptide having DNA polymerase activity. , Including. Therefore, it can be said that the fusion polypeptide of the present invention is a DNA polymerase mutant.
- the “peptide that binds to PCNA” constituting the fusion polypeptide is not particularly limited as long as it has the ability to bind to PCNA.
- the peptide include peptides containing a PIP box which is a peptide present in various PCNA binding proteins.
- the PIP box is an amino acid sequence present in a protein that interacts with PCNA, and functions to keep the protein on the DNA strand via PCNA.
- all peptides having the above-mentioned functions belong to the category of PIP boxes even if their names are different. For example, it is known that a thermophilic bacterial protein involved in DNA replication or the like (for example, replication factor C large subunit) has a PIP box.
- a suitable PIP box is an oligopeptide consisting of at least 8 amino acids.
- A1 is a glutamine residue.
- Groups, A2 and A3 are arbitrary amino acid residues
- A4 is an amino acid residue selected from the group consisting of leucine residues, isoleucine residues and methionine residues
- A5 and A6 are arbitrary amino acid residues
- A7 is phenylalanine residue
- Examples are groups or tryptophan residues
- A8 is an amino acid residue selected from the group consisting of phenylalanine residues, tryptophan residues or leucine residues.
- amino acid may further contain 9 amino acids having a lysine residue on the N-terminal side of the 8 amino acid oligopeptide.
- Table 1 shows examples of amino acid sequences of PIP boxes that can be used in the present invention.
- the PIP box used in the present invention is not particularly limited, and examples thereof include those derived from proteins produced by thermophilic bacteria.
- a PIP box derived from a replication factor C large subunit of a thermophilic bacterium is preferred, and a PIP box derived from a replication factor C large subunit of Pyrococcus furiosus is more preferred. Alternatively, it may be a functional equivalent having substantially the same activity.
- a plurality of these PIP boxes may be present in the fusion polypeptide of the present invention.
- the number of PIP boxes contained in the fusion polypeptide is not particularly limited, but is 1 to 6, preferably 2 to 4.
- These plural PIP boxes may have different amino acid sequences as long as they fulfill their functions. Further, another amino acid sequence such as a linker peptide described later may be sandwiched between the plurality of PIP boxes.
- linker peptide may be present on the C-terminal side of the PIP box.
- the “linker peptide” constituting the fusion polypeptide of the present invention refers to the polypeptides to be fused with each other or the peptide and the polypeptide in order to avoid inhibiting the function and folding of the fusion polypeptide of the present invention. It is a peptide inserted between The length of the linker peptide is not particularly limited, but a peptide having 3 to 100 amino acids, preferably 5 to 50 amino acids is exemplified.
- linker peptide there are no particular restrictions on the type of amino acid that constitutes the linker peptide, but it is better to avoid those in which the linker itself forms a complex higher order structure, and it contains many amino acids with relatively small side chains such as serine and glycine Peptides are often used.
- the linker peptide in the present invention is preferably an amino acid consisting of serine and glycine.
- polypeptide having DNA polymerase activity exists on the C-terminal side of the linker peptide.
- a known DNA polymerase or a variant thereof can be used as the “polypeptide having DNA polymerase activity” constituting the fusion polypeptide of the present invention.
- a thermostable DNA polymerase and a variant thereof preferably a thermostable family A (pol I type) DNA polymerase and a variant thereof, more preferably a Thermus bacterium Or a mutant thereof is used as “polypeptide having DNA polymerase activity”.
- the present invention is not particularly limited, the present invention can dramatically improve the performance of Taq DNA polymerase.
- “Taq polymerase” or “Taq DNA polymerase” refers to a thermus aquaticus Pol I type DNA polymerase.
- the amino acid sequence possessed by this DNA polymerase and the base sequence encoding the amino acid sequence are shown as SEQ ID NOS: 1 and 2, respectively, in the sequence listing which is a part of this specification.
- thermus thermophilus and Thermus flavus pol I type DNA polymerases can also be used in the present invention.
- examples of the polypeptide having DNA polymerase activity include a full-length polypeptide of PolI type DNA polymerase or a fragment thereof, and preferably a full-length polypeptide of Taq DNA polymerase or a fragment thereof.
- these polymerase fragments may be of a natural type or a mutant type as long as they have DNA polymerase activity.
- these polymerase fragments may be fragments of a polymerase that does not have a PIP box in a natural type, or may be fragments of a polymerase that has a PIP box in a natural type. Further, in the case of a fragment of a polymerase having a PIP box in its natural form, the PIP box may be removed.
- P.I. Examples include a fusion polypeptide comprising a PIP box derived from the furiosus replication factor C large subunit and Taq DNA polymerase.
- the amino acid sequence of this fusion polypeptide is shown in SEQ ID NOs: 4, 6, 8, 10, 12, 20, 22, 24, 26.
- the fusion polypeptide of the present invention has a dissociation constant (Kd) with PCNA of 1 ⁇ 10 ⁇ 8 to 25 ⁇ 10 ⁇ 7 M, preferably 3 ⁇ 10 ⁇ 8 to 15 ⁇ 10 ⁇ 7 M, more preferably 5 ⁇ Those in the range of 10 ⁇ 8 to 10 ⁇ 10 ⁇ 7 M are preferred.
- Kd dissociation constant
- the fusion polypeptide of the present invention can amplify longer DNA than a polypeptide having DNA polymerase activity that does not have a peptide that binds to PCNA.
- the fusion polypeptide of the present invention can amplify DNA in a shorter time than a polypeptide having DNA polymerase activity that does not have a peptide that binds to PCNA.
- the fusion polypeptide of the present invention is a DNA polymerase excellent in rapidity. That is, the fusion polypeptide of the present invention can shorten the time required for DNA elongation compared to the polypeptide before fusion. Therefore, it is very useful in a nucleic acid amplification reaction in which the DNA extension time is set shorter than before. Furthermore, since the DNA extension time for each cycle can be shortened, the total time required for the nucleic acid amplification method can also be shortened compared to the conventional method. For example, it is possible to amplify DNA of 8 kb in length by performing 30 cycles of shuttle PCR for 5 seconds at 99 ° C. and 4 minutes at 66 ° C. It is possible to amplify DNA having a length of preferably 12 kb or more, more preferably 15 kb or more by carrying out 30 cycles of shuttle PCR for 5 seconds at 99 ° C. and 12 minutes at 66 ° C.
- the present invention provides a nucleic acid encoding the fusion polypeptide of (1) above.
- the fusion polypeptide of the present invention can be produced by methods well known to those skilled in the art.
- nucleotide sequence of the nucleic acid of the present invention nucleotides may be substituted so that they are optimal codons for expression of the fusion polypeptide in the host cell.
- the nucleic acid of the present invention is inserted downstream of the promoter of an appropriate expression vector to prepare an expression vector. The nucleic acid needs to be incorporated into a vector so that the fusion polypeptide of the present invention is expressed in a host.
- the vector includes a promoter, a ribosome binding sequence (for example, SD sequence: Shine-Dalgarno sequence), It can contain cis elements such as terminators and enhancers, selectable markers (eg, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene) and the like.
- a transformant capable of producing the fusion polypeptide of the present invention can be obtained by introducing the expression vector into an appropriate host cell.
- the nucleic acid of the present invention may further include a nucleic acid encoding an affinity tag in order to facilitate purification of the expressed protein.
- an affinity tag examples include a histidine (His) tag, a glutathione S-transferase (GST) tag, a maltose binding protein (MBP) tag, 8 It is a nucleic acid that encodes a Strep (II) tag composed of the amino acid of a residue (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys).
- the tag may be added at any position on the 5 'end and / or 3' end side of the nucleic acid encoding the fusion polypeptide of the present invention, and may be appropriately added at a position that does not hinder expression and tag function.
- the tag is preferably a tag that can be cleaved during purification of the expressed protein.
- a vector capable of autonomous replication in a host cell or a vector that can be integrated into a host chromosome can be used.
- vectors for example, plasmid vectors, phage vectors, virus vectors and the like can be used.
- plasmid vectors plasmids suitable for the host used, for example, plasmids derived from E. coli, plasmids derived from bacteria belonging to the genus Bacillus, and plasmids derived from yeasts are well known to those skilled in the art, and many are commercially available. In the present invention, these known plasmids and their modifications can be used.
- ⁇ phage for example, Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP
- viral vector examples include animal viruses such as retrovirus and vaccinia virus. Insect viruses such as baculovirus can be used.
- any of prokaryotic cells, yeast, animal cells, insect cells, plant cells and the like may be used as long as the fusion polypeptide of the present invention can be expressed.
- prokaryotic cells for example, Escherichia coli (Escherichia coli), etc., Bacillus subtilis, Bacillus genus, Pseudomonas putida, Pseudomonas putida, etc.
- Bacteria belonging to the genus Rhizobium such as Rhizobium meliloti can be used as host cells.
