JP2006180886A - Method for producing DNA polymerase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly heat-resistant DNA polymerase having 3'-5 'exonuclease activity which is a proofreading function of DNA chain elongation and strong primer extension activity.
SOLUTION: It consists of a small subunit and a large subunit having molecular weights of about 70 ± 7 kDa and about 144 ± 14.4 kDa, respectively, the large subunit does not contain an intein sequence, and is heated at least at 85 ° C. for 1 hour. It is preferable that it has about 50% activity, retains at least about 20% activity at 90 ° C. for 1 hour, and has DNA polymerase activity and 3′-5 ′ exonuclease activity. A thermostable heterodimeric enzyme derived from a thermoarchaebacterium, DNA encoding the enzyme, and a method for producing the enzyme.
[Selection figure] None

Description

The present invention relates to a method for producing a DNA polymerase having improved properties.

  DNA polymerase is an enzyme useful for DNA sequencing reaction, gene amplification reaction (PCR reaction), radioactive labeling of DNA, in vitro synthesis of mutant genes, and the like. DNA polymerases known to date can be roughly classified into four families based on the commonality of amino acid sequences. Among them, the family A (pol I type enzyme) represented by Escherichia coli DNA polymerase I and thermophilic bacterial thermus aquaticus DNA polymerase (Taq DNA polymerase) is generally used as a reagent for genetic manipulation experiments. , Belonging to family B (α-type enzyme) represented by T4 phage DNA polymerase.

Various DNA polymerases with different optimal temperatures have been discovered so far from bacteria and animals and plants, but most are derived from cold organisms, so they have poor heat resistance and PCR that includes heat denaturation reaction of template DNA above 94 ° C It was inappropriate for the reaction. In addition, enzymes derived from thermophilic bacteria such as Taq DNA polymerase are commercially available as thermostable DNA polymerases, but all of them lack 3'-5 'proofreading exonuclease activity, so errors occur during polymerase reactions such as PCR reactions. It is easy to induce and is not suitable for PCR reactions.
Furthermore, α-type enzyme with high heat resistance and 3'-5 'proofreading exonuclease activity is isolated and marketed from hyperthermophilic archaea such as Pyrococcus and Thermococcus, but has low primer extension activity. Not suitable for PCR reaction of long DNA.

  There is a strong need to find a DNA polymerase having high heat resistance, 3'-5 'exonuclease activity, which is a calibration function for DNA strand elongation, and strong primer extension activity, and stably supplying it at a low price.

  In order to solve the above problems, the present inventor has focused on a hyperthermophilic archaea that grows at 90 to 100 ° C., and has found a gene presumed to exhibit the present enzyme activity from its gene sequence. Furthermore, we have produced an enzyme from the gene using bacteria, confirmed that this enzyme exists relatively stably even at a high temperature of 85 ° C., and exhibits DNA polymerase activity. The present invention was completed by finding a method for producing a large amount of the present enzyme having a high activity by incorporating it into the microorganism.

That is, the present invention is as follows.
(1) In a thermostable DNA polymerase derived from a thermophilic archaea comprising a small subunit and a large subunit, and the large subunit includes an intein sequence, the intein sequence in the large subunit is removed. An improved method for producing a DNA polymerase.

(2) The production method according to (1) above, wherein the produced DNA polymerase has 3′-5 ′ exonuclease activity and primer length-dependent primer extension activity.

(3) The production method according to (1) or (2) above, wherein the thermophilic archaea are archaea belonging to the genus Pyrococcus.

(4) The small subunit is a protein having the amino acid sequence shown in SEQ ID NO: 4, or includes substitution, deletion or addition of one or several amino acid residues in the amino acid sequence, The production method according to any one of the above (1) to (3), which is a protein that exhibits DNA polymerase activity when a dimer is formed.

(5) The large subunit of the DNA polymerase to be produced is a protein having a sequence in which the amino acid (955th to 1120th) encoded by the intein sequence is removed from the amino acid sequence shown in SEQ ID NO: 2, or A protein that exhibits improved DNA polymerase activity when formed into a dimer with a small subunit, comprising substitution, deletion or addition of one or several amino acid residues in the sequence; (1) The manufacturing method in any one of (4).

(6) A method for producing an improved DNA polymerase, comprising the following steps a) to c):
a) incorporating the DNA encoding the small subunit according to claim 4 and the DNA encoding the large subunit according to claim 5 into the same or different vectors;
b) transforming a host cell with the obtained recombinant vector to obtain a transformant;
c) A step of culturing the transformant to co-express these DNAs and collecting DNA polymerase from the culture.
(7) The production method according to (6) above, wherein the host cell is a bacterium.

