WO2011055737A1 - Adn polymérase à déplacement de brin thermostable et procédé de fabrication de l'adn polymérase - Google Patents

Adn polymérase à déplacement de brin thermostable et procédé de fabrication de l'adn polymérase Download PDF

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WO2011055737A1
WO2011055737A1 PCT/JP2010/069560 JP2010069560W WO2011055737A1 WO 2011055737 A1 WO2011055737 A1 WO 2011055737A1 JP 2010069560 W JP2010069560 W JP 2010069560W WO 2011055737 A1 WO2011055737 A1 WO 2011055737A1
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dna polymerase
strand displacement
amino acid
activity
acid sequence
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Japanese (ja)
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泰郎 大島
文典 牧
正一 山村
雅和 木谷
真樹 伊澤
祐康 米田
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株式会社ニッポンジーン
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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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  • the present invention relates to a heat-resistant strand displacement type DNA polymerase and a method for producing the DNA polymerase.
  • strand displacement type DNA amplification such as SDA (Strand® Displacement® Amplification) method and LAMP method has attracted attention and has been widely used as a DNA amplification method.
  • a DNA polymerase having strand displacement activity is used.
  • DNA polymerase having strand displacement activity for example, Bst DNA polymerase derived from Geobacillus stearothermophilus, Bpa DNA polymerase derived from Bacillus pallidus [Patent Document 1] and the like have been reported.
  • a Klenow-type enzyme that does not contain a 5′-3 ′ exonuclease domain on the N-terminal side of a protein is also known [Patent Document 2].
  • a DNA polymerase derived from the genus Bacillus grows in a medium high temperature range (temperature around 60 ° C.), has heat resistance and has a strand displacement type DNA polymerase activity.
  • the DNA polymerase of Phi29 phage that infects Bacillus subtilis has a strong 3'-5 'exonuclease activity as well as T4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ DNA polymerase, and also has a strand displacement type DNA polymerase activity.
  • these strand displacement DNA polymerases have been used for sample preparation and base sequence decoding in large-scale sequencing using an ultrafast parallel DNA sequencer, and their usefulness is high.
  • Patent Documents 1 to 5 and Non-Patent Documents 1 to 6 are specifically incorporated herein by reference.
  • DNA sequences with a high GC content include DNA sequences that are extremely difficult to amplify by PCR.
  • a method in which amplification is possible by adding a solvent that affects hydrogen bonding for example, DMSO, betaine, formamide
  • a solvent that affects hydrogen bonding for example, DMSO, betaine, formamide
  • Non-Patent Document 1 a solvent that affects hydrogen bonding
  • strand displacement type DNA amplification is easy even for a DNA sequence having a high GC content as described above. Therefore, strand displacement type DNA amplification is a promising technique in the field of gene analysis, testing and diagnosis in the future.
  • strand displacement type DNA polymerases reported so far have lower heat resistance than Taq ⁇ ⁇ DNA polymerase, Pfu ⁇ DNA polymerase, etc., which are widely used in the PCR method.
  • DNA polymerases having new functions such as better protein stability and higher resistance to inhibitors.
  • an object of the present invention is to provide a novel strand-displacement DNA polymerase having heat resistance and a production method thereof.
  • the present inventors investigated and examined DNA polymerase derived from microorganisms present in compost obtained by fermenting organic waste at a temperature of 85 ° C. or higher.
  • the present invention has been completed by finding a novel thermostable DNA polymerase having heat resistance that achieves the above.
  • a strand displacement DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • a temperature comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, having a strand displacement type DNA polymerase activity, and having a maximum strand displacement type DNA polymerase activity by the RCA method.
  • It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has a strand displacement type DNA polymerase activity, and has a strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 70 ° C.
  • a strand displacement DNA polymerase A strand displacement type consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, having strand displacement type DNA polymerase activity, and after heat treatment at a temperature of 70 to 80 ° C. for 5 minutes A strand displacement type DNA polymerase having a heat resistance of 80% or more of the strand displacement type DNA polymerase activity by the LAMP method when the DNA polymerase activity is not heat-treated.
  • the strand displacement DNA polymerase according to [4], wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 60 to 75 ° C.
  • [6] The strand displacement DNA polymerase according to [4], which has strand displacement DNA polymerase activity according to the LAMP method at a temperature of 70 ° C.
  • [7] The strand displacement DNA polymerase according to any one of [2] to [6], wherein the pH at which the strand displacement DNA polymerase activity is maximized is 8.0 or more.
  • Any one of [2] to [7], wherein the amino acid sequence shown in SEQ ID NO: 1 or 2 comprises at least one or more biologically acceptable amino acid substitutions, amino acid deletions, and / or amino acid insertions The strand displacement type DNA polymerase as described.
  • the amino acid sequence has an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has a strand displacement type DNA polymerase activity, and has a temperature at which the strand displacement type DNA polymerase activity by the RCA method is maximum at 45 to 45 Strand displacement DNA polymerase in the range of 55 ° C.
  • a strand displacement DNA polymerase comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement type DNA polymerase activity, and subjected to a heat treatment at a temperature of 55 to 60 ° C. for 5 minutes
  • the amino acid sequence shown in SEQ ID NO: 3 contains at least one or more biologically acceptable amino acid substitutions, amino acid deletions, and / or amino acid insertions, according to any one of [11] to [16] Strand displacement DNA polymerase.
  • the strand-displacement DNA polymerase according to [17] comprising 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.
  • the strand displacement DNA polymerase according to any one of [1] to [18] which is used for nucleic acid synthesis in a LAMP (Loop-mediated isothermal amplification) method.
  • [20] [1] A gene encoding the DNA polymerase according to any one of [18].
  • [21] [20] The gene described in [20] is mounted on a vector, and after the host cell is transformed with this vector, the transformed host cell is cultured, and the DNA polymerase encoded by the gene is accumulated in the culture, A method for producing a strand displacement DNA polymerase, comprising collecting accumulated DNA polymerase. [22] The production method according to [20], wherein the collected DNA polymerase includes the strand displacement type DNA polymerase according to any one of [1] to [18]. [23] The production method according to [20] or [21], wherein an E. coli expression vector using a co-expression method with a leader sequence is used.