- E. coli that can be used for the production of heterologous proteins are well known to those skilled in the art and many are commercially available (for example, Escherichia coli BL21, E. coli XL1-Blue, E. coli XL2-Blue, E. coli).
- DH1 E. coli JM109, E.
- Bacillus subtilis MI114 which is a bacterium belonging to the genus Bacillus.
- Subtilis 207-21 and the like, Brevibacillus choshinensis which is a bacterium belonging to the genus Brevibacillus, are known as hosts for producing heterologous proteins.
- These host cells can be combined with an appropriate expression vector and used to produce the fusion polypeptide of the present invention.
- the promoter mounted on the expression vector can be selected according to the host.
- a promoter derived from E. coli such as trp promoter, lac promoter, PL promoter, PR promoter, etc.
- an expression system for example, a pET expression system
- a phage-derived promoter and an RNA polymerase gene may be used.
- many heterologous protein expression systems using yeast, insect cells and mammalian cells as hosts have been constructed and are already on the market. These expression systems may be used for the production of the fusion polypeptide of the present invention.
- the method of introducing the expression vector into the host is not particularly limited as long as it can introduce a nucleic acid into the host.
- a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, etc. Can be used.
- the method for introducing a recombinant vector into insect cells is not particularly limited as long as DNA can be introduced into insect cells, and for example, calcium phosphate method, lipofection method, electroporation method and the like can be used.
- Phage vectors and virus vectors can be used to infect host cells by a method corresponding to these vectors to obtain transformants that express the fusion polypeptide of the present invention.
- the transformant into which an expression vector incorporating a DNA encoding the fusion polypeptide of the present invention has been introduced is cultured.
- the transformant can be cultured according to a usual method used for culturing host cells. Depending on the type of promoter loaded in the expression vector, an appropriate induction operation (induction agent addition or change in culture temperature) is performed.
- the fusion polypeptide of the present invention is collected from a culture of transformants.
- the “culture” includes any of culture supernatant, cultured cells, cultured cells, cells or disrupted cells.
- the fusion polypeptide of the present invention accumulates in the cells of the transformant, the cells are collected by centrifuging the culture, washed, and then disrupted to obtain the target protein. Extract and use as starting material for purification.
- the culture supernatant is used as it is, or the culture supernatant from which cells have been removed by centrifugation or the like is used as a starting material for purification.
- the fusion polypeptide of the present invention is extracted from the above starting materials by solvent extraction, salting out with ammonium sulfate, precipitation with organic solvent, various chromatography (ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, etc.) It can refine
- Nucleic acid amplification method using the fusion polypeptide of the present invention can be used for amplification of long-chain nucleic acids and nucleic acid amplification with reduced reaction time by combining with PCNA.
- the nucleic acid amplification method of the present invention can be used for either isothermal nucleic acid amplification method or variable temperature nucleic acid amplification method.
- PCR polymerase chain reaction
- MALBAC method Multiple Annealing and Looping-based amplification chain type
- MDA method MDA method
- SDA DNA elongation reaction
- RCA rolling circle amplification
- RCA rolling circle amplification
- loop priming amplification loop-mediated isothermal amplification
- LAMP loop-mediated isothermal amplification
- the combination of the fusion polypeptide of the present invention and PCNA can be expected to be highly extensible, it is effective in the preparation of long-chain DNA for genome analysis and genome editing, and can be used in isothermal nucleic acid amplification methods.
- the combination of the fusion polypeptide of the present invention and PCNA can be used for high-speed PCR with a shortened reaction time because it has excellent high-speed reactivity for improving the DNA synthesis rate.
- the present invention is not particularly limited, PCNA introduced with mutations that reduce the stability of the cyclic structure is suitable for combination with the fusion polypeptide of the present invention.
- PCNA As the PCNA used in the present invention, known PCNA or a variant thereof may be mentioned, and preferably, heat-resistant PCNA or a variant thereof is used. Although not particularly limited, P.I. furiosus PCNA or T. Kodakarensis PCNA is exemplified. Furthermore, mutant PCNA can also be used in the nucleic acid amplification composition of the present invention. As the mutant PCNA, for example, the mutant PCNA described in International Publication No. WO2007 / 004654, that is, P.I. Examples are mutant PCNAs having sequences in which the 82nd, 84th, 109th, 139th, 143rd, and 147th amino acid residues of furiosus PCNA are substituted with other amino acids.
- mutant PCNA having a sequence in which the 143rd amino acid residue is changed from aspartic acid to arginine (D143R) is exemplified.
- D143R aspartic acid to arginine
- the mutant PCNA of this embodiment exhibits a particularly excellent assisting action in a well-balanced manner with respect to the elongation and reaction rate of the DNA replication reaction.
- a DNA polymerase different from the fusion polypeptide of the present invention may be combined.
- the fusion polypeptide of the present invention produced using a pol I type DNA polymerase may be used in a nucleic acid amplification method in combination with an ⁇ type DNA polymerase having 3 ′ ⁇ 5 ′ exonuclease activity.
- a technique for performing PCR using a reaction solution containing two kinds of DNA polymerases having different 3 ′ ⁇ 5 ′ exonuclease activities is known as LA-PCR (Long and Accurate PCR). Further, it may be a combination of two types of fusion polypeptides of the present invention having different polypeptides having DNA polymerase activity.
- the oligonucleotide used as a primer in the nucleic acid amplification method of the present invention has a sequence complementary to the nucleic acid sequence of the template nucleic acid, and hybridizes to the template nucleic acid under the reaction conditions used. If it is a thing, it will not specifically limit.
- the primer chain length is preferably 6 nucleotides or more, more preferably 10 nucleotides or more from the viewpoint of hybridization specificity, and preferably 100 nucleotides or less, more preferably from the viewpoint of oligonucleotide synthesis. Is 30 nucleotides or less.
- the oligonucleotide can be chemically synthesized, for example, by a known method. Moreover, it may be an oligonucleotide derived from a biological sample, for example, isolated from a restriction endonuclease digest of DNA prepared from a natural sample.
- the nucleic acid amplification method of the present invention may be combined with a real-time detection technique.
- a real-time detection technique an amplification product is detected over time in parallel with the amplification reaction using an intercalator or a fluorescently labeled probe.
- intercalators include SYBR (registered trademark) Green I and other nucleic acid binding dyes
- fluorescently labeled probes include TaqMan (registered trademark) probes, CyCleave (registered trademark) probes, and molecular beacon probes. .
- composition for nucleic acid amplification containing the fusion polypeptide of the present invention is used as a component of a composition for nucleic acid amplification that can be used in the nucleic acid amplification method of (3) above. Can do.
- the nucleic acid amplification composition may contain elements necessary for the activity of the DNA polymerase, such as a divalent metal salt (magnesium salt, etc.), dNTP, and a buffer component for maintaining pH.
- the composition for nucleic acid amplification of the present invention can further contain PCNA in addition to the fusion polypeptide of the present invention.
- PCNA contained in the nucleic acid amplification composition PCNA described in the above (3) or a variant thereof can be used.
- composition of the present invention may contain a DNA polymerase different from the fusion polypeptide of the present invention.
- the fusion polypeptide of the present invention produced using pol I type DNA polymerase may be combined with an ⁇ type DNA polymerase having 3 ′ ⁇ 5 ′ exonuclease activity to prepare a composition.
- divalent metal ion constituting the divalent metal salt contained in the composition of the present invention magnesium ion, manganese ion, and cobalt ion are exemplified. Suitable divalent metal ions and their concentrations for each DNA polymerase are known in the art.
- the divalent metal ion can be supplied in the form of a salt such as chloride, sulfate, or acetate.
- concentration of the divalent metal ion in the composition of the present invention include 0.5 to 20 mM.
- dNTP deoxyribonucleotide triphosphates
- deoxyribonucleotide triphosphates for example, dATP, dCTP, dGTP, and dTTP
- Preferred examples of the deoxyribonucleotide triphosphate contained in the composition of the present invention include four types of mixtures of dATP, dCTP, dGTP, and dTTP.
- the composition of the present invention may contain a buffer component.
- the said component is not specifically limited, For example, it means the compound or mixture which has the effect
- a mixed solution of a weak acid and its salt or a weak base and its salt has a strong buffering action, and is therefore widely used as a reaction buffer for the purpose of pH control.
- the present invention is not particularly limited, it is appropriate that the pH of the composition of the present invention is set in a normal range in which PCR is performed, for example, in the range of pH 8.0 to 9.5.
- composition of the present invention may contain a component for real-time detection. Although not particularly limited, it can be combined with an intercalator or a fluorescently labeled probe.
- Kit containing the fusion polypeptide of the present invention is one embodiment of the present invention.
- a kit containing PCNA in addition to the fusion polypeptide described in (1) above is exemplified.