Further, the present invention, in the first embodiment, comprises a small subunit and a large subunit having molecular weights of about 70 ± 7 kDa and about 144 ± 14.4 kDa, respectively, the large subunit does not contain an intein sequence, 85 ° C. Retain at least about 50% activity after 1 hour heat treatment, retain at least about 20% activity at 90 ° C for 1 hour heat treatment, and DNA polymerase activity and 3'-5 'exonuclease activity A thermostable heterodimeric enzyme derived from a thermophilic archaea characterized by comprising:

In an embodiment of the present invention, the thermophilic archaea is an archaea belonging to the genus Pyrococcus.
In another embodiment of the invention, the enzyme further exhibits primer length dependent primer extension activity.
In another embodiment of the invention, the enzyme further has an optimum pH of about 8.0 to about 9.0.
In another embodiment of the invention, the enzyme further has an optimal Mg2 + concentration of about 12 mM or greater.

In the second aspect, the present invention has the following properties (1): DNA polymerase activity and 3′-5 ′ exonuclease activity;
(2) consisting of a small subunit and a large subunit having a molecular weight of about 70 ± 7 kDa and about 144 ± 14.4 kDa, respectively, and the large subunit does not contain an intein sequence;
(3) The suitable pH is about 8.0 to about 9.0;
(4) The optimal Mg2 + concentration is about 12 mM or more;
(5) Retain at least about 50% activity at 85 ° C for 1 hour heat treatment and at least about 20% activity at 90 ° C for 1 hour heat treatment; and (6) Primer length dependence A thermostable heterodimer enzyme having the primer extension activity of

In an embodiment of the invention, the enzymes of the invention are derived from thermophilic archaea, preferably sulfur metabolizing thermophilic archaea, such as archaea belonging to the genus Pyrococcus.
In another embodiment of the present invention, the small subunit of the enzyme of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 4, or substitution of one or several amino acid residues in the amino acid sequence, It is a protein that contains the deletion or addition and expresses the property when it forms a dimer with the large subunit.

  In yet another embodiment of the present invention, the large subunit of the enzyme of the present invention is a protein having a sequence obtained by removing the amino acid sequence (955th to 1120th) encoded by the intein sequence from the amino acid sequence shown in SEQ ID NO: 2. Or a protein that contains a substitution, deletion or addition of one or several amino acid residues in the sequence and expresses the property when formed into a dimer with the small subunit.

In the third aspect, the present invention provides DNA encoding any of the enzymes defined above.
In the fourth embodiment, the present invention further removes DNA encoding a small subunit having the amino acid sequence shown in SEQ ID NO: 4 and the intein sequence (955th to 1120th) in the amino acid sequence shown in SEQ ID NO: 2. Provided is a one or two recombinant vector comprising a DNA encoding a large subunit having a sequence so that it can be coexpressed.

  Here, “consisting of one or two” means that both DNAs may be inserted into a single vector DNA as long as the DNAs encoding the large and small subunits can be co-expressed. Or each of the DNAs may be inserted in a different vector.

  According to an embodiment of the present invention, in the recombinant vector, the DNA encoding the small subunit has the nucleotide sequence shown in SEQ ID NO: 3, and the DNA encoding the large subunit is in the nucleotide sequence shown in SEQ ID NO: 1. It has a nucleotide sequence obtained by removing the sequence encoding the intein sequence (2863rd to 3360th).

  The present invention further provides, in a fifth aspect, a method for producing any of the above enzymes, comprising transforming a host cell with a DNA or a recombinant vector as defined above, and transforming the host cell in a medium. There is provided a method comprising culturing, co-expressing DNA encoding a small subunit and DNA encoding a large subunit, and recovering the enzyme composed of the small subunit and the large subunit.

In an embodiment of the invention, the host cell is a bacterium.
As used herein, “intein” refers to a proteinaceous intron. The “primer length-dependent primer extension activity” refers to a tendency that the primer extension activity of a DNA polymerase enzyme increases depending on the length of the primer. Further, “one or several” as used herein means 10 or less, preferably 7 or less, more preferably 5 or less.

The vector of the present invention can take the form of a plasmid, phage, phagemid (for example, cosmid), virus, etc., preferably a plasmid. And the DNA encoding the small subunit and / or the large subunit can be present on the same or different vector DNA, preferably on the same vector. Alternatively, DNAs encoding these large and small subunits may be integrated into the genome of the host by homologous recombination methods so that they can be co-expressed, in which case the host may be a bacterium from which these DNAs are derived or its close relatives. Species are preferred. The recombinant vector of the present invention can usually contain the same or different promoters so that the two DNAs of interest can be co-expressed. In the present specification, “co-expressible” means that two DNAs can be expressed simultaneously in a host to produce a protein encoded by each DNA.

According to the present invention, it is possible to provide a DNA polymerase that replicates long-chain DNA at high speed and accurately in a high-temperature environment, which is considered useful for Long-PCR. In addition, it will be possible to develop new methods for gene sequence analysis using this enzyme.

