JPH09252776A - Thermally stable dna polymerase derived from thermococcus peptonophilus, gene encoding the same enzyme and use therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA polymerase, derived from Theremococcus peptonophilus, excellent in thermal stability, high in DNA synthesis rate, and suitable for the amplification of nucleic acids such as a polymerase chain reaction(PCR), etc. SOLUTION: This thermally stable DNA polymerase, high in DNA synthesis rate and suitable for a nucleic acid amplifying method, etc., such as a polymerase chain reaction(PCR), is derived from Thermococcus peptonophilus, catalyzes a polymerase reaction for forming a complementary nucleic acid chain to a nucleic acid mold chain from a nucleoside triphosphate, and has 3'-5' exonuclease activity and an approximately 60% residual activity by treating at 100 deg.C for 30min and amino acid sequences of the formula. The DNA polymerase is obtained by isolating a chromosomal DNA from an OG-1 strain of Thermococcus peptonophilus, performing the PCR amplification of the DNA, and expressing the obtained gene in host cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel Thermococcus peptonophilus.
Derived thermostable DNA polymerase and said thermostable D
Gene encoding NA polymerase, recombinant DNA expression vector containing the gene, recombinant host cell transformed with the recombinant expression vector, and method for producing thermostable DNA polymerase using the recombinant host cell Regarding

[0002]

2. Description of the Related Art Conventionally, DNA polymerases of mesophilic bacteria such as Escherichia coli and mesogenic bacteria that infect mesophilic bacteria.
Many studies have already been conducted on DNA polymerases derived from di. Also, recently, recombinant DN using nucleic acid amplification such as polymerase chain reaction (abbreviated as PCR)
There have been many studies on thermostable DNA polymerases useful in the A technique. Thermostable DNA used for PCR
The polymerase is mainly used in Thermus thermophilus (T
DNA polymerase from hermus thermophilus (Tth D
NA polymerase) and Thermus aquaticus (Thermus
Aquaticus-derived DNA polymerase (Taq DNA polymerase) and the like have been used.

[0003]

However, conventionally known thermostable DNA polymerases have heat resistance, but have some problems in their thermal stability and stability against organic solvents. There is. In addition, there is a lack of accuracy when incorporating nucleic acids, and there remain problems to be solved when using these enzymes in DNA sequencing and polymerase chain reaction. Therefore, a novel thermostable DNA polymerase that solves these problems has been desired.

In addition, Pyrococcus furiosus (Pyroc
occus furiosus) thermostable DNA polymerase (Pfu
Polymerase, WO92 / 09689, JP-A-5-328969), Thermococcus litoralis (Termococcus litor)
A heat-resistant DNA polymerase derived from alis (Tli polymerase, JP-A-6-7160) is also known. However, although these thermostable DNA polymerases are superior to Taq DNA polymerase and Tth DNA polymerase in the accuracy of nucleic acid uptake,
A novel thermostable DNA polymerase has been desired, which is not perfect as compared with aq DNA polymerase and has a slower synthesis rate.

[0005]

Means for Solving the Problems As a result of intensive investigations by the present inventors, a novel hyperthermophilic archaea which produces a thermostable DNA polymerase having high thermostability and high DNA synthesis rate is obtained. And succeeded in elucidating the gene, and arrived at the present invention.

That is, the present invention is a thermostable DNA polymerase derived from Thermococcus peptonophilus.

The present invention is also an isolated DNA encoding a DNA polymerase derived from Thermococcus peptonophilus.

Further, the present invention provides a DNA derived from Thermococcus peptonophilus.
It is a DNA recombinant expression vector in which an isolated DNA encoding a polymerase is inserted into the vector.

The present invention is a recombinant host cell transformed with a DNA recombinant expression vector in which an isolated DNA encoding a DNA polymerase derived from Thermococcus peptonophilus is inserted into a vector. .

The present invention also relates to a recombinant host cell transformed with a DNA recombinant expression vector in which the isolated DNA encoding a DNA polymerase derived from Thermococcus peptonophilus is inserted into the vector. A method for producing a thermostable DNA polymerase, which comprises culturing in a nutrient medium and collecting the thermostable DNA polymerase from the culture.

Further, the present invention provides a DNA derived from Thermococcus peptonophilus.
A recombinant host cell transformed with the DNA recombinant expression vector in which the isolated DNA encoding the polymerase is inserted into the vector is cultured in a nutrient medium, and (a) after collecting the recombinant host cell, A method for producing a thermostable DNA polymerase, which comprises the steps of crushing, preparing a cell extract, and (b) removing an impure protein derived from a recombinant host cell.