- the PCNA contained in the kit is particularly preferably a mutant PCNA in which the above-mentioned cyclic structure is further destabilized.
- the kit of the present invention further contains, as individual components, components used for preparing the nucleic acid amplification composition of the present invention, such as a divalent metal salt (magnesium salt, etc.), dNTP, and a buffer component for maintaining pH. It may also contain components made by combining a plurality of these.
- a component for real-time detection may be included as a component.
- examples include an intercalator and a fluorescently labeled probe.
- a composition containing the fusion polypeptide of the present invention and PCNA can be used in a method for producing DNA complementary to a template DNA.
- the nucleic acid amplification method (3) above can be used as a method for producing DNA.
- Example 1 Purification of Taq DNA Polymerase with PIP Box Replication of Pyrococcus furiossis having the amino acid sequence described in SEQ ID NO: 3 in the N-terminal or C-terminal of Taq DNA polymerase having the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing
- a PIP box of factor C large subunit (hereinafter referred to as PfuRFCL) was added via a linker peptide.
- Taq81 to Taq85 PIP-L5-Taq, PIP-L10-Taq, PIP-L15-Taq, PIP-L35-Taq, PIP-L47-Taq
- Taq92 to Taq94 Taq-L5-PIP, Taq-L10-PIP, Taq-L15-PIP
- Taq DNA polymerase with 2-5 PIP boxes added to the N-terminus was prepared.
- Taq95 (PIP-L14-PIP-L15-Taq)
- Taq96 (PIP-L14-PIP-L14-PIP-L15-Taq)
- Taq97 (PIP-L14-PIP-L14-PIP-L14-PIP-L15- Taq)
- Taq98 (PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq).
- Table 2 shows the names and structures of Taq DNA polymerases with PIP boxes in this specification.
- the numbers given after “L” in the “Structure” column of Table 2 indicate the length (number of amino acid residues) of a linker peptide consisting of repeating serine residues and glycine residues.
- Taq81 (PIP-L5-Taq) expression plasmid Using a Taq DNA polymerase gene having a base sequence described in SEQ ID NO: 2 in the sequence listing as a template, a TaqNPIP-5 primer having a base sequence described in SEQ ID NO: 28 in the sequence listing; PCR was performed with Taq-3 primer having the base sequence shown in SEQ ID NO: 29 in the Sequence Listing.
- the PCR enzyme was KOD Plus Neo DNA polymerase (manufactured by Toyobo Co., Ltd.), and the PCR conditions were 95 cycles at 30 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 3 minutes.
- the amplified fragment was purified by agarose gel electrophoresis, and then cleaved with restriction enzymes NdeI (Takara Bio) and NotI (Takara Bio). This fragment was ligated to pET21a (Novagen) cleaved with the same restriction enzyme.
- Escherichia coli JM109 strain (manufactured by Takara Bio Inc.) was transformed with the ligation product and then spread on LB-ampicillin plates. The plasmid was purified from the resulting colonies, the base sequence was read, and it was confirmed that the base sequence of SEQ ID NO: 5 was contained. This plasmid was named pTaq81.
- E. coli strain JM109 was transformed with 1 ⁇ L of the reaction solution digested with DpnI, and then plated on LB-ampicillin plates.
- the plasmid was purified from the resulting colonies, the base sequence was read, and it was confirmed that the base sequence of SEQ ID NO: 7 was included. This plasmid was named pTaq82.
- Taq83 (PIP-L15-Taq) expression plasmid Taq83 (PIP-L15) in which a sequence corresponding to 10 amino acids is inserted between the PIP box of Taq81 and the linker peptide by the mutagenesis method described in Example 1 (2).
- -Taq) expression plasmid was constructed.
- pTaq81 is used as a template
- the primers used are changed to taqN15-5 and taqN15-3 having the base sequences described in SEQ ID NOs: 32 and 33 in the sequence listing, respectively, and PCR is performed.
- a plasmid containing was obtained. This plasmid was named pTaq83.
- Taq84 (PIP-L35-Taq) expression plasmid Taq84 (PIP-L35) in which a sequence corresponding to 20 amino acids is inserted between the PIP box of Taq83 and the linker peptide by the mutagenesis method described in Example 1 (2).
- -Taq) expression plasmid was constructed.
- pTaq83 is used as a template, PCR is performed using two primers, taq-plus 20-5 and taq-plus 20-3, each having the base sequences described in SEQ ID NOs: 34 and 35 in the sequence listing, and SEQ ID NO: 11
- a plasmid containing the nucleotide sequence was obtained. This plasmid was named pTaq84.
- Taq85 (PIP-L47-Taq) expression plasmid In the mutation introducing method described in Example 1 (2), a sequence corresponding to 12 amino acids was inserted between the 14th and 15th amino acids of the linker peptide of Taq84. A Taq85 (PIP-L47-Taq) expression plasmid was constructed. In this mutagenesis, pTaq84 is used as a template, PCR is performed using two primers Taq-plus12-5 and Taq-plus12-3 having the nucleotide sequences described in SEQ ID NOs: 36 and 37 in the sequence listing, respectively, and SEQ ID NO: 13 A plasmid containing the nucleotide sequence was obtained. This plasmid was named pTaq85.
- the plasmid thus obtained containing the nucleotide sequence of SEQ ID NO: 15 was named pTaq92.
- Taq93 Taq-L10-PIP expression plasmid Taq- having Taq DNA polymerase gene having the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing as a template and the nucleotide sequences described in SEQ ID NO: 38 and 40 in the sequence listing, respectively.
- PCR, ligation, transformation and plasmid purification were performed under the conditions described in Example 1 (1) using two primers 5 and Tq-L10-PIP-3.
- the plasmid thus obtained containing the nucleotide sequence of SEQ ID NO: 17 was named pTaq93.
- Taq94 (Taq-L15-PIP) expression plasmid Taq- having a base sequence described in SEQ ID NO: 38 and 41 in the sequence using a Taq DNA polymerase gene having the base sequence described in SEQ ID NO: 2 in the sequence listing as a template PCR, ligation, transformation and plasmid purification were performed using 5 and Tq-L15-PIP-3 under the conditions described in Example 1 (1).
- the plasmid thus obtained containing the nucleotide sequence of SEQ ID NO: 19 was designated as pTaq94.
- Taq95 (PIP-L14-PIP-L15-Taq) expression plasmid In the mutation introduction method described in Example 1 (2), a sequence corresponding to the PIP box and linker peptide is inserted between the PIP box and linker peptide of Taq83. The inserted Taq95 (PIP-L14-PIP-L15-Taq) expression plasmid was constructed. In this mutagenesis, pTaq83 was used as a template, PCR was performed using two primers Taq95-5 and Taq-3 having the nucleotide sequences described in SEQ ID NOs: 48 and 29 in the sequence listing, respectively, and the nucleotide sequence of SEQ ID NO: 21 was The contained plasmid was obtained. This plasmid was named pTaq95.
- Taq96 (10) Taq96 (PIP-L14-PIP-L14-PIP-L15-Taq) expression plasmid
- a PIP box and a linker peptide are inserted between the PIP box and the linker peptide of Taq95.
- pTaq95 was used as a template
- PCR was performed using two primers Taq96-5 and Taq-3 having the nucleotide sequences described in SEQ ID NOs: 49 and 29 in the sequence listing, and the nucleotide sequence of SEQ ID NO: 23 was The contained plasmid was obtained.
- This plasmid was named pTaq96.
- Taq97 (PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) expression plasmid
- a PIP box between the PIP box of Taq96 and the linker peptide And a Taq97 (PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) expression plasmid in which the sequence corresponding to the linker peptide was inserted.
- pTaq96 was used as a template, PCR was performed using two primers, Taq97-5 and Taq-3, having the nucleotide sequences described in SEQ ID NOs: 50 and 29 in the sequence listing, respectively, and the nucleotide sequence of SEQ ID NO: 25 was The contained plasmid was obtained. This plasmid was named pTaq97.
- Taq98 (PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) expression plasmid
- the Taq97 PIP box and linker peptide A Taq98 (PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) expression plasmid having a PIP box and a sequence corresponding to a linker peptide inserted therebetween was constructed.
- PCR was performed using pTaq97 as a template and two primers, Taq98-5 and Taq-3, having the nucleotide sequences described in SEQ ID NOs: 51 and 29 in the sequence listing, respectively.
- the contained plasmid was obtained. This plasmid was named pTaq98.
- the cells were collected, washed with a PBS solution (150 mM NaCl, 20 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , pH 7.5), then collected again at ⁇ 80 ° C. Saved with.
- a PBS solution 150 mM NaCl, 20 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , pH 7.5
- the obtained precipitate was suspended in a solution B containing 1M ammonium sulfate (50 mM Tris-HCl, 10% glycerol, pH 8.0), and 5 mL of a hydrophobic column HiTrap Phenyl HP using AKTA Explorer (manufactured by GE Healthcare) (GE Healthcare). (Made by company). Solution B was used to elute the protein with an ammonium sulfate gradient from 1M to 0M.