  • the present invention it is an object to provide a novel heat-resistant strand displacement type DNA polymerase and a production method thereof. Furthermore, the strand displacement type DNA polymerase of the present invention has a good strand displacement type DNA polymerase even under basic conditions, and also has excellent storage stability of the strand displacement type DNA polymerase activity.
  • thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (fYMO81 DNA polymerase) and the DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). It is explanatory drawing which shows the comparison of the amino acid sequence of the thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (fYMO81 DNA polymerase) and the DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611).
  • thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (tYMO81 DNA polymerase) and DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611). It is explanatory drawing which shows the comparison of the amino acid sequence of thermostable DNA polymerase derived from Caldothrix satsumae YMO81 (tYMO81 DNA polymerase) and DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611).
  • FIG. 5 is an explanatory diagram showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from an unidentified microorganism 96-7 (t96-7 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611).
  • FIG. 5 is an explanatory diagram showing a comparison of amino acid sequences between a thermostable DNA polymerase derived from an unidentified microorganism 96-7 (t96-7 DNA polymerase) and a DNA polymerase derived from Geobacillus stearothermophilus (GenBank Accession No. AAB52611).
  • FIG. 2 is a gel electrophoresis photograph showing the optimum reaction temperature of the fYMO81 ⁇ DNA polymerase and tYMO81 DNA polymerase in the RCA method, and a gel electrophoresis photograph showing the optimum reaction temperature of the t96-7 DNA polymerase in the RCA method.
  • the present invention relates to a strand displacement type DNA polymerase, a strand displacement type DNA polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2 (the strand displacement type DNA polymerase according to the first aspect of the present invention) and SEQ ID NO: 3
  • the present invention relates to a strand displacement type DNA polymerase comprising the described amino acid sequence (the strand displacement type DNA polymerase according to the second aspect of the present invention).
  • Geobacillus stearothermophilus that grows at moderately high temperatures is common as a thermophilic aerobic bacterium, but its classification is very ambiguous, and several DNA polymerases derived from Geobacillus stearothermophilus have been reported so far [Non-patent Document 2 ].
  • DNA polymerases having the same structure as those of the same Geobacillus genus but with the reported stealothermophilus and caldotenax have been discovered. This indicates that the genus Geobacillus, which is a moderately thermophilic bacterium, still has a wide variety, and is an attractive microorganism for exploring new DNA polymerases.
  • Compost is a fertilizer obtained by fermenting organic matter such as livestock excrement, manure, sludge, and municipal waste using microorganisms.
  • thermophilic microorganisms have been used in the fermentation process.
  • the upper limit of the fermentation temperature by heat of fermentation is about 80 ° C., and spore-forming germs could not be completely removed.
  • bacterial cultures grown at a temperature of 85 ° C or higher obtained from soil in the Kirishima volcanic area are mixed with raw sludge, mixed, aerated and fermented in the sludge at a fermentation temperature of 85 ° C or higher.
  • a method has been found for killing germs, seeds, etc. present in sucrose, purifying sludge, and obtaining a fermented sludge containing only a large number of useful cells [Patent Documents 3 and 4].
  • thermophilic aerobic spore bacteria and thermophilic bacteria have been found [Patent Documents 3 and 4].
  • the present inventors pay attention to the above-mentioned compost obtained by fermenting organic waste at a temperature of 85 ° C. or higher, and the strand-substituted DNA polymerase having the heat resistance aimed by the present invention can be obtained from this compost. I tried to find out. As a result, a DNA polymerase gene derived from the thermophile Caldothrix satsumae YMO81 (FERM BP-8233) isolated from the compost was isolated.
  • the aforementioned Geobacillus stearothermophilus is a gram-positive gonococcus having a spore-forming ability
  • Caldothrix satsumae YMO81 is a gram-negative bacterium and does not have a spore-forming ability.
  • the obtained DNA polymerase derived from Caldothrix satsumae YMO81 since there was no report of a base sequence or an amino acid sequence, the recombinant protein was expressed in Escherichia coli and the activity was verified.
  • the chain of the first aspect of the present invention It led to substitutional DNA polymerase.
  • the DNA polymerase gene was also isolated directly from the metagenome of the microorganism group present in the compost.
  • the microorganism obtained here was named 96-7, and for the DNA polymerase derived from 96-7, there was no report of the base sequence or amino acid sequence, so the recombinant protein was expressed in E. coli and the activity was verified.
  • the strand-displacing DNA polymerase according to the second aspect of the present invention has been reached.
  • the classification of the microorganism 96-7 has not yet been identified, the DNA polymerase derived from 96-7 has no report of nucleotide sequence or amino acid sequence, and the DNA polymerase derived from 96-7 is also a novel DNA polymerase.
  • the strand displacement type DNA polymerase comprising the amino acid sequence shown in SEQ ID NO: 1 comprises 890 amino acids as shown in Examples 1 to 3 and Test Example 1, and has the maximum strand displacement type DNA polymerase activity by the RCA method.
  • the temperature is in the range of 60 to 75 ° C., and has a strand displacement type DNA polymerase activity by the LAMP method even at a temperature of 70 ° C., and has a strand displacement type DNA polymerase activity after heat treatment at a temperature of 70 to 80 ° C. for 5 minutes. It has a heat resistance of 80% or more of the strand displacement type DNA polymerase activity when not heat-treated, and the pH at which the strand displacement type DNA polymerase activity is maximized is 8.0 or more.
  • the strand displacement type DNA polymerase comprising the amino acid sequence represented by SEQ ID NO: 2 comprises 608 amino acids and comprises the amino acid sequence represented by SEQ ID NO: 1.
  • the temperature at which the strand displacement DNA polymerase activity by the RCA method is maximized is in the range of 60 to 75 ° C., and has the strand displacement DNA polymerase activity by the LAMP method even at a temperature of 70 ° C.
  • the strand displacement type DNA polymerase activity after heat treatment at a temperature of ⁇ 80 ° C. has a heat resistance of 80% or more of the strand displacement type DNA polymerase activity when not heat-treated, and the strand displacement type DNA polymerase activity is maximum.