The present invention will be specifically described below.
The enzyme of the present invention has the properties described above and can be obtained from nature or artificially. For example, the enzyme can be obtained from a hyperthermophilic bacterium, preferably a thermophilic archaea, more preferably a sulfur metabolizing thermophilic archaea. Examples of thermophilic archaea include the genus Pyrococcus (eg, P. horikoshii, P. furiosus, P. woesei, etc.), Thermococcus Thermococcus genus [such as Thermococcus litoralis, Thermococcus cellar (T. celer), Thermococcus profundas (T. profundus), Thermococcus peptonopjilus (P.) (Pyrobaculum) genus [Pyrobaculum islandicum, etc.] and the like. Alternatively, the enzyme may be made using DNA recombination techniques.

As a method for obtaining the enzyme of the present invention, after culturing and growing a bacterium as described above, which is considered to contain the enzyme, in an appropriate medium, the cultured cells are destroyed by cell destruction such as homogenization, for example. The target enzyme can be obtained using any combination of general techniques for enzyme purification. Examples of such methods include salting out, heat treatment, solvent extraction, solvent precipitation, ultrafiltration, immunochemical methods, column chromatography (gel filtration, ion exchange chromatography, hydrophobic interaction) Chromatography, affinity chromatography, etc.), HPLC, electrophoresis, isoelectric focusing, and the like. As specific descriptions of these methods, reference can be made to, for example, the Japan Biochemical Society, New Chemistry Experiment Course 1, Protein I, Separation / Purification / Property, 1990, Tokyo Kagaku Dojin. The culture of bacteria varies depending on the type of bacteria and can be performed under known culture conditions.
For example, in the case of thermophilic archaea, many of them are anaerobic bacteria, so that nitrogen gas or the like is continuously aerated during culture, or a substance such as liquid paraffin is overlaid to block air, For about 1 to 4 days at 100 ° C, in the presence of NaCl 0 to 4%, NaSO2, KCl, Na2SO4, KBr, H3BO3, MgCl2, CaCl2, SrCl2, NH4Cl, K2HPO4 and other inorganic substances, CH3COONa, tryptone, yeast extract, etc. It can be cultured in a medium containing trace elements such as organic substances, MnSO4, FeSO4, ZnSO4 (see, for example, Kojo Chitosu et al., Biochemistry, Vol. 72, No. 3, pages 203-206, 2000).

  Alternatively, another method for obtaining the enzyme of the present invention includes a method using a DNA recombination technique. From the gene sequence of a hyperthermophilic bacterium, a gene that is likely to exhibit this enzyme activity is screened from, for example, a genomic library or cDNA library derived from the bacterium, and a target clone is obtained by a conventional method, or an appropriate primer is used. After amplification of the target DNA by the polymerase chain reaction (PCR) used, DNA is prepared and isolated by treatment with an organic solvent such as phenol or chloroform, or sucrose or cesium chloride density gradient centrifugation, and a protein expression plasmid (for example, pET11a Alternatively, the plasmid is inserted into an appropriate vector such as pET15b) so that the DNA can be expressed, and the plasmid is introduced into an appropriate host (for example, bacteria such as Escherichia coli and Bacillus subtilis, and optionally fungi such as yeast and basidiomycete). The enzyme of the present invention can be produced through culturing the host. In general, in addition to a promoter and a selectable marker that can be selected depending on the host, vectors include, for example, an origin of replication, a ribosome binding site (or Shine-Dalgarno sequence), a transcription initiation site, a terminator, and a polylinker (from multiple restriction enzyme sites). And the like) are included as appropriate. The target enzyme produced is isolated and purified by appropriately combining the above-mentioned conventional enzyme purification techniques.

  The enzyme of the present invention is a heterodimeric protein composed of two subunits having molecular weights of about 144 ± 14.4 kDa and about 70 ± 7 kDa, and has an activity of synthesizing a complementary strand using DNA as a template, and a 3′-5 ′ proofreading exonuclease activity. Is an enzyme classified as a so-called DNA-dependent DNA polymerase. Compared to this enzyme, it retains at least about 50% of its pre-treatment activity even when treated at 85 ° C for 1 hour in 50m Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and 10 mM MgCl2. In addition, when this DNA is used to extend a DNA strand using this enzyme, the extension activity is not observed when the primer length is about 15 mer, and the extension activity is observed when the primer length exceeds about 30 mer. The extension activity increases as the primer length increases. It also has a unique property of showing a tendency to increase.