- 1M ammonium sulfate 50 mM Tris-HCl, 10% glycerol, pH 8.0
- AKTA Explorer manufactured by GE Healthcare
- Solution B was used to elute the protein with an ammonium sulfate gradient from 1M to 0M.
- the obtained elution fraction was dialyzed overnight against Solution C (50 mM Tris-HCl, 50 mM NaCl, pH 8.0), and then the dialyzed solution was applied to an affinity column HiTrap Heparin HP 5 mL (GE Healthcare), 50 mM.
- the protein was eluted by a sodium chloride concentration gradient from 1 to 1 M, and this eluted fraction was used as the final purified product.
- the final purified product was subjected to 10% SDS-PAGE and detected by CBB staining. The results are shown in FIGS.
- Taq DNA polymerase mutants (Taq 81 to Taq 85 and Taq 92 to Taq 94), which are fusion proteins fused with wild type Taq DNA polymerase and PIP box, could be purified to a single.
- Taq95 to Taq98 could also be purified to a single by the same method.
- Example 2 Physical Interaction Analysis Using SPR Method (1)
- Method BIAcore J (manufactured by BIACORE) was used for surface plasmon resonance (SPR) analysis.
- Pyrococcus furiosus PCNA J. Bacteriology, Vol.181, pages 6591-6599, 1999: hereinafter referred to as PfuPCNA
- CM5 sensor chip manufactured by GE Healthcare
- the measurement was performed under the conditions of a flow rate of 30 ⁇ L / min and solution E (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , pH 8.3).
- the dissociation constant (Kd) of Taq94 and PfuPCNA was calculated to be 2.5 ⁇ 10 ⁇ 6 M. This is about 10 times the dissociation constant of Taq81 to Taq85, and from this result, it was confirmed that the interaction between Taq DNA polymerase with a PIP box added to the C terminus and PfuPCNA was weak.
- Example 3 Amplification of DNA by PCR
- 1 kb DNA amplification PCR was performed using Taq81 to Taq85 prepared in Example 1 (13) using lambda DNA as a template.
- the reaction solution composition was 1 nM Taq81 to Taq85, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM dNTP, 1 ng lambda DNA, 0.4 ⁇ M each, and the final reaction solution The volume was 50 ⁇ L.
- the reaction was carried out for 30 cycles at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds.
- the composition of the reaction solution was 1 nM wild-type Taq DNA polymerase or Taq81 to Taq85, International Publication WO 2007/004654 Pamphlet 40 nM PufPCNA D143R mutant (PCNA13), 10 mM Tris-HCl (pH 8.3) prepared by the method described in Examples. ), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM dNTP, 1 ng lambda DNA, 0.4 ⁇ M each, and the final volume of the reaction solution was 50 ⁇ L. First, the reaction solution was incubated at 95 ° C.
- Taq81 to Taq85 almost no band was seen in the absence of PCNA (lanes 3, 5, 7, 9, 11), but a dark band was seen at the 8 kb position in the presence of PCNA (lanes). 4, 6, 8, 10, 12).
- Taq81 to Taq85 which are Taq DNA polymerases with a PIP box added to the N-terminus, can amplify DNA of 8 kb in the presence of PCNA.
- Taq92 to Taq93 which are Taq DNA polymerases with a PIP box added to the C-terminus, were unable to amplify DNA of 8 kb even in the presence of PCNA.
- PCR conditions were as follows. First, the reaction solution was incubated at 95 ° C. for 1 minute, and then 30 cycles of shuttle PCR were performed at 99 ° C. for 5 seconds and at 66 ° C. for 12 minutes. The obtained product was analyzed in the same manner as in Example 3 (2). The results are shown in FIGS.
- the shuttle PCR conditions employed in this example are shorter in the time required for primer annealing and complementary strand DNA extension than in conventional shuttle PCR conditions. Nevertheless, it was confirmed that the DNA having the same long chain length as the conventional one could be amplified, and thus it was excellent in rapidity.
- Example 4 Taq DNA Polymerase with Plural PIP Boxes Added Taq95 (PIP-L14-PIP-L15-Taq), Taq96 (PIP-L14-PIP-L14-PIP-L15-) Prepared in Example 1 (13) Taq), Taq97 (PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) and Taq98 (PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq) In the same manner as in Example 3 (3), 12 kb PCR was performed in the presence of PCNA. The result is shown in FIG.
- amplification products were quantified using LAS-3000mini (manufactured by GE Healthcare). The PCR to quantification was repeated three times, and the relative values when Taq83 (PIP-L15-Taq) amplification product was set to 1 were graphed. The result is shown in FIG.
- Taq95 to Taq98 to which a plurality of PIP boxes were added confirmed more amplification products than Taq83 with one PIP box.
- Taq96 to which three PIP boxes were added had the largest number of amplification products. This indicates that efficient DNA amplification is possible by adding a plurality of PIP boxes to the N-terminus of Taq DNA polymerase.
- Example 5 Taq DNA polymerase to which a PIP box having various sequences was added Instead of the PIP box having the amino acid sequence described in SEQ ID NO: 3 in the sequence listing used in Example 1, the sequence listing in Table 1 Using any peptide having the amino acid sequence set forth in SEQ ID NOs: 52 to 91 (ie, PIP box), any PIP box is added to the N-terminus according to the method described in Example 1. Purify Taq DNA polymerase. Using such Taq DNA polymerase, DNA amplification is carried out in the presence of PCNA according to the method described in Example 3. When any of the peptides listed in Table 1 is used instead of the PIP box having the amino acid sequence described in SEQ ID NO: 3, the same effect as the former can be expected.
- SEQ ID NO: 52 in the Sequence Listing described in Table 1 PCR is carried out in the presence of PCNA using Taq DNA polymerase in which any one of the peptides having the amino acid sequences of ⁇ 91 is added to the N-terminus.
- any of the peptides listed in Table 1 is used instead of the PIP box having the amino acid sequence described in SEQ ID NO: 3, the same effect as the former can be expected.
- the present invention provides a family A (pol I type) DNA polymerase mutant that is more convenient, easy to use, and excellent in extensibility and rapidity, and a nucleic acid amplification method using the polymerase.
- the DNA polymerase mutant of the present invention is particularly useful for nucleic acid amplification in the presence of PCNA.
- SEQ ID NO: 1 Taq DNA polymerase amino acid sequence
- SEQ ID NO: 2 Taq DNA polymerase nucleic acid sequence
- SEQ ID NO: 3 Pyrococcus furiosus RFC-L PIP-box amino acid sequence
- SEQ ID NO: 4 DNA polymerase variant Taq81 amino acid sequence
- SEQ ID NO: 5 DNA polymerase variant Taq81 nucleic acid sequence
- SEQ ID NO: 6 DNA polymerase variant Taq82 amino acid sequence
- SEQ ID NO: 7 DNA polymerase variant Taq82 nucleic acid sequence
- SEQ ID NO: 8 DNA polymerase variant Taq83 amino acid sequence
- SEQ ID NO: 9 DNA polymerase variant Taq83 nucleic acid sequence
- SEQ ID NO: 10 DNA polymerase variant Taq84 amino acid sequence
- SEQ ID NO: 12 DNA polymerase variant Taq85 amino acid sequence
- SEQ ID NO: 13 DNA polymerase variant Taq
- Fen1 PIP-box amino acid sequence SEQ ID NO: 66 A.fulgidus Fen1 PIP-box amino acid sequence SEQ ID NO: 67: H.sapiens Fen1 PIP-box amino acid sequence SEQ ID NO: 68: D.melanogaster Fen1 PIP-box amino acid sequence SEQ ID NO: 69: S. cerevisiae Fen1 PIP-box amino acid sequence SEQ ID NO: 70: S.pombe Fen1 PIP-box amino acid sequence SEQ ID NO: 71: H.
