WO2007099737A1 - Dna polymerase mutant - Google Patents

Dna polymerase mutant Download PDF

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WO2007099737A1
WO2007099737A1 PCT/JP2007/051928 JP2007051928W WO2007099737A1 WO 2007099737 A1 WO2007099737 A1 WO 2007099737A1 JP 2007051928 W JP2007051928 W JP 2007051928W WO 2007099737 A1 WO2007099737 A1 WO 2007099737A1
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dna
amino acid
acid sequence
activity
hetero
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PCT/JP2007/051928
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French (fr)
Japanese (ja)
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Ikuo Matsui
Yuji Urushibata
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National Institute Of Advanced Industrial Science And Technology
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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

Definitions

  • the present invention relates to a DNA polymerase D (PolD) mutant enzyme gene and a DNA polymerase D (PolD) mutant enzyme encoded by the gene.
  • DNA polymerase is an enzyme useful for DNA sequencing reaction, gene amplification reaction (PCR reaction), radioactivity labeling of DNA, in vitro synthesis of mutant genes, and the like.
  • DNA polymerases known to date can be broadly classified into four families based on the commonality of amino acid sequences. Among them, it is generally used as a reagent for genetic manipulation experiments! The family A represented by Escherichia coli DNA polymerase I and thermophile Thermus aquaticus DN A polymerase (Taq DNA polymerase), It belongs to family B represented by T4 phage DNA polymerase.
  • various DNA polymerases with different optimum temperatures have been discovered in bacteria, animals and plants. Most of them are derived from room temperature organisms.
  • thermostable DNA polymerases have been marketed as thermostable DNA polymerases, however, since they lack 3'-5 'proofreading exonuclease activity, PCR reactions, etc. It is not suitable for PCR reactions that induce errors during the polymerase reaction and require immediate accuracy.
  • PolyB family B enzyme
  • It is isolated from a hyperthermophilic archaea such as Pyrococcus thermococcus and is commercially available, but primer extension It is not suitable for PCR reaction of long DNA with weak activity.
  • thermostable family that has 3'-5 'exonuclease activity, a heat-resistant proofreading function for DNA strand elongation, and can replicate the target DNA region in a short time even under high salt concentrations where the primer extension activity is strong. D There was a strong demand for a cheap and stable supply. Therefore, the subject of this invention is providing the novel enzyme which satisfy
  • PolD has a high molecular weight hetero-oligomer structure composed of two subunits (L and S), and was extremely unstable when expressed alone.
  • L and S two subunits
  • PolD obtained by the recombination method has extremely high heat resistance, DNA synthesis activity, 3, -5, exonuclease activity.
  • the present inventor has further studied and created deletion mutants of the two PolD subunits (L and S), which are essential for the expression of DNA synthesis activity and exonuclease activity.
  • L and S two PolD subunits
  • the deletion mutant enzyme that retains high DNA synthesis ability even under high salt concentration (salt concentration of 0.2M or more)! I came to the present invention.
  • the present invention is as follows.
  • the small subunit is a DNA synthesis activity control region, and the 3'-5, exonuclease activity suppression region is deleted, The thermostable hetero-oligomer enzyme mutant.
  • the region deleted from the small subunit includes at least the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having 45% or more homology with the amino acid sequence.
  • thermostable hetero-oligomer enzyme mutant that has the power of the following large subunits and small subunit mutants and has DNA polymerase activity and 3,15, exonuclease activity.
  • thermostable hetero-oligomer enzyme derived from Pyrococcus archaea and comprising a large subunit not containing a small subunit and an intin sequence And a region containing at least an amino acid sequence having 45% or more homology with the large subunit.
  • a small subunit mutant of a thermostable hetero-oligomer enzyme characterized by having a DNA polymerase activity and a 3'-5 'exonuclease activity when a telo-oligomer is constituted.
  • (6) a) the ability to have an amino acid sequence in which at least the 167th to 200th region is deleted in the amino acid sequence shown in SEQ ID NO: 1, or b) one or several amino acids in the amino acid sequence of a) When the amino acid residue has an amino acid sequence deleted, substituted or added, and does not contain a tin sequence, it constitutes a large subunit and a hetero-oligomer, and DNA polymerase activity and 3, 1 ' The small subunit mutant of the thermostable hetero-oligomer enzyme according to (5) above, which has exonuclease activity.
  • DNA encoding a small subunit in a thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′-5 ′ exonuclease activity,
  • thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′-5 ′ exonuclease activity. Or a DNA that hybridizes under stringent conditions with the DNA described in) or a DNA having a sequence complementary to the DNA.
  • a recombinant vector comprising the DNA according to any one of the above claims (7) to (9) and the DNA encoding the large subunit according to claim 2 so as to be capable of co-expression.
  • a transformant wherein the recombinant vector according to (10) or (11) is introduced. The invention's effect
  • a DNA polymerase capable of accurately replicating long-chain DNA at high speed under high temperature and high salt concentration environment can be provided, and the enzyme is useful for Long-PCR and the like.
  • FIG. L Base sequence of primer pair necessary for amplification of DPI genes ( ⁇ S (140-200) and ⁇ S (167-200)) lacking internal sequence by overlap PCR method and DPI gene FIG.
  • FIG. 2 is a diagram showing SDS-PAGE patterns of PolD hetero-oligomer wild type and DP1 (1-200) deletion mutant.
  • M represents a molecular weight marker.
  • FIG. 3 is a graph showing the effects of a salt concentration of PolD heterooligomer wild type and DPK1-200) deletion mutant on 3 and 5 ′ exonuclease activity (A) and DNA polymerase activity (B).
  • -Enzyme is a control sample obtained by removing DNA polymerase from the enzyme reaction system.
  • FIG. 4 is a graph showing changes over time in DNA polymerase activity of PolD wild type and PolD A S (1-200) in the presence and absence of 0.2 M sodium chloride.
  • FIG. 5 is a diagram showing the results of a quantitative test of DNA polymerase activity of PolD wild type and PolD A S (1-200) in the presence and absence of 0.2 M sodium chloride.
  • the left shows the electrophoresis pattern of the product of the primer extension reaction in the presence and absence of 0.2 M salt, and the right shows the ratio of the extension product to the total primer amount calculated from the electrophoresis pattern. Show.
  • FIG. 6 shows changes in the DNA polymerase activity of PolD A S (1-200) by DP1 (1-200) supplementation.
  • FIG. 7 is a diagram showing SDS-PAGE patterns of DPI (1 200) and its various deletion mutants. is there. M represents a molecular weight marker.
  • FIG. 8 is a graph showing the results of measuring the ability of PolD A S (1-200) to inhibit DNA polymerase activity using DP1 (1-200) and various deletion mutants thereof.
  • FIG. 9 is a diagram showing the N-terminus and deletion position (A) of DPI constituting the PolD hetero-oligomer mutant and the SDS-PAGE pattern (B) of these enzymes.
  • M represents a molecular weight marker.
  • FIG. 10 The results of measuring the primer extension activity of the hetero-oligomer mutants, PolD AS (1-200), PolD AS (1-65), PolD AS (140-200), PolD AS (167-200) FIG.
  • FIG. 12 is a view showing amino acid sequence alignments in each PolD enzyme for the DP1 (167-200) region.
  • the present invention relates to a thermostable hetero-oligomer derived from Pyrococcus archaea having a DNA polymerase activity and 3,15, exonuclease activity, comprising a small subunit and a large subunit not containing an intin sequence.
  • the enzyme by removing the 3'-5 'exonuclease activity suppression region, which is the DNA synthesis activity control region in the small subunit, the DNA synthesis activity (primer extension activity) of the enzyme under high salt concentration And 3′—5 ′ exonuclease activity is significantly enhanced.
  • thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit from which the intin sequence has been deleted, and having a DNA polymerase activity and 3,15, exonuclease activity, is a prior application of the present inventors. (Described in Japanese Patent Application No. 2000-116257 (Japanese Patent Application Laid-Open No. 2001-299348). The invention of this application has a DNA polymerase activity and 3,15, exonuclease activity derived from Pyrococcus horicosi.
  • thermostable hetero-oligomer enzyme For the first time, the presence of an intin sequence was found in the large subunit of a thermostable hetero-oligomer enzyme, but it was found that removing the intin sequence significantly improved the primer extension activity of the enzyme.
  • the amino acid sequence of the small subunit in the thermostable heteroligomer enzyme derived from Pyrococcus horikoshi and Nucleotide sequence of the gene The amino acid sequence of the same subunit and the sequence of the gene are shown in SEQ ID NOs: 3 and 4, respectively, and the amino acid sequence of the intin portion is 955 to 1120, respectively.
  • the base sequence is the 2863-3360th region.
  • amino acid sequence of the large subunit from which the intin moiety has been removed and the base sequence of the gene are shown in SEQ ID NOs: 5 and 6, respectively.
  • thermostable hetero-oligomer enzyme mutant of the present invention a large sub that does not originally contain an intin sequence can be obtained by removing the intin sequence. It may be a Pyrococcus heat-resistant heterodimer enzyme having a unit. Examples of such an enzyme include thermostable DNA polymerase derived from Pyrococcus' Friosus. The amino acid sequences and gene sequences of the large subunit and small subunit of this enzyme are listed in the “Microbial Genome Database (http: // mbgd. Genome, ad. JpZ)”.
  • thermostable heterodimer enzyme thermostable DNA polymerase; hereinafter sometimes referred to as PolD
  • PolD thermostable DNA polymerase
  • DPI amino group terminal 200 residues
  • the sequence homologous to DPI (167-200) is widely present in the small subunit of the PolD enzyme derived from Pyrococcus archaea, and the DNA synthesis activity control region is widely conserved in Pyrococcus bacteria.
  • FIG. 12 shows a sequence alignment of the region.
  • the ability to delete the DNA polymerase activity control region of the small subunit is as long as the range of deletion does not impair the DNA polymerase activity and 3'-5 'exonuclease activity. At least.
  • the small subunit mutant of the present invention includes, for example, a region at least 167-200 in the amino acid sequence shown in SEQ ID NO: 1 derived from Pyrococcus horicosi.
  • small subunit mutants derived from Pyrococcus horikoshii having an amino acid sequence deleted from the amino acid sequence wherein the amino acid sequence has an amino acid sequence in which one to several amino acid residues have been deleted, substituted or added
  • small subunit mutants are included as long as they have DNA polymerase activity and 3′-5 ′ exonuclease activity when they constitute a hetero-oligomer with a large subunit.
  • the large subunit constituting the hetero-oligomer enzyme with these small subunits has the amino acid sequence shown in SEQ ID NO: 5 above, and the sequence and one to several amino acid residues are deleted, substituted or added.
  • Those having a defined amino acid sequence are also included as long as they have DNA polymerase activity and 3, -5, exonuclease activity when they constitute a hetero-oligomer.
  • means for obtaining DNA encoding a small subunit mutant from which the DNA polymerase activity control region is deleted is based on a conventional method without any particular limitation.
  • a DNA that encodes the small subunit of Pyrococcus or a vector such as a plasmid containing the DNA is used as a cage and amplified by PCR using a primer that is synthesized based on the base sequence of the region that remains as appropriate.
  • the means to make is mentioned.
  • DNA encoding the small subunit mutant of the present invention include, for example, a portion encoding at least the above-mentioned DNA synthesis activity control region in the gene sequence shown in SEQ ID NO: 2 derived from Pyrococcus horikoshi (499 600)
  • a DNA that has a nucleotide sequence deleted from the DNA A DNA that can be hybridized under stringent conditions with a DNA having a nucleotide sequence complementary to the DNA, and that has DNA polymerase activity and 3'1 5 'exonuclease activity. It may be capable of expressing a protein that can be a small subunit of the thermostable hetero-oligomer enzyme.
  • the stringent conditions in the present invention are 42 ° C or higher, for example, 50 ° C to 65 ° C in 6 X SSC containing 0.5% SDS, 5 X Denharz and 100 ⁇ g / ml salmon sperm DNA. Incubate at room temperature for more than 4 hours under more severe conditions of ° C or 65-70 ° C, then in 6 X SSC, Wash for 10 minutes at 2 ° SSC containing 0.1% SDS for 10 minutes at room temperature, for 10 minutes at room temperature, and in 0.2 X SSC containing 0.1% SDS for 30 minutes at 42 ° C.
  • DNA having a base sequence from which at least the portion encoding the DNA polymerase activity control region (499 600) is deleted 1 to several nucleotides are deleted, substituted or added. Any DNA that can express a protein that can be a small subunit of a thermostable hetero-oligomer enzyme having DNA polymerase activity and 3′-5, exonuclease activity may be used.
  • the ability to combine DNA encoding these small subunit mutants and DNA encoding the above large subunit and to introduce them into a host microorganism so that they can be co-expressed. May be introduced into the host microorganism by ligating the DNA encoding the small subunit mutant and the DNA encoding the large subunit to separate expression vectors such as plasmids. It is preferable that the DNA variant encoding and the DNA encoding the large subunit are introduced into the same expression vector and expressed under the same or two promoters.
  • co-expression as used herein means that two DNAs can be expressed simultaneously in a host and a protein encoded by them can be produced. Examples of host microorganisms to be used include bacteria such as Escherichia coli and Bacillus subtilis, and yeast.
  • thermostable hetero-oligomer enzyme derived from Pyrococcus' holicosi
  • a small subunit a large subunit not containing an intin sequence
  • a vector pET 15b / PolSL (-Intein) co-expressing these and
  • the method for producing the transformant is described in detail in the above-mentioned previous application of the present inventor (Japanese Patent Application No. 2000-116257 (Japanese Patent Laid-Open No. 2001-299348)). As shown below.
  • This culture solution was cultured at 95 ° C. for 2 to 4 days, and then centrifuged and collected.
  • the chromosomal DNA of JCM9974 was prepared by the following method. Collect the cells by centrifugation at 5000rpm for 10 minutes after completion of the culture. The cells are washed twice with 10 mM Tris (pH 7.5) -ImM EDTA solution and then enclosed in an InCert Agarose (FMC) block. The block 1% N-la ⁇ acryloyl sarcosine (l auroy l sarcos ine), by treatment with lmg / ml protease K solution, the chromosomal DNA is separated prepared in Agarose block.
  • FMC InCert Agarose
  • the chromosomal DNA obtained in (2) was partially decomposed with the restriction enzyme Hindlll, and then a fragment of about 40 kb was prepared by agarose gel electrophoresis. This DNA fragment was ligated with Bac vectors pBAC108L and pFOSl completely digested with the restriction enzyme Hindll using T4 ligase. When the former vector P BAC108L was used, the DNA after completion of the binding was immediately introduced into E. coli by electroporation. When the latter vector pFOSl is used, the DNA after the completion of binding is packed into ⁇ phage particles in a test tube with GIGA Pack Gold (manufactured by Stratagene). Introduced. The E. coli population resistant to the chloramphee-chol antibiotics obtained by these methods was designated as one of the C and Fosmid libraries. Clones suitable for covering the chromosome of JCM9974 were selected from the library, and the clones were aligned.
  • restriction enzyme Ndel and BamHI
  • DNA primers were synthesized for the purpose of constructing restriction enzyme (Ndel and BamHI) sites before and after the small subunit structural gene region, and restriction enzyme sites were introduced before and after the gene by PCR: upper primer: PdSl; 5 TTTTGTCGACGTACATATGGATGAATTC GTAAAG-3 ′ (SEQ ID NO: 9; underlined indicates Ndel site);
  • the full-length large subunit gene was cloned into the pGEMEX- ⁇ vector (Promega) in two steps.
  • the first half of the DNA fragment was obtained by PCR using the following two primers: Upper primer: PolLl; 5'-CTCGACTTTAGCATATGGCTCTGATGGAGC-3 '(ia column number 11; underlined indicates Ndel site);
  • This PCR product was completely digested with Ndel and Sail, cloned into the pGEMEX- ⁇ vector, and abbreviated as pGEM / PolL1-2.
  • This PCR product was completely digested with Sail and Nsil and then cloned into the previously prepared pGEM / PolLl-2 to obtain pGEM / PolL containing the full-length large subunit gene.
  • the large subunit gene of p. Horikoshii DNA polymerase contains one intin (encoding a protein intron), so PCR using primers PolL3 and PolL6 (below)
  • the DNA fragment upstream of the intin was amplified by the PCR method, and the DNA fragment downstream of the intin was amplified by the PCR method using the primers PolL5 (below) and PolL4.
  • primers PolL3 and PolL4 the DNA fragment from which intin was removed was amplified by overlap PCR.
  • a plasmid that co-expresses both subunits was constructed.
  • PCR was performed using primers PolSl and PolS3 (below) to introduce a new multicloning site immediately upstream of the BamHI site of pET15b / PolS.
  • BamHI, Nsil, Sail, and SacII sites are encoded in PolS3 in order from the 5'-end.
  • the PCR product was treated with Ndel and BamHI and inserted into pET15b to construct pET15b / PolS (M) containing a multicloning site between the stop codon of the small subunit and the BamHI site.
  • PCR was performed using pGEM / PolL (-Intein) as a truncated DNA and primers PolL7 (below) and PolL2.
  • the resulting product has a new SacII site at the 5'-end, and includes the protein expression unit of pGEM / PolL (-Intein) from the ribosome binding site to the Sail site in the coding region.
  • PolL7 5'-GGTGTCCGCGGCTC ACTATAGGGAGACC AC-3 '(SEQ ID NO: 18; underlined indicates SacII site, bold indicates ribosome binding site of pGEMEX- ⁇ vector) [0037] (10) Expression of recombinant gene E. coli BL21- CodonPlus (DE3)-RIL,
  • the obtained expression plasmids were pET15b / DPl (l-200), pET15b / DPl (33-200), pET15b / DP1 (49-200), pET15b / DP1 (66-200), pET15b / DP1 (85-200), respectively. ), PET15b / DP1 (1-139), pET15b / DP1 (1-166), pET15b / DP1 (33-166), pET15b / DP1 (33-180). The absence of random mutations on the structural gene was confirmed by DNA sequencing.
  • Example 2 is a hetero-oligomer mutant in which the internal sequence is deleted at the N-terminal side of DPI, Po 1D AS (1-200), PolD AS (1-65), PolD AS (140-200 ), PolD AS (167-200), plasmid construction
  • GAG-3 (SEQ ID NO: 29; underlined parts indicate BamHI, Sail, and SacII sites from the left, respectively, bold letters indicate the complementary strand sequence at the 3' end of the DPI gene)
  • Lower primer S2M Furthermore, the amplification product was inserted into pET15b using Ndel and BamHI sites. Next, 2n d PCR was performed using the pET15b vector containing the target insert as a saddle, and the upper primer pET-Sp h; 5 '-C AAGGAATGGTGCATGC AAGGAGATGGC-3' (SEQ ID NO: 30; the underlined part is the Ndel site of the pE T15b vector. (The Sphl site existing at 354 bp upstream is shown) and the lower primer S2M.
  • This amplified product was digested with Sphl and SacII, and replaced with the SpW-SacII fragment of pET15b / PolSL (-Intein), so that two co-expression vectors pET15b / S ⁇ (1-200) L and pET15b / S ⁇ (1-65) L was obtained.
  • Figure 1 shows the base sequence of the primer pair and the position on the DPI gene required for amplification of the DPI gene with the internal sequence deleted by overlap PCR.
  • the co-expression vector pET15b / Pol SL (-Intein) was made into a saddle type, and the following two kinds of DNA primer pairs were used, and the 5 'and 3' fragments of DPI were PCR amplified separately. Since these two fragments are designed so that the former 3 'region and the latter 5' region overlap, the two fragments are then mixed and ligated by overlap PCR using the outer primer. It was.