  Specifically, the enzyme of the present invention is obtained by DNA recombination technology based on DNA derived from Pyrococcus horikoshi (particularly strain JCM9974 deposited at RIKEN) as shown below. Can do. However, unexpectedly, when preparing the enzyme of the present invention having DNA synthesis activity, a sequence encoding an intein sequence from a nucleotide sequence encoding a large subunit constituting the enzyme (for example, 2863 to 3360 of SEQ ID NO: 1). It was found necessary to be deleted. Thus, the enzyme thus obtained is, for example, a small subunit having the amino acid sequence shown in SEQ ID NO: 4, and an amino acid sequence encoded by an intein sequence in the amino acid sequence shown in SEQ ID NO: 2, for example (955th to 1120). Consists of heterodimers with large subunits having sequences removed. If the intein sequence is present in the large subunit, even if it forms a heterodimer with the small subunit, it shows very low DNA synthesis activity (see Table 1 in Example 11 below). Such low activity is also observed in the case of only the small subunit, in the case of only the large subunit, and in the case of only the large subunit from which the intein sequence has been removed. Therefore, in order for the enzyme of the present invention to exhibit practical DNA synthesis activity, it is necessary to remove the intein sequence (if present) from the large subunit.

The present invention also includes an enzyme consisting of a subunit protein containing substitution, deletion or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 4. Such a mutation operation comprising substitution, deletion or addition can be carried out using techniques well known to those skilled in the art, such as site-directed mutagenesis and PCR.
Specific examples of the DNA recombination technique, PCR method, site-directed mutagenesis method and the like used to synthesize the enzyme of the present invention include, for example, J. Sambrook et al., Molecular Cloning A Laboratory Manual, second edition, See Cold Spring Harbor Laboratory Press, 1989.

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.
<Example 1> Culture of fungus Pyrococcus horikoshi (RIKEN accession number JCM9974) was cultured by the following method.
13.5 g sodium chloride, 4 g Na2SO4, 0.7 g KCl, 0.2 g NaHCO3, 0.1 g KBr, 30 mg H3BO3, 10 g MgCl2-6H2O, 1.5 g CaCl2, 25 mg SrCl2, 1.0 ml resalin solution ( 0.2 g / L), 1.0 g of yeast extract and 5 g of 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 was added to 0.2%, and the medium was saturated with argon to make anaerobic, and then JCM9974 was inoculated. Whether or not the medium became anaerobic was confirmed by adding a Na2S solution and confirming that the pink color of the resazurin solution with Na2S was not colored in the culture solution. This culture solution was cultured at 95 ° C. for 2 to 4 days, and then centrifuged to collect bacteria.

<Example 2> Preparation of chromosomal DNA
The chromosomal DNA of JCM9974 was prepared by the following method.
After culturing, the cells are collected by centrifugation at 5000 rpm for 10 minutes. The cells are washed twice with 10 mM Tris (pH 7.5) -1 mM EDTA solution and then enclosed in an InCert Agarose (FMC) block. By treating this block in 1% N-lauroylsarcosine, 1 mg / ml protease K solution, chromosomal DNA is isolated and prepared in an Agarose block.

<Example 3> Preparation of library clone containing chromosomal DNA The chromosomal DNA obtained in Example 2 was partially decomposed with the restriction enzyme HindIII, and then a fragment of about 40 kb was prepared by agarose gel electrophoresis. This DNA fragment and Bac vectors pBAC108L and pFOS1 completely digested with the restriction enzyme HindII were ligated using T4 ligase. When the former vector pBAC108L was used, the DNA after the completion of binding was immediately introduced into E. coli by electroporation. When the latter vector pFOS1 is used, the DNA after completion of binding is packed into λ phage particles in a test tube with GIGA Pack Gold (manufactured by Stratagene), and the DNA is transferred into E. coli by infecting the particles with E. coli. Introduced. The E. coli population resistant to the antibiotic chloramphenicol obtained by these methods was used as BAC and Fosmid libraries. A clone suitable for covering the chromosome of JCM9974 was selected from the library, and the clones were aligned.

<Example 4> Determination of nucleotide sequence of each BAC or Fosmid clone The nucleotide sequence of the aligned BAC or Fosmid clones was sequentially determined by the following method. Each BAC or Fosmid clone DNA recovered from E. coli was fragmented by sonication, and 1 kb and 2 kb long DNA fragments were recovered by agarose gel electrophoresis. 500 shotgun clones were prepared for each BAC or Fosmid clone in which this fragment was inserted into the HincII restriction enzyme site of plasmid vector pUC118. The base sequence of each shotgun clone was determined using an automatic base sequence reader 373 or 377 manufactured by PerkinElmer, ABI. The base sequence obtained from each shotgun clone was ligated and edited using the base sequence automatic linking software Sequencher, and the entire base sequence of each BAC or Fosmid clone was determined.

<Example 5> Identification of DNA polymerase gene The base sequence of each BAC or Fosmid clone determined above was analyzed with a large computer, and the gene encoding the large subunit of DNA polymerase (Fig. 1) and the small subunit were identified. The encoding gene (Figure 3) was identified.