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Abstract
Description
[1]N末側からC末側の方向に、
a)PCNAと結合する1又は複数のペプチド
および、
b)DNAポリメラーゼ活性を有するポリペプチド、
を含む融合ポリペプチド、
[2]PCNAと結合するペプチドがPIPボックスを有するペプチドであることを特徴とする、[1]に記載の融合ポリペプチド、
[3]PIPボックスが、配列表の配列番号52~91のいずれかで示されるアミノ酸配列からなるペプチドであることを特徴とする、[2]に記載の融合ポリペプチド、
[4]PCNAと結合するペプチドがDNAポリメラーゼ関連因子由来のPIPボックスを有するペプチドであることを特徴とする、[1]または[2]に記載の融合ポリペプチド、
[5]PCNAと結合するペプチドが複製因子Cラージサブユニット由来のPIPボックスを有するペプチドであることを特徴とする、[1]、[2]及び[4]のいずれかに記載の融合ポリペプチド、
[6]PCNAと結合するペプチドが配列表の配列番号3で示されるアミノ酸配列、もしくは前記アミノ酸配列を含むペプチドであることを特徴とする、[1]、[2]、[4]及び[5]のいずれかに記載の融合ポリペプチド、
[7]PCNAと結合するペプチドとDNAポリメラーゼ活性を有するポリペプチドの間に5~50アミノ酸のリンカーペプチドを有することを特徴とする、[1]~[6]のいずれかに記載の融合ポリペプチド、
[8]リンカーペプチドがセリンとグリシンから成るアミノ酸配列であることを特徴とする、[7]に記載の融合ポリペプチド、
[9]DNAポリメラーゼ活性を有するポリペプチドがPolI型DNAポリメラーゼもしくはそのフラグメントであることを特徴とする、[1]~[8]のいずれかに記載の融合ポリペプチド、
[10]DNAポリメラーゼ活性を有するポリペプチドがTaq DNAポリメラーゼもしくはそのフラグメントであることを特徴とする、[1]~[9]のいずれかに記載の融合ポリペプチド、
[11][1]~[10]のいずれかに記載の融合ポリペプチドをコードする核酸、
[12][1]~[10]のいずれかに記載の融合ポリペプチドを含有する核酸増幅用組成物、
[13]さらに、PCNAを含有する、[12]に記載の核酸増幅用組成物、
[14][1]~[10]のいずれかに記載の融合ポリペプチドを含有するキット、
[15]さらに、PCNAを含有する、[14]に記載のキット、
[16][1]~[10]のいずれかに記載の融合ポリペプチドならびにPCNAを含有する組成物を使用することを特徴とする、鋳型DNAに相補的なDNAの製造方法、
に関する。
本明細書において「ペプチド」とは、2個以上のアミノ酸分子が、一方のアミノ基と他方のカルボキシル基とから1分子の水がとれて結合した化合物をいう。一般的には、約10個以下のアミノ酸から成るものをオリゴペプチド、それ以上のアミノ酸から成るものをポリペプチドというが、厳密な境界はない。
本発明の融合ポリペプチドは、N末側からC末側の方向に、a)PCNAと結合する1又は複数のペプチドおよび、b)DNAポリメラーゼ活性を有するポリペプチド、を含んでいる。よって、本発明の融合ポリペプチドはDNAポリメラーゼ変異体であると言うことができる。
また、これらのPIPボックスは、本発明の融合ポリペプチド内に複数個存在してもよい。融合ポリペプチドに含まれるPIPボックスの数は、特に限定はないが、1~6個、好ましくは2~4個が例示される。これら複数のPIPボックスは、その機能を果たす限りにおいては、それぞれのアミノ酸配列が異なっていてもよい。また、複数のPIPボックス同士の間には、例えば後述のリンカーペプチドなど、別のアミノ酸配列が挟まっていてもよい。
本発明の融合ポリペプチドは、PCNAと組み合わせることにより長鎖長の核酸の増幅や反応時間を短縮した核酸増幅に使用することができる。本発明の核酸増幅法は、等温核酸増幅法や変温核酸増幅方法のいずれにも使用できる。特に限定はされないが、ポリメラーゼ連鎖反応(polymerase chain reaction:PCR)、リガーゼ連鎖反応(ligase chain reaction)、MALBAC法 (Multiple Annealing and Looping-Based Amplification Cycles)、MDA法(Multiple Displacement Amplification)、鎖置換型DNA伸長反応(strand displacement amplification:SDA)、ローリングサークル増幅法(rolling circle amplification:RCA)、交差プライミング増幅法(cross priming amplification)、ループ介在等温増幅法(loop-mediated isothermal amplification:LAMP)、ICAN法(isothermal and chimeric primer-initiated amplification of nucleic acids)等のいずれにも好適に使用できる。例えば、本発明の融合ポリペプチドとPCNAの組み合わせは高い伸長性が期待できることからゲノム解析やゲノム編集用の長鎖長のDNA調製において有効であり、等温核酸増幅法に利用できる。また、本発明の融合ポリペプチドとPCNAの組み合わせは、DNA合成速度を向上させる高速反応性に優れていることから反応時間を短縮させた高速PCRに使用できる。特に本発明を限定するものではないが、環状構造の安定性が低下するような変異を導入されたPCNAは本発明の融合ポリペプチドと組み合わせるのに好適である。
本発明により得られる融合ポリペプチドは、上記(3)の核酸増幅法に使用可能な核酸増幅用組成物のコンポーネントとして使用する事ができる。さらに、核酸増幅用組成物はDNAポリメラーゼの活性に必要な要素、例えば二価金属塩(マグネシウム塩等)、dNTP、pH維持のための緩衝成分等を含んでいてもよい。
本発明の融合ポリペプチドを含有するキットは、本発明の一態様である。好適には、上記(1)に記載の融合ポリペプチドに加え、さらにPCNAを含有するキットが例示される。キットに含有されるPCNAとして、特に好適には上述の環状構造をより不安定化させた変異型PCNAが挙げられる。本発明のキットは、さらに二価金属塩(マグネシウム塩等)、dNTP、pH維持のための緩衝成分等、本発明の核酸増幅用組成物の調製に使用される成分を個別のコンポーネントとして含有してもよく、これらのうちの複数を組み合わせて作製されたコンポーネントを含有してもよい。
本発明の融合ポリペプチドならびにPCNAを含有する組成物は、鋳型DNAに相補的なDNAの製造方法に使用することができる。DNAの製造方法には、上記(3)の核酸増幅方法が利用できる。これらの核酸増幅方法で製造したDNAを利用することにより、標的核酸の塩基配列を決定したり、標的核酸を標識化したり、標的核酸に部位特異的変異を導入することも可能である。
配列表の配列番号1記載のアミノ酸配列を有するTaq DNAポリメラーゼのN末端またはC末端に、配列表の配列番号3記載のアミノ酸配列を有するPyrococcus furiosisの複製因子Cラージサブユニット(以下、PfuRFCLとする)のPIPボックスを、リンカーペプチドを介して付加した。N末端にPIPボックスが付加されたTaq DNAポリメラーゼはTaq81~Taq85(PIP-L5-Taq、PIP-L10-Taq、PIP-L15-Taq、PIP-L35-Taq、PIP-L47-Taq)の5種類であり、C末端にPIPボックスが付加されたTaq DNAポリメラーゼはTaq92~Taq94(Taq-L5-PIP、Taq-L10-PIP、Taq-L15-PIP)の3種類である。さらに、N末端にPIPボックスが2~5個付加されたTaq DNAポリメラーゼを作成した。すなわち、Taq95(PIP-L14-PIP-L15-Taq)、Taq96(PIP-L14-PIP-L14-PIP-L15-Taq)、Taq97(PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)、Taq98(PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)の4種類である。本明細書におけるPIPボックス付きTaq DNAポリメラーゼの名称及び構造を表2に示す。表2の「構造」欄の「L」の後ろに付された数字は、セリン残基とグリシン残基の繰り返しから成るリンカーペプチドの長さ(アミノ酸残基数)を示す。
配列表の配列番号2記載の塩基配列を有するTaq DNAポリメラーゼ遺伝子を鋳型として、配列表の配列番号28記載の塩基配列を有するTaqNPIP-5プライマーおよび配列表の配列番号29記載の塩基配列を有するTaq-3プライマーでPCRを行った。
PCR酵素はKOD Plus Neo DNA polymerase(東洋紡社製)を用い、PCRの条件は、95℃で30秒、55℃で30秒、72℃で3分を1サイクルとした30サイクル反応で行った。増幅した断片をアガロースゲル電気泳動により精製した後、制限酵素NdeI(タカラバイオ社製)およびNotI(タカラバイオ社製)で切断した。この断片を同制限酵素で切断されたpET21a(ノバジェン社製)にライゲーションした。大腸菌JM109株(タカラバイオ社製)をライゲーション産物で形質転換した後、LB-アンピシリンプレートに撒いた。生じたコロニーからプラスミドを精製し、塩基配列を読み、配列番号5の塩基配列が含まれていることを確認した。このプラスミドをpTaq81と命名した。
QuickChange site-directed mutagenesis kit(Agilent Technologies社製)を用いた部位特異的変異法によって、Taq81のPIPボックスとリンカーペプチドの間に5アミノ酸が挿入されたポリペプチドをコードするDNA(配列番号7)を作製した。
鋳型としてpTaq81、プライマーに、配列表の配列番号30及び31記載の塩基配列をそれぞれ有するtaqN10-5およびtaqN10-3を用いて、95℃で30秒、55℃で60秒、68℃で8分を1サイクルとした14サイクルのPCRを行った。続いて、DpnIで消化した反応液1μLで大腸菌JM109株を形質転換した後、LB-アンピシリンプレートに撒いた。生じたコロニーからプラスミドを精製し、塩基配列を読み、配列番号7の塩基配列が含まれていることを確認した。このプラスミドをpTaq82と命名した。
実施例1(2)記載の変異導入方法で、Taq81のPIPボックスとリンカーペプチドの間に10アミノ酸に該当する配列を挿入したTaq83(PIP-L15-Taq)発現プラスミドを構築した。
本変異導入では、pTaq81を鋳型とし、使用するプライマーを配列表の配列番号32及び33記載の塩基配列をそれぞれ有するtaqN15-5およびtaqN15-3に変更してPCRを行い、配列番号9の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq83と命名した。