  • the pET15b vector containing the target insert is made into a saddle shape, and the upper primer pET-Sph; 5'-C AAGGAATGGTGCATGC AAGGAGATGGC-3 '(SEQ ID NO: 30; the underlined part is 354 bp upstream of the Ndel site of the pET15 b vector.
  • PCR reaction was performed using Sp2 site and lower primer S2M.
  • This amplified product was digested with Sphl and SacII, and replaced with the SpW-SacII fragment of pET15b / PolsL (-Intein), so that two co-expression vectors pET15b / S ⁇ (140-200) L and pET15b / SA (167-200) L was obtained.
  • the transformant was cultured in 2YT medium (2 liters) containing ampicillin until the absorption at 600 nm reached 0.6, and then IPTG (Isopropyl- ⁇ -D-thiogalactopyranoside) was added and further cultured at 37 ° C for 3 hours. After incubation, the cells were collected by centrifugation (6,000 rpm, 20 min).
  • the collected cells were suspended in A buffer (50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl) and sonicated in ice. The mixture was further heated at 75 ° C for 15 minutes and then centrifuged (12,000 g, 10 minutes) to obtain a supernatant. This was used as a crude enzyme solution. Next, this crude enzyme solution was added to a Hi trap Q column (5 ml, manufactured by Amersham Biosciences) equilibrated with buffer A, washed with buffer A, and NaCl gradient (0.1-1M). Eluted with.
  • a buffer 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl
  • the elution fraction obtained here was concentrated with Centricon YM-10 (Millipore), added to Superose 12 column (HR 10/30, Amersham Biosciences), and B buffer (50 mM Tris-HCl buffer). (pH 8.0), 200 mM NaCl). The peak fraction was then concentrated with Microcon YM-10 (Millipore) and stored at 4 ° C.
  • Example 5 is a hetero-oligomer mutant lacking an internal sequence at the N-terminal side of DPI, Po ID AS (1-200), PolD AS (1-65), PolD AS (140-200 ), Purification of PolD AS (167-200)
  • the collected cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and disrupted with a French press. The mixture was further heated at 85 ° C for 30 minutes and then centrifuged (27,000 g, 20 minutes) to obtain a supernatant. This was used as a crude enzyme solution.
  • this crude enzyme solution was added to a Hitrap Q column (5 ml, manufactured by Amersham Biosciences) equilibrated with 50 mM Tris-HCl buffer (pH 8.0), washed with the same buffer, Elute with a gradient (0-1M).
  • This eluate is added to a Ni-column (1 ml, Novagen) equilibrated with C buffer (20 mM Tris-HCl buffer (pH 7.9), 0.5 M NaCl, 5 mM imidazole) and contains 25 mM imidazole.
  • the column was washed with 20 mM Tris-HCl buffer (pH 7.9) and 0.5 M Na CI, and eluted with 20 mM Tris-HCl buffer (pH 7.9) and 0.5 M Na CI containing 200 mM imidazole.
  • the elution fraction obtained here was concentrated with Centricon YM-10 (Millipore), loaded onto a Superose 12 column (HR 10/30, Amersham Biosciences), and B buffer (50 mM Tris-HCl buffer). Elution was performed with a solution (pH 8.0) and 200 mM NaCl).
  • the peak fraction containing PolD heteroligomer enzyme was then mixed with an equal volume of 50% glycerol and stored at -20 ° C.
  • the substrate adjustment for measuring the primer extension activity is as follows. First, in 25 1 anneal buffer (10 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl, 1 mM DTT, 50 mM NaCl)
  • the reaction was carried out at 60 ° C with DNA polymerase. The reaction was stopped by adding 40 Stop solutions (containing 95% formamide, 10 mM EDTA, 0.5 mg / ml bromophenol blue) after a predetermined time. The reaction products were boiled for 5 minutes, quenched in ice water, and separated by 12% polyacrylamide gel electrophoresis (PAGE) containing 7M urea using lx TBE buffer. The PAGE pattern was visualized and analyzed with a Fluoro Imager 585 (Amersham Neuroscience).
  • the reaction conditions were the same as those for the primer extension activity measurement except that dNTP was not included in the reaction system.
  • DNA polymerase was added and reacted at 60 ° C for 15 minutes. After a predetermined time, the reaction was stopped by adding 40 1 S top solution (containing 95% formamide, 10 mM EDTA, 0.5 mg / ml bromophenol blue). The reaction products were boiled for 5 minutes, quenched in ice water, and separated by 12% polyacrylamide gel electrophoresis (PAGE) containing 7M urea using lx TBE buffer. The PAGE pattern was visualized and analyzed with a Fluoro Imager 585 (Amersham Biosciences).
  • the amount of protein was quantified using a protein assay system (Bio-Rad) and bovine serum anolebumin as a standard protein. SDS-PAGE was performed according to the Laemmli method, and the protein band was stained with Coomassie Brilliant Blue R-250, and a wide range protein marker (Bio-Rad) was used as a molecular weight marker.
  • Figure 2 shows the SDS-PAGE pattern of the purified enzyme preparation obtained in Example 5.
  • the wild type and DPK1-200) deletion mutant (PolD AS (1-200)) was completely purified.
  • 3'-5 'exonuclease activity was measured using 84 / 34-mer DNA substrate and changing the salt concentration from 0 to 300 mM (0, 100, 150, 200, 300 mM) as shown in Figure 3A. .
  • PolD A S (1-200) showed higher 3'-5 'exonuclease activity compared to the wild type.
  • the effect of sodium chloride is inhibitory, and for both enzymes, 3'-5 'exonuclease activity decreases with increasing salt concentration and is almost undetectable at 0.2% concentration.
  • the DNA polymerase activity was determined as the primer extension activity in the presence of 0.2 M salt using an 84 / 34-mer FAM-labeled double-stranded DNA substrate. It was measured by changing the salt concentration from 0 to 300 mM. In the absence of salt, PolD AS (1-200) had a lower DNA polymerase activity due to the degradation of the substrate by 3 and 5 'exonuclease activity compared to the wild type.
  • DNA polymerase activity of wild-type PolD is sensitive to salt concentration, and the force that decreases with increasing salt concentration is that of PolD AS (1-200), which is resistant to salt concentration, and DNA polymerase activity remains high up to 0.2 M. It was. The level was equivalent to that of wild-type PolD DNA polymerase activity without salt. This indicates that deletion of 0-1 (1-200) confers salt tolerance to the deletion mutant 1 3 010 3 (1-200).
  • PolD wild-type and PolD ⁇ S DNA polymerase reaction (primer extension reaction) in the presence of 0.2 M salt was converted to 60 ° C2 using 84 / 34-mer FAM-labeled double-stranded DNA.
  • the DNA polymerase activity (primer extension activity) of PolD wild type and PolD AS (1-200) in the presence of 0.2 M salt was evaluated by the product length and product amount. As shown in Figure 4, 5PolD AS (1-200) was able to synthesize a full-length product (84-mer) in 5 minutes even in the presence of 0.2 M salt. Then, even after 12.5 minutes, the full-length product could not be synthesized.
  • Primer extension reaction was performed using PolD wild type and PolD A S (1-200) in the presence or absence of 0.2 M salt to quantify the amount of DNA synthesis product.
  • the primer extension reaction was performed at 60 ° C. for 2 minutes using an 84 / 34-mer FAM-labeled double-stranded DNA substrate.
  • the results are shown in FIG. As is clear from the results in Fig. 5, PolD A S (1-200) was able to synthesize twice the product of PolD wild type in the presence of salt. This result indicates that the DPK1-200) deletion from PolD confers DNA polymerase activity equivalent to PolD wild-type in the absence of salt, even in the presence of 0.2 M salt. It was.
  • Example 5 In the presence of 0.2 M NaCl, PolD ⁇ S (1-200) obtained in Example 5 was added to DPl (l-200) and bovine serum albumin (BSA) obtained in Example 4 at each ratio. The mixture was mixed and a primer extension reaction was performed at 60 ° C. for 2 minutes using 84 / 34-mer FAM-labeled double-stranded DNA substrate.
  • the molar ratio of 0-1 (1-200) and 83 to! 3 ⁇ 410 3 (1-200) is 0: 1, 15: 1, 30: 1, 60: 1, 90: 1, 120: 1, respectively. It was.
  • FIG. 7 shows the SDS pattern of the various deletion mutants of DPI (1-200) obtained in Example 4 from which each region of DPI (1-200) has been deleted. As shown in FIG. 7, DPI (1-200) and various deletion mutants of DPI were completely purified.
  • the molar ratios of DPK1-200) wild type and its various deletion mutants to PolD ⁇ S (1-200) are 15: 1, 30: 1, 60: 1, 90: 1, 120: 1.
  • Example 5 Heteroligomer mutants obtained in Example 5, lacking the N-terminal side or internal sequence of various DPIs, PolD AS (1-200), PolD AS (1-65), PolD AS (140- 200), PolD AS (167-200), SDS-PAGE pattern.
  • the hetero-oligomer variants As shown in Figure 9, the hetero-oligomer variants, PolD AS (1-200), PolD AS (1-65), Pol DAS (140-200), PolD AS (167-200) were completely purified and DP2 And two protein bands corresponding to the deleted DPI. From this, it was clarified that all the mutants can form a hetero-oligomer structure and are resistant to heat treatment at 85 ° C for 30 minutes. In this study, 1.6 g of heterooligomer variants were analyzed by SDS-PAGE.
  • a primer extension reaction was performed at 60 ° C for 2 minutes using an 84 / 34-mer FAM-labeled double-stranded DNA substrate, and the primer extension activity was measured. It was.
  • the elongation DNA product was quantified by multiplying the fluorescence intensity measured using Fluor Imager. The standard deviation (SD) value was calculated based on the results of three independent experiments.
  • DPK1-200 DPK1-200 and its eight deletion mutants (DP1 (33-200), (DP1 (49-200), DP1 (66-200), (DP1 (85-200), (DP1 (1- 139), DP1 (1-166), DP1 (33-180, DP1 (33-166), respectively, with PolD AS (1-200) at a molar ratio of 60: 1 to give a 51-mer FAM
  • PolD AS (1-200 a molar ratio of 60: 1 to give a 51-mer FAM
  • the labeled single-stranded DNA was used as a substrate and reacted at 60 ° C for 15 minutes to measure the 3'-5 'exonuclease activity, and the various hetero-oligomer mutants used in D.
  • salt acts to inhibit 3 'and 5' exonuclease activity, so 3'-5 'exonuclease activity was measured in this test. There was no salt.
  • accession number of each PolD sequence is P. horikoshii, BA000001; P. abyssi, AJ 248283; P. foriosus, AE010127.
  • DPl 34-residue polypeptide, DPl (167), which is a DNA polymerase activity (primer extension activity) control region and a region that suppresses 3′-5 ′ exonuclease activity in the absence of NaCl. -200) is clearly conserved in the DPI subunit of PolD from the genus Pyrococcus. Therefore, if this homologous region is deleted, the DNA synthesis activity and functional modification to 3'_5 'exonuclease activity observed in P. horikoshi Yurai PolD can be achieved with P. abyssi and P. foriosus PolD enzymes. It can be reproduced.

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Abstract

Disclosed is a novel thermostable family-D DNA polymerase (PolD) mutant which is thermostable, has a 3'-5' exonuclease activity (i.e., a proofreading function for DNA chain elongation), has a potent primer elongation activity, and can replicate a DNA region of interest within a short period even under high salt concentration conditions. The mutant can be produced from a thermostable heterooligomeric enzyme which is composed of a small subunit and a large subunit containing no intein sequence and has a DNA polymerase activity and a 3'-5' exonuclease activity, and can be produced by removing a DNA synthesis activity-regulating region from the small subunit in the thermostable heterooligomeric enzyme.

Description

DNAポリメラーゼ変異体  DNA polymerase mutant
技術分野  Technical field
[0001] 本発明は DNAポリメラーゼ D(PolD)変異酵素遺伝子及び該遺伝子にコードされる D NAポリメラーゼ D(PolD)変異酵素に関する。  The present invention relates to a DNA polymerase D (PolD) mutant enzyme gene and a DNA polymerase D (PolD) mutant enzyme encoded by the gene.
背景技術 Background art
[0002] DNAポリメラーゼは DNAシークェンシング反応、遺伝子増幅反応 (PCR反応)、 DNA の放射活性標識、変異遺伝子の試験管内合成等に有用な酵素である。現在までに 知られている DNAポリメラーゼは、アミノ酸配列の共通性から、大きく 4つのファミリー に分類することができる。その中で一般的に遺伝子操作実験用試薬として利用され て!、るのは、大腸菌 DNAポリメラーゼ Iや好熱菌サーマスアクアティカス DN Aポリメラ ーゼ（Taq DNAポリメラーゼ）に代表されるファミリー Aと、 T4ファージ DNAポリメラー ゼに代表されるファミリー Bに属するものである。今までに至適温度の異なる種々の D NAポリメラーゼが細菌や動植物から発見されている力 多くが常温生物由来のため 、耐熱性に乏しぐ铸型 DNAの 94°C以上での熱変性反応を含む PCR反応等には不 適当であった。また、耐熱性 DNAポリメラーゼとして Taq DNAポリメラーゼ等のファミリ 一 A酵素 (PolA)が巿販されて 、るが、 、ずれも 3'- 5'校正ェキソヌクレアーゼ活性を欠 くため、 PCR反応等のポリメラーゼ反応中にエラーを誘発しやすぐ正確性を求めら れる PCR反応等には不向きである。また、高耐熱性で 3'-5'校正ェキソヌクレアーゼ活 性を持つファミリー B酵素 (PolB)力 パイロコッカスゃサーモコッカス等の超好熱性古 細菌より単離され、市販されているがプライマー伸長活性が弱ぐ長鎖 DNAの PCR反 応には適さない。さらに、 PolAや PolBを含む既存の DNAポリメラーゼは铸型 DNAの認 識にイオン結合等の相互作用を用いるため、高塩濃度下では DNA合成能は低ぐ高 塩濃度下での遺伝子増幅反応には不向きであった。 発明の開示 発明が解決しょうとする課題 [0002] DNA polymerase is an enzyme useful for DNA sequencing reaction, gene amplification reaction (PCR reaction), radioactivity labeling of DNA, in vitro synthesis of mutant genes, and the like. DNA polymerases known to date can be broadly classified into four families based on the commonality of amino acid sequences. Among them, it is generally used as a reagent for genetic manipulation experiments! The family A represented by Escherichia coli DNA polymerase I and thermophile Thermus aquaticus DN A polymerase (Taq DNA polymerase), It belongs to family B represented by T4 phage DNA polymerase. To date, various DNA polymerases with different optimum temperatures have been discovered in bacteria, animals and plants. Most of them are derived from room temperature organisms. It was inappropriate for the PCR reaction. Furthermore, Taq DNA polymerase and other family 1A enzymes (PolA) have been marketed as thermostable DNA polymerases, however, since they lack 3'-5 'proofreading exonuclease activity, PCR reactions, etc. It is not suitable for PCR reactions that induce errors during the polymerase reaction and require immediate accuracy. In addition, it is a family B enzyme (PolB) with high heat resistance and 3'-5 'proofreading exonuclease activity. It is isolated from a hyperthermophilic archaea such as Pyrococcus thermococcus and is commercially available, but primer extension It is not suitable for PCR reaction of long DNA with weak activity. In addition, existing DNA polymerases, including PolA and PolB, use interactions such as ion binding for the recognition of vertical DNA. Therefore, DNA synthesis capacity is low at high salt concentrations, and gene amplification reactions at high salt concentrations are low. Was unsuitable. Disclosure of the invention Problems to be solved by the invention
[0003] 遺伝子増幅法としては、市販の耐熱性 PolAと PolBを用いる従来型 PCR法や、 DNA 合成鎖置換活性 (Displacement活性)の強い φ 29DNAポリメラーゼを用いる常温 PCR 法が開発されているが、既存の PolAと PolB酵素は複製できる領域は短ぐ数 10kbが 上限である。また、合成速度も 30b/secと遅い。一方、 φ 29DNAポリメラーゼは常温で 遺伝子増幅ができるため、増幅反応に高価な装置は必要なぐ簡便に行うことができ るが、ランダムプライマーを用いるため、複製される領域は比較的短ぐ長鎖の DNA 合成産物は得難い。さらに、上記の既存酵素を用いて、血液、体液等から直接的に DNAを増幅することは困難である。それは、いずれの既存酵素も高塩濃度で DNA合 成活性が低下するためであり、反応液中の塩濃度を下げるための、脱塩操作が必須 である。耐熱性で、 DNA鎖伸長の校正機能である 3'-5'ェキソヌクレアーゼ活性を有 し、かつプライマー伸長活性が強ぐ高塩濃度下でも目的 DNA領域を短時間で複製 できる新規耐熱性ファミリー D
Figure imgf000003_0001
廉価で安定に供給 する事が強く求められていた。したがって、本発明の課題は、このような要求を満たす 新規酵素を提供する点にある。 [0003] As gene amplification methods, conventional PCR methods using commercially available thermostable PolA and PolB, and room temperature PCR methods using φ29DNA polymerase with strong DNA synthetic strand displacement activity (Displacement activity) have been developed. The existing PolA and PolB enzymes can be replicated within a short range of several tens of kb. The synthesis speed is also slow at 30b / sec. On the other hand, φ29 DNA polymerase can amplify genes at room temperature, so an expensive apparatus for amplification reaction can be performed as easily as necessary.However, since a random primer is used, the region to be replicated is a relatively short long chain. DNA synthesis products are difficult to obtain. Furthermore, it is difficult to directly amplify DNA from blood, body fluids, etc. using the above existing enzymes. This is because the DNA synthesis activity of any existing enzyme decreases at high salt concentrations, and desalting is essential to reduce the salt concentration in the reaction solution. A new thermostable family that has 3'-5 'exonuclease activity, a heat-resistant proofreading function for DNA strand elongation, and can replicate the target DNA region in a short time even under high salt concentrations where the primer extension activity is strong. D
Figure imgf000003_0001
There was a strong demand for a cheap and stable supply. Therefore, the subject of this invention is providing the novel enzyme which satisfy | fills such a request | requirement.
課題を解決するための手段  Means for solving the problem
[0004] PolDは 2つのサブユニット（Lと S)力 構成される高分子量へテロオリゴマー構造を 有し、サブユニット単独発現では極めて不安定であった。我々は、先に両サブュ-ッ トの共発現系を開発し、 PolDの高発現に成功している。組み換え法で得られた PolD は極めて高!ヽ耐熱性と DNA合成活性、 3， -5，ェキソヌクレアーゼ活性を有して 、た。  [0004] PolD has a high molecular weight hetero-oligomer structure composed of two subunits (L and S), and was extremely unstable when expressed alone. We have previously developed a co-expression system for both subunits and succeeded in high expression of PolD. PolD obtained by the recombination method has extremely high heat resistance, DNA synthesis activity, 3, -5, exonuclease activity.
本発明者は、このような成果をふまえ、さらに研究を進め、 PolDの 2つのサブュ- ット（Lと S)の欠失変異体を作成し、 DNA合成活性ゃェキソヌクレア-ゼ活性発現に必 須な領域の探索を行った。そして PolDヘテロオリゴマー野生型分子および欠失分子 等の生化学的機能解析の結果、高塩濃度下 (0.2M以上の食塩濃度)でも高い DNA 合成能を保持して!/ヽる欠失変異酵素を見!ヽだし、本発明に至った。  Based on these results, the present inventor has further studied and created deletion mutants of the two PolD subunits (L and S), which are essential for the expression of DNA synthesis activity and exonuclease activity. We searched for a new area. And as a result of biochemical functional analysis of PolD hetero-oligomer wild-type molecule and deletion molecule, etc., the deletion mutant enzyme that retains high DNA synthesis ability even under high salt concentration (salt concentration of 0.2M or more)! I came to the present invention.