[0012]

The preferred thermostable DNA polymerase of the present invention is Thermococcus peptonophilus, which is one of the hyperthermophilic archaea.
OG1 (JCM No. 9653) or Thermococcus peptonophilus S
It is a DNA polymerase available from M-2 (JCM No. 9654).

Thermococcus peptonophyllus (Ther
mococcus peptonophilus) OG1 is a strain isolated from the hydrothermal deposit off Izu-Ogasawara, and Thermococcus peptonophilus SM-2 is a strain isolated from the hydrothermal deposit of the South Mariana Trough. The mycological properties of these strains are described in Arch Microbiol (1995) 164: 159-16.
4, Gonzalez et al. In addition, these strains are available from JCM (RIKEN Institute for Preservation of Microbial Strains).

The thermostable DNA polymerase gene of the present invention is cloned by the following method. The cloning method was performed using Pfu DNA polymerase (Nucleic Ac
ids Research, 1993, vol.21, 259-265), TliDNA
Polymerase (Vent DNA Polymerase) (Proc. Natl.
Acad. Sci. USA, 1992, vol.89, 5577-5581), based on the amino acid sequence of the conserved region, a primer is designed and synthesized.

First, Thermococcus peptonophilus
(Thermococcus peptonophilus) chromosomal DNA as a template and PCR using the above-prepared primers (for example, described in SEQ ID NOs: 6 and 7, 8 and 9), and D
Amplify the NA fragment. DNA of amplified DNA fragment
After determining the sequence and confirming that it encodes the initially set amino acid sequence, the DNA fragment is used as a probe,
Southern hybridization is carried out on the restriction enzyme degradation product of the chromosomal DNA. It is preferable to limit the approximate size of the DNA fragment containing the DNA polymerase gene of interest to about 4 to 7 Kbp.

Further, a DNA fragment of about 4 to 7 Kbp is recovered from the gel, and using this, a DNA library is prepared in Escherichia coli, and the above PCR amplified DNA fragment is used as a probe for plaque hybridization or colony hybridization. To obtain a clone strain.

Thermococcus peptonophilus cloned in the present invention
The DNA polymerase gene of the OG1 strain is composed of 2325 bases (774 deduced amino acids) (SEQ ID NO: 1). In addition, Thermococcus peptonophyllus (Th
ermococcus peptonophilus) SM-2 is composed of 3492 bases (1164 deduced amino acids) (SEQ ID NO: 3). There is one intervening sequence in the gene derived from the SM-2 strain, and the open reading frame (ORF) is connected in a conserved form. Other DN
Compared to A polymerase, these genes contain a conserved region of eukaryotic type α-DNA polymerase,
Regions 1 to 5 are present. In addition, EXO1 to 3 which are 3 ′ → 5 ′ exonuclease motifs are present on the N-terminal side of these genes.

Thermococcus peptonophilus (Therm
ococcus peptonophilus) OG-1 strain and Thermococcus peptonophilus S
The thermostable DNA polymerase gene of the M-2 strain was compared with a Tli (Vent) DNA polymerase gene derived from Thermococcus litoralis, which is a known enzyme derived from the same Thermococcus (Termococcus) genus. , TliDNA polymerase gene,
Although there are two intervening sequences, the gene derived from the Thermococcus peptonophilus OG-1 strain does not have such intervening sequences, and the Thermococcus peptonophilus (Thermococcus) is not present.
There is one intervening sequence in the gene derived from peptonophilus) SM-2 strain.

The gene of the present invention is DN derived from Thermococcus peptonophilus.
It is a DNA encoding A polymerase. An example of the DNA contains a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or 5. Also, such D
NA contains the base sequence shown in SEQ ID NO: 1 or 3, or a part thereof. Thermococcus peptonophilu which is one example of the present invention
s) In order to express the thermostable DNA polymerase derived from the SM-2 strain in Escherichia coli, 1 of the nucleotide sequence shown in SEQ ID NO: 3
The 621 to 2790 bp intervening sequence is removed by PCR fusion to construct a complete DNA polymerase gene. Specifically, a cloned gene containing an intervening sequence is subjected to PCR with a combination of two sets of primers to amplify two fragments that are separated by the intervening sequence. Furthermore, by joining each fragment, the complete form of Thermococcus peptonophyllus (Therm
A DNA polymerase gene derived from the ococcus peptonophilus) SM-2 strain is obtained (see SEQ ID NO: 4).