<Example 6> Construction of small subunit expression plasmid The following DNA primers were synthesized for the purpose of constructing restriction enzyme (NdeI and BamHI) sites before and after the small subunit structural gene region, and restricted by PCR before and after the gene. Enzyme site introduced:
Upper primer: PolS1; 5'-TTTTGTCGACGTACATATGGATGAATTCGTAAAG-3 '
(SEQ ID NO: 5; underlined indicates NdeI site);
Bottom primer: PolS2; 5'-TTTTGAGCTCTTTGGATCCTTAGAAGCTCCATCAGCACCACCT-3 '
(SEQ ID NO: 6; underlined indicates BamHI site).
After the PCR reaction, it was completely digested with restriction enzymes (NdeI and BamHI) (2 hours at 37 ° C.), and then its structural gene was purified. Further, pET11a or pET15b (both from Novagen) were cleaved and purified with restriction enzymes NdeI 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 competent cells of E. coli XL1-BlueMRF1 (Stratagene) to obtain transformant colonies. The expression plasmid was purified from the obtained colonies by the alkaline method.
The resulting expression plasmids were abbreviated as pET11a / PolS or pET15b / PolS, respectively. The absence of random mutations on the structural gene was confirmed by DNA sequencing.

<Example 7> Construction of plasmid for expressing full-length large subunit The full-length large subunit gene was cloned into the pGEMEX-1 vector (Promega) in two steps. The DNA fragment in the first half was obtained by PCR using the following two primers:
Upper primer: PolL1; 5'-CTCGACTTTAGCATATGGCTCTGATGGAGC-3 '
(SEQ ID NO: 7; underlined indicates NdeI site);
Bottom primer: PolL2; 5'-GCTTGTCGACGCCATAAACTTTGACATTATCCATTGCGCGCTTAAGCAAC-3 '
(SEQ ID NO: 8; underlined indicates SalI site).
This PCR product was completely digested with NdeI and SalI, then cloned into the pGEMEX-1 vector, and abbreviated as pGEM / PolL1-2.

The latter half of the DNA fragment was obtained by PCR using the following two primers:
Upper primer: PolL3; 5'-TTTATGGCGTCGACAAGCTGAAGG-3 '
(SEQ ID NO: 9; underlined indicates SalI site);
Bottom primer: PolL4; 5'-TATAACTTATGCATTGTGGTTATTTCGCTGAGAAG-3 '
(SEQ ID NO: 10; underlined indicates NsiI site).
This PCR product was completely digested with SalI and NsiI and then cloned into the previously prepared pGEM / PolL1-2 to obtain pGEM / PolL containing the full-length large subunit gene.

<Example 8> Construction of plasmid for expressing large subunit from which intein has been removed As shown in FIG. 2, some of the large subunit genes of P. horikoshii DNA polymerase (proteinaceous) Since one intein (which encodes an intron) is included, a DNA fragment upstream of the intein is amplified by PCR using primers PolL3 and PolL6 (below), and intein is obtained by PCR using primers PolL5 (below) and PolL4 A downstream DNA fragment was amplified. Using these two fragments and primers PolL3 and PolL4, the DNA fragment from which the intein was removed was amplified by overlap PCR. Next, this product was completely digested with restriction enzymes SalI and NsiI, and then cloned into the previously prepared pGEM / PolL1-2 to obtain pGEM / PolL (-Intein) containing the large subunit gene from which the intein was removed. .
PolL5: 5'-CACGCTGCAAAGAGGAGAAATTGCGATGGTGATGAAGATGCT-3 '
(SEQ ID NO: 11)
PolL6: 5'-AGCATCTTCATCACCATCGCAATTTCTCCTCTTTGCAGCGTG-3 '
(SEQ ID NO: 12)

<Example 9> Construction of plasmid co-expressing small subunit and large subunit from which intein was removed In order to achieve stable production of the target heterodimeric DNA polymerase, a plasmid co-expressing both subunits was constructed. . First, PCR was performed using primers PolS1 and PolS3 (below) to introduce a new multicloning site immediately upstream of the BamHI site of pET15b / PolS. As described below, PolS3 encodes BamHI, NsiI, SalI, and SacII sites in order from the 5′-end. The PCR product was treated with NdeI 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 template DNA and primers PolL7 (described below) and PolL2. The obtained 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 SalI site in the coding region. This was treated with SacII and SalI and inserted into the multicloning site constructed in pET15b / PolS (M). This plasmid is abbreviated as pET15b / PolSL1-2. Furthermore, pGEM / PolL (-Intein) was treated with SalI and NsiI, and the gene of the latter half of the large subunit (without intein) was isolated and remained at the end of the protein expression unit of pET15b / PolSL1-2. Inserted into SalI and NsiI 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 added to the amino terminus and a large subunit not containing intein.
PolS3: 5'-CGGGATCCATGCATGGTCGACACCGCGGTCAGCACCACCTACTAAAGTCGAG-3 '
(SEQ ID NO: 13; underlined parts indicate BamHI, NsiI, SalI, and SacII sites in order from the 5′-end.)
PolL7: 5'-GGTGTCCGCGGCTCACTATAGGGAGACCAC-3 '
(SEQ ID NO: 14; the underlined portion indicates the SacII site, and the bold type indicates the ribosome binding site of the pGEMEX-1 vector.)