[0005] すなわち本発明は以下のとおりである。  That is, the present invention is as follows.
(1) 小サブユニットとインティン配列を含有しない大サブユニットとからなり、 DNAポ リメラーゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有するパイロコッカス属古細菌 由来の耐熱性へテロオリゴマー酵素において、小サブユニットが、 DNA合成活性制 御領域であって、かつ 3 '—5，ェキソヌクレアーゼ活性抑制領域が削除されて 、るこ とを特徴とする、上記耐熱性へテロオリゴマー酵素変異体。 (1) Pyrococcus archaea consisting of a small subunit and a large subunit that does not contain an intin sequence, and has DNA polymerase activity and 3, 15, exonuclease activity In the heat-resistant hetero-oligomer enzyme derived from the enzyme, the small subunit is a DNA synthesis activity control region, and the 3'-5, exonuclease activity suppression region is deleted, The thermostable hetero-oligomer enzyme mutant.
(2) 小サブユニットから削除される領域が、配列番号 33に示されるアミノ酸配列ある いは該アミノ酸配列と 45%以上の相同性を有するアミノ酸配列を少なくとも含むこと を特徴とする。上記（1)に記載の耐熱性へテロオリゴマー酵素変異体。 (2) The region deleted from the small subunit includes at least the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having 45% or more homology with the amino acid sequence. The thermostable hetero-oligomer enzyme mutant according to (1) above.
(3) 以下に示される大サブユニットと小サブユニット変異体と力 なり、かつ DNAポ リメラーゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有する耐熱性へテロオリゴマー 酵素変異体。 (3) A thermostable hetero-oligomer enzyme mutant that has the power of the following large subunits and small subunit mutants and has DNA polymerase activity and 3,15, exonuclease activity.
1) a)配列番号 5に示されるアミノ酸配列を有するか、あるいは b) a)のアミノ酸配列 にお ヽて 1または数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を 有する大サブユニット。 1) a) having the amino acid sequence shown in SEQ ID NO: 5 or b) having a large amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence of a) Sub unit.
2) a)配列番号 1に示されるアミノ酸配列において、少なくとも 167〜200番目の領 域が削除されたアミノ酸配列を有する力、あるいは b) a)のアミノ酸配列において、 1 乃至数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を有する小サブ ユニット変異体。  2) a) the ability to have an amino acid sequence in which at least the region from 167 to 200 is deleted in the amino acid sequence shown in SEQ ID NO: 1, or b) one to several amino acid residues in the amino acid sequence of a) Is a small subunit variant having an amino acid sequence deleted, substituted or added.
(4) 上記（3)に記載の大サブユニットをコードする DNAと、同小サブユニット変異体 をコードする DNAと力 なる組み合わせ。 (4) A powerful combination of the DNA encoding the large subunit described in (3) above and the DNA encoding the mutant small subunit.
(5) パイロコッカス属古細菌由来の、小サブユニットとインティン配列を含有しない 大サブユニットからなる耐熱性へテロオリゴマー酵素における小サブユニットにおい て、配列番号 33に示されるアミノ酸配列あるいは該アミノ酸配列と 45%以上の相同 性を有するアミノ酸配列を少なくとも含む領域が削除され、かつ、大サブユニットとへ テロオリゴマーを構成したとき、 DNAポリメラーゼ活性及び 3'— 5'ェキソヌクレア一 ゼ活性を有するものであることを特徴とする、耐熱性へテロオリゴマー酵素の小サブ ユニット変異体。 (5) The amino acid sequence shown in SEQ ID NO: 33 or the amino acid sequence thereof in the small subunit of the thermostable hetero-oligomer enzyme derived from Pyrococcus archaea and comprising a large subunit not containing a small subunit and an intin sequence And a region containing at least an amino acid sequence having 45% or more homology with the large subunit. A small subunit mutant of a thermostable hetero-oligomer enzyme characterized by having a DNA polymerase activity and a 3'-5 'exonuclease activity when a telo-oligomer is constituted.
(6) a)配列番号 1に示されるアミノ酸配列において、少なくとも 167〜200番目の領 域が削除されたアミノ酸配列を有する力、あるいは b) a)のアミノ酸配列において、 1ま たは数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を有し、かつ、ィ ンティン酉己列を含まな 、大サブユニットとヘテロオリゴマーを構成したとき、 DNAポリ メラーゼ活性及び 3， 一 5 'ェキソヌクレアーゼ活性を有するものであることを特徴とす る、上記（5)に記載の耐熱性へテロオリゴマー酵素の小サブユニット変異体。 (6) a) the ability to have an amino acid sequence in which at least the 167th to 200th region is deleted in the amino acid sequence shown in SEQ ID NO: 1, or b) one or several amino acids in the amino acid sequence of a) When the amino acid residue has an amino acid sequence deleted, substituted or added, and does not contain a tin sequence, it constitutes a large subunit and a hetero-oligomer, and DNA polymerase activity and 3, 1 ' The small subunit mutant of the thermostable hetero-oligomer enzyme according to (5) above, which has exonuclease activity.
(7) 上記（6)に記載の小サブユニット変異体をコードする DNA。 (7) A DNA encoding the small subunit mutant according to (6) above.
(8) 小サブユニットと大サブユニットからなり、 DNAポリメラーゼ活性及び 3'— 5'ェ キソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素における小サブユニット をコードする DNAであって、 配列番号 2に示される塩基配列において、 499〜600 番目の領域が削除された塩基配列を有するか、あるいは、該塩基配列において 1又 は数個のヌクレオチドが欠失、置換、又は付加された塩基配列を有する DNA。 (8) DNA encoding a small subunit in a thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′-5 ′ exonuclease activity, In the base sequence shown, DNA having the base sequence from which the 499th to 600th region has been deleted, or having one or several nucleotides deleted, substituted, or added in the base sequence .
(9) 小サブユニットと大サブユニットからなり、 DNAポリメラーゼ活性及び 3'— 5'ェ キソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素における小サブユニット をコードする DNAであって、請上記（8)に記載の DNA、あるいは該 DNAと相補の配 列を有する DNAとストリンジェントな条件下、ハイブリダィズする DNA。 (9) A DNA encoding a small subunit in a thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′-5 ′ exonuclease activity. Or a DNA that hybridizes under stringent conditions with the DNA described in) or a DNA having a sequence complementary to the DNA.
(10) 上記（7)〜（9)の 、ずれかに記載の DNAを含む組み換えベクター (10) A recombinant vector comprising the DNA according to any one of (7) to (9) above
(11) 上記請求項（7)〜（9)のいずれかに記載の DNA及び請求項 2に記載の大サ ブユニットをコードする DNA力 共発現可能に含むことを特徴とする、組み換えべクタ (12) 上記（10)又は（11)に記載の組み換えベクターが導入されていることを特徴 とする、形質転換体。 発明の効果 (11) A recombinant vector comprising the DNA according to any one of the above claims (7) to (9) and the DNA encoding the large subunit according to claim 2 so as to be capable of co-expression. (12) A transformant, wherein the recombinant vector according to (10) or (11) is introduced. The invention's effect
[0006] 本発明により、高温かつ高塩濃度環境下で長鎖 DNAを高速で正確に複製する DN Aポリメラーゼが提供でき、該酵素は Long-PCR等に有用である。また、当該酵素を用 いた遺伝子配列解析に関する新手法の開発が可能になる。 図面の簡単な説明  [0006] According to the present invention, a DNA polymerase capable of accurately replicating long-chain DNA at high speed under high temperature and high salt concentration environment can be provided, and the enzyme is useful for Long-PCR and the like. In addition, it will be possible to develop new methods for gene sequence analysis using the enzyme. Brief Description of Drawings
[0007] [図 l]overlap PCR法による内部配列が欠失した DPI遺伝子（ Δ S (140-200)及び Δ S (167-200) )の増幅に必要なプライマー対の塩基配列と DPI遺伝子上の位置を示 す図である。  [0007] [Fig. L] Base sequence of primer pair necessary for amplification of DPI genes (ΔS (140-200) and ΔS (167-200)) lacking internal sequence by overlap PCR method and DPI gene FIG.
[図 2]PolDヘテロオリゴマー野生型と DP1(1- 200)の欠失変異体の SDS-PAGEパター ンを示す図である。 Mは分子量マーカーを示す。  FIG. 2 is a diagram showing SDS-PAGE patterns of PolD hetero-oligomer wild type and DP1 (1-200) deletion mutant. M represents a molecular weight marker.
[図 3]食塩濃度の PolDヘテロオリゴマー野生型と DPK1-200)の欠失変異体の 3し 5'ェ キソヌクレアーゼ活性 (A)と DNAポリメラーゼ活性 (B)へ与える影響を示す図である。- Enzymeは酵素反応系から DNAポリメラーゼを除いたコントロールサンプルを示す。  FIG. 3 is a graph showing the effects of a salt concentration of PolD heterooligomer wild type and DPK1-200) deletion mutant on 3 and 5 ′ exonuclease activity (A) and DNA polymerase activity (B). -Enzyme is a control sample obtained by removing DNA polymerase from the enzyme reaction system.
[図 4]0.2 M食塩存在下と非存在下での、 PolD野生型と PolD A S (1-200)の DNAポリ メラーゼ活性の経時変化を示す図である。  FIG. 4 is a graph showing changes over time in DNA polymerase activity of PolD wild type and PolD A S (1-200) in the presence and absence of 0.2 M sodium chloride.
[図 5]0.2 M食塩存在下と非存在下での、 PolD野生型と PolD A S (1-200)の DNAポリ メラーゼ活性の定量試験の結果を示す図である。左は 0.2 M食塩存在下と非存在下 でプライマー伸長反応を行った産物の電気泳動パターンを示し、右は該電気泳動パ ターンカゝら計算された、全プライマー量に対する伸長産物の割り合 ヽを示す。  FIG. 5 is a diagram showing the results of a quantitative test of DNA polymerase activity of PolD wild type and PolD A S (1-200) in the presence and absence of 0.2 M sodium chloride. The left shows the electrophoresis pattern of the product of the primer extension reaction in the presence and absence of 0.2 M salt, and the right shows the ratio of the extension product to the total primer amount calculated from the electrophoresis pattern. Show.
[図 6]DP1(1- 200)添カ卩による、 PolD A S (1-200)の DNAポリメラーゼ活性の変化を示 す図である。  FIG. 6 shows changes in the DNA polymerase activity of PolD A S (1-200) by DP1 (1-200) supplementation.
[図 7]DPI ( 1 200)及びその各種欠失変異体の SDS— PAGEパターンを示す図で ある。 Mは分子量マーカーを示す。 FIG. 7 is a diagram showing SDS-PAGE patterns of DPI (1 200) and its various deletion mutants. is there. M represents a molecular weight marker.
[図 8]DP1 (1— 200)及びその各種欠失変異体による、 PolD A S (1-200)の DNAポリ メラーゼ活性の抑制能を測定した結果を示す図である。  FIG. 8 is a graph showing the results of measuring the ability of PolD A S (1-200) to inhibit DNA polymerase activity using DP1 (1-200) and various deletion mutants thereof.
[図 9]PolDヘテロオリゴマー変異体を構成する DPIの N末端と欠失位置 (A) 及びこ れら酵素の SDS-PAGEパターン（B)を示す図である。 Mは分子量マーカーを示す。  FIG. 9 is a diagram showing the N-terminus and deletion position (A) of DPI constituting the PolD hetero-oligomer mutant and the SDS-PAGE pattern (B) of these enzymes. M represents a molecular weight marker.
[図 10]ヘテロオリゴマー変異体、 PolD A S (1-200)、 PolD A S (1-65)、 PolD A S (140-2 00)、 PolD A S (167-200)のプライマー伸長活性を測定した結果を示す図である。  [Fig. 10] The results of measuring the primer extension activity of the hetero-oligomer mutants, PolD AS (1-200), PolD AS (1-65), PolD AS (140-200), PolD AS (167-200) FIG.
[図 ll]DPl(l-200)及びその各種欠失置換体の 3し5'ェキソヌクレアーゼ活性抑制機 能、及び各種へテロオリゴマーの 3'-5'ェキソヌクレアーゼ活性を測定した結果を示 す図である。  [Figure ll] The results of measuring the 3'-5 'exonuclease activity inhibitory function of DPl (l-200) and its various deletions and the 3'-5' exonuclease activity of various heterooligomers. FIG.
[図 12]DP1 (167— 200)領域についての各 PolD酵素におけるアミノ酸配列ァラィメ ントを示す図である。  FIG. 12 is a view showing amino acid sequence alignments in each PolD enzyme for the DP1 (167-200) region.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0008] 本発明は、小サブユニットとインティン配列を含有しない大サブユニットとからなり、 DNAポリメラーゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有するパイロコッカス属 古細菌由来の耐熱性へテロオリゴマー酵素において、小サブユニットにおける DNA 合成活性制御領域であって、かつ 3'— 5'ェキソヌクレアーゼ活性抑制領域を削除 することにより、該酵素の高塩濃度下における DNA合成活性 (プライマー伸長活性） を向上させるとともに、 3 '— 5 'ェキソヌクレアーゼ活性を著しく高めたものである。  [0008] The present invention relates to a thermostable hetero-oligomer derived from Pyrococcus archaea having a DNA polymerase activity and 3,15, exonuclease activity, comprising a small subunit and a large subunit not containing an intin sequence. In the enzyme, by removing the 3'-5 'exonuclease activity suppression region, which is the DNA synthesis activity control region in the small subunit, the DNA synthesis activity (primer extension activity) of the enzyme under high salt concentration And 3′—5 ′ exonuclease activity is significantly enhanced.
[0009] 小サブユニットとインティン配列を削除した大サブユニットからなり、 DNAポリメラー ゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素は 、本発明者等の先の出願 (特願 2000— 116257 (特開 2001— 299348号公報）に 記載されている。この出願の発明は、パイロコッカス'ホリコシ由来の、 DNAポリメラー ゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素の 大サブユニットにお 、てインティン配列が存在することを初めて見 、だし、該インティ ン配列を除去することにより、該酵素のプライマー伸長活性が著しく向上するという知 見に基づきなされたものである。このパイロコッカス ·ホリコシ由来の耐熱性へテロオリ ゴマー酵素における小サブユニットのアミノ酸配列及びその遺伝子の塩基配列は、 それぞれ、配列番号 1及び 2に示され、また、同大サブユニットのアミノ酸配列及びそ の遺伝子の配列は配列番号 3及び 4にそれぞれ示され、インティン部分のアミノ酸配 列はその 955〜1120番目、同塩基配列は 2863〜3360番目の領域である。 [0009] A thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit from which the intin sequence has been deleted, and having a DNA polymerase activity and 3,15, exonuclease activity, is a prior application of the present inventors. (Described in Japanese Patent Application No. 2000-116257 (Japanese Patent Application Laid-Open No. 2001-299348). The invention of this application has a DNA polymerase activity and 3,15, exonuclease activity derived from Pyrococcus horicosi. For the first time, the presence of an intin sequence was found in the large subunit of a thermostable hetero-oligomer enzyme, but it was found that removing the intin sequence significantly improved the primer extension activity of the enzyme. The amino acid sequence of the small subunit in the thermostable heteroligomer enzyme derived from Pyrococcus horikoshi and Nucleotide sequence of the gene, The amino acid sequence of the same subunit and the sequence of the gene are shown in SEQ ID NOs: 3 and 4, respectively, and the amino acid sequence of the intin portion is 955 to 1120, respectively. The base sequence is the 2863-3360th region.
インティン部分が除去された大サブユニットのアミノ酸配列及びその遺伝子の塩基 配列を配列番号 5及び 6にそれぞれ示す。  The amino acid sequence of the large subunit from which the intin moiety has been removed and the base sequence of the gene are shown in SEQ ID NOs: 5 and 6, respectively.
[0010] また、本発明の耐熱性へテロオリゴマー酵素変異体の作成するために用いる酵素 としては、このようにインティン配列を除去したものばカゝりでなぐもともとインティン配 列を含有しない大サブユニットを有するパイロコッカス属の耐熱性へテロダイマー酵 素であってもよい。このような酵素としては、例えば、パイロコッカス'フリオサス由来の 耐熱性 DNAポリメラーゼを挙げることができる。この酵素における大サブユニット及 び小サブユニットのアミノ酸配列、遺伝子配列は、それぞれ「Microbial Genome Database (http： / / mbgd. genome, ad. jpZ)」に掲載されている。  [0010] In addition, as the enzyme used for preparing the thermostable hetero-oligomer enzyme mutant of the present invention, a large sub that does not originally contain an intin sequence can be obtained by removing the intin sequence. It may be a Pyrococcus heat-resistant heterodimer enzyme having a unit. Examples of such an enzyme include thermostable DNA polymerase derived from Pyrococcus' Friosus. The amino acid sequences and gene sequences of the large subunit and small subunit of this enzyme are listed in the “Microbial Genome Database (http: // mbgd. Genome, ad. JpZ)”.
[0011] ノイロコッカス 'ホリコシにおける、上記耐熱性へテロダイマー酵素（耐熱性 DNAポリ メラーゼ;以下 PolDという場合がある）の小サブユニットにおける、 DNA合成活性制 御領域は、該小サブユニットのァミノ末端側の 167— 200番目の領域であり、該領域 は 3，一 5'ェキソヌクレアーゼ活性抑制領域でもある。このことは、以下の実験により 解明された。  [0011] The control region of DNA synthesis activity in the small subunit of the above-mentioned thermostable heterodimer enzyme (thermostable DNA polymerase; hereinafter sometimes referred to as PolD) in Neurococcus' holicosi is the amino terminal of the small subunit. The 167th-200th region on the side, which is also a 3, 15 'exonuclease activity suppression region. This was clarified by the following experiment.
すなわち、パイロコッカス'ホリコシ由来で、ァミノ末端にヒスチジンタグが付加した小 サブユニットとインティンを含まな ヽ大サブユニットを共発現させる発現プラスミド、 pE T15b/PolSL(-Intein)を基に、 PolDの大小サブユニット（DP2と DPI)の欠失変異体を 作成し、 DNAポリメラーゼ活性ゃェキソヌクレア-ゼ活性発現に必須な領域の探索を 行った。なお、 pET15b/P0lSL(-Intein)は、上記出願明細書にその製法とともに詳述さ れている。 That is, based on pE T15b / PolSL (-Intein), an expression plasmid derived from Pyrococcus' holikoshi that co-expresses a small subunit with a histidine tag added to the amino terminal and a large subunit that does not contain intin. We created deletion mutants of large and small subunits (DP2 and DPI), and searched for regions essential for the expression of DNA polymerase activity and exonuclease activity. Note that pET15b / P 0 1SL (-Intein n ) is described in detail in the above application specification together with its production method.
[0012] その結果、まず、 DPIのァミノ末端側ドメイン、 200残基が欠失したヘテロオリゴマー 変異体、 PolD A S(l-200)は野生型 PolDに較べ、著しくェキソヌクレア-ゼ活性が向上 することが明ら力となった。また、この反応系に欠失分子、小サブユニット（DPI)のアミ ノ末端側ドメインの 1〜200番目の領域が欠失したタンパク質 DPK1-200)を大過剰に カロえると、ェキソヌクレア-ゼ活性が野生型 PolDレベルまで阻害された。このことは、 D PIサブユニットのァミノ末端側ドメイン、 DP1(1- 200)がカルボキシル末端側 Mrell様ド メイン、 DP1(201- 622)のェキソヌクレアーゼ活性を負に制御して ヽることを示す。 As a result, first, the DPI amino-terminal domain, a hetero-oligomer mutant lacking 200 residues, PolD AS (l-200) has significantly improved exonuclease activity compared to wild-type PolD. Clearly became a force. In addition, when a deletion molecule, a protein DPK1-200 in which the 1st to 200th region of the amino-terminal domain of the small subunit (DPI) is deleted, is excessively removed, exonuclease activity is observed. Was inhibited to wild-type PolD levels. This means that D It is shown that DP1 (1-200), an amino-terminal domain of the PI subunit, negatively regulates the exonuclease activity of the carboxyl-terminal Mrell-like domain, DP1 (201-622).