  • the pH is 8.0 or more.
  • the strand displacement DNA polymerase according to the first aspect of the present invention includes the following strand displacement DNA polymerase in addition to the strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • strand displacement DNA polymerase in addition to the strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, has strand displacement DNA polymerase activity, and has maximum strand displacement DNA polymerase activity by the RCA method
  • the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 60 to 70 ° C.
  • An amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2 has a strand displacement type DNA polymerase activity, and is a strand displacement type by LAMP method even at a temperature of 70 ° C.
  • a strand displacement DNA polymerase having DNA polymerase activity It preferably has strand displacement DNA polymerase activity by the LAMP method even at a temperature of 75 ° C., more preferably at a temperature of 80 ° C.
  • It comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 2 has a strand displacement type DNA polymerase activity, and is subjected to a heat treatment at a temperature of 70 to 80 ° C. for 5 minutes.
  • it has a heat resistance of 90% or more, more preferably 95% or more of the strand displacement type DNA polymerase activity by the LAMP method when not heat-treated.
  • the strand displacement DNA polymerase according to (3), wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 60 to 75 ° C.
  • the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 60 to 70 ° C.
  • the strand displacement DNA polymerase according to (3) which has strand displacement DNA polymerase activity according to the LAMP method at a temperature of 70 ° C. It preferably has strand displacement DNA polymerase activity by the LAMP method even at a temperature of 75 ° C., more preferably at a temperature of 80 ° C.
  • the strand displacement type DNA polymerase according to any one of (1) to (5) which has a high basic activity with a pH of 8.0 or more at which the strand displacement DNA polymerase activity is maximized.
  • the strand displacement DNA polymerase activity is maximized when the pH is in the range of 8.0 to 10.0.
  • amino acid sequence shown in SEQ ID NO: 1 or 2 is at least one biologically acceptable amino acid substitution or amino acid deletion And / or amino acid insertions, preferably 1-10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.
  • the amino acid sequence having an identity of 99% or more has high activity at high temperature of the strand displacement DNA polymerase, high heat resistance of the strand displacement DNA polymerase activity, and high basic activity. Appropriate from the viewpoint.
  • the strand displacement DNA polymerase according to the second aspect of the present invention includes the following strand displacement DNA polymerase in addition to the strand displacement DNA polymerase consisting of the amino acid sequence shown in SEQ ID NO: 3.
  • a temperature consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, having strand displacement DNA polymerase activity, and maximizing strand displacement DNA polymerase activity by the RCA method
  • the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 50 to 55 ° C.
  • An amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, has strand displacement DNA polymerase activity, and strand displacement DNA polymerase activity by the LAMP method at a temperature of 60 ° C A strand displacement DNA polymerase.
  • the strand displacement DNA polymerase according to (10), wherein the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is in the range of 45 to 55 ° C.
  • the temperature at which the strand displacement DNA polymerase activity is maximized by the RCA method is preferably in the range of 50 to 55 ° C.
  • the strand displacement DNA polymerase activity by the LAMP method even at a temperature of 60 ° C. (13)
  • the strand displacement DNA polymerase activity is maximized when the pH is in the range of 8.0 to 9.0.
  • amino acid sequence shown in SEQ ID NO: 3 has at least one biologically acceptable amino acid substitution, amino acid deletion, and An amino acid insertion can be included, and preferably includes 1 to 10 amino acid substitutions, amino acid deletions, and / or amino acid insertions.
  • the strand displacement type DNA polymerase has high activity at high temperature, the heat resistance of the strand displacement type DNA polymerase activity is high, and the basic activity is also high. Is appropriate.
  • the strand displacement type DNA polymerase of the present invention can be used for various nucleic acid synthesis to which the strand displacement type DNA polymerase activity is applied, and is preferably used for nucleic acid synthesis in a LAMP (Loop-mediated isothermal amplification) method.
  • LAMP Loop-mediated isothermal amplification
  • the present invention includes a gene encoding the strand displacement DNA polymerase of the present invention. Representative examples of the gene of the present invention are shown in SEQ ID NOs: 4-6.
  • the method for obtaining the strand displacement type DNA polymerase of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique.
  • a gene (DNA) encoding the protein is obtained as described later.
  • the protein of the present invention strand displacement DNA polymerase
  • the strand-displacing DNA polymerase of the present invention has a gene encoding the strand-displacing DNA polymerase of the present invention mounted on a vector, transforms the host cell with this vector, and then cultures the transformed host cell.
  • the DNA polymerase encoded by the gene can be accumulated in the culture, and the accumulated DNA polymerase can be collected by a production method.
  • the method for obtaining a gene encoding the strand displacement DNA polymerase of the present invention is not particularly limited.
  • the gene encoding the strand displacement type DNA polymerase of the present invention can be obtained by, for example, chemical synthesis or genetic engineering techniques based on the amino acid sequences set forth in SEQ ID NOs: 1 to 3 and the nucleotide sequences set forth in SEQ ID NOs: 4-6. Alternatively, it can be produced by any method known to those skilled in the art, such as mutagenesis.
  • Caldothrixthrsatsumae YMO81 The gene of the present invention can be isolated by screening a cDNA library.
  • a cDNA library can be prepared from Caldothrix satsumae YMO81 by a conventional method.
  • a gene encoding the strand displacement DNA polymerase of the present invention can also be obtained by PCR.
  • PCR is carried out using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 4 or 5.
  • PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified.
  • the amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.
  • the gene of the present invention can be used by inserting it into an appropriate vector.
  • the type of vector used in the present invention is not particularly limited.
  • the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes.
  • the vector used in the present invention is an expression vector.
  • the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like).
  • a promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.
  • Promoters that can operate in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, and the Bacillus amyloliquefaction.
  • Enns-BAN amylase gene Bacillus amyloliquefaciens BAN amylase gene
  • Bacillus subtilis alkaline protease gene Bacillus subtilis alkaline protease gene
  • promoters of the Bacillus pumilus-xylosidase gene Bacillus pumilus-xylosidase gene
  • phage lambda P R or P L promoters
  • lac of E.coli such as trp or tac promoter and the like.
  • promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, and the adenovirus 2 major late promoter.
  • promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene.
  • Examples of a promoter operable in a yeast host cell include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, an ADH2-4c promoter, and the like.
  • promoters that can operate in filamentous fungal cells include the ADH3 promoter or the tpiA promoter.
  • the gene of the present invention may be operably linked to an appropriate terminator as necessary.
  • the recombinant vector containing the gene of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like.
  • the recombinant vector containing the gene of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). Is mentioned.
  • the recombinant vector containing the gene of the present invention may further contain a selection marker.
  • Selectable markers include, for example, genes that lack their complement in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin. Methods for ligating the gene, promoter, and, if desired, terminator and / or secretory signal sequence of the present invention and inserting them into an appropriate vector are well known to those skilled in the art.
  • a transformant can be prepared by introducing a recombinant vector containing the gene of the present invention into an appropriate host.
  • the host cell into which the recombinant vector containing the gene of the present invention is introduced may be any cell as long as it can express the gene of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.
  • Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces or Gram-negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method.
  • Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.
  • yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevis 1ae or Saccharomyces kluyveri.
  • Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.
  • filamentous fungi examples include Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.
  • the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells.
  • protein can be expressed (for example, as described in Baculovirus Expression Vectors, Laboratory Laboratory Mana1; Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).
  • baculovirus for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.
  • Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company), New York, which is an ovarian cell of Spodoptera frugiperda (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.
  • Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include the calcium phosphate method and the lipofection method.
  • the above transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene.
  • ordinary protein isolation and purification methods may be used. For example, when the protein of the present invention is expressed in a dissolved state in the cell, after the culture is completed, the cell is collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher or the like. A cell-free extract is obtained.
  • an ordinary protein isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Using the hydrophobic chromatography method used, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination, the strand displacement of the present invention Type DNA polymerase can be obtained as a purified preparation.
  • a solvent extraction method such as diethylaminoethyl (DEAE) Sepharose
  • cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia)
  • E. coli recombinant protein expression vector can be used for expression of the above recombinant protein.
  • Expression Vector pLEAD5 DNA manufactured by Nippon Gene [ The use of Patent Document 5] is preferable from the viewpoint of a vector optimized for expressing a heterologous gene with a biased GC or AT content in E. coli.
  • genes with high GC content derived from thermophiles and actinomycetes are often difficult to express in E. coli, and the cause is thought to be due to low translation efficiency.
  • Expression Vector pLEAD5 is constructed based on the results of screening an expression vector with good protein expression efficiency from a gene with a high GC content, and may be effective for the expression of genes with a high AT content. It is shown.
  • Example 1 [Cloning of DNA polymerase I from Caldothrix satsumae YMO81]
  • DNA polymerase I a degenerate primer is designed from the amino acid sequence of a DNA polymerase domain that is highly conserved among different organisms, and a gene is obtained by PCR using the already obtained Caldothrix satsumae YMO81 genomic DNA as a template DNA.
  • Non-patent Document 3 Sommer, R., Tautz, D. Nucleic Acids Research, 17: 6749 (1989)
  • the sequence of the obtained DNA fragment was analyzed by the Sanger method, and homology with DNA polymerase I was confirmed.
  • Non-patent Document 4 Cold-patent Document 4 (Collins, F. S., and Weissman, S. M. Proc Natl Acad Sci USA 81: 6812 (1984)), Non-Patent Document 5 (Triglia, T., Peterson, M. G., and Kemp, D. J. Nucleic Acids Reseach, 16: 8186 (1988)), Non-Patent Document 6 ( Xu, S. Y., Xiao, J.
  • the predicted sequence of the isolated DNA polymerase I gene was 2,673 bp, and it was estimated to encode a protein composed of 890 amino acids.
  • a comparison between the obtained amino acid sequence and the amino acid sequence (SEQ ID NO: 23) of DNA polymerase I (GenBank Accession No. AAB52611) derived from Geobacillus stearothermophilus is shown in Fig. 1, and a domain presumed to be a 5'-3 'exonuclease domain
  • FIG. 2 shows a comparison between the amino acid sequence obtained by removing E. coli and the amino acid sequence (SEQ ID NO: 23) of Geobacillus Ge stearothermophilus-derived DNA polymerase I (GenBank Accession No. AAB52611).
  • Example 2 [Construction of a DNA polymerase expression vector derived from Caldothrix satsumae YMO81] PCR primer (fYMO81_N: 5'-GTG ATG GAG AAG CTG GTC) for introducing a 890 amino acid residue, 2,670 bp gene corresponding to the full length of the DNA polymerase gene from Caldothrix satsumae YMO81 into the protein expression vector Expression Vector pLEAD5 DNA CT-3 ', tYMO81_C_SacI: 5'-GCT GAG CTC TAT TTC GCG TCG TAC CAC-3') was designed. The base sequences of the designed primers (fYMO81_N, tYMO81_C_SacI) are shown in SEQ ID NOs: 7 and 8.
  • FIG. 4 is an acrylamide gel electrophoresis photograph showing expression in Escherichia coli of a DNA polymerase derived from Caldothrix satsumae YMO81.
  • Lane 2 confirms the expression of the recombinant protein from the full length gene of the cloned DNA polymerase.
  • the target DNA fragment is obtained by PCR method, which is 35 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds and 68 ° C for 3 minutes. Amplified. Since a region that is difficult to amplify by PCR was included, a DNA fragment was obtained by PCR in the presence of 5% formamide.
  • the 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, and one end was further digested with SacI. Then, only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene). Escherichia coli JM109 was transformed with this DNA, and the expression of the protein in the transformant was confirmed. As a result, it was confirmed that a recombinant protein having an expected molecular weight of about 113 kDa was expressed (FIG. 4, lane 2). ). The sequence of 890 amino acids encoding this protein is shown in SEQ ID NO: 1 described later.