The vector used in the present invention is a Thermococcus peptonop
(hilus) -derived thermostable DNA polymerase can be cloned and expressed, and examples thereof include phages or plasmids. As a plasmid, pUC18, pBR32
2, pBluescript, pLED-M1, p7
3, pGW7, pET3A, pET8c and the like. Examples of phages include λgt11 and λZAPI.
I, etc.

Examples of the host cell used in the present invention include Escherichia coli and yeast. As Escherichia coli,
For example, JM109, HB101, XL1Blue, PR
1, LE392, BL21 (DE3) and the like. In the present invention, a gene encoding a thermostable DNA polymerase derived from Thermococcus peptonophilus is inserted into the above vector to give a recombinant expression vector, and a host cell is transformed with this recombinant expression vector.

In the production method of the present invention, the above recombinant host cell is cultured to obtain Thermococcus peptonophyllus (Th
(ermococcus peptonophilus) -derived thermostable DNA polymerase gene is inducibly expressed. The medium and conditions used for culturing the recombinant host cell follow conventional methods. As a specific example, Thermococcus peptonophilus (Thermococc
us peptonophilus) OG-1 strain-derived DNA polymerase gene-containing pBluescript plasmid or Thermococcus peptonophilus (Thermococcus
peptonophilus) pLED containing a complete form of DNA polymerase gene containing no intervening sequence derived from strain SM-2
Escherichia coli transformed with the M1 plasmid is cultured in, for example, TB medium.

In one of the production methods of the present invention, (a) the recombinant hosts are collected and then crushed to prepare a cell extract, and (b)
The step of removing the impure protein derived from the host cell is included. The thermostable DNA polymerase produced from the recombinant host cell is separated and collected from the culture solution by centrifugation or the like after culturing the host cell in the medium. After resuspending the bacterial cells in a buffer solution, the bacterial cells are disrupted by ultrasonic treatment, dynomill, French presence and the like. Then, heat treatment is performed to recover the thermostable DNA polymerase from the supernatant. As a method for disrupting the cells, ultrasonic treatment, dynomill, French press method, etc. are preferable. Heat treatment is preferred as one of the steps for removing host-derived impure proteins. The heat treatment condition is 70 ° C. or higher, preferably 85 ° C. or higher. Other methods for removing impure proteins include various types of chromatography.

Thermococcus peptonophilus (Ther
mococcus peptonophilus) OG-1 derived thermostable DN
A polymerase has the following physicochemical properties. Action: (1) Catalyze a polymerase reaction that forms a nucleic acid strand complementary to a nucleic acid template strand from nucleoside triphosphate. (2) It has a 3′-5 ′ exonuclease activity. Thermal stability: Residual activity of about 60% after treatment at 100 ° C. for 30 minutes. Molecular weight: about 86 to 92 KDa Optimum pH: about 6.5 to 9 Optimum temperature: about 70 to 80 ° C. DNA synthesis rate: about 60 bases / second or more.

Thermococcus peptonophyllus (Ther
mococcus peptono philus) SM-2 derived thermostability D
NA polymerase has the following physicochemical properties. Action: (1) Catalyze a polymerase reaction that forms a nucleic acid strand complementary to a nucleic acid template strand from nucleoside triphosphate. (2) It has a 3′-5 ′ exonuclease activity. Thermal stability: Residual activity of about 60% after treatment at 100 ° C. for 30 minutes. Molecular weight: about 86 to 92 KDa Optimum pH: about 6.5 to 9 Optimum temperature: about 70 to 80 ° C. DNA synthesis rate: about 40 bases / second or more.

[0026]

INDUSTRIAL APPLICABILITY Thermostable DN derived from Thermococcus peptonophilus of the present invention
A polymerase is 60% at 100 ° C for 30 minutes.
It shows the above residual activity and shows the polymerase chain reaction (PC
R) and the like (see FIG. 3).

[0027]

EXAMPLES The present invention will be described below with reference to examples. Example 1 Thermococcus peptonophylla (Thermo
Cloning of DNA polymerase gene derived from OG-1 strain of Thermococcus peptonophilus (Thermococcus pep)
tonophilus) OG-1 strain (JCM No. 9653) 85
After culturing at ℃, the bacterial cells were collected. Chromosomal DNA was prepared from the obtained bacterial cells according to a conventional method (Arch Microbiol (199
5) 164: 159-164, Gonzalez et al.).