[0013] 次に、この 200残基力 なるドメイン DPK1-200)のどこの領域が直接的にェキソヌク レアーゼ活性阻害に働いている力を明らかにするために、 DP1(1- 200)分子をさらに 小分子化した、欠失タンパク質を造成した。これを DPIサブユニットのァミノ末端側ドメ イン、 200残基が欠失したヘテロオリゴマー変異体、 PolD A S(l-200)の反応系に加え 、ェキソヌクレアーゼ活性阻害の程度を評価し、阻害機能を有するペプチド領域を調 ベた結果、 DP1 1-200)ドメインの C末端に局在する 34アミノ酸残基領域、 DP1(167 - 20 0；配列番号 33)が酸性かつ両親媒性の性質を有しており、そのイオン性相互作用と 疎水性相互作用を介して PolDヘテロオリゴマーの 3，一 5，ェキソヌクレア-ゼ活性と D NAポリメラーゼ活性を各々コントロールすることが明らかになった。  [0013] Next, in order to elucidate the force of which region of this 200-residue domain DPK1-200) directly acts on inhibition of exonuclease activity, the DP1 (1-200) molecule was further modified. A deletion protein with a small molecule was constructed. In addition to the DPI subunit domain at the amino terminal side, the hetero-oligomer mutant lacking 200 residues, and PolD AS (l-200) reaction system, the degree of inhibition of exonuclease activity was evaluated and its inhibitory function As a result of investigating the peptide region having a DP1 1-200) domain, the 34 amino acid residue region located at the C-terminus of the domain, DP1 (167-200; SEQ ID NO: 33) has acidic and amphiphilic properties. It was revealed that the 3,5,5 exonuclease activity and DNA polymerase activity of PolD hetero-oligomers were controlled through their ionic and hydrophobic interactions.
[0014] さらに、 DP 167-200)領域を欠失した PolDヘテロオリゴマー変異体の生化学的機 能解析の結果、高塩濃度下 (0.2M以上の食塩濃度）でも高い DNAポリメラーゼ活 性を保持していることを確認し、一方、野生型 PolDヘテロオリゴマー酵素は 0.2M食塩 存在下では DNAポリメラーゼ活性は大きく低下した。  [0014] Furthermore, as a result of biochemical functional analysis of a PolD hetero-oligomer mutant lacking the DP 167-200) region, it retains high DNA polymerase activity even under high salt concentrations (salt concentration of 0.2M or higher). On the other hand, the wild-type PolD hetero-oligomer enzyme significantly decreased the DNA polymerase activity in the presence of 0.2 M sodium chloride.
以下に上記小サブユニットのアミノ基末端 200残基 (DPI (1— 200) )のアミノ酸配 列及び塩基配列を示す。 DPI (167— 200 ;配列番号 33)は下線部で示される。  The amino acid sequence and base sequence of the amino group terminal 200 residues (DPI (1-200)) of the above small subunit are shown below. DPI (167-200; SEQ ID NO: 33) is underlined.
[0015] 本酵素の小サブユニットのァミノ末端側ドメイン、 DPl(l-200)、の遺伝子配列（配列 番号 7)  [0015] The gene sequence of the amino subunit side domain DPl (l-200) of the small subunit of the enzyme (SEQ ID NO: 7)
[化 1]  [Chemical 1]
atggatgaattcgtaaagggattaatgaaaaacgggtaccttataactccttctgcctat tacctcttagttggccatttcaatgagggaaagttctcgctcatagaattgataaaattt gcaaaatccagggaaacgttcatcatagatgatgagattgctaatgaattccttaagtcc atcggggctgaagttgaacttccacaggaaataaaggagggttacatttccactggagaa ggttcacagaaggttccagatcatgaagaactggaaaaaataacgaatgaatctagtgta gagagttctatttccactggagaaactccaaaaactgaggaactacagcctactttagat atattagaggaagaaataggggacattgaaggtggagagagttctatttccactggagat gaagtccccgaagtggaaaataataatggaggtacggtggtagttttcgataaatacggc tatcccttcacgtatgttccagaggaaattgaggaagaactagaagagtatcctaagtat gaagatgtaacaattqagatcaatcctaacctcgaagtcgttccgatagaaaaagactat [0016] 本酵素の小サブユニットのァミノ末端側ドメイン、 DPK1-200)のアミノ酸配列（配列 番号 8) atggatgaattcgtaaagggattaatgaaaaacgggtaccttataactccttctgcctat tacctcttagttggccatttcaatgagggaaagttctcgctcatagaattgataaaattt gcaaaatccagggaaacgttcatcatagatgatgagattgctaatgaattccttaagtcc atcggggctgaagttgaacttccacaggaaataaaggagggttacatttccactggagaa ggttcacagaaggttccagatcatgaagaactggaaaaaataacgaatgaatctagtgta gagagttctatttccactggagaaactccaaaaactgaggaactacagcctactttagat atattagaggaagaaataggggacattgaaggtggagagagttctatttccactggagat gaagtccccgaagtggaaaataataatggaggtacggtggtagttttcgataaatacggc tatcccttcacgtatgttccagaggaaattgaggaagaactagaagagtatcctaagtat gaagatgtaacaattqagatcaatcctaacctcgaagtcgttccgatagaaaaagactat [0016] Amino-terminal sequence of the small subunit of this enzyme, DPK1-200) (SEQ ID NO: 8)
[化 2]  [Chemical 2]
mdef vkglmk ngylitpsay yl lvghf neg kf sl iel ikf aks retr i id aeianef Iks igaevelpqe ikegyistge gsqkvpdhee lekitnessv essistgetp kteelqpt ld i leeeigdie ggessistgd evpevennng gtvvvf dkyg ypf tyvpeei eeeleeypky edvtieinpn le vpiekdy  mdef vkglmk ngylitpsay yl lvghf neg kf sl iel ikf aks retr i id aeianef Iks igaevelpqe ikegyistge gsqkvpdhee lekitnessv essistgetp kteelqpt ld i leeeigdie ggessistgd evpevennng gtvvypvkydy
[0017] 上記 DPI (167— 200)と相同する配列は、パイロコッカス属古細菌由来の PolD酵 素の小サブユニットに広く存在し、上記 DNA合成活性制御領域はパイロコッカス属 細菌に広く保存されており、図 12に、該領域の配列ァライメントを示す。 [0017] The sequence homologous to DPI (167-200) is widely present in the small subunit of the PolD enzyme derived from Pyrococcus archaea, and the DNA synthesis activity control region is widely conserved in Pyrococcus bacteria. FIG. 12 shows a sequence alignment of the region.
したがって、少なくとも、パイロコッカス属菌由来の PolDにおける DPI (167- 200) と相同する領域を欠失させれば、ノイロコッカス ·ホリコシ以外のパイロコッカス属菌由 来の PolD酵素においても、高塩濃度下における DNAポリメラーゼ活性、及び 3'— 5 ，ェキソヌクレアーゼ活性を向上させることが可能である。 DPI (167— 200)との相同 率は、少なくとも 45%以上、好ましくは 55%以上、さらに好ましくは 80%以上であれ ば、 DNAポリメラーゼ活性制御領域であるといえ、本発明においては、このような DP 1 (167- 200)と相同性を有する領域を欠失した他のパイロコッカス属菌由来の小サ ブユニット変異体、あるいは該変異体を有する耐熱性へテロオリゴマー酵素も含む。  Therefore, if at least the region homologous to DPI (167-200) in PolD derived from Pyrococcus is deleted, high salt concentration is also found in PolD enzymes derived from Pyrococcus other than Neurococcus horicosi Underlying DNA polymerase activity and 3'-5 exonuclease activity can be improved. If the homology rate with DPI (167-200) is at least 45% or more, preferably 55% or more, more preferably 80% or more, it can be said that it is a DNA polymerase activity control region. Also included are small subunit mutants derived from other Pyrococcus species lacking a region homologous to DP 1 (167-200), or thermostable hetero-oligomer enzymes having such mutants.
[0018] 本発明においては、小サブユニットの DNAポリメラーゼ活性制御領域を欠失させる 力 欠失させる範囲は DNAポリメラーゼ活性及び 3'— 5'ェキソヌクレアーゼ活性を 損なわない限り、 DNAポリメラーゼ活性制御領域を少なくとも含めばよい。このため には、 DNAポリメラーゼ活性制御領域を含むように小サブユニットの N末端側を欠失 させることが好ましい。例えば、パイロコッカス 'ホリコシ由来の PolDの場合、上記小サ ブユニットの 167— 200残基を欠失したものに加え、 1 200残基、 140— 200残基 を欠失した小サブユニット変異体にっ 、て、上記配列番号 5に示すインティン配列を 欠失させた大サブユニットとヘテロオリゴマー酵素を形成した場合に高塩濃度下の D NAポリメラーゼ活性及び 3，一 5，ェキソヌクレアーゼ活性向上効果を奏することを確 認している。 [0019] すなわち、本発明の小サブユニット変異体をより具体的に挙げれば、例えば、パイ ロコッカス ·ホリコシ由来の、配列番号 1に示されるアミノ酸配列にお 、て少なくとも 16 7— 200番目の領域が欠失したアミノ酸配列を有するパイロコッカス'ホリコシ由来の 小サブユニット変異体が挙げられる力 該アミノ酸配列において、 1乃至数個のァミノ 酸残基が欠失、置換または付加されたアミノ酸配列を有する小サブユニット変異体で あっても、大サブユニットとヘテロオリゴマーを構成したとき、 DNAポリメラーゼ活性及 び 3'— 5 'ェキソヌクレアーゼ活性を有するものである限り、これらを包含する。また、 これら小サブユニットとヘテロオリゴマー酵素を構成する大サブユニットは、上記配列 番号 5に示すアミノ酸配列を有するものの他に、該配列と 1乃至数個のアミノ酸残基 が欠失、置換または付加されたアミノ酸配列を有するものもへテロオリゴマーを構成し たとき、 DNAポリメラーゼ活性及び 3，—5，ェキソヌクレアーゼ活性を有するものであ る限り、これらを包含する。 [0018] In the present invention, the ability to delete the DNA polymerase activity control region of the small subunit is as long as the range of deletion does not impair the DNA polymerase activity and 3'-5 'exonuclease activity. At least. For this purpose, it is preferable to delete the N-terminal side of the small subunit so as to include the DNA polymerase activity control region. For example, in the case of PolD derived from Pyrococcus horikoshi, in addition to the above-mentioned small subunits lacking 167-200 residues, small subunit mutants lacking 1 200 residues and 140-200 residues Thus, when a hetero-oligomer enzyme is formed with a large subunit from which the intin sequence shown in SEQ ID NO: 5 is deleted, DNA polymerase activity and 3,15, exonuclease activity are improved under high salt concentration. It is confirmed that That is, more specifically, the small subunit mutant of the present invention includes, for example, a region at least 167-200 in the amino acid sequence shown in SEQ ID NO: 1 derived from Pyrococcus horicosi. The ability to include small subunit mutants derived from Pyrococcus horikoshii having an amino acid sequence deleted from the amino acid sequence, wherein the amino acid sequence has an amino acid sequence in which one to several amino acid residues have been deleted, substituted or added Even small subunit mutants are included as long as they have DNA polymerase activity and 3′-5 ′ exonuclease activity when they constitute a hetero-oligomer with a large subunit. In addition, the large subunit constituting the hetero-oligomer enzyme with these small subunits has the amino acid sequence shown in SEQ ID NO: 5 above, and the sequence and one to several amino acid residues are deleted, substituted or added. Those having a defined amino acid sequence are also included as long as they have DNA polymerase activity and 3, -5, exonuclease activity when they constitute a hetero-oligomer.
[0020] また、本発明における、 DNAポリメラーゼ活性制御領域を削除した小サブユニット 変異体をコードする DNAを得るための手段には特に制限がなぐ常法による。例え ばパイロコッカス属菌の小サブユニットをコードする DNAあるいは該 DNAを含むプ ラスミド等のベクターを铸型とし、適宜残存させる領域の塩基配列を基に合成したプ ライマーを使用し、 PCRより増幅させる手段が挙げられる。 [0020] In the present invention, means for obtaining DNA encoding a small subunit mutant from which the DNA polymerase activity control region is deleted is based on a conventional method without any particular limitation. For example, a DNA that encodes the small subunit of Pyrococcus or a vector such as a plasmid containing the DNA is used as a cage and amplified by PCR using a primer that is synthesized based on the base sequence of the region that remains as appropriate. The means to make is mentioned.
本発明の小サブユニット変異体をコードする DNAの具体例は、例えば、パイロコッ カス'ホリコシ由来の配列番号 2に示される遺伝子配列中、少なくとも上記 DNA合成 活性制御領域をコードする部分 (499 600)を欠失した塩基配列を有する DNAが 挙げられる力 該 DNAと相補の塩基配列を有する DNAとストリンジヱントな条件でノヽ イブリダィズ可能な DNAであって、 DNAポリメラーゼ活性及び 3 '一 5 'ェキソヌクレ ァーゼ活性を有する耐熱性へテロオリゴマー酵素の小サブユニットとなりうるタンパク 質を発現可能なものであってもよ 、。  Specific examples of the DNA encoding the small subunit mutant of the present invention include, for example, a portion encoding at least the above-mentioned DNA synthesis activity control region in the gene sequence shown in SEQ ID NO: 2 derived from Pyrococcus horikoshi (499 600) A DNA that has a nucleotide sequence deleted from the DNA. A DNA that can be hybridized under stringent conditions with a DNA having a nucleotide sequence complementary to the DNA, and that has DNA polymerase activity and 3'1 5 'exonuclease activity. It may be capable of expressing a protein that can be a small subunit of the thermostable hetero-oligomer enzyme.
[0021] 本発明におけるストリンジヱントな条件とは、 0. 5%SDS、 5 Xデンハルツ及び 100 μ g/mlサケ精子 DNAを含む 6 X SSC中で、 42°C以上、例えば、 50°C〜65°C、ある いは 65〜70°Cのよりシビアな条件で 4時間〜ー晚保温し、次いで、 6 X SSC中、室 温で 10分間、 0. 1% SDSを含む 2 X SSC中、室温で 10分間、 0. 1% SDSを含む 0. 2 X SSC中、 42°Cで 30分間洗浄する条件である。 [0021] The stringent conditions in the present invention are 42 ° C or higher, for example, 50 ° C to 65 ° C in 6 X SSC containing 0.5% SDS, 5 X Denharz and 100 µg / ml salmon sperm DNA. Incubate at room temperature for more than 4 hours under more severe conditions of ° C or 65-70 ° C, then in 6 X SSC, Wash for 10 minutes at 2 ° SSC containing 0.1% SDS for 10 minutes at room temperature, for 10 minutes at room temperature, and in 0.2 X SSC containing 0.1% SDS for 30 minutes at 42 ° C.
[0022] また、上記少なくとも DNAポリメラーゼ活性制御領域をコードする部分 (499 600 )を欠失した塩基配列を有する DNA該塩基配列において、 1乃至数個のヌクレオチ ドが欠失、置換または付加された DNAであっても、 DNAポリメラーゼ活性及び 3'— 5，ェキソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素の小サブユニットと なりうるタンパク質を発現可能なものであればよい。  [0022] Further, in the DNA having a base sequence from which at least the portion encoding the DNA polymerase activity control region (499 600) is deleted, 1 to several nucleotides are deleted, substituted or added. Any DNA that can express a protein that can be a small subunit of a thermostable hetero-oligomer enzyme having DNA polymerase activity and 3′-5, exonuclease activity may be used.
[0023] 本発明においては、これら小サブユニット変異体をコードする DNA及び上記大サ ブユニットをコードする DNAを組み合わせて、これらが共発現可能なように宿主微生 物に導入する力 この導入においては、上記小サブユニット変異体をコードする DN Aと大サブユニットをコードする DNAとを別々のプラスミド等の発現ベクターに連結し て、宿主微生物に導入してもよいが、これら小サブユニットをコードする DNA変異体 と大サブユニットをコードする DNAとを同一の発現ベクターに導入し、同一のあるい は 2つのプロモータ下で発現させることが好ましい。本明細書にいう共発現とは宿主 内で 2つの DNAが同時に発現し、これらによりコードするタンパク質が生成可能なこ とをいう。また、使用する宿主微生物としては、大腸菌、枯草菌等の細菌類、あるいは 酵母等が挙げられる。  [0023] In the present invention, the ability to combine DNA encoding these small subunit mutants and DNA encoding the above large subunit and to introduce them into a host microorganism so that they can be co-expressed. May be introduced into the host microorganism by ligating the DNA encoding the small subunit mutant and the DNA encoding the large subunit to separate expression vectors such as plasmids. It is preferable that the DNA variant encoding and the DNA encoding the large subunit are introduced into the same expression vector and expressed under the same or two promoters. The term “co-expression” as used herein means that two DNAs can be expressed simultaneously in a host and a protein encoded by them can be produced. Examples of host microorganisms to be used include bacteria such as Escherichia coli and Bacillus subtilis, and yeast.
[0024] 上記のように小サブユニット変異体をコードする DNAと大サブユニットをコードする DNAを導入することにより得られた形質転換体を、それぞれの宿主微生物にとって 好適な培地で培養することにより、菌体を含む培養物カゝら耐熱性へテロオリゴマー酵 素を採取する。該酵素は高塩濃度下における DNAポリメラーゼ活性、及び 3'— 5' ェキソヌクレアーゼ活性を著しく高められた酵素である。  [0024] By transforming the transformant obtained by introducing the DNA encoding the small subunit mutant and the DNA encoding the large subunit as described above in a medium suitable for each host microorganism, Then, heat-resistant hetero-oligomer enzyme is collected from the culture containing the cells. The enzyme is an enzyme with significantly enhanced DNA polymerase activity and 3′-5 ′ exonuclease activity under high salt concentration.
[0025] なお、パイロコッカス 'ホリコシ由来の耐熱性へテロオリゴマー酵素における、小サブ ユニット、インティン配列を含まない大サブユニット、これらを共発現するベクター pET 15b/PolSL(-Intein)、及びこれを含む形質転換体の製法については、上記本発明者 の先の出願（特願 2000— 116257号 (特開 2001— 299348号公報））に詳述されて いるが、これらの製法については、参考例として以下に示す。  [0025] It should be noted that, in the thermostable hetero-oligomer enzyme derived from Pyrococcus' holicosi, a small subunit, a large subunit not containing an intin sequence, a vector pET 15b / PolSL (-Intein) co-expressing these, and The method for producing the transformant is described in detail in the above-mentioned previous application of the present inventor (Japanese Patent Application No. 2000-116257 (Japanese Patent Laid-Open No. 2001-299348)). As shown below.