  • Lane 5 confirms the expression of a DNA polymerase from which the 5'-3 'exonuclease domain has been removed. That is, in order to remove the 5'-3 'exonuclease domain, the PCR primer (tYMO81_N: 5'-GTG was used to introduce a 608 amino acid residue, 1,824 ⁇ ⁇ ⁇ bp gene in which the 284th amino acid was replaced by methionine.
  • ATG CCC CAT GTG CCG-3 ', tYMO81_C_SacI was designed and heat-treated at 94 ° C for 2 minutes, then 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 2 minutes in total
  • the DNA fragment of interest was amplified by PCR using 35 cycles.
  • the 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, one end was further digested with SacI, and only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene).
  • the base sequence of the designed primer (tYMO81_N) is shown in SEQ ID NO: 9.
  • Escherichia coli JM109 was transformed with this DNA, and the expression of the transformant was confirmed. As a result, it was confirmed that a recombinant protein having an expected molecular weight of approximately 77 kDa was expressed (FIG. 4, lane 5).
  • the sequence of 608 amino acids encoding this protein is shown in SEQ ID NO: 2 described later.
  • fYMO81 DNA polymerase proteins of approximately 113 kDa
  • tYMO81 DNA polymerase proteins of approximately 113 kDa
  • fYMO81 DNA polymerase proteins of approximately 113 kDa
  • tYMO81 DNA polymerase proteins of approximately 77 kDa
  • RCA Rolling Circle Amplification
  • 5'-3 'exonuclease-deficient DNA-polymerase derived from Geobacillus-stearothermophilus was used (Bst-DNA Polymerase, Large-Fragment; New-England Biolabs).
  • the nucleotide sequence of Universal primer is shown in SEQ ID NO: 10.
  • Example 3 [Purification of recombinant DNA polymerase from Caldothrix satsumae YMO81]
  • the E. coli obtained in Example 2 is cultured and collected.
  • E. coli is suspended in Sonication Buffer (200 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol, 0.1 mM Benzamidine, 10 ⁇ g / ml Bacitracin, 30 ⁇ g / ml Phenylmethylsulfonyl fluoride, 20 mM Tris-HCl (pH 7.5 at 25 ° C.))
  • the suspension is heated in a 55 ° C. water bath for 20 minutes. Immediately lower the temperature on ice, centrifuge and collect the supernatant.
  • the supernatant was added to an anion exchange column (HiTrap® Q®HP; manufactured by GE Healthcare Bioscience), washed thoroughly, and then separated by a linear concentration gradient of NaCl.
  • HiTrap® Q®HP manufactured by GE Healthcare Bioscience
  • RCA method and SDS-PAGE were performed to confirm fractions containing DNA polymerase.
  • the desired fractions were collected, added to a heparin affinity column (HiTrap Heparin HP; manufactured by GE Healthcare Biosciences), washed thoroughly, and separated by a NaCl linear concentration gradient.
  • HiTrap Heparin HP manufactured by GE Healthcare Biosciences
  • Example 1 SDS-PAGE was performed to confirm fractions containing DNA polymerase (FIG. 4, lane 3 and lane 6). Collected fractions were tested using storage buffer (0.1 ⁇ mM EDTA, 1mM Dithiothreitol, 10mM Tris-HCl (pH 7.5 at 25 °C), 0.1% Tween 20, 50% Glycerol). The analysis in Example 1 was performed.
  • Example 4 [Cloning of 96-7-derived DNA polymerase I]
  • many organisms such as bacteria are symbiotic in nature, and pure culture of specific bacteria is often difficult. Therefore, the present inventors tried a method of extracting metagenomic DNA from composting during fermentation without isolating bacteria and isolating DNA polymerase I gene by PCR.
  • the present inventors include a bacterial culture grown at a temperature of 85 ° C. or higher obtained from soil in the Kirishima volcanic area, and a high temperature portion around 90 ° C. from compost during fermentation using sewage sludge as a main raw material.
  • the DNA was extracted using QIAamp DNA Stool Mini Kit (Qiagen).
  • DNA polymerase I a degenerate primer was designed from the amino acid sequence of a DNA polymerase domain that is particularly highly conserved among different organisms, and a part of the gene was obtained by PCR using the obtained metagenomic DNA as a template DNA. The sequence of the obtained DNA fragment was analyzed by the Sanger method, and homology with DNA polymerase I was confirmed. The microorganism presumed to have this DNA fragment in the genomic DNA was designated as 96-7. The classification of this microorganism 96-7 has not been identified yet.
  • metagenomic DNA was digested with an arbitrary restriction enzyme, and the DNA fragment was circularized by self-ligation.
  • the sequence before and after the obtained DNA fragment was sequentially isolated by the inverse PCR method using this as a template DNA.
  • the sequence of the region excluding the sequence presumed to encode the 5′-3 ′ exonuclease domain of the DNA polymerase I gene was determined.
  • FIG. 3 shows a comparison between the obtained amino acid sequence and the amino acid sequence (SEQ ID NO: 23) of DNA polymerase I (GenBank Accession No. AAB52611) derived from Geobacillus stearothermophilus.
  • Example 5 [Construction of DNA polymerase expression vector derived from 96-7] PCR primers (t967_N: 5'-GTG ATG TCC GGA CAA AAG GAT G-3 ', t967_C_SacI: 5'-GAG GAG CTC TAT to introduce the 96-7-derived DNA polymerase gene into the protein expression vector Expression Vector pLEAD5 DNA TTT GCA TCG AAC CAG-3 ') was designed. The nucleotide sequences of the designed primers (t967_N, t967_C_SacI) are shown in SEQ ID NOs: 11 and 12, respectively.
  • FIG. 5 is an acrylamide gel electrophoresis photograph showing the expression of 96-7-derived DNA polymerase in E. coli.
  • lane 2 confirms the expression of DNA polymerase from which the 5′-3 ′ exonuclease domain has been removed. That is, in order to remove the 5'-3 'exonuclease domain, the 285 amino acid was replaced with methionine from leucine to 595 amino acid residues, and the above primer (t967_N, t967_C_SacI) was introduced to introduce a 1,785 bp gene.