PfuDNA polymerase, TliDNA
Based on the amino acid sequence of the conserved region of polymerase, four primers (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9) was synthesized. The primers SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 were set as one set, respectively, and the prepared chromosomal DNA was used as a template for PC
R (94 ℃, 30 seconds, 55 ℃, 30 seconds, 75 ℃, 2 minutes 3
0 seconds, 25 cycles. 100 pmol of primer and 100 ng of template DNA were used. As the enzyme, 5 units of Taqplus (manufactured by Stratagene) was used. As a result, when using SEQ ID NO: 6 and SEQ ID NO: 7, about 7
With the 40 bp expected length amplicon, SEQ ID NO: 8 and SEQ ID NO: 9, an expected length amplicon of approximately 400 bp was obtained.

The partial nucleotide sequence of this PCR-amplified DNA fragment was determined, and it was confirmed that it was an amplification product of the target region. Using this amplified fragment as a probe, the restriction enzyme Ec was used.
As a result of Southern hybridization performed on the chromosomal DNA treated with oRI (manufactured by Toyobo), about 4.4
It was confirmed that a fragment encoding the DNA polymerase was present in the kb fragment. Furthermore, a DNA fragment of this size was recovered from agarose gel, inserted into λZAPII (manufactured by Stratagene), and E. coli (E. coli XL1blue) was transduced with a mixture of these to prepare a library.

Next, plaque hybridization was carried out using the probe used in Southern hybridization. As a result of screening about 5000 plaques, 10 positive clones were obtained. The plasmid was rescued from the positive clone using the helper phage R408 in accordance with a conventional method, and the nucleotide sequence was partially determined. As a result, a clone (pO5'1, insert fragment about 4) encoding the 5'side of the DNA polymerase gene was obtained. .4 kbp) and a clone encoding the 3'side (pOG
7, insert fragment 4.4 kbp) was obtained.

Example 2 Construction of Recombinant Expression Vector In order to prepare a complete DNA polymerase gene, the coding portions of the DNA polymerase genes of the two clones obtained above were ligated according to the construction diagram shown in FIG. did. p
O5′1 was cleaved with restriction enzymes SalI and EcoRI, and fractionated by agarose gel electrophoresis.
Was recovered. Next, pOG7 is used as a restriction enzyme Ec.
After cutting with oRI and BglII and fractionating by agarose gel electrophoresis, a DNA fragment of about 1.8 kbp was recovered. The obtained two kinds of fragments were digested with restriction enzymes SalI and B
Plasmid vector pBluesc cleaved with amHI
By ligating to ript SK (-) (manufactured by Stratagene), an expression recombinant vector pOGSB (6.64 kb) containing a complete DNA polymerase gene was obtained.

Example 3 Determination of Nucleotide Sequence of Cloned DNA Fragment The plasmid DNA pOGSB obtained in Example 2 was prepared in a large amount and the nucleotide sequence was determined by a conventional method. Furthermore, the amino acid sequence was deduced from the determined base sequence (Sequence Listing 1 and Sequence Listing 2). DNA from Thermococcus peptonophilus OG-1 strain
The polymerase gene consisted of 2325 bases and was coded for 774 amino acids. The molecular weight of the protein estimated from the amino acid sequence was about 90,000 daltons.

Example 4 Preparation of Transformant The competent cell of Escherichia coli JM109 was transformed with pOGSB prepared in Example 2 to obtain the transformant Escherichia coli JM1.
09 (pOGSB) was obtained.

Example 5 Activity Confirmation of DNA Polymerase Derived from Thermococcus peptonophilus OG-1 Strain 5 ml of TB medium containing 100 μg / ml ampicillin (Molecular Cloning, described in pA 2)
Was dispensed into a 20 ml test tube. 100μ in this medium
LB agar medium containing 1 g / ml of ampicillin (1% bactotryptone, 0.5% yeast extract,
(0.5% sodium chloride, 1.5% agar, manufactured by Gibco)
Escherichia coli JM109 (pOGSB) grown in 1. was inoculated for 1 platinum and cultured at 35 ° C. for 16 hours with shaking. 1.5 ml of this culture medium was transferred to an Eppendorf tube, and cells were collected by centrifugation. 0.6 ml of crushing buffer (10 mM Tris-HCl (pH8.0), 80 mM KCl, 5 mM 2-
After suspending in mercaptoethanol and 1 mM EDTA), the cells were disrupted by ultrasonic treatment to obtain a cell extract. Furthermore, in order to inactivate the DNA polymerase derived from the host cell,
The cell lysate was treated at 85 ° C for 30 minutes. The insoluble fraction was removed by centrifugation and the DN of the heat-treated liquid was removed by the method described below.
A polymerase activity was measured.