[0026] 《参考例》 ( 1) 菌の焙着 [0026] << Reference Example >> (1) Fungal roasting
パイロコッカス'ホリコシ (理ィ匕学研究所受託番号 JCM9974)は次の方法で培養した 。 13.5gの食塩、 4gの Na SO , 0.7 gの KC1, 0.2gの NaHCO O.lgの KBr、 30 mgの H B  Pyrococcus' Horikoshi (Science Research Institute Accession No. JCM9974) was cultured by the following method. 13.5 g salt, 4 g Na SO, 0.7 g KC1, 0.2 g NaHCO O.lg KBr, 30 mg H B
2 4 3 3 2 4 3 3
O、 lOgの MgCl -6H 0、 1.5gの CaCl 、 25mgの SrCl、 1.0mlのレザスリン溶液（0.2g/O, lOg MgCl -6H 0, 1.5 g CaCl 2, 25 mg SrCl, 1.0 ml resazurin solution (0.2 g /
3 2 2 2 2 3 2 2 2 2
L)、 l.Ogの酵母エキス、 5gのバクトペプトンを 1Lに溶かし、この溶液の pHを 6.8に調整 し加圧殺菌した。ついで、乾熱滅菌した元素硫黄を 0.2%となるようにカ卩え、この培地 をアルゴンで飽和して嫌気性とした後、 JCM9974を植菌した。培地が嫌気性となつ たか否かは Na S溶液をカ卩えて、培養液中で Na2Sによるレザスリン溶液のピンク色が  L), l.Og yeast extract and 5 g bactopeptone were dissolved in 1 L, and the pH of this solution was adjusted to 6.8 and pasteurized under pressure. Next, elemental sulfur sterilized by dry heat sterilization was adjusted to 0.2%, and this medium was made anaerobic by saturation with argon, and then JCM9974 was inoculated. Whether the medium has become anaerobic or not is determined by the NaS solution and the pink color of the resalin solution with Na2S in the culture medium.
2  2
着色しないことにより確認した。この培養液を 95°Cで 2〜4日間培養し、その後遠心分 離し集菌した。  This was confirmed by not coloring. This culture solution was cultured at 95 ° C. for 2 to 4 days, and then centrifuged and collected.
[0027] (2) 染色体 DNAの調製  [0027] (2) Preparation of chromosomal DNA
JCM9974の染色体 DNAは以下の方法により調製した。培養終了後 5000rpm、 10分間 の遠心分離により菌体を集菌する。菌体を 10mM Tris(pH 7.5)- ImM EDTA溶液で 2 回洗浄後 InCert Agarose (FMC社製）ブロック中に封入する。このブロックを 1%N-ラ ゥロイルサルコシン（lauroylsarcosine) , lmg/mlプロテアーゼ K溶液中で処理すること により、染色体 DNAは Agaroseブロック中に分離調製される。 The chromosomal DNA of JCM9974 was prepared by the following method. Collect the cells by centrifugation at 5000rpm for 10 minutes after completion of the culture. The cells are washed twice with 10 mM Tris (pH 7.5) -ImM EDTA solution and then enclosed in an InCert Agarose (FMC) block. The block 1% N-la © acryloyl sarcosine (l auroy l sarcos ine), by treatment with lmg / ml protease K solution, the chromosomal DNA is separated prepared in Agarose block.
[0028] (3) ¾色体 DNAを含す eライブラリークローンの作製  [0028] (3) Preparation of e-library clones containing ¾-chromosome DNA
十.記 (2)で得られた染色体 DNAを制限酵素 Hindlllで部分分解後、ァガロースゲル電 気泳動により約 40kb長の断片を調製した。この DNA断片と制限酵素 Hindllによって 完全分解した Bacベクター pBAC108L及び pFOSlとを T4リガーゼを用いて結合させた 。前者のベクター PBAC108Lを用いた場合には結合終了後の DNAをただちに大腸菌 内へ電気孔窄法により導入した。後者のベクター pFOSlを用いた場合には結合終了 後の DNAを GIGA Pack Gold (ストラタジーン社製）により試験管内で λファージ粒子 内に詰め込み、この粒子を大腸菌に感染させることにより DNAを大腸菌内に導入した 。これらの方法により得られた抗生物質クロラムフエ-コール耐性の大腸菌集団を ΒΑ C及び Fosmidライブラリ一とした。ライブラリーから JCM9974の染色体をカバーするの に適したクローンを選択して、クローンの整列化を行った。 10. The chromosomal DNA obtained in (2) was partially decomposed with the restriction enzyme Hindlll, and then a fragment of about 40 kb was prepared by agarose gel electrophoresis. This DNA fragment was ligated with Bac vectors pBAC108L and pFOSl completely digested with the restriction enzyme Hindll using T4 ligase. When the former vector P BAC108L was used, the DNA after completion of the binding was immediately introduced into E. coli by electroporation. When the latter vector pFOSl is used, the DNA after the completion of binding is packed into λ phage particles in a test tube with GIGA Pack Gold (manufactured by Stratagene). Introduced. The E. coli population resistant to the chloramphee-chol antibiotics obtained by these methods was designated as one of the C and Fosmid libraries. Clones suitable for covering the chromosome of JCM9974 were selected from the library, and the clones were aligned.
[0029] (4) 各 BAC或いは Fosmidクローンの塩某配列決定 整列化された BAC或いは Fosmidクローンにつ!/、て順次以下の方法で塩基配列を決 定した。大腸菌より回収した各 BAC或 、は Fosmidクローンの DNAを超音波処理する ことにより断片化し、ァガロースゲル電気泳動により lkb及び 2kb長の DNA断片を回収 した。この断片をプラスミドベクター pUC118の Hindi制限酵素部位に挿入したショット ガンクローンを各 BAC或いは Fosmidクローン当たり 500クローン作製した。各ショットガ ンクローンの塩基配列をパーキンエルマ一、 ABI社製自動塩基配列読み取り装置 37 3または 377を用いて決定した。各ショットガンクローン力も得られた塩基配列を塩基 配列自動連結ソフト Sequencherを用いて連結編集し、各 BAC或いは Fosmidクローン の全塩基配列を決定した。 [0029] (4) Determination of salmon salt sequence of each BAC or Fosmid clone The sequence was determined for the aligned BAC or Fosmid clones by the following method. Each BAC or Fosmid clone DNA recovered from E. coli was fragmented by sonication, and lkb and 2kb long DNA fragments were recovered by agarose gel electrophoresis. 500 shotgun clones in which this fragment was inserted into the Hindi restriction enzyme site of plasmid vector pUC118 were prepared for each BAC or Fosmid clone. The base sequence of each shotgun clone was determined using Perkin Elma I, an automatic base sequence reader 373 or 377 manufactured by ABI. The base sequences obtained for each shotgun clone were linked and edited using the automatic base sequence linking software Sequencher, and the entire base sequence of each BAC or Fosmid clone was determined.
[0030] (5) DNAポリメラーゼ遣伝子の同定  [0030] (5) Identification of DNA polymerase gene
上記で決定された各 BAC或いは Fosmidクローンの塩基配列の大型計算機による解 析を行い、 DNAポリメラーゼの大サブユニットをコードする遺伝子 (配列番号 4)と小サ ブユニットをコードする遺伝子 (配列番号 2)が同定された。  Analyzing the base sequence of each BAC or Fosmid clone determined above with a large computer, the gene encoding the large subunit of DNA polymerase (SEQ ID NO: 4) and the gene encoding the small subunit (SEQ ID NO: 2) Was identified.
[0031] (6) 小サブユニット発現プラスミドの構篛  [0031] (6) Structure of small subunit expression plasmid
小サブユニット構造遺伝子領域の前後に制限酵素 (Ndelと BamHI)サイトを構築する 目的で下記の DNAプライマーを合成し、 PCRでその遺伝子の前後に制限酵素サイト を導入した：上部プライマー： PdSl; 5し TTTTGTCGACGTACATATGGATGAATTC GTAAAG-3' (配列番号 9 ;下線部は Ndelサイトを示す）；  The following DNA primers were synthesized for the purpose of constructing restriction enzyme (Ndel and BamHI) sites before and after the small subunit structural gene region, and restriction enzyme sites were introduced before and after the gene by PCR: upper primer: PdSl; 5 TTTTGTCGACGTACATATGGATGAATTC GTAAAG-3 ′ (SEQ ID NO: 9; underlined indicates Ndel site);
下部プライマー： PolS2; 5 -TTTTGAGCTCTTTGGATCCTTAGAAGCTCCATCA GCACCACCT-3' (配列番号 10；下線部は BamHIサイトを示す）。  Lower primer: PolS2; 5 -TTTTGAGCTCTTTGGATCCTTAGAAGCTCCATCA GCACCACCT-3 '(SEQ ID NO: 10; underlined indicates BamHI site).
PCR反応後、制限酵素 (Ndelと BamHI)で完全分解 (37°Cで 2時間）した後、その構造 遺伝子を精製した。さらに、 pETlla或いは pET15b (ともに Novagen社製)を制限酵素 Ndelと BamHIで切断'精製した後、上記の構造遺伝子と T4リガーゼで 16°C、 2時間 反応させ連結した。連結した DNAの一部を大腸菌 E. coli XLl-BlueMRFl (Stratage ne社製)のコンビテントセルに導入し形質転換体のコロニーを得た。得られたコロニー 力も発現プラスミドをアルカリ法で精製した。得られた発現プラスミドは各々 pETl la/P olS或 、は pET15b/PolSと略記された。構造遺伝子上にランダム変異がな!、ことは DN A配列決定により確認された。 [0032] (7) 完全長の大サブユニットを発現するためのプラスミドの構築 After the PCR reaction, complete digestion with restriction enzymes (Ndel and BamHI) (2 hours at 37 ° C) was followed by purification of the structural gene. Further, pETlla or pET15b (both manufactured by Novagen) was cleaved and purified with restriction enzymes Ndel and BamHI and then ligated by reacting with the above structural gene and T4 ligase at 16 ° C for 2 hours. A part of the ligated DNA was introduced into a competent cell of E. coli XLl-BlueMRFl (Stratagene) to obtain a transformant colony. The obtained colony force was also purified from the expression plasmid by the alkaline method. The resulting expression plasmids were abbreviated as pETlla / PolS or pET15b / PolS, respectively. It was confirmed by DNA sequencing that there was no random mutation on the structural gene! [0032] (7) Construction of plasmid for expressing full-length large subunit
完全長の大サブユニット遺伝子を 2段階で pGEMEX-Ιベクター（プロメガ社製）にクロ 一ユングした。前半部の DNA断片は以下の二つのプライマーを用い、 PCR法で得た: 上部プライマー： PolLl; 5'-CTCGACTTTAGCATATGGCTCTGATGGAGC-3' (ia 列番号 11；下線部は Ndelサイトを示す）；  The full-length large subunit gene was cloned into the pGEMEX-Ι vector (Promega) in two steps. The first half of the DNA fragment was obtained by PCR using the following two primers: Upper primer: PolLl; 5'-CTCGACTTTAGCATATGGCTCTGATGGAGC-3 '(ia column number 11; underlined indicates Ndel site);
下部プライマー： PolL2; 5 -GCTTGTCGACGCCATAAACTTTGACATTATCCATT GCGCGCTTAAGCAAC-3' (配列番号 12；下線部は Sailサイトを示す）。  Lower primer: PolL2; 5 -GCTTGTCGACGCCATAAACTTTGACATTATCCATT GCGCGCTTAAGCAAC-3 ′ (SEQ ID NO: 12; underlined indicates Sail site).
この PCR産物を Ndelと Sailで完全消化した後、 pGEMEX-Ιベクターにクローユングし、 pGEM/PolLl- 2と略記した。  This PCR product was completely digested with Ndel and Sail, cloned into the pGEMEX-Ι vector, and abbreviated as pGEM / PolL1-2.
[0033] 後半部の DNA断片は以下の二つのプライマーを用い、 PCR法で得た：上部プライ マー： PolL3; 5'- TTTATGGC^!C ACAAGCTGAAGG- 3' (配列番号 13 ;下線部は Sailサイトを示す）； [0033] The DNA fragment in the latter half was obtained by PCR using the following two primers: Upper primer: PolL3; 5'-TTTATGGC ^! C ACAAGCTGAAGG-3 '(SEQ ID NO: 13; underlined on the Sail site) );
下部プライマー： PolL4; 5 -TATAACTTATGCATTGTGGTTATTTCGCTGAGAAG -3' (配列番号 14；下線部は Nsilサイトを示す)。  Lower primer: PolL4; 5 -TATAACTTATGCATTGTGGTTATTTCGCTGAGAAG-3 '(SEQ ID NO: 14; underlined indicates Nsil site).
この PCR産物は Sailと Nsilで完全消化した後、先に調製した pGEM/PolLl-2にクロー ユングし、完全長の大サブユニット遺伝子を含む pGEM/PolLを得た。  This PCR product was completely digested with Sail and Nsil and then cloned into the previously prepared pGEM / PolLl-2 to obtain pGEM / PolL containing the full-length large subunit gene.
[0034] (8) インティンを除去した大サブユニットを発現するためのプラスミドの構築 [8] (8) Construction of plasmid for expressing large subunit from which intin is removed
ノイロコッカス .ホリコシ (p. horikoshii)の DNAポリメラーゼの大サブユニット遺伝子の 中には（蛋白質性のイントロンをコードする)インティンが一つ含まれるので、プライマ 一 PolL3と PolL6 (下記）を用いた PCR法でインティンの上流の DNA断片を増幅し、プ ライマー PolL5 (下記）と PolL4を用いた PCR法でインティンの下流の DNA断片を増幅 した。この 2断片とプライマー PolL3と PolL4を用い、インティンの除かれた DNA断片を オーバーラップ PCRで増幅した。次にこの産物を制限酵素 Sailと Nsilで完全消化した 後、先に調製した pGEM/PolLl-2にクローユングし、インティンの除かれた大サブュ ニット遺伝子を含む pGEM/PolL(- Intein)を得た。 配列番号 15) 配列番号 16) The large subunit gene of p. Horikoshii DNA polymerase contains one intin (encoding a protein intron), so PCR using primers PolL3 and PolL6 (below) The DNA fragment upstream of the intin was amplified by the PCR method, and the DNA fragment downstream of the intin was amplified by the PCR method using the primers PolL5 (below) and PolL4. Using these two fragments and primers PolL3 and PolL4, the DNA fragment from which intin was removed was amplified by overlap PCR. Next, this product was completely digested with the restriction enzymes Sail and Nsil and then cloned into the previously prepared pGEM / PolLl-2 to obtain pGEM / PolL (-Intein) containing the large subunit gene from which intin was removed. . (SEQ ID NO: 15) (SEQ ID NO: 16)
[0035] (9) 小サブユニットとインティンを除去した大サブユニットを共発現するプラスミドの 嫌  [0035] (9) Aversion of plasmids that co-express small subunits and large subunits with intin removed
目的のへテロダイマー DNAポリメラーゼの安定した生産を図るために両サブユニット を共発現するプラスミドの構築を行った。まず、 pET15b/PolSの BamHIサイトの直上流 域に新しいマルチクローユングサイトを導入するためにプライマー PolSlと PolS3 (下記 )を用い PCR反応を行った。なお、下記のように PolS3には 5'-末端より順番に BamHI、 Nsil、 Sail, SacIIサイトがコードされている。 PCR産物を Ndelと BamHI処理し、 pET15b に挿入することにより、小サブユニットの終止コドンと BamHIサイトの間にマルチクロー ユングサイトを含む pET15b/PolS(M)を造成した。次に、 pGEM/PolL(- Intein)を铸型 D NAとし、プライマー PolL7 (下記）と PolL2を用い PCR反応を行った。得られた産物は新 し!ヽ SacIIサイトを 5'-末端にもち、 pGEM/PolL(- Intein)のタンパク質発現ユニットのうち 、リボソーム結合サイトからコード領域中の Sailサイトまでを含んでいる。  In order to achieve stable production of the desired heterodimer DNA polymerase, a plasmid that co-expresses both subunits was constructed. First, PCR was performed using primers PolSl and PolS3 (below) to introduce a new multicloning site immediately upstream of the BamHI site of pET15b / PolS. As shown below, BamHI, Nsil, Sail, and SacII sites are encoded in PolS3 in order from the 5'-end. The PCR product was treated with Ndel and BamHI and inserted into pET15b to construct pET15b / PolS (M) containing a multicloning site between the stop codon of the small subunit and the BamHI site. Next, PCR was performed using pGEM / PolL (-Intein) as a truncated DNA and primers PolL7 (below) and PolL2. The resulting product has a new SacII site at the 5'-end, and includes the protein expression unit of pGEM / PolL (-Intein) from the ribosome binding site to the Sail site in the coding region.
[0036] これを SacIIと Sail処理し、 pET15b/PolS(M)に造成されたマルチクローユングサイトに 挿入した。このプラスミドは pET15b/PolSLl- 2と略記される。さらに、 pGEM/PolL(- Inte in)を Sailと Nsil処理し、大サブユニットの後半部分の遺伝子 (インティンを含まな!/、）を 単離し、これを pET15b/PolSLl-2のタンパク質発現ユニットの最後に残った Sailと Nsil サイトに挿入した。得られたプラスミドは pET15b/PolSL(- Intein)と略記される。この発 現プラスミド pET15b/PolSL(-Intein)によりァミノ末端にヒスチジンタグが付カ卩した小サ ブユニットとインティンを含まない大サブユニットの共発現が可能になった。 [0036] This was treated with SacII and Sail, and inserted into the multicloning site constructed in pET15b / PolS (M). This plasmid is abbreviated as pET15b / PolSL1-2. In addition, pGEM / PolL (-Inte in) was treated with Sail and Nsil to isolate the second half of the large subunit (without intin! /), And this was expressed in the protein expression unit of pET15b / PolSLl-2. Inserted into the last remaining Sail and Nsil sites. The resulting plasmid is abbreviated as pET15b / PolSL (-Intein). This expression plasmid pET15b / PolSL (-Intein) enables co-expression of a small subunit with a histidine tag at the amino end and a large subunit without intin.
AGTCGAG- 3' (配列番号 17 ;下線部は 5'-末端より順番に BamHI, Nsil, Sail, SacIIサ イトを示す。 ) AGTCGAG-3 '(SEQ ID NO: 17; underlined parts indicate BamHI, Nsil, Sail, SacII sites in order from the 5'-end)
PolL7: 5 ' -GGTGTCCGCGGCTC ACTATAGGGAGACC AC-3 ' (配列番号 18；下線 部は SacIIサイトを、太字は pGEMEX-Ιベクターのリボソーム結合サイトを示す。） [0037] (10) 組換え遣伝子の発現大腸菌（E. coli BL21- CodonPlus(DE3)- RIL,  PolL7: 5'-GGTGTCCGCGGCTC ACTATAGGGAGACC AC-3 '(SEQ ID NO: 18; underlined indicates SacII site, bold indicates ribosome binding site of pGEMEX-Ι vector) [0037] (10) Expression of recombinant gene E. coli BL21- CodonPlus (DE3)-RIL,
Stratagene社製）のコンビテントセルを融解して、ファルコンチューブに O.lmL移した。 その中に発現プラスミド溶液 0.005mLを加え氷中に 30分間放置した後 42°Cでヒート ショックを 30秒間行い、 SOCmedium 0.9mLをカ卩え、 37°Cで 1時間振とう培養する。そ の後アンピシリンを含む 2YT寒天プレートに適量まき、 37°Cで一晩培養し、形質転換 体を得た。この开質転換体は E. coli BL21-CodonPlus(DE3)-RIL/ pET15b/PolSL(- 1 ntein)と命名され、茨城県つくば巿東 1 1 1中央第 6に所在の独立行政法人産業 技術総合研究所、特許生物寄託センターに受託番号 FERM ABP-10752として寄託 されている。 以下に本発明の実施例を示すが、本発明はこれら実施例により限定されるもので はない。 (Commercially available from Stratagene) was thawed and transferred to a Falcon tube at O.lmL. Add 0.005 mL of the expression plasmid solution to it, leave it on ice for 30 minutes, and heat at 42 ° C. Shock for 30 seconds, add 0.9 mL of SOCmedium, and incubate for 1 hour at 37 ° C. Thereafter, a suitable amount was spread on a 2YT agar plate containing ampicillin and cultured overnight at 37 ° C to obtain a transformant. This transformant is named E. coli BL21-CodonPlus (DE3) -RIL / pET15b / PolSL (-1 ntein), and is an independent administrative agency of industrial technology in Tsukuba Tohoku, Ibaraki Prefecture 1 1 1 Deposited at the Institute and Patent Biological Depositary Center under the deposit number FERM ABP-10752. Examples of the present invention are shown below, but the present invention is not limited to these examples.