  • the DNA fragment of interest is obtained by PCR using a total of 35 cycles of 94 ° C for 15 seconds, 50 ° C for 30 seconds, and 68 ° C for 2 minutes. Amplified. The 5 ′ end of the DNA fragment was phosphorylated with T4 polynucleotide kinase, and one end was further digested with SacI. Then, only the target DNA fragment was excised from an agarose gel, and Expression Vector pLEAD5 DNA, pre-digested (Inc. Nippon Gene). Escherichia coli JM109 was transformed with this DNA, and the expression of the protein in the transformant was confirmed.
  • t96-7 DNA polymerase This approximately 75 kDa protein (referred to as t96-7 DNA polymerase) was purified, and its DNA polymerase activity was examined by the RCA (Rolling Circle Amplification) method.
  • a circular single-stranded DNA derived from Escherichia coli phage was used as a template, and primers were added to this DNA to verify the presence or absence of DNA polymerase activity and strand displacement activity.
  • As an enzyme control 5'-3 'exonuclease-deficient DNA-polymerase derived from Geobacillus-stearothermophilus was used (Bst DNA-Polymerase, Large-Fragment; New New England Biolabs).
  • Example 6 [Purification of 96-7-derived recombinant DNA polymerase]
  • the E. coli obtained in Example 5 is cultured and collected.
  • E. coli is suspended in Sonication Buffer (200 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol, 0.1 mM Benzamidine, 10 ⁇ g / ml Bacitracin, 30 ⁇ g / ml Phenylmethylsulfonyl fluoride, 20 mM Tris-HCl (pH 7.5 at 25 ° C.))
  • the suspension is heated in a 55 ° C. water bath for 20 minutes. Immediately lower the temperature on ice, centrifuge and collect the supernatant.
  • the supernatant was added to an anion exchange column (HiTrap Q HP; made by GE Healthcare Bioscience), washed thoroughly, and separated by a linear NaCl concentration gradient.
  • HisTrap Q HP made by GE Healthcare Bioscience
  • RCA method and SDS-PAGE were performed to confirm fractions containing DNA polymerase.
  • the desired fractions were collected, added to a heparin affinity column (HiTrap Heparin HP; manufactured by GE Healthcare Biosciences), washed thoroughly, and separated by a NaCl linear concentration gradient.
  • HiTrap Heparin HP manufactured by GE Healthcare Biosciences
  • Test Example 1-1 [Evaluation of optimum reaction temperature by RCA method] The optimum reaction temperature of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by the RCA method. That is, the RCA method was performed at each temperature of 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, and 75 ° C. After reacting for 30 minutes, agarose gel electrophoresis was performed, and it was confirmed that the DNA band was shifted to the polymer side (FIG. 8). For each reaction, 4 units of Bst DNA Polymerase and Large Fragment, 400 ng of fYMO81 DNA polymerase, tYMO81 DNA polymerase, and t96-7 DNA polymerase were used.
  • the strength of DNA polymerase activity and strand displacement activity was evaluated by the mobility of the band shift to the polymer side within a certain time.
  • Bst ⁇ DNA Polymerase and Large Fragment showed DNA polymerase activity in the temperature range of 50-65 °C.
  • the tYMO81 DNA polymerase had the maximum activity at 60 to 75 ° C
  • the t96-7 DNA polymerase had the maximum activity at 45 to 55 ° C.
  • fYMO81 DNA polymerase showed high molecular weight of DNA suggesting strand displacement DNA polymerase activity, but formation of a large amount of small nucleic acid, which is thought to be due to endogenous 5'-3 'exonuclease activity. As a result, subsequent analysis on the reaction characteristics was not performed.
  • Test Example 1-2 The optimal reaction temperature of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by real-time turbidity measurement using the LAMP method. Specifically, positive control plasmid DNA, LAMP primer set, 0.25 mM each dNTPs, 20 mM Tris-HC (pH 8.8 at 25 ° C.), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Tween 20, and a total of 25 ⁇ l of DNA polymerase were added to the Loopamp Real-time Turbidimeter LA-200 (manufactured by Teramex for 1 hour, and the increase in turbidity was observed.
  • Real-time turbidity using LAMP method In the degree measurement, the reaction time (Tt) required until the turbidity reached 0.1 was compared, and the lower the Tt value, the more efficiently the reaction progresses.
  • the positive control plasmid DNA used here contains sequences characteristic of Candidatus Liberibacter asiaticus which is the causative microorganism of citrus greening disease or tobacco whitefly biotype Q which is a vector insect of plant disease virus.
  • the one designed for the purpose of specifically detecting the sequence of was used.
  • As a LAMP primer set for specifically detecting Candidatus Liberibacter asiaticus SEQ ID NO: 13 for FIP, SEQ ID NO: 14 for BIP, SEQ ID NO: 15 for F3, SEQ ID NO: 16 for B3, SEQ ID NO: 17 for LF, SEQ ID NO: 17 for LB A combination using 18 each primer was used.
  • LAMP primer set for specifically detecting tobacco whitefly biotype Q a combination using SEQ ID NO: 19 for FIP, SEQ ID NO: 20 for BIP, SEQ ID NO: 21 for F3, and SEQ ID NO: 22 for B3 Using.
  • tYMO81 DNA polymerase For tYMO81 DNA polymerase, real-time turbidity measurement was performed at 60 ° C., 65 ° C., and 70 ° C. using a LAMP primer set for detecting Candidatus Liberibacter asiaticus and a positive control plasmid DNA. In addition, Bst DNA Polymerase and Large Fragment per reaction used 8 unit, and tYMO81 DNA polymerase used 800 ng.
  • t96-7 DNA polymerase For t96-7 DNA polymerase, real-time turbidity measurement was performed at 55 ° C. and 60 ° C. using a LAMP primer set for specifically detecting tobacco whitefly biotype Q and a positive control plasmid DNA. For each reaction, 8 units were used for Bst DNA Polymerase and Large Fragment, and 600 ng was used for t96-7 DNA polymerase.