(Reagent) A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / ml activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ³ H ] dTTP) D: 20% trichloroacetic acid (2 mM pyrophosphate) E: 1 μg / ml carrier DNA

(Method) Solution A (25 μl), solution B and solution C (5 μl) and sterilized water (10 μl) were added to an Eppendorf tube and mixed by stirring, and then the heat treatment solution (5 μl) was added and reacted at 75 ° C. for 10 minutes. Then, cool with ice, and use solution E 5
Add 0 μl and 100 μl of D solution, stir, and then add 10
Cool on ice for a minute. This solution was filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard), and template DN was used.
The incorporation of nucleotides into A was measured. One unit of enzyme activity is 10 nanomoles (n
The amount of the enzyme that incorporated all the nucleotides of M) into the acid-insoluble fraction was used.

When the enzyme activity of the heat-treated solution was measured by the above method, 75 units of DNA polymerase activity were obtained per 1 ml of the heat-treated solution. This titer indicates that 30 units of DNA polymerase were expressed per ml of culture. For comparison, a vector pBluescript SK (-) that does not encode a thermostable DNA polymerase.
The Escherichia coli JM109 transformed with 1. was subjected to the same operation as above to measure the DNA polymerase activity, but it was not detected.

Example 6 D from Thermococcus peptonophilus OG-1 strain
Purification of NA polymerase 6 L of TB medium (Molecular Cloning, described in pA2) containing 100 μg / ml ampicillin that had been sterilized was dispensed into a 10 L jar fermenter. 50 ml of LB medium containing 100 μg / ml of ampicillin in advance in this medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, manufactured by Gibco)
E. coli JM109 (pOG
SB) (using 500 ml Sakaguchi flask), 35
The culture was performed at 12 ° C. for 12 hours with aeration and stirring. The disruption buffer solution 40 used in Example 5 was prepared by collecting bacterial cells from the culture solution by centrifugation.
After suspending in 0 ml, the cells were disrupted by ultrasonic treatment to obtain a cell disruption solution.

Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Nucleic acid removal treatment with polyethyleneimine, ammonium sulfate fractionation, and heparin sepharose chromatography were performed, and finally storage buffer (50 mM Tris-pH8.0, 50 mM potassium chloride, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% nonideto). P40, 50% glycerin) and highly purified Thermococcus peptonophyllus (Thermococcus pe
A thermostable DNA polymerase derived from ptonophilus OG-1 was obtained. The DNA polymerase activity in the above purification step was the same as in Example 5. When the enzyme activity was high, the sample was diluted with a storage buffer and measured.

Example 7 DN from Thermococcus peptonophilus SM-2 strain
Cloning of the A polymerase gene Thermococcus peptonophilus
onophilus) SM-2 strain (JCM No. 9654) at 85 ° C.
After culturing in, the bacterial cells were collected. Chromosomal DNA was prepared from the obtained bacterial cells by a conventional method (Arch Microbiol (1995)).
164: 159-164, Gonzalez et al.). PCR was carried out using the prepared chromosomal DNA as a template and the primers used in Example 1. As a result, when using SEQ ID NO: 6 and SEQ ID NO: 7, an amplification product of an expected length of about 740 bp, and when using SEQ ID NO: 8 and SEQ ID NO: 9, about 400 were used.
An amplification product of the expected length of bp was obtained.

The partial base sequence of this PCR-amplified DNA fragment was determined, and it was confirmed that it was an amplification product of the desired region. Using this amplified fragment as a probe, the restriction enzyme Ec was used.
As a result of Southern hybridization performed on the chromosomal DNA treated with oRI (manufactured by Toyobo), about 5 kb
It was confirmed that a fragment encoding the DNA polymerase was present in the p fragment. Further, a DNA fragment of this size was recovered from agarose gel, inserted into λZAPII (manufactured by Stratagene), and mixed with E. coli.
(E.coli XL1blue) and the library
Was produced. Next, plaque hybridization was carried out using the probe used in Southern hybridization. As a result of screening about 5000 plaques, 8 positive clones were obtained. A plasmid was rescued from a positive clone using a helper phage R408 according to a conventional method, and the base sequence was partially determined. A clone (pSM3, insert about 5 kbp) covering the region used for the probe was obtained. However, this clone is a DNA polymerase.
About 100 bp at the 3'end of the ze gene was not covered.