実施例1(2)記載の変異導入方法で、Taq83のPIPボックスとリンカーペプチドの間に20アミノ酸に該当する配列を挿入したTaq84(PIP-L35-Taq)発現プラスミドを構築した。
本変異導入では、pTaq83を鋳型とし、配列表の配列番号34及び35記載の塩基配列をそれぞれ有するtaq-plus20-5およびtaq-plus20-3の2つのプライマーを用いてPCRを行い、配列番号11の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq84と命名した。
実施例1(2)記載の変異導入方法で、Taq84のリンカーペプチドの14番目と15番目のアミノ酸の間に12アミノ酸に該当する配列を挿入したTaq85(PIP-L47-Taq)発現プラスミドを構築した。
本変異導入では、pTaq84を鋳型とし、配列表の配列番号36及び37記載の塩基配列をそれぞれ有するTaq-plus12-5およびTaq-plus12-3の2つのプライマーを用いてPCRを行い、配列番号13の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq85と命名した。
配列表の配列番号2記載の塩基配列を有するTaq DNAポリメラーゼ遺伝子を鋳型として、配列表の配列番号38及び39記載の塩基配列をそれぞれ有するTaq-5およびTq-L5-PIP-3の2つのプライマーを使用し、実施例1(1)記載の操作でPCR、ライゲーション、形質転換及びプラスミド精製を行った。こうして得られた、配列番号15の塩基配列が含まれているプラスミドをpTaq92と命名した。
配列表の配列番号2記載の塩基配列を有するTaq DNAポリメラーゼ遺伝子を鋳型として、配列表の配列番号38及び40記載の塩基配列をそれぞれ有するTaq-5およびTq-L10-PIP-3の2つのプライマーを使用し、実施例1(1)記載の条件でPCR、ライゲーション、形質転換及びプラスミド精製を行った。こうして得られた、配列番号17の塩基配列が含まれているプラスミドをpTaq93と命名した。
配列表の配列番号2記載の塩基配列を有するTaq DNAポリメラーゼ遺伝子を鋳型として、配列表の配列番号38及び41記載の塩基配列をそれぞれ有するTaq-5およびTq-L15-PIP-3を使用して実施例1(1)記載の条件でPCR、ライゲーション、形質転換及びプラスミド精製を行った。こうして得られた、配列番号19の塩基配列が含まれているプラスミドをpTaq94と命名した。
実施例1(2)記載の変異導入方法で、Taq83のPIPボックスとリンカーペプチドの間にPIPボックスとリンカーペプチドに該当する配列を挿入したTaq95(PIP-L14-PIP-L15-Taq)発現プラスミドを構築した。
本変異導入では、pTaq83を鋳型とし、配列表の配列番号48及び29記載の塩基配列をそれぞれ有するTaq95-5およびTaq-3の2つのプライマーを用いてPCRを行い、配列番号21の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq95と命名した。
実施例1(2)記載の変異導入方法で、Taq95のPIPボックスとリンカーペプチドの間にPIPボックスとリンカーペプチドに該当する配列を挿入したTaq96(PIP-L14-PIP-L14-PIP-L15-Taq)発現プラスミドを構築した。
本変異導入では、pTaq95を鋳型とし、配列表の配列番号49及び29記載の塩基配列をそれぞれ有するTaq96-5およびTaq-3の2つのプライマーを用いてPCRを行い、配列番号23の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq96と命名した。
実施例1(2)記載の変異導入方法で、Taq96のPIPボックスとリンカーペプチドの間にPIPボックスとリンカーペプチドに該当する配列を挿入したTaq97(PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)発現プラスミドを構築した。
本変異導入では、pTaq96を鋳型とし、配列表の配列番号50及び29記載の塩基配列をそれぞれ有するTaq97-5およびTaq-3の2つのプライマーを用いてPCRを行い、配列番号25の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq97と命名した。
実施例1(2)記載の変異導入方法で、Taq97のPIPボックスとリンカーペプチドの間にPIPボックスとリンカーペプチドに該当する配列を挿入したTaq98(PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)発現プラスミドを構築した。
本変異導入では、pTaq97を鋳型とし、配列表の配列番号51及び29記載の塩基配列をそれぞれ有するTaq98-5およびTaq-3の2つのプライマーを用いてPCRを行い、配列番号27の塩基配列が含まれているプラスミドを得た。このプラスミドをpTaq98と命名した。
大腸菌BL21-CodonPlus(DE3)-RIL(アジレント社製)を前記それぞれの発現プラスミド(pTaq81~pTaq85及びpTaq92~pTaq94)で形質転換し、得られた形質転換体を50μg/mLアンピシリン及び34μg/mLクロラムフェニコールを含むLB培地1Lで振盪培養した。OD600が0.2-0.3に達したところでIPTGを終濃度1mMになるように加えてTaq DNAポリメラーゼを発現誘導した後、さらに25℃で約18時間振盪培養した。培養後に菌体を回収し、PBS溶液(150mM NaCl、20mM Na2HPO4、2mM NaH2PO4、pH7.5)を用いて菌体を洗浄した後、再び菌体を回収し、-80℃で保存した。
(1)方法
表面プラズモン共鳴(SPR)解析は、BIAcore J(BIACORE社製)を用いた。Pyrococcus furiosusのPCNA(J. Bacteriology、第181巻、第6591~6599頁、1999年:以下PfuPCNAと記載する)をアミンカップリングによりCM5センサーチップ(GE Healthcare社製)上に固定し、25℃、流速30μL/分、溶液E(10mM Tris-HCl、50mM KCl、1.5mM MgCl2、pH8.3)の条件下で測定を行った。10、20、40、80、160、320nMに希釈したTaq81~Taq85の試料溶液、もしくは125nM、250nM、500nM、1μM、2μMに希釈したTaq94の試料溶液をそれぞれ2分間添加した。得られたセンサーグラムをBIAevaluation programにより解析し、Taq81~Taq85とPfuPCNAおよびTaq94とPfuPCNAの解離定数(Kd)を算出した。
SPR解析の結果を図3に示す。リンカーペプチドの長さが違う、N末端にPIPボックスが付加されたTaq DNAポリメラーゼ5種(Taq81、Taq82、Taq83、Taq84およびTaq85)とPfuPCNAの結合に関する解離定数(Kd)を算出したところ、それぞれ7.1×10-7M、4.4×10-7M、2.4×10-7M、2.5×10-7M、2.0×10-7Mであり、リンカーペプチドの長さに関わらずほぼ同等の値となった。この結果は、リンカーペプチドの長さに関わらず、N末端にPIPボックスが付加されたTaq DNAポリメラーゼとPfuPCNAとの相互作用の強さがほぼ一定であることを示す。
(1)1kbのDNA増幅
実施例1(13)で調製したTaq81~Taq85を用いてラムダDNAを鋳型としてPCRを行った。反応液組成は、1nMのTaq81~Taq85、10mM Tris-HCl(pH8.3)、50mM KCl、1.5mM MgCl2、0.2mM dNTP、1ng lambda DNA、各0.4μM プライマーとし、反応液の最終容量は50μLとした。反応は95℃で30秒、55℃で30秒、72℃で60秒を1サイクルとして30サイクル行った。
本PCRでは、配列表の配列番号42及び43記載の塩基配列をそれぞれ有するF1およびR2-2の2つのプライマーを用いた。得られた生成物を1%アガロースゲルで分離し、エチジウムブロマイドで染色した。その結果、図4に示したように、市販のTaq DNAポリメラーゼ(レーン1)、野生型のTaq DNAポリメラーゼ(レーン2)およびTaq81~Taq85(レーン3~7)とも1kbの位置にバンドが見られた。この結果から、N末端に付加したPIPボックスおよびリンカーペプチドがPCRに影響しないことが確認できた。
次に、PCNA非存在下および存在下でPCRを行った。反応液組成は、1nMの野生型Taq DNAポリメラーゼ又はTaq81~Taq85、国際公開WO2007/004654号パンフレット 実施例記載の方法で調製した40nMのPufPCNA D143R変異体(PCNA13)、10mM Tris-HCl(pH8.3)、50mM KCl、1.5mM MgCl2、0.2mM dNTP、1ng lambda DNA、各0.4μM プライマーとし、反応液の最終容量は50μLとした。最初に反応液を95℃で1分インキュベートした後、99℃で5秒、66℃で4分のシャトルPCRを30サイクル行った。本PCRでは、配列表の配列番号44及び45記載の塩基配列をそれぞれ有するLF-35およびLR8-35の2つのプライマーを用いた。得られた生成物を1%アガロースゲルで分離し、エチジウムブロマイドで染色した。その結果、図5に示したように、野生型のTaq DNAポリメラーゼでは、PCNA非存在下(レーン1)でもPCNA存在下(レーン2)でも、8kbの位置にバンドはほとんど見られなかったのに対し、Taq81~Taq85では、PCNA非存在下ではバンドはほとんど見られなかった(レーン3、5、7、9、11)が、PCNA存在下で、8kbの位置に濃いバンドが見られた(レーン4、6、8、10、12)。
このことは、N末端にPIPボックスが付加されたTaq DNAポリメラーゼであるTaq81~Taq85がPCNA存在下で8kbのDNA増幅が可能であることを示す。
一方、図6に示したように、C末端にPIPボックスが付加されたTaq DNAポリメラーゼであるTaq92~Taq93は、PCNA存在下でも8kbのDNA増幅はできなかった。
12kb及び15kbのDNA増幅について検討した。12kb増幅においては、配列表の配列番号44及び46記載の塩基配列をそれぞれ有するLF-35およびLR12-35の2つのプライマー、15kbのDNA増幅においては、配列表の配列番号44及び47記載の塩基配列をそれぞれ有するLF-35およびLR15-35の2つのプライマー、を使用した点を除き、実施例3-(2)同様に反応液を調製した。
このことは、Taq81~Taq85がPCNA存在下で12kbおよび15kbのDNA増幅が可能であることを示す。
また、本実施例で採用したシャトルPCRの条件は、従来のシャトルPCRの条件に比べてプライマーのアニーリングと相補鎖DNAの伸長にかかる時間が短い。それにもかかわらず従来と同じ長鎖長のDNAを増幅できたことから迅速性においても優れていることが確認できた。