  • Test Example 1-3 Evaluation of heat resistance of tYMO81 DNA polymerase
  • tYMO81 DNA polymerase was suggested to have higher heat resistance than Bst DNA Polymerase, Large Fragment, tYMO81 DNA polymerase and Bst DNA for 5 minutes in advance at 70 ° C, 75 ° C, 80 ° C, 85, 90 ° C.
  • real-time turbidity measurement using LAMP method was performed to evaluate whether the activity was maintained.
  • Real-time turbidity measurement was performed at 65 ° C for 1 hour using a LAMP primer set and a positive control plasmid DNA for specifically detecting Candidatus Liberibacter asiaticus.
  • 8 units of Bst DNA Polymerase and Large Fragment and 800 ng of tYMO81 DNA polymerase were used.
  • Test Example 1-4 Evaluation of optimum reaction pH by LAMP method
  • the optimal reaction pH of tYMO81 DNA polymerase and t96-7 DNA polymerase was evaluated by real-time turbidity measurement using the LAMP method. Specifically, the change in the Tt value was observed when the pH of Tris-HCl contained in the LAMP method was changed from the original 8.8 up and down. The pH of Tris-HCl was adjusted under the temperature condition of 25 ° C. (Table 5).
  • the tYMO81 DNA polymerase was compared with Bst DNA Polymerase and Large Fragment using a LAMP primer set for specifically detecting Candidatus Liberibacter asiaticus and a positive control plasmid DNA. That is, real-time turbidity measurement was performed at 65 ° C. for 1 hour at each pH of 7.5, 8.0, 8.3, 8.8, 9.0, 9.5, and 10.0.
  • Bst DNA Polymerase and Large Fragment per reaction used 8 unit
  • tYMO81 DNA polymerase used 800 ng.
  • tYMO81 DNA polymerase had the lowest Tt at pH around 8.3, whereas tYMO81 DNA polymerase had the lowest Tt in a wide pH range from 8.8 to 10.0.
  • the pH of the reaction solution tends to be acidic due to the generation of reaction by-products and the temperature dependence of the Tris buffer, and the solubility of the protein may significantly reduce the DNA polymerase activity.
  • tYMO81 DNA polymerase is expected to solve this problem by using a high pH buffer in advance, and to perform a stable reaction while maintaining the activity of DNA polymerase. .
  • T96-7 DNA polymerase was compared with Bst Polymerase, aseLarge Fragment using LAMP primer set and positive control plasmid DNA for specifically detecting tobacco whitefly biotype Q. That is, real-time turbidity measurement was performed at 60 ° C. for 1 hour at each pH of 7.5, 8.0, 8.3, 8.8, and 9.0. For each reaction, 8 units were used for Bst DNA Polymerase and Large Fragment, and 600 ng was used for t96-7 DNA polymerase.
  • Bst DNA Polymerase and Large Fragment had the lowest Tt at a pH around 8.3, whereas t96-7 DNA polymerase had the lowest Tt in the pH range of 8.3 to 9.0.
  • t96-7 DNA polymerase does not reach tYMO81 DNA polymerase, but is expected to be usable on the basic side and in a wider range of pH conditions than Bst DNA Polymerase and Large Fragment.
  • Test Example 1-5 Evaluation of base resistance by LAMP method
  • the target nucleic acid to be amplified is extracted from a biological sample.
  • a method of denaturing cellular proteins using a strongly basic solution such as sodium hydroxide is known. It has been. However, the remaining of the basic solution unintentionally increases the pH of the reaction solution and causes hydrolysis of protein molecules such as DNA polymerase. As a result, the DNA synthesis reaction by DNA polymerase is inhibited, so nucleic acid molecules are extracted from biological samples.
  • a protocol for extracting genomic DNA of CandidatusandLiberibacter asiaticus infected with citrus leaves was used. That is, extraction of nucleic acid from citrus leaf specimens with 250 mM sodium hydroxide, neutralization with 2.5 M acetic acid followed by isopropanol precipitation, modification of the method for concentration and purification of nucleic acids, and extraction with reduced acetic acid addition Real-time turbidity measurement using the LAMP method was performed under the condition where the solution was added directly.
  • citrus leaves are put in 250 ⁇ l of 250 ⁇ mM sodium hydroxide and boiled, and then neutralized by adding 50 ⁇ l of 2.5 ⁇ m acetic acid.
  • the inventors prepared a mixed solution of sodium hydroxide and acetic acid in a pseudo manner, and positively added a LAMP primer set for specifically detecting Candidatus Liberibacter asiaticus under the condition that 2 ⁇ l was added to the reaction solution.
  • Real-time turbidity measurement was performed using control plasmid DNA.
  • the above mixed solution stock solution was used, no increase in turbidity was observed in tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment. This is presumably because the ionic strength and pH of the solution greatly fluctuate due to high concentrations of sodium hydroxide or acetic acid, and the DNA polymerase reaction is inhibited.
  • the present inventors diluted the above mixed solution (referred to as diluted extraction solution) twice with sterilized distilled water and then added it, and both tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment have turbidity.
  • diluted extraction solution twice with sterilized distilled water and then added it
  • tYMO81 DNA polymerase a DNA polymerase that polymerizes a diluted extract solution
  • Bst DNA Polymerase Bst DNA Polymerase
  • Large Fragment Large Fragment have turbidity.
  • Real-time turbidity measurement was performed at 65 ° C for 1 hour using the LAMP primer set and positive control plasmid DNA to specifically detect CandidatusidaLiberibacter asiaticus .
  • 8 units were used for Bst DNA Polymerase and Large Fragment, and 500 ng was used for tYMO81 DNA polymerase.
  • 500 ng was used for tYMO81 DNA polymerase.
  • a control for the diluted extract solution a reaction in which only sterile distilled water was added was added instead.
  • Tt was the lowest when the added amount of acetic acid was 5 ⁇ l or around 0 ⁇ l. Therefore, when tYMO81 DNA polymerase is used, the nucleic acid extraction sample using a strong base solution can be directly input into the nucleic acid amplification reaction by omitting operations such as neutralization, concentration, and solution removal. In the field of testing and diagnosis, it is expected to simplify the testing method by applying to a wider range of applications and automation than the conventional DNA polymerase.