Then, D of pOGSB obtained in Example 2
After performing plaque hybridization again using the NA polymerase gene region as a probe and performing the same operation as above, a clone (pS3'1 insertion fragment of about 3 kb) covering the 3'side of the DNA polymerase gene was obtained. did.

Example 8 Determination of nucleotide sequence of cloned DNA fragment Plasmid DNAs pSM3 and pS obtained in Example 7
A large amount of 3'1 was prepared and the nucleotide sequence of the DNA polymerase gene region was determined according to a conventional method. Furthermore, the amino acid sequence was deduced from the determined nucleotide sequence (Sequence Listing 3, Sequence Listing 4, Sequence Listing 5). DNA derived from Thermococcus peptonophilus SM-2 strain
The polymerase gene has 3492 bases (deduced amino acid 11
64). DNA derived from SM-2 strain
The polymerase gene has one intervening sequence (ivs2) and has an open reading frame (OR).
F) was connected in a preserved form. The molecular weight of the complete DNA polymerase protein deduced from the amino acid sequence was about 90,000 daltons.

Example 9 Construction of Recombinant Expression Vector To construct a complete DNA polymerase gene, FIG.
A recombinant expression vector pSpolMT with the intervening sequence removed was constructed according to the flow shown in FIG. After cleaving pSM3 with restriction enzyme EcoRV and fractionating by agarose gel electrophoresis, a DNA fragment of about 3.5 kbp was recovered,
Self-ligation was performed to construct the plasmid polV. Next, insert about 3 kb of the pSM3 'insert into polV
Was inserted into the EcoRI site to construct polEV.
The insert fragment and insertion direction were confirmed by partially determining the nucleotide sequence.

Next, PCR was carried out using pSM3 as a template and the primers of SEQ ID NO: 10 and SEQ ID NO: 11.
The amplified product was cleaved with restriction enzymes NdeI and EcoRV and fractionated by agarose gel electrophoresis to give about 1.6
The kbp DNA was recovered. Next, PCR was performed using polEV as a template and the primers of SEQ ID NO: 12 and SEQ ID NO: 13. This amplification product was blunted with T4 DNA polymerase, cleaved with restriction enzyme SalI, and fractionated by agarose gel electrophoresis to give about 0.7 kb.
p DNA was recovered. In PCR, 1 μg of template DNA was used to suppress mutations during PCR, and the number of cycles was 10
I went there.

A plasmid vector p obtained by digesting the two DNA fragments obtained above with restriction enzymes NdeI and SalI
Expression recombinant vector pSpolMT (5. ligation to LED-M1 and containing the complete DNA polymerase gene).
02 kb) was obtained.

Example 10 Preparation of Transformant The competent cell of Escherichia coli JM109 was transformed with pSolMT prepared in Example 9 to obtain the transformant Escherichia coli JM.
109 (pSpolMT) was obtained.

Example 11 Activity Confirmation of DNA Polymerase from Thermococcus peptonophilus SM-2 Strain 5 ml of TB medium (Molecular Cloning, described in pA2) containing 100 μg / ml of ampicillin sterilized
Was dispensed into a 20 ml test tube. 100 μg in this medium
Agar containing 1 ml / ml ampicillin (1% bactotryptone, 0.5% yeast extract, 0.
Escherichia coli JM109 (pSpolMT) grown in 5% sodium chloride, 1.5% agar, manufactured by Gibco) was inoculated for 1 platinum and cultured at 35 ° C. for 16 hours with shaking. 1.5 ml of this culture medium was transferred to an Eppendorf tube, and cells were collected by centrifugation. 0.6 ml of crushing buffer (10 mM Tris-HCl (pH8.0), 80 mM KCl, 5 mM 2-
After suspension in mercaptoethanol), the cells were disrupted by ultrasonication to obtain a cell extract. Further, in order to inactivate the DNA polymerase derived from the host cell, the cell lysate was treated at 85 ° C. for 30 minutes. The insoluble fraction was removed by centrifugation, and the DNA polymerase activity of the heat-treated solution was measured by the same method as in Example 5.