Example 10 Expression of Recombinant Gene A competent cell of E. coli BL21-CodonPlus (DE3) -RIL, manufactured by Stratagene was melted and transferred to a Falcon tube by 0.1 mL. Then, 0.005 mL of the expression plasmid solution is added and left in ice for 30 minutes, followed by heat shock at 42 ° C. for 30 seconds, 0.9 mL of SOC medium is added, and cultured with shaking at 37 ° C. for 1 hour. Thereafter, an appropriate amount was spread on a 2YT agar plate containing ampicillin and cultured overnight at 37 ° C. to obtain a transformant. This transformant was named E. coli BL21-CodonPlus (DE3) -RIL / pET15b / PolSL (-Intein) and was established by the Institute of Biotechnology, Institute of Industrial Science and Technology (1-3 East 1-chome, Tsukuba, Ibaraki Prefecture). Deposited on April 14, 2012 and given the deposit number FERM P-17816.
The obtained transformant was cultured in a 2YT medium (2 liters) containing ampicillin until the absorption at 600 nm reached 1, and then IPTG (Isopropyl-β-D-thiogalactopyranoside) was added and further cultured at 30 ° C. for 8 hours. After culture, the cells were collected by centrifugation (6,000 rpm, 20 minutes).

<Example 11> Purification of thermostable enzyme The collected cells were frozen and thawed at -20 ° C, and twice as much 10 mM Tris-HCl buffer (pH 8.0) and 1 mg DNase were added to obtain a suspension. It was. The resulting suspension was kept at 37 ° C. for 30 minutes and then irradiated with ultrasonic waves for 10 minutes. Further, after heating at 85 ° C. for 30 minutes, the mixture was centrifuged (11,000 rpm, 20 minutes) to obtain a supernatant. This was used as a crude enzyme solution. Next, this crude enzyme solution was added to a Ni-column (using Novagen, His • Bind metal chelation resin & His • Bind buffer kit), and affinity chromatography was performed. The 60 mM imidazole elution fraction obtained here was replaced with 100 mM phosphate buffer (pH 6.0) with Centriprep 30 (Amicon). Further, this was adsorbed on a HiTrap SP (Pharmacia) column and eluted with a NaCl concentration gradient.
Next, SDS-electrophoresis of each fraction was performed, and the molecular weight of the contained protein was measured. Since the molecular weight of the DNA polymerase subunit was predicted to be about 144,000 Da and about 70,000 Da from the gene sequence, fractions containing proteins of this molecular weight were collected and 50 mM Tris-HCl buffer (pH 7. To 0). This was further adsorbed to a HiTrap heparin (Pharmacia) column, which is the next affinity chromatography column, and eluted with a NaCl concentration gradient to obtain a purified enzyme.

<Example 12> Enzyme reaction conditions and enzyme properties
1. Enzyme reaction conditions (1) PCR reaction In order to detect the activity of the target DNA polymerase, the expression vector pET15b / encoding the two DNA oligomers (Upper primer and Lower primer) and the small subunit of the DNA polymerase. PCR reaction was performed using PolS as template DNA. The reaction temperature condition consisted of 3 steps (94 ° C: 1 minute, 61 ° C: 2 minutes, 70 ° C: 3 minutes), and 35 cycles were repeated. The reaction solution composition (100 ml) is 20 mM Tris-HCl buffer (pH 8.8), 10 mM KCl, 4 mM MgSO4, 0.1% Triton X-100, 0.375 mM dNTP mix, 100 pmol Upper primer, 100 pmol Lower primer, 0.1 μg DNA polymerase.