実施例1(13)で調製したTaq95(PIP-L14-PIP-L15-Taq)、Taq96(PIP-L14-PIP-L14-PIP-L15-Taq)、Taq97(PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)およびTaq98(PIP-L14-PIP-L14-PIP-L14-PIP-L14-PIP-L15-Taq)を用いて、実施例3(3)と同様にして、PCNA存在下で12kbのPCRを行った。その結果を図9に示す。さらに、LAS-3000mini(GEヘルスケア社製)を用いて増幅産物の定量を行った。上記PCRから定量までを3回繰り返し、Taq83(PIP-L15-Taq)の増幅産物を1とした時の相対値をグラフ化した。その結果を図10に示す。
このことは、Taq DNAポリメラーゼのN末端にPIPボックスを複数個付加することにより、効率的なDNA増幅が可能となることを示す。
実施例1で使用した配列表の配列番号3に記載のアミノ酸配列を有するPIPボックスに代えて、表1に記載の配列表の配列番号52~91に記載のアミノ酸配列を有するいずれかのペプチド(即ち、PIPボックス)を使用して、実施例1に記載された方法に準じて、いずれかのPIPボックスがN末端に付加されたTaq DNAポリメラーゼを精製する。かかるTaq DNAポリメラーゼを用いて、実施例3に記載された方法に準じて、PCNA存在下でDNA増幅を実施する。
配列番号3に記載のアミノ酸配列を有するPIPボックスに代えて、表1に記載のいずれかのペプチドを使用する場合、前者と同様の効果が期待できる。
配列番号3に記載のアミノ酸配列を有するPIPボックスに代えて、表1に記載のいずれかのペプチドを使用する場合、前者と同様の効果が期待できる。
SEQ ID NO: 2: Taq DNA polymerase nucleic acid sequence
SEQ ID NO: 3: Pyrococcus furiosus RFC-L PIP-box amino acid sequence
SEQ ID NO: 4: DNA polymerase variant Taq81 amino acid sequence
SEQ ID NO: 5: DNA polymerase variant Taq81 nucleic acid sequence
SEQ ID NO: 6: DNA polymerase variant Taq82 amino acid sequence
SEQ ID NO: 7: DNA polymerase variant Taq82 nucleic acid sequence
SEQ ID NO: 8: DNA polymerase variant Taq83 amino acid sequence
SEQ ID NO: 9: DNA polymerase variant Taq83 nucleic acid sequence
SEQ ID NO: 10: DNA polymerase variant Taq84 amino acid sequence
SEQ ID NO: 11: DNA polymerase variant Taq84 nucleic acid sequence
SEQ ID NO: 12: DNA polymerase variant Taq85 amino acid sequence
SEQ ID NO: 13: DNA polymerase variant Taq85 nucleic acid sequence
SEQ ID NO: 14: DNA polymerase variant Taq92 amino acid sequence
SEQ ID NO: 15: DNA polymerase variant Taq92 nucleic acid sequence
SEQ ID NO: 16: DNA polymerase variant Taq93 amino acid sequence
SEQ ID NO: 17: DNA polymerase variant Taq93 nucleic acid sequence
SEQ ID NO: 18: DNA polymerase variant Taq94 amino acid sequence
SEQ ID NO: 19: DNA polymerase variant Taq94 nucleic acid sequence
SEQ ID NO: 20: DNA polymerase variant Taq95 amino acid sequence
SEQ ID NO: 21: DNA polymerase variant Taq95 nucleic acid sequence
SEQ ID NO: 22: DNA polymerase variant Taq96 amino acid sequence
SEQ ID NO: 23: DNA polymerase variant Taq96 nucleic acid sequence
SEQ ID NO: 24: DNA polymerase variant Taq97 amino acid sequence
SEQ ID NO: 25: DNA polymerase variant Taq97 nucleic acid sequence
SEQ ID NO: 26: DNA polymerase variant Taq98 amino acid sequence
SEQ ID NO: 27: DNA polymerase variant Taq98 nucleic acid sequence
SEQ ID NO: 28: TaqNPIP-5 primer
SEQ ID NO: 29: Taq-3 primer
SEQ ID NO: 30: taqN10-5 primer
SEQ ID NO: 31: taqN10-3 primer
SEQ ID NO: 32: taqN15-5 primer
SEQ ID NO: 33: taqN15-3 primer
SEQ ID NO: 34: taq-plus20-5 primer
SEQ ID NO: 35: taq-plus20-3 primer
SEQ ID NO: 36: Taq-plus12-5 primer
SEQ ID NO: 37: Taq-plus12-3 primer
SEQ ID NO: 38: Taq-5 primer
SEQ ID NO: 39: Tq-L5-PIP-3 primer
SEQ ID NO: 40: Tq-L10-PIP-3 primer
SEQ ID NO: 41: Tq-L15-PIP-3 primer
SEQ ID NO: 42: F1 primer
SEQ ID NO: 43: R2-2 primer
SEQ ID NO: 44: LF-35 primer
SEQ ID NO: 45: LR8-35 primer
SEQ ID NO: 46: LR12-35 primer
SEQ ID NO: 47: LR15-35 primer
SEQ ID NO: 48: Taq95-5 primer
SEQ ID NO: 49: Taq96-5 primer
SEQ ID NO: 50: Taq97-5 primer
SEQ ID NO: 51: Taq98-5 primer
SEQ ID NO: 52: P. furiosus RFC-L PIP-box amino acid sequence
SEQ ID NO: 53: M.jannaschii RFC-L PIP-box amino acid sequence
SEQ ID NO: 54: P. furiosus PolBI PIP-box amino acid sequence
SEQ ID NO: 55: T.litoralis PolBI PIP-box amino acid sequence
SEQ ID NO: 56: A.fulgidus PolBI PIP-box amino acid sequence
SEQ ID NO: 57: P.occultum PolBII PIP-box amino acid sequence
SEQ ID NO: 58: M.jannaschii DP2 PIP-box amino acid sequence
SEQ ID NO: 59: M.thermoautotrophicum DP2 PIP-box amino acid sequence
SEQ ID NO: 60: H.sapiens Polδ p66 PIP-box amino acid sequence
SEQ ID NO: 61: S.cerevisiae Pol32 PIP-box amino acid sequence
SEQ ID NO: 62: S.pombe Cdc27 PIP-box amino acid sequence
SEQ ID NO: 63: S.cerevisiae Pol2 PIP-box amino acid sequence
SEQ ID NO: 64: P.furiosus Fen1 PIP-box amino acid sequence
SEQ ID NO: 65: M.jannaschii Fen1 PIP-box amino acid sequence
SEQ ID NO: 66: A.fulgidus Fen1 PIP-box amino acid sequence
SEQ ID NO: 67: H.sapiens Fen1 PIP-box amino acid sequence
SEQ ID NO: 68: D.melanogaster Fen1 PIP-box amino acid sequence
SEQ ID NO: 69: S.cerevisiae Fen1 PIP-box amino acid sequence
SEQ ID NO: 70: S.pombe Fen1 PIP-box amino acid sequence
SEQ ID NO: 71: H.sapiens DNA ligase I PIP-box amino acid sequence
SEQ ID NO: 72: X.laevis DNA ligase I PIP-box amino acid sequence
SEQ ID NO: 73: S.cerevisiae DNA ligase I PIP-box amino acid sequence
SEQ ID NO: 74: S.pombe DNA ligase I PIP-box amino acid sequence
SEQ ID NO: 75: H.sapiens MSH3 PIP-box amino acid sequence
SEQ ID NO: 76: S.cerevisiae MSH3 PIP-box amino acid sequence
SEQ ID NO: 77: H.sapiens MSH6 PIP-box amino acid sequence