実施例 Example
〔実施例 1〕小サブユニットのァミノ末端側ドメイン、 DP1(1- 200)、の野生型および欠失 変異体の発現プラスミドの構築 [Example 1] Construction of wild type and deletion mutant expression plasmids of the amino subunit domain of the small subunit, DP1 (1-200)
DPK1-200)の野生型および N-末端、 C-末端欠失変異体の構造遺伝子領域の前 後に制限酵素 (Ndelと BamHI)サイトを導入し、各遺伝子を増幅する目的で、以下の D NAプライマー対を合成した。  In order to amplify each gene by introducing restriction enzyme (Ndel and BamHI) sites before and after the structural gene region of wild type and N-terminal and C-terminal deletion mutants of DPK1-200) Primer pairs were synthesized.
1) DP1(1- 200)に関して：  1) Regarding DP1 (1-200):
上部プライマー F1; Upper primer F1;
5 ' - GGGGGC ATATGGATGAATTCGTAAAGGGATTA- 3 ' (配列番号 19；下線部 は Ndelサイトを示す）  5'-GGGGGC ATATGGATGAATTCGTAAAGGGATTA-3 '(SEQ ID NO: 19; underlined indicates Ndel site)
下部プライマー R4; Lower primer R4;
5'- GGGGGATCCTCAATAGTCTTTTTCTATCGGAACGAC -3' (配列番号 20 ; 下線部は BamHIサイトを示す）  5'- GGGGGATCCTCAATAGTCTTTTTCTATCGGAACGAC -3 '(SEQ ID NO: 20; underlined indicates BamHI site)
2) DP1(33- 200)に関して： 2) Regarding DP1 (33-200):
上部プライマー F2; Upper primer F2;
5 ' - TTC AATGAGGGAC ATATGTCGCTCATAGAA- 3 ' (配列番号 21；下線部は Nd elサイトを示す）  5 '-TTC AATGAGGGAC ATATGTCGCTCATAGAA-3' (SEQ ID NO: 21; underlined indicates Nd el site)
下部プライマー R4 [0039] 3) DP1(49- 200)に関して： Lower primer R4 [0039] 3) Regarding DP1 (49-200):
上部プライマー F3;  Upper primer F3;
5 -TCCAGGGAAACGCATATGATAGATGATGAG-3 ' (配列番号 22；下線部は Nd elサイトを示す）  5 -TCCAGGGAAACGCATATGATAGATGATGAG-3 '(SEQ ID NO: 22; underlined indicates Nd el site)
下部プライマー R4  Lower primer R4
4) DP1(66- 200)に関して： 4) Regarding DP1 (66-200):
上部プライマー F4; 5し TCCATCGGGGCTCATATGGAACTTCCACAG- 3' (配列 番号 23；下線部は Ndelサイトを示す）  Upper primer F4; 5 and TCCATCGGGGCTCATATGGAACTTCCACAG-3 '(SEQ ID NO: 23; underlined indicates Ndel site)
下部プライマー R4  Lower primer R4
[0040] 5) DP1(85- 200)に関して： [0040] 5) Regarding DP1 (85-200):
上部プライマー F5;  Upper primer F5;
5し GGAGAAGGTTCACAIAIGGTTCCAGATCAT- 3' (配列番号 24；下線部は Nd elサイトを示す）  5 GGAGAAGGTTCACAIAIGGTTCCAGATCAT-3 '(SEQ ID NO: 24; underlined indicates Nd el site)
下部プライマー R4  Lower primer R4
6) DPK1-139)に関して： 6) Regarding DPK1-139):
上部プライマー F1  Upper primer F1
下部プライマー R1;  Lower primer R1;
5'- GGGGGATCCTCATCCAGTGGAAATAGAACTCTC -3' (配列番号 25；下線 部は BamHIサイトを示す）  5'-GGGGGATCCTCATCCAGTGGAAATAGAACTCTC -3 '(SEQ ID NO: 25; underlined indicates BamHI site)
[0041] 7) DPK1-166)に関して： [0041] 7) Regarding DPK1-166):
上部プライマー F1  Upper primer F1
下部プライマー R2;  Lower primer R2;
5'- GGGGGATCCTCAAACATACGTGAAGGGATAGC -3' (配列番号 26；下線部 は BamHIサイトを示す）  5'-GGGGGATCCTCAAACATACGTGAAGGGATAGC -3 '(SEQ ID NO: 26; underlined indicates BamHI site)
8) DPK33-166)に関して: 上部プライマー F2 8) Regarding DPK33-166): Upper primer F2
下部プライマー R2  Lower primer R2
9) DP1(33- 180)に関して： 9) Regarding DP1 (33-180):
上部プライマー F2  Upper primer F2
下部プライマー R3;  Lower primer R3;
5'- GGGGGATCCTCAATACTTAGGATACTCTTCTAGTT  5'- GGGGGATCCTCAATACTTAGGATACTCTTCTAGTT
-3' (配列番号 27；下線部は BamHIサイトを示す）  -3 '(SEQ ID NO: 27; underlined indicates BamHI site)
[0042] PCR反応後、制限酵素 (Ndelと BamHI)で完全分解 (37°Cで 2時間）した後、その構 造遺伝子を精製した。さらに、 pET15b (Novagen社製)を制限酵素 Ndelと BamHIで切 断 ·精製した後、上記の構造遺伝子と T4リガーゼで 16°C、 2時間反応させ連結した。 連結した DNAの一部を大月昜菌 E. coli XLl-BlueMRFlのコンビテントセノレに導入し 形質転換体のコロニーを得た。得られたコロニー力も発現プラスミドをアルカリ法で精 製した。得られた発現プラスミドは各々 pET15b/ DPl(l-200)、 pET15b/ DPl(33-200) 、 pET15b/ DP1(49— 200)、 pET15b/ DP1(66— 200)、 pET15b/ DP1(85— 200)、 pET15b/ DP1(1— 139)、 pET15b/ DP1(1— 166)、 pET15b/ DP1(33— 166)、 pET15b/ DP1(33— 180) 、と略記する。構造遺伝子上にランダム変異が無い事は DNAシーケンスイングにより 確認された。 [0042] After the PCR reaction, complete digestion with restriction enzymes (Ndel and BamHI) (at 37 ° C for 2 hours) was followed by purification of the structural gene. Furthermore, pET15b (manufactured by Novagen) was cut and purified with restriction enzymes Ndel and BamHI, and then reacted with the above structural gene and T4 ligase at 16 ° C. for 2 hours for ligation. A part of the ligated DNA was introduced into a competent senore of E. coli XLl-BlueMRFl to obtain transformant colonies. The obtained colony strength was also obtained by purifying the expression plasmid by the alkaline method. The obtained expression plasmids were pET15b / DPl (l-200), pET15b / DPl (33-200), pET15b / DP1 (49-200), pET15b / DP1 (66-200), pET15b / DP1 (85-200), respectively. ), PET15b / DP1 (1-139), pET15b / DP1 (1-166), pET15b / DP1 (33-166), pET15b / DP1 (33-180). The absence of random mutations on the structural gene was confirmed by DNA sequencing.
[0043] 〔実施例 2〕 DPIの N末端側ある 、は内部配列が欠失したヘテロオリゴマー変異体、 Po 1D A S(1- 200)、 PolD A S (1-65)、 PolD A S (140-200)、 PolD A S (167-200)、を発現す る為のプラスミドの構築  [0043] [Example 2] is a hetero-oligomer mutant in which the internal sequence is deleted at the N-terminal side of DPI, Po 1D AS (1-200), PolD AS (1-65), PolD AS (140-200 ), PolD AS (167-200), plasmid construction
(1) DPIの N末端側が欠失したヘテロオリゴマー変異体を発現するプラスミドの構 築法を以下に示す。まず、共発現ベクター pET15b/PolSL(-Intein) (参考例 9, 10参 照）を铸型にし、以下の DNAプライマー対を使用し、 DPIの 3'領域が PCR増幅された (1) The following shows how to construct a plasmid that expresses a hetero-oligomer mutant lacking the N-terminal side of DPI. First, the co-expression vector pET15b / PolSL (-Intein) (see Reference Examples 9 and 10) was made into a saddle shape, and the 3 'region of DPI was PCR amplified using the following DNA primer pair:
1) PolD A S (1-200)に関して： 1) Regarding PolD A S (1-200):
上部プライマー F6; 5 -TGGATACCTCATATGGAGATAAAGTTCGACGTTAGACG-3' (配列番号 28 ；下線部は Ndelサイトを示す。太字は DPI遺伝子からの配列を示す） Upper primer F6; 5 -TGGATACCTCATATGGAGATAAAGTTCGACGTTAGACG-3 '(SEQ ID NO: 28; underlined indicates Ndel site. Bold indicates sequence from DPI gene)
下部プライマー S2M;  Lower primer S2M;
GAG -3' (配列番号 29；下線部は左から各々、 BamHI、 Sail, SacIIサイトを示す。太 字は DPI遺伝子 3'端の相補鎖配列を示す） GAG-3 '(SEQ ID NO: 29; underlined parts indicate BamHI, Sail, and SacII sites from the left, respectively, bold letters indicate the complementary strand sequence at the 3' end of the DPI gene)
[0044] 2) PolD A S (1-65)に関して： [0044] 2) Regarding PolD A S (1-65):
上部プライマー F4  Upper primer F4
下部プライマー S2M さらに、その増幅産物は Ndelと BamHIサイトを用い、 pET15bに挿入された。次に、 2n d PCRを、目的インサートを含む pET15bベクターを铸型とし、上部プライマー pET-Sp h; 5 '-C AAGGAATGGTGCATGC AAGGAGATGGC-3 ' (配列番号 30 ;下線部は pE T15bベクターの Ndelサイトの 354 bp上流に存在する Sphlサイトを示す）と下部プライ マー S2Mを用いて行なった。この増幅産物を Sphlと SacIIで消化し、 pET15b/PolSL(- In tein)の SpW-SacII断片とすげ替えることにより、 2種の共発現ベクター pET15b/ S Δ (1 - 200)Lと pET15b/ S Δ (1- 65)Lを得た。  Lower primer S2M Furthermore, the amplification product was inserted into pET15b using Ndel and BamHI sites. Next, 2n d PCR was performed using the pET15b vector containing the target insert as a saddle, and the upper primer pET-Sp h; 5 '-C AAGGAATGGTGCATGC AAGGAGATGGC-3' (SEQ ID NO: 30; the underlined part is the Ndel site of the pE T15b vector. (The Sphl site existing at 354 bp upstream is shown) and the lower primer S2M. This amplified product was digested with Sphl and SacII, and replaced with the SpW-SacII fragment of pET15b / PolSL (-Intein), so that two co-expression vectors pET15b / S Δ (1-200) L and pET15b / S Δ (1-65) L was obtained.
[0045] (2) DPIの内部配列が欠失したヘテロオリゴマー変異体を発現するプラスミドの構築 法を以下に示す。 [0045] (2) A method for constructing a plasmid expressing a hetero-oligomer mutant lacking the internal sequence of DPI is shown below.
図 1に overlap PCR法による内部配列が欠失した DPI遺伝子の増幅に必要なプライ マー対の塩基配列と DPI遺伝子上の位置を示す。まず、共発現ベクター pET15b/Pol SL(-Intein)を铸型にし、以下の 2種の DNAプライマー対を使用し、 DPIの 5'断片と 3'断 片を別々に PCR増幅した。これらの 2断片は、前者の 3'領域と後者の 5'領域が重複さ れるようにプライマー設計がなされているので、次に 2断片は混合され、外側プライマ 一を用いて overlap PCRで連結された。  Figure 1 shows the base sequence of the primer pair and the position on the DPI gene required for amplification of the DPI gene with the internal sequence deleted by overlap PCR. First, the co-expression vector pET15b / Pol SL (-Intein) was made into a saddle type, and the following two kinds of DNA primer pairs were used, and the 5 'and 3' fragments of DPI were PCR amplified separately. Since these two fragments are designed so that the former 3 'region and the latter 5' region overlap, the two fragments are then mixed and ligated by overlap PCR using the outer primer. It was.
[0046] 1) A S (140- 200)に関して： [0046] 1) Regarding A S (140-200):
DPIの 5'断片； 上部プライマー F1と下部プライマー R5  5 'fragment of DPI; upper primer F1 and lower primer R5
DPIの 3'断片； 上部プライマー F7と下部プライマー S2M 2) A S (167-200) に関して： 3 'fragment of DPI; upper primer F7 and lower primer S2M 2) Regarding AS (167-200):
DPIの 5'断片； 上部プライマー Flと下部プライマー R6  5 'fragment of DPI; upper primer Fl and lower primer R6
DPIの 3'断片； 上部プライマー F8と下部プライマー S2M  3 'fragment of DPI; upper primer F8 and lower primer S2M
[0047] さらに、その overlap PCR産物は Ndelと BamHIサイトを用い、 pET15bに挿入された。  [0047] Furthermore, the overlap PCR product was inserted into pET15b using Ndel and BamHI sites.
次に、目的インサートを含む pET15bベクターを铸型とし、上部プライマー pET-Sph; 5' -C AAGGAATGGTGCATGC AAGGAGATGGC-3 ' (配列番号 30；下線部は pET15 bベクターの Ndelサイトの 354 bp上流に存在する Sphlサイトを示す）と下部プライマー S2Mを用いて PCR反応を行なった。この増幅産物を Sphlと SacIIで消化し、 pET15b/Po lSL(-Intein)の SpW-SacII断片とすげ替えることにより、 2種の共発現ベクター pET15b/ S Δ (140- 200)Lと pET15b/ S A (167- 200)Lを得た。  Next, the pET15b vector containing the target insert is made into a saddle shape, and the upper primer pET-Sph; 5'-C AAGGAATGGTGCATGC AAGGAGATGGC-3 '(SEQ ID NO: 30; the underlined part is 354 bp upstream of the Ndel site of the pET15 b vector. PCR reaction was performed using Sp2 site and lower primer S2M. This amplified product was digested with Sphl and SacII, and replaced with the SpW-SacII fragment of pET15b / PolsL (-Intein), so that two co-expression vectors pET15b / S Δ (140-200) L and pET15b / SA (167-200) L was obtained.
[0048] 〔実施例 3〕組換え遺伝子の発現  [Example 3] Expression of recombinant gene
大腸菌 . coli BL21-CodonPlus(DE3)-RIL, Stratagene社製）のコンビテントセルを 融解して、ファルコンチューブに O.lmL移す。その中に発現プラスミド溶液 0.005mL を加え氷中に 30分間放置した後 42度でヒートショックを 30秒間行 、、 SOCmedium0.9 mLをカ卩え、 37度で 1時間振とう培養する。その後アンピシリンを含む 2YT寒天プレー トに適量まき、 37度で一晩培養し、形質転換体を得た。  Thaw a competent cell of E. coli BL21-CodonPlus (DE3) -RIL, Stratagene) and transfer to O.lmL in a Falcon tube. Add 0.005 mL of the expression plasmid solution to it and leave it on ice for 30 minutes. Then heat shock at 42 degrees for 30 seconds, add 0.9 mL of SOCmedium, and incubate with shaking at 37 degrees for 1 hour. Thereafter, an appropriate amount was spread on a 2YT agar plate containing ampicillin and cultured overnight at 37 ° C. to obtain transformants.
当形質転換体をアンピシリンを含む 2YT培地（2リットル）で 600nmの吸収が 0.6に 達するまで培養した後、 IPTG (Isopropyl- β -D- thiogalactopyranoside)を加え 37°Cで さらに 3時間培養した。培養後遠心分離 (6,000rpm, 20min)で集菌した。  The transformant was cultured in 2YT medium (2 liters) containing ampicillin until the absorption at 600 nm reached 0.6, and then IPTG (Isopropyl-β-D-thiogalactopyranoside) was added and further cultured at 37 ° C for 3 hours. After incubation, the cells were collected by centrifugation (6,000 rpm, 20 min).
[0049] 〔実施例 4〕小サブユニットのァミノ末端側ドメイン、 DP1(1- 200)の野生型および欠失 変異体の精製  [0049] [Example 4] Amino-terminal domain of small subunit, purification of wild-type and deletion mutants of DP1 (1-200)
集菌した菌体を A緩衝液（50mMトリス塩酸緩衝液 (pH8.0) , 100 mM NaCl)で懸濁 し、氷中で超音波破砕した。さらに 75°Cで 15分加熱後、遠心分離 (12,000 g、 10分)し 上澄液を得た。これを粗酵素液とした。次にこの粗酵素液を A緩衝液で平衡ィ匕した Hi trap Qカラム（5ml,アマシャムバイオサイエンス社製）に添カ卩し、 A緩衝液で洗浄後、 N aCl濃度勾配 (0.1-1M)で溶出した。ここで得られた溶出画分はセントリコン YM-10 (ミ リポア社）で濃縮し、 Superose 12カラム（HR 10/30、アマシャムバイオサイエンス社製 )に添加し、 B緩衝液（50mMトリス塩酸緩衝液 (pH8.0) , 200 mM NaCl)で溶出した。 その後ピーク画分はマイクロコン YM- 10 (ミリポア社)で濃縮され、 4°Cで保存された。 The collected cells were suspended in A buffer (50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl) and sonicated in ice. The mixture was further heated at 75 ° C for 15 minutes and then centrifuged (12,000 g, 10 minutes) to obtain a supernatant. This was used as a crude enzyme solution. Next, this crude enzyme solution was added to a Hi trap Q column (5 ml, manufactured by Amersham Biosciences) equilibrated with buffer A, washed with buffer A, and NaCl gradient (0.1-1M). Eluted with. The elution fraction obtained here was concentrated with Centricon YM-10 (Millipore), added to Superose 12 column (HR 10/30, Amersham Biosciences), and B buffer (50 mM Tris-HCl buffer). (pH 8.0), 200 mM NaCl). The peak fraction was then concentrated with Microcon YM-10 (Millipore) and stored at 4 ° C.