(2) DNA synthesis reaction
The DNA synthesis reaction was performed according to the method of Kornberg et al. (Aposhian, HV and Kornberg, A., (1962) J. Biol. Chem., 237: 519-525, and Kornberg, RS, Zimmerman, BS and Kornberg, A., (1961). ) J. Biol. Chem., 236: 1487-1493). The composition of the reaction solution (200 μl) was 20 mM Tris-HCl buffer (pH 8.8), 10 mM KCl, 10 mM (NH4) 2SO4, 2 mM MgSO4, 0.1% Triton X-100, 0.25 mM dNTP mix, 0.37 Mbq. (α-32P) dATP, 20 μg of heat-quenched salmon testis DNA, and 0.1 μg of DNA polymerase. The reaction was carried out at 75 ° C for 30 minutes. After the reaction, 0.5 mg of ice-cold salmon testis DNA was added, 500 μl of ice-cold 1N perchloric acid and 500 μl of ice-cold water were added, and the acid-insoluble fraction was centrifuged ( 9000xg for 5 minutes). This precipitate was dissolved in 300 μl of 0.2N NaOH, 300 μl of ice-cold 1N perchloric acid and 300 μl of ice-cold water were added again, and an acid-insoluble fraction was obtained by centrifugation. This precipitate was washed with 1 ml of 1N acetic acid, and after centrifugation, the precipitate was dissolved in 0.4 ml of 2N aqueous ammonia, and the radioactivity was measured by a Cherenkov effect using a liquid scintillation counter. In addition, the amount of enzyme that took in 10 nmol of dNTP in a synthesis reaction at 75 ° C. for 30 minutes was defined as 1 unit.

(3) Optimum pH
The optimum pH is fixed at a reaction temperature of 75 ° C under the above measurement conditions. The pH of the enzyme reaction solution is changed from pH 5.8 to 9.5 using phosphate buffer in the acidic range and Tris-HCl buffer in the alkaline range. Determined by incorporation of radioactivity into the acid-insoluble fraction.
(4) Optimal Mg2 + concentration The optimal Mg2 + concentration is determined by incorporating the radioactivity into the acid-insoluble fraction after fixing the reaction temperature at 75 ° C in the above DNA synthesis reaction and changing the MgSO4 concentration from 0 mM to 20 mM. did.

(5) Thermal stability The enzyme solution for heat treatment (100 ml) contains 20 mM Tris-HCl buffer (pH 8.0, 25 ° C), 500 mM NaCl, 10 mM MgSO4 and the target enzyme at a concentration of 0.1 mg / ml. This was heat-treated with a GeneAmp PCR System 2400 (Perkin Elmer) for 1 hour from 60 ° C. to 95 ° C., and the residual activity was measured by incorporation of radioactivity into the acid-insoluble fraction using the above DNA synthesis reaction.

(6) Primer extension activity Primer extension activity was measured as follows. M13 phage single-stranded DNA (0.2 mg) and 0.5 pmol primer (15 mer, 34 mer, 50 mer) 5'-labeled with 32P, each with 20 mM Tris-HCl buffer ( In the presence of 10 mM MgCl2, 0.05 Unit of DNA polymerase was added and reacted at 75 ° C. The reaction was stopped after 2 minutes and 10 minutes by adding a Stop solution. The reaction products were analyzed by 15% polyacrylamide gel electrophoresis (PAGE) containing 8M urea.

(7) 3'-5 'exonuclease activity
3'-5 'exonuclease activity was measured using a 50mer primer (0.5 pmol) 5'-labeled with 32P. The reaction solution contained 20 mM Tris-HCl buffer (pH 8.5), 12 mM MgCl 2, and 4 ng of labeled DNA in a 20 ml reaction solution, and 0.05 Unit of DNA polymerase was added thereto and reacted at 75 ° C. The reaction was stopped after 30 minutes by adding Stop solution. The reaction products were analyzed by 15% polyacrylamide gel electrophoresis (PAGE) containing 8M urea.

2. Properties of the enzyme (1) Protein chemical properties The large subunit of the target enzyme is composed of 1434 amino acid residues and 1268 amino acid residues before and after intein removal, respectively, and its molecular weight is 163,000 Da and 144,000 Da, respectively. Met. The small subunit was composed of 623 amino acid residues, and its molecular weight was 70,000 Da.

(2) High-efficiency expression of active enzyme by co-expressing a small subunit and a large subunit from which intein has been removed When each subunit is expressed alone, it is extremely unstable and stable high expression is impossible. Therefore, we decided to construct a co-expression system. As a result, high expression of the active enzyme was enabled, and the physicochemical properties and the heat stabilization mechanism were elucidated.

(3) Detection of DNA synthesis activity and PCR reaction DNA synthesis activity was examined using a crude enzyme solution from an E. coli recombinant. As shown in Table 1, no activity was detected with the subunit alone. The combination of a small subunit and a large subunit containing intein was also inactive.
From these results, it became clear that a heterodimer structure consisting of a large subunit from which a small subunit and an intein were removed was essential for the expression of activity.

Furthermore, the heterodimeric enzyme pET15b / PolSL (-Intein) consisting of the large subunit from which the small subunit and the intein were removed was purified, and the DNA synthesis activity was examined using purified enzyme (0.1 μg) and 10 mM MgSO4. An increase in radioactivity of 175 times or more was detected in the enzyme-added system compared to the non-added system. Next, as a result of PCR reaction using the purified enzyme, an amplification reaction product having the same length (1.9 kb) as the target DNA fragment was confirmed by agarose gel electrophoresis. In addition, no PCR reaction product was detected in the reaction system not containing the enzyme. Furthermore, the above facts revealed that the heterodimeric DNA polymerase composed of a large subunit from which a small subunit and an intein were removed was sufficiently active.