SEQ ID NO: 78: S.cerevisiae MSH6 PIP-box amino acid sequence
SEQ ID NO: 79: H.sapiens UNG2 PIP-box amino acid sequence
SEQ ID NO: 80: S.cerevisiae UNG PIP-box amino acid sequence
SEQ ID NO: 81: H.sapiens hMYH PIP-box amino acid sequence
SEQ ID NO: 82: H.sapiens XPG PIP-box amino acid sequence
SEQ ID NO: 83: C.elegans XPG PIP-box amino acid sequence
SEQ ID NO: 84: S.cerevisiae XPG PIP-box amino acid sequence
SEQ ID NO: 85: S.pombe XPG PIP-box amino acid sequence
SEQ ID NO: 86: S.cerevisiae Cac1 PIP-box amino acid sequence
SEQ ID NO: 87: H.sapiens hRECQ5 PIP-box amino acid sequence
SEQ ID NO: 88: S.cerevisiae Rrm3 PIP-box amino acid sequence
SEQ ID NO: 89: H.sapiens Cdc25C PIP-box amino acid sequence
SEQ ID NO: 90: H.sapiens p15 PIP-box amino acid sequence
SEQ ID NO: 91: H.sapiens DNA-dependent protein kinase PIP-box amino acid sequence
Claims (16)
- N末側からC末側の方向に、
a)PCNAと結合する1又は複数のペプチド
および、
b)DNAポリメラーゼ活性を有するポリペプチド、
を含む融合ポリペプチド。 - PCNAと結合するペプチドがPIPボックスを有するペプチドであることを特徴とする、請求項1に記載の融合ポリペプチド。
- PIPボックスが、配列表の配列番号52~91のいずれかで示されるアミノ酸配列からなるペプチドであることを特徴とする、請求項2に記載の融合ポリペプチド。
- PCNAと結合するペプチドがDNAポリメラーゼ関連因子由来のPIPボックスを有するペプチドであることを特徴とする、請求項1または2に記載の融合ポリペプチド。
- PCNAと結合するペプチドが複製因子Cラージサブユニット由来のPIPボックスを有するペプチドであることを特徴とする、請求項1、2及び4のいずれか1項に記載の融合ポリペプチド。
- PCNAと結合するペプチドが配列表の配列番号3で示されるアミノ酸配列、もしくは前記アミノ酸配列を含むペプチドであることを特徴とする、請求項1、2、4及び5のいずれか1項に記載の融合ポリペプチド。
- PCNAと結合するペプチドとDNAポリメラーゼ活性を有するポリペプチドの間に5~50アミノ酸のリンカーペプチドを有することを特徴とする、請求項1~6のいずれか1項に記載の融合ポリペプチド。
- リンカーペプチドがセリンとグリシンから成るアミノ酸配列であることを特徴とする、請求項7に記載の融合ポリペプチド。
- DNAポリメラーゼ活性を有するポリペプチドがPolI型DNAポリメラーゼもしくはそのフラグメントであることを特徴とする、請求項1~8のいずれか1項に記載の融合ポリペプチド。
- DNAポリメラーゼ活性を有するポリペプチドがTaq DNAポリメラーゼもしくはそのフラグメントであることを特徴とする、請求項1~9のいずれか1項に記載の融合ポリペプチド。
- 請求項1~10のいずれか1項に記載の融合ポリペプチドをコードする核酸。
- 請求項1~10のいずれか1項に記載の融合ポリペプチドを含有する核酸増幅用組成物。
- さらに、PCNAを含有する、請求項12に記載の核酸増幅用組成物。
- 請求項1~10のいずれか1項に記載の融合ポリペプチドを含有するキット。
- さらに、PCNAを含有する、請求項14に記載のキット。
- 請求項1~10のいずれか1項に記載の融合ポリペプチドならびにPCNAを含有する組成物を使用することを特徴とする、鋳型DNAに相補的なDNAの製造方法。
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JP2017552698A JP7067737B2 (ja) | 2015-11-27 | 2016-11-24 | Dnaポリメラーゼ変異体 |
CN201680069042.8A CN108350087A (zh) | 2015-11-27 | 2016-11-24 | Dna聚合酶变体 |
EP16868628.5A EP3381947A4 (en) | 2015-11-27 | 2016-11-24 | DNA Polymerase VARIANT |
US15/772,614 US20190055527A1 (en) | 2015-11-27 | 2016-11-24 | Dna polymerase variant |
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JP2015231974 | 2015-11-27 |
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PCT/JP2016/084808 WO2017090685A1 (ja) | 2015-11-27 | 2016-11-24 | Dnaポリメラーゼ変異体 |
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US (1) | US20190055527A1 (ja) |
EP (1) | EP3381947A4 (ja) |
JP (1) | JP7067737B2 (ja) |
CN (1) | CN108350087A (ja) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020013058A1 (ja) | 2018-07-13 | 2020-01-16 | タカラバイオ株式会社 | Rnaからの核酸増幅反応に適したdnaポリメラーゼ変異体 |
EP3653729A4 (en) * | 2017-07-12 | 2021-06-09 | Genecast Co., Ltd. | DNA POLYMERASE WITH INCREASED GENE MUTATIONAL SPECIFICITY AND PCR BUFFER COMPOSITION TO INCREASE THE ACTIVITY OF IT |
Families Citing this family (4)
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CN112725299B (zh) * | 2020-12-30 | 2023-10-10 | 苏州白垩纪生物科技有限公司 | 改善Taq DNA聚合酶耐受性的突变体及制备方法和应用 |
CN113186175B (zh) * | 2021-06-04 | 2023-08-04 | 翌圣生物科技(上海)股份有限公司 | 突变型Taq DNA聚合酶、编码DNA序列、重组载体、重组表达细胞及其应用 |
WO2023059361A1 (en) * | 2021-10-06 | 2023-04-13 | 5Prime Biosciences, Inc. | Polymerases for mixed aqueous-organic media and uses thereof |
CN114958800B (zh) * | 2022-06-24 | 2023-08-25 | 北京脉道生物药品制造有限公司 | 一种耐受血液或者血液制品抑制的Taq DNA聚合酶突变体及其应用 |
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- 2016-11-24 WO PCT/JP2016/084808 patent/WO2017090685A1/ja active Application Filing
- 2016-11-24 JP JP2017552698A patent/JP7067737B2/ja active Active
- 2016-11-24 CN CN201680069042.8A patent/CN108350087A/zh active Pending
- 2016-11-24 US US15/772,614 patent/US20190055527A1/en not_active Abandoned
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3653729A4 (en) * | 2017-07-12 | 2021-06-09 | Genecast Co., Ltd. | DNA POLYMERASE WITH INCREASED GENE MUTATIONAL SPECIFICITY AND PCR BUFFER COMPOSITION TO INCREASE THE ACTIVITY OF IT |
WO2020013058A1 (ja) | 2018-07-13 | 2020-01-16 | タカラバイオ株式会社 | Rnaからの核酸増幅反応に適したdnaポリメラーゼ変異体 |
JPWO2020013058A1 (ja) * | 2018-07-13 | 2021-07-15 | タカラバイオ株式会社 | Rnaからの核酸増幅反応に適したdnaポリメラーゼ変異体 |
JP7324200B2 (ja) | 2018-07-13 | 2023-08-09 | タカラバイオ株式会社 | Rnaからの核酸増幅反応に適したdnaポリメラーゼ変異体 |
US12018294B2 (en) | 2018-07-13 | 2024-06-25 | Takara Bio Inc. | DNA polymerase mutant suited to nucleic acid amplification from RNA |
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US20190055527A1 (en) | 2019-02-21 |
JPWO2017090685A1 (ja) | 2018-09-13 |
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EP3381947A4 (en) | 2019-04-10 |
CN108350087A (zh) | 2018-07-31 |
JP7067737B2 (ja) | 2022-05-16 |
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