[0050] 〔実施例 5〕 DPIの N末端側ある 、は内部配列が欠失したヘテロオリゴマー変異体、 Po ID A S (1-200)、 PolD A S (1-65)、 PolD A S (140-200)、 PolD A S (167-200)、の精製 集菌した菌体を 50mMトリス塩酸緩衝液 (pH8.0)で懸濁し、フレンチプレスで細胞 破砕した。さらに 85°Cで 30分加熱後、遠心分離 (27,000 g、 20分)し上澄液を得た。こ れを粗酵素液とした。次にこの粗酵素液を 50mMトリス塩酸緩衝液 (pH8.0)で平衡ィ匕 した Hitrap Qカラム（5ml,アマシャムバイオサイエンス社製）に添カ卩し、同緩衝液で洗 浄後、 NaCl濃度勾配 (0-1M)で溶出した。この溶出液を C緩衝液 (20mMトリス塩酸緩 衝液 (pH7.9), 0.5 M NaCl, 5 mMイミダゾール）で平衡化した Ni-カラム（1 ml, Novag en)に添加し、 25 mMイミダゾールを含む 20mMトリス塩酸緩衝液 (pH7.9), 0.5 M Na CIで洗浄し、 200 mMイミダゾールを含む 20mMトリス塩酸緩衝液 (pH7.9), 0.5 M Na CIで溶出した。ここで得られた溶出画分はセントリコン YM-10 (ミリポア社)で濃縮し、 S uperose 12カラム（HR 10/30、アマシャムバイオサイエンス社製）に添カロし、 B緩衝液（ 50mMトリス塩酸緩衝液（pH8.0) , 200 mM NaCl)で溶出した。その後、 PolDヘテロォ リゴマー酵素を含むピーク画分は等量の 50%グリセロールと混和され、 -20°Cで保存さ れた。 [0050] [Example 5] is a hetero-oligomer mutant lacking an internal sequence at the N-terminal side of DPI, Po ID AS (1-200), PolD AS (1-65), PolD AS (140-200 ), Purification of PolD AS (167-200) The collected cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and disrupted with a French press. The mixture was further heated at 85 ° C for 30 minutes and then centrifuged (27,000 g, 20 minutes) to obtain a supernatant. This was used as a crude enzyme solution. Next, this crude enzyme solution was added to a Hitrap Q column (5 ml, manufactured by Amersham Biosciences) equilibrated with 50 mM Tris-HCl buffer (pH 8.0), washed with the same buffer, Elute with a gradient (0-1M). This eluate is added to a Ni-column (1 ml, Novagen) equilibrated with C buffer (20 mM Tris-HCl buffer (pH 7.9), 0.5 M NaCl, 5 mM imidazole) and contains 25 mM imidazole. The column was washed with 20 mM Tris-HCl buffer (pH 7.9) and 0.5 M Na CI, and eluted with 20 mM Tris-HCl buffer (pH 7.9) and 0.5 M Na CI containing 200 mM imidazole. The elution fraction obtained here was concentrated with Centricon YM-10 (Millipore), loaded onto a Superose 12 column (HR 10/30, Amersham Biosciences), and B buffer (50 mM Tris-HCl buffer). Elution was performed with a solution (pH 8.0) and 200 mM NaCl). The peak fraction containing PolD heteroligomer enzyme was then mixed with an equal volume of 50% glycerol and stored at -20 ° C.
[0051] 〔実施例 6〕酵素活性の解析  [Example 6] Analysis of enzyme activity
以下 A〜Eにおける実験において用いた手法、試薬は以下のとおりである。 (1) DNAポリメラーゼ活性 (プライマー伸長活性）  Hereinafter, the methods and reagents used in the experiments in A to E are as follows. (1) DNA polymerase activity (primer extension activity)
プライマー伸長活性測定用の基質調整は以下の様である。まず、 25 1ァニール緩 衝液中 (10 mMトリス塩酸緩衝液 (pH7.5), 10 mM MgCl , 1 mM DTT, 50 mM NaCl)  The substrate adjustment for measuring the primer extension activity is as follows. First, in 25 1 anneal buffer (10 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl, 1 mM DTT, 50 mM NaCl)
2  2
で、 20 μ Mの 84— merオリゴマー  20 μM 84-mer oligomer
TTTACAACGTCGTGACTGGGAAAACCCTGGCGTTAC-3' (配列番号 31) )と 20 μ Μの 5し FAMラベル化された 34- merオリゴマー TTTACAACGTCGTGACTGGGAAAACCCTGGCGTTAC-3 '(SEQ ID NO: 31)) and 20 μΜ 5 and FAM labeled 34-mer oligomer
(5'- GTAACGCCAGGGTTTTCCCAGTCACGACGTTGTA- 3' (配列番号 32) )を混 和し、 100°C、 5分間加熱し、室温まで overnightで徐冷しすることによりァニールさせ [0052] プライマー伸長反応は、 10 μ 1反応液中（20 mMトリス塩酸緩衝液 (pH8.8), 6 mM MgCl , 10 mM KC1, 10 mM (NH ) SO , 0.1% Triton, 0.25 mM dNTP, 200 nM ァニー(5'-GTAACGCCAGGGTTTTCCCAGTCACGACGTTGTA-3 '(SEQ ID NO: 32)) is mixed, heated at 100 ° C for 5 minutes, and then annealed by overnight cooling to room temperature. [0052] Primer extension reaction was performed in 10 μl reaction solution (20 mM Tris-HCl buffer (pH 8.8), 6 mM MgCl 2, 10 mM KC1, 10 mM (NH 2) SO, 0.1% Triton, 0.25 mM dNTP, 200 nM
2 4 2 4 2 4 2 4
ル基質を含む)で行われた。 反応は DNAポリメラーゼをカ卩え、 60°Cで行われた。所定 時間後に 40 の Stop液（95%ホルムアミド、 10 mM EDTA、 0.5 mg/mlのブロモフエノ ールブルーを含む）を加える事で反応停止させた。反応産物は 5分間の煮沸後、氷 水中で急冷され、 lx TBE緩衝液を用いた 7M尿素含有 12%ポリアクリルアミドゲル電 気泳動 (PAGE)で分離された。 PAGEパターンはフルォロイメージヤー 585 (アマシャム ノィォサイエンス社製)で可視化され、分析された。  Containing the substrate). The reaction was carried out at 60 ° C with DNA polymerase. The reaction was stopped by adding 40 Stop solutions (containing 95% formamide, 10 mM EDTA, 0.5 mg / ml bromophenol blue) after a predetermined time. The reaction products were boiled for 5 minutes, quenched in ice water, and separated by 12% polyacrylamide gel electrophoresis (PAGE) containing 7M urea using lx TBE buffer. The PAGE pattern was visualized and analyzed with a Fluoro Imager 585 (Amersham Neuroscience).
[0053] (2) 3'- 5'ェキソヌクレアーゼ活性  [0053] (2) 3'-5 'exonuclease activity
反応系に dNTPを含まな 、こと以外はプライマー伸長活性測定と同様な反応条件を 用いた。 DNAポリメラーゼを加え、 60°Cで 15分間反応させた。所定時間後に 40 1の S top液（95%ホルムアミド、 10 mM EDTA、 0.5 mg/mlのブロモフエノールブルーを含む） を加える事で反応停止させた。反応産物は 5分間の煮沸後、氷水中で急冷され、 lx TBE緩衝液を用いた 7M尿素含有 12%ポリアクリルアミドゲル電気泳動 (PAGE)で分離 された。 PAGEパターンはフルォロイメージヤー 585 (アマシャムバイオサイエンス社製 )で可視化され、分析された。  The reaction conditions were the same as those for the primer extension activity measurement except that dNTP was not included in the reaction system. DNA polymerase was added and reacted at 60 ° C for 15 minutes. After a predetermined time, the reaction was stopped by adding 40 1 S top solution (containing 95% formamide, 10 mM EDTA, 0.5 mg / ml bromophenol blue). The reaction products were boiled for 5 minutes, quenched in ice water, and separated by 12% polyacrylamide gel electrophoresis (PAGE) containing 7M urea using lx TBE buffer. The PAGE pattern was visualized and analyzed with a Fluoro Imager 585 (Amersham Biosciences).
[0054] (3)タンパク質定量と SDS-ポリアクリルアミドゲル電気泳動（SDS-PAGE)  [0054] (3) Protein quantification and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
タンパク質量は Protein assay system (Bio- Rad社)を用い、牛血清ァノレブミンを標準タ ンパク質として定量された。 SDS-PAGEは Laemmli法に従い、タンパク質バンドはクマ シーブリリアントブルー R-250で染色され、分子量マーカーとして広レンジタンパク質 マーカー (Bio- Rad社)を用いた。  The amount of protein was quantified using a protein assay system (Bio-Rad) and bovine serum anolebumin as a standard protein. SDS-PAGE was performed according to the Laemmli method, and the protein band was stained with Coomassie Brilliant Blue R-250, and a wide range protein marker (Bio-Rad) was used as a molecular weight marker.
A. PolDヘテロオリゴマーの DP1(1- 200)の欠失変異体の 0.2 M NaCl存在下におけ る DNAポリメラーゼ活性 A. DNA polymerase activity in the presence of 0.2 M NaCl of DP1 (1-200) deletion mutant of PolD hetero-oligomer
[0055] (1) PolDヘテロオリゴマー野生型と DPK1-200)の欠失変異体。 [0055] (1) PolD hetero-oligomer wild type and DPK1-200) deletion mutant.
図 2.に実施例 5で得られた精製酵素標品の SDS-PAGEパターンを示す。野生型と DPK1-200)の欠失変異体 (PolD A S (1-200))は完全に精製された。 (2)食塩濃度変化による PolDヘテロオリゴマー野生型と、実施例 5で得られた DP1(1- 200)欠失変異体（PolD Δ S (1-200))の 3'-5'ェキソヌクレアーゼ活性と DNAポリメラー ゼ活性 (プライマー伸長活性)への影響。 Figure 2 shows the SDS-PAGE pattern of the purified enzyme preparation obtained in Example 5. The wild type and DPK1-200) deletion mutant (PolD AS (1-200)) was completely purified. (2) 3'-5 'exonuclease of PolD hetero-oligomer wild type by salt concentration change and DP1 (1-200) deletion mutant (PolD Δ S (1-200)) obtained in Example 5 Effects on DNA activity and DNA polymerase activity (primer extension activity).
3'- 5'ェキソヌクレアーゼ活性は図 3Aに示すように、 84/34-mer DNA基質を用い、 食塩濃度を 0から 300mM (0, 100, 150, 200, 300mM)まで変化させて測定した。食塩 が存在しないと、 PolD A S (1-200)は野生型に較べると高い 3'- 5'ェキソヌクレアーゼ 活性を示した。食塩の効果は阻害的で、両酵素において、塩濃度が上昇すると 3'-5' ェキソヌクレアーゼ活性は低下し、 0.2Μ濃度でほとんど検出出来ない。  3'-5 'exonuclease activity was measured using 84 / 34-mer DNA substrate and changing the salt concentration from 0 to 300 mM (0, 100, 150, 200, 300 mM) as shown in Figure 3A. . In the absence of salt, PolD A S (1-200) showed higher 3'-5 'exonuclease activity compared to the wild type. The effect of sodium chloride is inhibitory, and for both enzymes, 3'-5 'exonuclease activity decreases with increasing salt concentration and is almost undetectable at 0.2% concentration.
[0056] 一方、 DNAポリメラーゼ活性 (プライマー伸長活性）は図 3Βに示すように、 84/34- mer FAMラベル化 2本鎖 DNA基質を用い、 0.2 M食塩存在下でのプライマー伸長活 性として、食塩濃度を 0から 300mMまで変化させて測定された。食塩が存在しないと、 PolD A S (1-200)は野生型に較べると、高い 3し5'ェキソヌクレアーゼ活性による基質 の分解のため、 DNAポリメラーゼ活性は低く抑えられた。しかし、野生型 PolDの DN Aポリメラーゼ活性は塩濃度に感受性で、塩濃度が上昇すると低下した力 PolD A S (1-200)のそれは塩濃度耐性で、 0.2 Mまで、 DNAポリメラーゼ活性は高く維持され た。そのレベルは食塩のな 、時の野生型 PolDの DNAポリメラーゼ活性レベルと同等 であった。このことは、 0?1(1-200)の欠失が食塩耐性を欠失変異体13010 3 (1-200) に付与することを示す。 [0056] On the other hand, as shown in Fig. 3 (b), the DNA polymerase activity (primer extension activity) was determined as the primer extension activity in the presence of 0.2 M salt using an 84 / 34-mer FAM-labeled double-stranded DNA substrate. It was measured by changing the salt concentration from 0 to 300 mM. In the absence of salt, PolD AS (1-200) had a lower DNA polymerase activity due to the degradation of the substrate by 3 and 5 'exonuclease activity compared to the wild type. However, the DNA polymerase activity of wild-type PolD is sensitive to salt concentration, and the force that decreases with increasing salt concentration is that of PolD AS (1-200), which is resistant to salt concentration, and DNA polymerase activity remains high up to 0.2 M. It was. The level was equivalent to that of wild-type PolD DNA polymerase activity without salt. This indicates that deletion of 0-1 (1-200) confers salt tolerance to the deletion mutant 1 3 010 3 (1-200).
[0057] (3) 0.2 M食塩存在下での PolD野生型と PolD Δ S (1-200)の DNAポリメラーゼ活性（ プライマー伸長活性)の比較。  [0057] (3) Comparison of PolD wild-type and PolD Δ S (1-200) DNA polymerase activity (primer extension activity) in the presence of 0.2 M salt.
0.2 M食塩存在下での PolD野生型と PolD Δ S(l-200)の DNAポリメラーゼ反応（プラ イマ一伸長反応）を、 84/34-mer FAMラベル化 2本鎖 DNAを用いて 60°C2分間行い 、 0.2 M食塩存在下での PolD野生型と PolD A S (1-200)の DNAポリメラーゼ活性（プ ライマー伸長活性)を産物の長さと産物量で評価した。図 4に示すように、 5PolD A S ( 1-200)は 0.2 M食塩存在下であろうと 5分間で全長 (84-mer)の産物を合成できた力 P ◦ID野生型は 0.2 M食塩存在下では 12.5分後でも全長産物は合成できな力つた。  PolD wild-type and PolD Δ S (l-200) DNA polymerase reaction (primer extension reaction) in the presence of 0.2 M salt was converted to 60 ° C2 using 84 / 34-mer FAM-labeled double-stranded DNA. The DNA polymerase activity (primer extension activity) of PolD wild type and PolD AS (1-200) in the presence of 0.2 M salt was evaluated by the product length and product amount. As shown in Figure 4, 5PolD AS (1-200) was able to synthesize a full-length product (84-mer) in 5 minutes even in the presence of 0.2 M salt. Then, even after 12.5 minutes, the full-length product could not be synthesized.
[0058] (4) 0.2 M食塩存在下での PolD野生型と PolD Δ S (1-200)の DNAポリメラーゼ活性（ プライマー伸長活性)の測定。 [0058] (4) PolD wild type and PolD Δ S (1-200) DNA polymerase activity in the presence of 0.2 M salt ( Measurement of primer extension activity.
0.2 M食塩存在、非存在下で PolD野生型と PolD A S (1-200)を用いてプライマー伸 長反応を行い、 DNA合成産物量を定量した。プライマー伸長反応は、 84/34-merの F AMラベル化 2本鎖 DNA基質を用い、 60°C、 2分間行った。結果を図 7に示す。図 5 の結果から明らかなように、 食塩存在下で PolD A S (1-200)は PolD野生型の 2倍の 産物を合成できた。この結果は、 PolDからの DPK1-200)の欠失は、その変異体に 0.2 M食塩存在下であろうと、食塩非存在下の PolD野生型と同等の DNAポリメラーゼ活 性を付与することを示した。  Primer extension reaction was performed using PolD wild type and PolD A S (1-200) in the presence or absence of 0.2 M salt to quantify the amount of DNA synthesis product. The primer extension reaction was performed at 60 ° C. for 2 minutes using an 84 / 34-mer FAM-labeled double-stranded DNA substrate. The results are shown in FIG. As is clear from the results in Fig. 5, PolD A S (1-200) was able to synthesize twice the product of PolD wild type in the presence of salt. This result indicates that the DPK1-200) deletion from PolD confers DNA polymerase activity equivalent to PolD wild-type in the absence of salt, even in the presence of 0.2 M salt. It was.
[0059] B. DP1(1- 200)による 0.2 M NaCl存在下での DNAポリメラーゼ活性（プライマー伸長 活件)の ¾制 [0059] B. Control of DNA polymerase activity (primer extension activity) in the presence of 0.2 M NaCl by DP1 (1-200)
0.2 M NaCl存在下で、実施例 5で得られた PolD Δ S (1-200)を、実施例 4で得られた DPl(l-200)、及び牛血清アルブミン (BSA)と各割合で添カ卩混合し、 84/34-mer FAM ラベル化 2本鎖 DNA基質を用い、プライマー伸長反応を 60°C、 2分間行った。  In the presence of 0.2 M NaCl, PolD Δ S (1-200) obtained in Example 5 was added to DPl (l-200) and bovine serum albumin (BSA) obtained in Example 4 at each ratio. The mixture was mixed and a primer extension reaction was performed at 60 ° C. for 2 minutes using 84 / 34-mer FAM-labeled double-stranded DNA substrate.
なお、 0?1(1-200)及び83 の!¾10 3 (1-200)に対するモル比は、それぞれ 0:1、 15 :1、 30:1、 60:1、 90:1、 120:1とした。  The molar ratio of 0-1 (1-200) and 83 to! ¾10 3 (1-200) is 0: 1, 15: 1, 30: 1, 60: 1, 90: 1, 120: 1, respectively. It was.
結果は、図 6に示すように、 0.2 M食塩存在下で高く維持された PolD A S (1-200)の DNAポリメラーゼ活性は過剰量の DP1(1- 200)の添加で抑制された。 0.2 M食塩存在 下で、 PolD A S (1-200)の反応液に DP1(1- 200)をカ卩えると、その添カ卩量の増加に伴い プライマー伸長産物量が減少し、産物の長さも短くなつた。 DPl(l-200;^PolD A S (1 -200)量の 120倍加えられた時に、その活性は PolD野生型とほぼ同等になった。すな わち、 DP1(1- 200)は DNAポリメラーゼ活性 (プライマー伸長活性)を抑制する。  As a result, as shown in FIG. 6, the DNA polymerase activity of PolD A S (1-200), which was maintained at a high level in the presence of 0.2 M sodium chloride, was suppressed by adding an excessive amount of DP1 (1-200). When DP1 (1-200) is added to the reaction solution of PolD AS (1-200) in the presence of 0.2 M sodium chloride, the amount of primer extension product decreases as the amount of the additive increases, and the product length increases. It was also shorter. When added 120 times the amount of DPl (l-200; ^ PolD AS (1 -200), its activity was almost the same as that of PolD wild type, that is, DP1 (1-200) is a DNA polymerase. Inhibits activity (primer extension activity).
[0060] C DPI (1— 200)の各種欠失変異体の使用に某づぐ DNAポリメラーゼ活性制御 領城の同定 [0060] Control of DNA polymerase activity based on the use of various deletion mutants of C DPI (1-200)
(1)各種欠失変異体  (1) Various deletion mutants
実施例 4で得られた、 DPI (1— 200)の各領域を欠失させた DPI (1— 200)の各 種欠失変異体の SDSパターンを図 7に示す。図 7に示されるように、 DPI (1 - 200) 及び DPIの各種欠失変異体は完全に精製された。  FIG. 7 shows the SDS pattern of the various deletion mutants of DPI (1-200) obtained in Example 4 from which each region of DPI (1-200) has been deleted. As shown in FIG. 7, DPI (1-200) and various deletion mutants of DPI were completely purified.
(2) DPK1-200)中の DNAポリメラーゼ活性制御領域の同定。 0.2 M NaCl存在下で、 PolD A S (1-200)を、各濃度の DPl(l-200)野生型及びその各 種変異体とそれぞれ混和し、プライマー伸長反応を、 84/34-mer FAMラベル化 2本 鎖 DNA基質を用いて 60°C、 2分間行い、プライマー伸長活性を測定した。 (2) Identification of DNA polymerase activity control region in DPK1-200). In the presence of 0.2 M NaCl, PolD AS (1-200) was mixed with each concentration of DPl (l-200) wild type and its various mutants, and primer extension reaction was performed using the 84 / 34-mer FAM label. The primer extension activity was measured at 60 ° C for 2 minutes using a double-stranded DNA substrate.
[0061] なお、 DPK1-200)野生型及びその各種欠失変異体の PolD Δ S (1-200)に対するモ ル比は、それぞれ 15:1、 30:1、 60:1、 90:1、 120:1とした。 [0061] The molar ratios of DPK1-200) wild type and its various deletion mutants to PolDΔS (1-200) are 15: 1, 30: 1, 60: 1, 90: 1, 120: 1.