  • Test Example 1-6 Evaluation of protein storage stability by LAMP method
  • the enzyme solution that is a component of the reaction is mostly a protein derived from Escherichia coli or any other microorganism, plant, or animal. Is often destabilized by factors such as proteolytic enzymes, temperature changes, and oxidation.
  • genetic diagnostic methods such as the LAMP method must be excellent in reproducibility, and how stable the quality of DNA polymerase can be ignored is an important property.
  • the present inventors focused on the fact that tYMO81 DNA polymerase does not lose its activity even at around 80 ° C., and compared the storage stability of tYMO81 DNA polymerase with Bst DNA Polymerase, Large Fragment. Specifically, no changes were observed in tYMO81 DNA polymerase, Bst DNA Polymerase, and Large Fragment in 24 hours during storage at 37 ° C. Therefore, under the conditions that increased the temperature load level, for example, the LAMP method is generally used here. Evaluation was made on the variation in residual activity at a temperature of 65 ° C., which is commonly used (Table 7). In Table 7, all were the average values of quadruple measurements.
  • Bst DNA Polymerase and Large Fragment were evaluated for storage stability under conditions containing a commercially available storage solution, that is, a buffer solution, salt, glycerol, and a surfactant. It should be noted that tYMO81 DNA polymerase does not show a significant change in activity by 24 hours even when stored at 65 ° C under conditions containing buffer, salt, glycerol, and surfactant. Increased the loading level and evaluated the storage stability in tYMO81 sterilized distilled water. The enzyme solution was stored at 65 ° C. for 3 hours, 6 hours, and 24 hours, respectively, and then evaluated using a LAMP primer set and a positive control plasmid DNA for specifically detecting Candidatus Liberibacter asiaticus. For each reaction, 8 units were used for Bst DNA Polymerase and Large Fragment, and 500 ng was used for tYMO81 DNA polymerase.
  • a commercially available storage solution that is, a buffer solution, salt, glycerol, and a
  • Tt In Bst DNA Polymerase and Large Fragment, a significant increase in Tt was confirmed by treatment at 65 ° C for 3 hours, and then Tt increased gradually after each of 6 hours and 24 hours. The decrease in activity at 3 hours is expected to be large. In contrast, with tYMO81 DNA polymerase, the change in Tt was extremely small even after treatment at 65 ° C for 3 hours, and the decrease in activity until 6 hours was gradual, with no decrease in activity until 24 hours thereafter. It was.
  • protein solutions are in the presence of protein stabilizers known as glycerol, bovine serum albumin, surfactants, protease inhibitors, etc., at a low temperature of about 2-8 ° C, or even at a low temperature range of -20 ° C.
  • the tYMO81 DNA polymerase which was stored as a nearly single protein molecule in sterile distilled water at -80 ° C, did not lose most of its activity even at 65 ° C. It suggests long-term storage stability and resistance to loading conditions, and is a useful property.
  • the present invention can be used in any field where a strand displacement type DNA polymerase is used.

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Abstract

L'invention porte sur une nouvelle ADN polymérase à déplacement de brin qui a une thermostabilité, et sur un procédé de fabrication de l'ADN polymérase à déplacement de brin. De façon spécifique, l'invention porte sur une ADN polymérase à déplacement de brin comprenant la séquence d'acide aminé énoncée dans SEQ ID NO: 1, 2 ou 3. Également de façon spécifique, l'invention porte sur une ADN polymérase à déplacement de brin comprenant une séquence d'acide aminé ayant une identité de 90 % ou davantage à la séquence d'acides aminés énoncée dans SEQ ID NO: 1 ou 2 et ayant une activité d'ADN polymérase à déplacement de brin. L'ADN polymérase à déplacement de brin est également caractérisée par le fait que : la température à laquelle l'activité d'ADN polymérase à déplacement de brin est rendue maximale par RCA se situe dans la plage de 60-75°C ; l'ADN polymérase à déplacement de brin a une activité d'ADN polymérase à déplacement de brin par LAMP à 70°C, ou l'activité d'ADN polymérase à déplacement de brin après un traitement thermique de 5 minutes à une température de 70-80°C a 80 % ou plus de la thermostabilité de l'activité ADN polymérase à déplacement de brin par LAMP sans traitement thermique. Egalement de façon spécifique, l'invention porte sur une ADN polymérase à déplacement de brin comprenant une séquence d'acides aminés ayant 90 % ou plus d'identité avec la séquence d'acides aminés énoncée dans SEQ ID NO: 3 et ayant une activité d'ADN polymérase à déplacement de brin. L'ADN polymérase à déplacement de brin est également caractérisée par le fait que : la température à laquelle l'activité d'ADN polymérase à déplacement de brin est rendue maximale par RCA se situe dans la plage de 45-55°C ; l'ADN polymérase à déplacement de brin a une activité d'ADN polymérase à déplacement de brin par LAMP à 55°C ; ou l'activité ADN polymérase à déplacement de brin après un traitement thermique de 5 minutes à une température de 55-60°C a 80 % ou plus de la thermostabilité de l'activité d'ADN polymérase à déplacement de brin par LAMP sans traitement thermique.
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JP2015116136A (ja) * 2013-12-17 2015-06-25 東ソー株式会社 核酸増幅方法および当該方法を利用した核酸増幅試薬
JP2017525376A (ja) * 2014-08-27 2017-09-07 ニユー・イングランド・バイオレイブス・インコーポレイテツド シントン形成
WO2017090684A1 (fr) * 2015-11-27 2017-06-01 国立大学法人九州大学 Mutant d'adn polymérase
CN108473970A (zh) * 2015-11-27 2018-08-31 国立大学法人九州大学 Dna聚合酶变体
JPWO2017090684A1 (ja) * 2015-11-27 2018-10-11 国立大学法人九州大学 Ｄｎａポリメラーゼ変異体
US11046939B2 (en) 2015-11-27 2021-06-29 Kyushu University, National University Corporation DNA polymerase variant
CN108473970B (zh) * 2015-11-27 2022-09-13 国立大学法人九州大学 Dna聚合酶变体

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