(4) Optimum pH
The optimum pH at 8.5C was 8.5 (Fig. 4).
(5) Optimal Mg2 + concentration The optimal Mg2 + concentration of this enzyme was 12 mM (FIG. 5).
(6) Thermostability As shown in FIG. 6, this enzyme retained 50% activity even after heat treatment at 85 ° C. for 1 hour. Furthermore, it had 20% activity even after heat treatment at 90 ° C. for 1 hour.

(7) Primer extension activity As shown in Fig. 7, the enzyme did not detect extension activity with the 15mer primer, but as the primer length increased to 34mer and 50mer, strong primer extension activity was observed. Such dependency of primer extension activity on primer length has not been reported for other DNA polymerases.

(8) 3′-5 ′ exonuclease activity As shown in FIG. 8, this enzyme showed strong 3′-5 ′ exonuclease activity against 50-mer oligonucleotides. This is why this enzyme is a heterodimeric protein consisting of two subunits with molecular weights of 144 kDa and 70 kDa. DNA-dependent DNA that synthesizes complementary strands using DNA as a template and retains 3'-5 'proofreading exonuclease activity. It turned out to be a polymerase.

The gene sequence (A) and amino acid sequence (B) of the large subunit of this enzyme are shown. The underlined portion indicates the intein sequence (encoding a proteinaceous intron). The identification of intein sequences by juxtaposition with known hyperthermophile homologs (from Metha. Ja and Py. Furi) is shown. The intein of this enzyme (derived from Py. Hori) starts with the 955th cysteine and ends with the 1120th glutamine. The gene sequence (A) and amino acid sequence (B) of the small subunit of this enzyme are shown. The pH profile (optimum pH) of this enzyme is shown. The optimal Mg2 + concentration of this enzyme is shown. It is an electrophoresis photograph which shows the residual activity after heat processing of this enzyme. It is an electrophoresis photograph showing the primer extension activity of this enzyme. Lanes 1 to 3 used 15mer primers, Lanes 4 to 6 used 34mer primers, and Lanes 7 to 9 used 50mer primers. Lanes 1, 4, and 7 were reacted for 2 minutes in the presence of the enzyme. Lanes 2, 5, and 8 were reacted for 10 minutes in the presence of the enzyme. Lanes 3, 6, and 9 are controls and no enzyme was added to the reaction system. 3'-5 'exonuclease activity of this enzyme is shown. A 50mer oligonucleotide was used as a substrate. Lane 1 is the reaction for 30 minutes in the presence of the enzyme, lane 2 is the control, and no enzyme is added to the reaction system.

SEQ ID NO: 1: Intein encoding proteinaceous intron.
SEQ ID NO: 2: intein.
SEQ ID NO: 4: Primer.
Sequence number 5: Primer.
Sequence number 6: Primer.
Sequence number 7: Primer.
Sequence number 8: Primer.
Sequence number 9: Primer.
SEQ ID NO: 10: primer.
Sequence number 11: Primer.
Sequence number 12: Primer.
SEQ ID NO: 13: primer.
SEQ ID NO: 14: primer.

Claims (7)

  An improved thermostable DNA polymerase comprising a small subunit and a large subunit, wherein the large subunit contains an intein sequence, characterized by removing the intein sequence in the large subunit A method for producing a DNA polymerase.   The production method according to claim 1, wherein the produced DNA polymerase has 3'-5 'exonuclease activity and primer length-dependent primer extension activity.   The method according to claim 1 or 2, wherein the thermophilic archaea is an archaea belonging to the genus Pyrococcus.   The small subunit is a protein having the amino acid sequence shown in SEQ ID NO: 4, or includes substitution, deletion or addition of one or several amino acid residues in the amino acid sequence, The method according to any one of claims 1 to 3, which is a protein that exhibits DNA polymerase activity when formed.   The large subunit of the DNA polymerase to be produced is a protein having a sequence obtained by removing the amino acid (955th to 1120th) encoded by the intein sequence from the amino acid sequence shown in SEQ ID NO: 2, or 1 in the sequence A protein that exhibits improved DNA polymerase activity when formed into dimers with small subunits, including substitution, deletion or addition of one or several amino acid residues. 4. The production method according to any one of 4 above. A method for producing an improved DNA polymerase, comprising the following steps a) to c):
a) incorporating the DNA encoding the small subunit according to claim 4 and the DNA encoding the large subunit according to claim 5 into the same or different vectors;
b) transforming a host cell with the obtained recombinant vector to obtain a transformant;
c) A step of culturing the transformant to co-express these DNAs and collecting DNA polymerase from the culture. The production method according to claim 6, wherein the host cell is a bacterium.