結果を図 8に示す。これによれば、 DPK1-200)の N末端欠失変異体が添加された時 に、プライマー伸長活性への抑制効果が見られ、欠失変異体量が増加するにつれ、 鎖長が減少した。一方、 DP1(1- 200)の C末端欠失変異体 (DP1(1- 139), DP1(1- 166), DP1(33-166))では、そのようなプライマー伸長活性への阻害効果は検出されなかつ た。  The results are shown in FIG. According to this, when the DPK1-200) N-terminal deletion mutant was added, an inhibitory effect on the primer extension activity was observed, and the chain length decreased as the amount of the deletion mutant increased. On the other hand, C1 deletion mutants of DP1 (1-200) (DP1 (1-139), DP1 (1-166), DP1 (33-166)) have such inhibitory effect on primer extension activity. It was not detected.
このことから、 34残基ポリペプチド、 DPK167-200)は DNA合成活性制御領域であり 、 PolD A S (1-200)の DNAポリメラーゼ活性 (プライマー伸長活性)を維持する機能を 阻害することが明らかになった。  This reveals that the 34-residue polypeptide, DPK167-200) is a DNA synthesis activity control region and inhibits the function of maintaining the DNA polymerase activity (primer extension activity) of PolD AS (1-200). became.
[0062] D.各糠へテロオリゴマー栾虽体のプライマー伸長活件の測定 [0062] D. Measurement of primer extension activity of each hetero-oligomer rod
(1)実施例 5で得られた、各種 DPIの N末端側あるいは内部配列が欠失したヘテロォ リゴマー変異体、 PolD A S (1-200)、 PolD A S (1-65)、 PolD A S (140-200)、 PolD A S (1 67-200)、の SDS- PAGEパターン。  (1) Heteroligomer mutants obtained in Example 5, lacking the N-terminal side or internal sequence of various DPIs, PolD AS (1-200), PolD AS (1-65), PolD AS (140- 200), PolD AS (167-200), SDS-PAGE pattern.
図 9に示すように、ヘテロオリゴマー変異体、 PolD A S (1-200)、 PolD A S (1-65)、 Pol D A S (140-200)、 PolD A S (167-200)は完全に精製され、 DP2と欠失 DPIに相当する 2 本のタンパク質バンドが確認された。このことから ヽずれの変異体もへテロオリゴマー 構造を形成することができ、 85°C、 30分間の加熱処理に耐性であることが明らかにな つた。なお、この試験においては各 1.6 gのへテロオリゴマー変異体が SDS-PAGEで 分析された。  As shown in Figure 9, the hetero-oligomer variants, PolD AS (1-200), PolD AS (1-65), Pol DAS (140-200), PolD AS (167-200) were completely purified and DP2 And two protein bands corresponding to the deleted DPI. From this, it was clarified that all the mutants can form a hetero-oligomer structure and are resistant to heat treatment at 85 ° C for 30 minutes. In this study, 1.6 g of heterooligomer variants were analyzed by SDS-PAGE.
[0063] (2) DPIの N末端側あるいは内部配列が欠失したヘテロオリゴマー変異体、 PolD Δ S (1-200)、 PolD A S (1-65)、 PolD A S (140-200)、 PolD A S (167- 200)のプライマー伸 長活性の測定。  [0063] (2) Hetero-oligomer mutant lacking the N-terminal side or internal sequence of DPI, PolD Δ S (1-200), PolD AS (1-65), PolD AS (140-200), PolD AS Measurement of primer extension activity of (167-200).
上記各へテロオリゴマー変異体を用いて、プライマー伸長反応を、 84/34-mer FAM ラベル化 2本鎖 DNA基質を用いて 60°C、 2分間行い、プライマー伸長活性を測定し た。なお、伸長 DNA産物の定量は、 Fluor Imagerを用いて計測される蛍光強度の積 算により行った。その際、標準偏差 (SD)値は 3回の独立した実験結果を基に計算さ れた。 Using each of the above hetero-oligomer variants, a primer extension reaction was performed at 60 ° C for 2 minutes using an 84 / 34-mer FAM-labeled double-stranded DNA substrate, and the primer extension activity was measured. It was. The elongation DNA product was quantified by multiplying the fluorescence intensity measured using Fluor Imager. The standard deviation (SD) value was calculated based on the results of three independent experiments.
図 10に示すように、食塩非存在下では PolD A S (1-200)、 PolD A S (140-200)、 PolD A S (167- 200)の DNA合成活性は野生型 PolDより低かった。 0.2M食塩存在下では Pol D A S (1-200)、 PolD Δ S (140-200)、 PolD Δ S (167- 200)の DNA合成活性は高く維持 されたが、逆に野生型 PolDと PolD A S (1-65)は活性が減少した。このことから 34残基 ポリペプチド、 DPK167-200)が食塩存在下での DNAポリメラーゼ活性制御に必須な 配列であることが明らかになった。また、この事実から、 34残基ポリペプチド、 DPK167 -200)を PolDから欠失させるならば、高食塩存在下でも、 DNA伸長活性を高く維持し た変異酵素の創成が可能であることが明らかである。  As shown in FIG. 10, in the absence of salt, the DNA synthesis activity of PolD A S (1-200), PolD A S (140-200), and PolD A S (167-200) was lower than that of wild-type PolD. In the presence of 0.2M salt, the DNA synthesis activity of Pol DAS (1-200), PolD Δ S (140-200), and PolD Δ S (167-200) was maintained high, but conversely, wild-type PolD and PolD AS (1-65) decreased in activity. This revealed that the 34-residue polypeptide, DPK167-200) is an essential sequence for controlling DNA polymerase activity in the presence of salt. In addition, it is clear from this fact that if a 34-residue polypeptide, DPK167-200) is deleted from PolD, it is possible to create a mutant enzyme that maintains high DNA elongation activity even in the presence of high salt. It is.
[0064] E. 34残某ポリペプチドの 3'-5'ェキソヌクレアーゼ活件の抑制 [0064] E. 34 Suppression of 3'-5 'exonuclease activity of residual polypeptide
DPK1-200)及びその 8種の欠失変異体（DP1(33- 200)、（DP1(49- 200)、 DP1(66- 20 0)、（DP1(85- 200)、（DP1(1- 139)、 DP1(1- 166)、 DP1(33- 180、 DP1(33- 166》を、それぞ れ PolD A S (1-200)と 60 : 1のモル比で混和し、 51-merの FAMラベル化一本鎖 DNAを 基質として、 60°C、 15分反応させ、 3'-5'ェキソヌクレアーゼ活性を測定した。また、 上記 D. (2)で用いた各種へテロオリゴマー変異体（PolD Δ S (1-200)、 PolD Δ S (1-6 5)、 PolD A S (140-200)、 PolD A S (167- 200))を、 DPI (1— 200)及びその 8種の欠 失置換体の非存在下、 84/34-merFAMラベル化 2本鎖 DNA基質、及び 51-merの FA Mラベルイ匕一本鎖 DNAを基質として、それぞれ 60。C、 15分反応させ、それぞれ 3'- 5'ェキソヌクレアーゼ活 ¾を測定した。  DPK1-200) and its eight deletion mutants (DP1 (33-200), (DP1 (49-200), DP1 (66-200), (DP1 (85-200), (DP1 (1- 139), DP1 (1-166), DP1 (33-180, DP1 (33-166), respectively, with PolD AS (1-200) at a molar ratio of 60: 1 to give a 51-mer FAM The labeled single-stranded DNA was used as a substrate and reacted at 60 ° C for 15 minutes to measure the 3'-5 'exonuclease activity, and the various hetero-oligomer mutants used in D. (2) above ( PolD Δ S (1-200), PolD Δ S (1-6 5), PolD AS (140-200), PolD AS (167-200)), DPI (1-200) and its 8 types In the absence of substitution, using 84 / 34-mer FAM-labeled double-stranded DNA substrate and 51-mer FAM-labeled single-stranded DNA as substrate, respectively, react for 60 C for 15 minutes, 3 ' -5 'exonuclease activity was measured.
なお、これらにおいては、上記 A. (2)に記載したように、 3し5'ェキソヌクレアーゼ活 性に対し食塩は阻害的に働くので、本試験の 3'-5'ェキソヌクレアーゼ活性測定には 食塩はカ卩えなかった。  In these cases, as described in A. (2) above, salt acts to inhibit 3 'and 5' exonuclease activity, so 3'-5 'exonuclease activity was measured in this test. There was no salt.
[0065] 結果を図 13に示す。これによれば、 C末端欠失タンパク質が加えられた場合は、 Po ID A S (1-200)のェキソヌクレアーゼ活性の抑制は観察されなかった。一方、 DPl(l-2 00)や N末端欠失タンパク質が加えられた場合は、 PolD A S (1-200)のェキソヌクレア ーゼ活性が減少した（図 11 A)。また、 PolD A S (167-200))は良好な 3し5'ェキソヌク レアーゼ活性を示した力 PolD A S (1-65)は野生型と同等であった（図 1 IB) 。 The results are shown in FIG. According to this, inhibition of exonuclease activity of Po ID AS (1-200) was not observed when C-terminal deletion protein was added. On the other hand, when DPl (l-200) or N-terminal deletion protein was added, the exonuclease activity of PolD AS (1-200) decreased (FIG. 11A). Also, PolD AS (167-200) is good 3 and 5 'exonuku The force PolD AS (1-65) that showed lyase activity was equivalent to that of the wild type (FIG. 1 IB).
これらのことから 34残基ポリペプチド、 DPl(167-200)が食塩非存在下での 3'-5'ェキ ソヌクレアーゼ活性制御に必須な配列であることが明らかになった。したがって、 34残 基ポリペプチド、 DP 167-200)を PolDから欠失させるならば、食塩非存在下で、 3'-5' ェキソヌクレアーゼ活性が高進した変異酵素の創成が可能であることが明らかである F.他の Pyrococcus属菌由来の PolD酵素における、 DPI (167— 200)と相同性を する纖  These results revealed that the 34-residue polypeptide, DPl (167-200), is an essential sequence for controlling 3'-5 'exonuclease activity in the absence of salt. Therefore, if 34 residue polypeptide, DP 167-200) is deleted from PolD, it is possible to create a mutant enzyme with enhanced 3'-5 'exonuclease activity in the absence of salt. F. F. 纖 Similar to DPI (167-200) in PolD enzymes from other Pyrococcus species
他の Pyrococcus属菌由来の PolD酵素のアミノ酸配列を抽出し、 DPI (167— 200) 領域について、 GeneWorks program (IntelliGenetics)により、その配列ァライメントを 作成した (配列番号 33〜35、図 12)。  The amino acid sequences of PolD enzymes derived from other Pyrococcus spp. Were extracted and their sequence alignments were created for the DPI (167-200) region using the GeneWorks program (IntelliGenetics) (SEQ ID NOs: 33-35, Fig. 12).
なお、各 PolD配列のァクセッション番号は、 P. horikoshii, BA000001; P. abyssi, AJ 248283; P. foriosus, AE010127である。  The accession number of each PolD sequence is P. horikoshii, BA000001; P. abyssi, AJ 248283; P. foriosus, AE010127.
これによれば、 DNAポリメラーゼ活性 (プライマー伸長活性)制御領域であり、かつ 、NaCl非存在下で 3'-5'ェキソヌクレアーゼ活性を抑制する領域である 34残基ポリべ プチド、 DPl(167-200)は Pyrococcus属の産する PolDの DPIサブユニットに広く保存さ れていることが明らかである。従って、この相同領域を欠失させるならば、 P. horikoshi 油来 PolDで観察された DNA合成活性と 3'_5'ェキソヌクレアーゼ活性への機能改変 が P. abyssiと P. foriosus由来 PolD酵素でも再現可能である。  According to this, a 34-residue polypeptide, DPl (167), which is a DNA polymerase activity (primer extension activity) control region and a region that suppresses 3′-5 ′ exonuclease activity in the absence of NaCl. -200) is clearly conserved in the DPI subunit of PolD from the genus Pyrococcus. Therefore, if this homologous region is deleted, the DNA synthesis activity and functional modification to 3'_5 'exonuclease activity observed in P. horikoshi Yurai PolD can be achieved with P. abyssi and P. foriosus PolD enzymes. It can be reproduced.

Claims

請求の範囲 The scope of the claims
[1] 小サブユニットとインティン配列を含有しない大サブユニットとからなる、 DNAポリメ ラーゼ活性及び 3'—5'ェキソヌクレアーゼ活性を有する、パイロコッカス属細菌由来 の耐熱性へテロオリゴマー酵素において、小サブユニットが、 DNA合成活性制御領 域であって、かつ 3 '—5，ェキソヌクレアーゼ活性抑制領域が削除されて 、ることを特 徴とする、上記耐熱性へテロオリゴマー酵素変異体。  [1] A thermostable hetero-oligomer enzyme derived from a Pyrococcus bacterium having a DNA polymerase activity and a 3′-5 ′ exonuclease activity comprising a small subunit and a large subunit not containing an intin sequence, The heat-stable hetero-oligomer enzyme mutant described above, wherein the small subunit is a DNA synthesis activity control region and the 3'-5, exonuclease activity suppression region is deleted.
[2] 小サブユニットから削除される領域力 配列番号 33に示されるアミノ酸配列あるい は該アミノ酸配列と 45%以上の相同性を有するアミノ酸配列を少なくとも含むことを 特徴とする。請求項 1に記載の耐熱性へテロオリゴマー酵素変異体。 [2] Region force deleted from small subunits The amino acid sequence shown in SEQ ID NO: 33 or at least an amino acid sequence having 45% or more homology with the amino acid sequence is characterized. The thermostable hetero-oligomer enzyme mutant according to claim 1.
[3] 以下に示される大サブユニットと小サブユニット変異体とからなり、かつ DNAポリメ ラーゼ活性及び 3，一 5，ェキソヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵 素変異体。 [3] A thermostable hetero-oligomer enzyme mutant consisting of the large subunit and small subunit mutants shown below and having DNA polymerase activity and 3,15, exonuclease activity.
(D a)配列番号 5に示されるアミノ酸配列を有するか、あるいは b) a)のアミノ酸配列 にお ヽて 1または数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を 有する大サブユニット。 (D a) has the amino acid sequence shown in SEQ ID NO: 5, or b) has a large amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence of a) Sub unit.
(2) a)配列番号 1に示されるアミノ酸配列において、少なくとも 167〜200番目の領 域が削除されたアミノ酸配列を有する力、あるいは b) a)のアミノ酸配列において、 1 乃至数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を有する小サブ ユニット変異体。  (2) a) the ability to have an amino acid sequence in which at least the 167th to 200th region is deleted in the amino acid sequence shown in SEQ ID NO: 1, or b) 1 to several amino acid residues in the amino acid sequence of a) A small subunit variant having an amino acid sequence in which a group is deleted, substituted or added.
[4] 請求項 3に記載の大サブユニットをコードする DNAと、同小サブユニット変異体をコ ードする DNAと力 なる組み合わせ。 [4] A powerful combination of DNA encoding the large subunit according to claim 3 and DNA encoding the small subunit mutant.
ノイロコッカス属細菌由来の、小サブユニットとインティン配列を含有しない大サブ ユニットからなる耐熱性へテロオリゴマー酵素における小サブユニットにおいて、配列 番号 33に示されるアミノ酸配列あるいは該アミノ酸配列と 45%以上の相同性を有す るアミノ酸配列を少なくとも含む領域が削除され、かつ、大サブユニットとヘテロオリゴ マーを構成したとき、 DNAポリメラーゼ活性及び 3'—5'ェキソヌクレアーゼ活性を有 するものであることを特徴とする、耐熱性へテロオリゴマー酵素の小サブユニット変異 体。 A large subunit derived from the genus Neurococcus that does not contain small subunits and intin sequences In the small subunit of the thermostable hetero-oligomer enzyme comprising a unit, the region containing at least the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having 45% or more homology with the amino acid sequence is deleted, A small subunit mutant of a thermostable hetero-oligomer enzyme characterized by having a DNA polymerase activity and a 3'-5 'exonuclease activity when a hetero-oligomer is composed of a large subunit.
[6] a)配列番号 1に示されるアミノ酸配列において、少なくとも 167〜200番目の領域 が削除されたアミノ酸配列を有する力、あるいは b) a)のアミノ酸配列において、 1また は数個のアミノ酸残基が欠失、置換または付加されたアミノ酸配列を有し、かつ、イン ティン配列を含まな ヽ大サブユニットとヘテロオリゴマーを構成したとき、 DNAポリメ ラーゼ活性及び 3， 一 5，ェキソヌクレアーゼ活性を有するものであることを特徴とする 、請求項 5に記載の耐熱性へテロオリゴマー酵素の小サブユニット変異体。 [6] a) the ability to have an amino acid sequence in which at least the 167th to 200th region is deleted in the amino acid sequence shown in SEQ ID NO: 1; or b) one or several amino acid residues in the amino acid sequence of a) DNA polymerase activity and 3,15, exonuclease activity when a hetero-oligomer with a large subunit having an amino acid sequence in which a group is deleted, substituted or added and does not contain an intin sequence The small subunit mutant of the thermostable hetero-oligomer enzyme according to claim 5, characterized in that
[7] 請求項 6に記載の小サブユニット変異体をコードする DNA。 [7] DNA encoding the small subunit variant according to claim 6.
[8] 小サブユニットと大サブユニットからなり、 DNAポリメラーゼ活性及び 3'— 5'ェキソ ヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素における小サブユニットをコ ードする DNAであって、 配列番号 2に示される塩基配列において、 499〜600番目 の領域が削除された塩基配列を有するか、あるいは、該塩基配列において 1又は数 個のヌクレオチドが欠失、置換、又は付加された塩基配列を有する DNA。 [8] A DNA encoding a small subunit in a thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′—5 ′ exonuclease activity, comprising SEQ ID NO: 2 DNA having the nucleotide sequence from which the 499th to 600th region is deleted or one or several nucleotides deleted, substituted, or added in the nucleotide sequence .
[9] 小サブユニットと大サブユニットからなり、 DNAポリメラーゼ活性及び 3'— 5'ェキソ ヌクレアーゼ活性を有する耐熱性へテロオリゴマー酵素における小サブユニットをコ ードする DNAであって、請求項 8に記載の DNA、あるいは該 DNAと相補の配列を有 する DNAとストリンジェントな条件下、ハイブリダィズする DNA。 [9] DNA encoding a small subunit in a thermostable hetero-oligomer enzyme comprising a small subunit and a large subunit and having DNA polymerase activity and 3′-5 ′ exonuclease activity, Or a DNA that hybridizes under stringent conditions with the DNA described in 1. or a DNA having a sequence complementary to the DNA.
[10] 請求項 7〜9の!、ずれかに記載の DNAを含む組み換えベクター 請求項 7〜9のいずれかに記載の DNA及び請求項 2に記載の大サブユニットをコ' ドする DNAが、共発現可能に含むことを特徴とする、組み換えベクター。 請求項 10又は 11に記載の組み換えベクターが導入されて 、ることを特徴とする、 形質転換体。 [10] A recombinant vector comprising the DNA according to any one of claims 7 to 9! A recombinant vector comprising the DNA according to any one of claims 7 to 9 and the DNA encoding the large subunit according to claim 2 so as to be capable of co-expression. A transformant, wherein the recombinant vector according to claim 10 or 11 is introduced.
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SHEN Y. ET AL.: "Invariant Asp-1122 and Asp-1124 are essential residues for polymerization catalysis of family D DNA polymerase from Pyrococcus horikoshii", J. BIOL. CHEM., vol. 276, no. 29, 2001, pages 27376 - 27383, XP003017377 *
SHEN Y. ET AL.: "Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii", J. BIOL. CHEM., vol. 278, no. 23, 2003, pages 21247 - 21257, XP003017376 *

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