When the enzyme activity of the heat-treated solution was measured by the above method, 100 units of DNA polymerase activity were obtained per 1 ml of the heat-treated solution. This titer indicates that 40 units of DNA polymerase were expressed per ml of culture. As a comparison, Escherichia coli JM109 transformed with the vector pLED-M1 which does not encode a thermostable DNA polymerase was subjected to the same operation as described above, and the DNA polymerase activity was measured but could not be detected.

Example 12 Purification of DNA polymerase derived from Thermococcus peptonophilus strain SM-2 6 L of TB medium (Molecular Cloning, described in pA2) containing 100 μg / ml ampicillin was sterilized. Dispensed to the fermenter. 50 ml of LB medium containing 100 μg / ml of ampicillin in advance in this medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, manufactured by Gibco)
E. coli JM109 (pSpo
1 MT) (using 500 ml Sakaguchi flask) and inoculate 35
The culture was performed at 12 ° C. for 12 hours with aeration and stirring. The cells were recovered from the culture solution by centrifugation, suspended in 400 ml of the disruption buffer used in Example 5, and then disrupted by sonication,
A cell lysate was obtained. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Nucleic acid removal treatment using polyethyleneimine, ammonium sulfate fractionation,
Heparin sepharose chromatography is performed, and finally storage buffer (50 mM Tris-pH8.0, 50 mM potassium chloride,
1 mM dithiothreitol, 0.1% Tween 20, 0.1% nonideto P40) and highly purified Thermococcus peptonophilus
A thermostable DNA polymerase derived from SM-2 was obtained.

Example 13 Confirmation of Thermostability Thermococcus peptonophi purified as described in Examples 5 and 12.
The thermal stability of the DNA polymerase of lus) was measured by the following method. Purified Thermococcus peptonophyllus (T
hermococcus peptonophilus strain SM-2 and Thermococcus peptonophilu
s) DNA polymerase derived from OG-1 strain (each SM
-2 polymerase and OG-1 polymerase)
5 units of each was added to 100 μl of buffer solution (20 mM Tris-HCl, pH 8.8,
25 ° C, 10 mM potassium chloride, 10 mM ammonium sulfate, 2 mM
Magnesium sulfate, 0.1% Triton X-100, 0.1mg / ml BS
A, 5mm 2-mercaptoethanol) and mix at 100 ℃
It was a pre-incubation. Samples were taken from this mixed solution over time, and the polymerase activity was measured by the method described in Example 5. For comparison, Taq polymerase (manufactured by Toyobo) and Tli polymerase (Vent polymerase, N
The same operation was performed for EB). As shown in FIG. 4, SM-2 polymerase and OG-1 polymerase had 60% or more of the activity remaining after treatment at 100 ° C. for 30 minutes like Tli polymerase, whereas Taq polymerase had only 5%. No activity remained.

Example 14 Measurement of DNA synthesis rate Thermococcus peptonophilus
onophilus) DNA polymerase rate of DNA polymerase was measured by the following method. SM-2 polymerase and OG
-1 Polymerase 5 units 10 μl of reaction solution (20 mM Tris
-HCl, (pH7.5), 8 mM magnesium chloride, 7.5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNTP, 0.2 μCi
0.2 µg of the primer of SEQ ID NO: 14 was annealed with [α- ³² P] dCTP) and reacted with M13mp181 single-stranded DNA at 75 ° C for 30 seconds, 60 seconds, and 90 seconds. To stop the reaction, use the same amount of reaction stop solution (50 mM sodium hydroxide, 10 mM
This was done by adding EDTA, 5% Ficoll, 0.05% bromphenol blue).

The gel was dried and subjected to autoradiography. Labeled λ / HindIII was used as a DNA size marker. The DNA synthesis rate was determined by measuring the size of the synthesized DNA using the band of this marker as an index. As a result, SM-2
The polymerase had a synthesis rate of about 40 bases / second, and the OG-1 polymerase had a synthesis rate of about 60 bases / second. This was approximately twice as fast as Tli polymerase from the same Thermococcus genus.

[0054]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3731 Sequence type: Nucleic acid (DNA) Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin: Thermococcus peptonophilus Strain name: OG-1 (JCM # 9653) Sequence features: 962-3283 CDS sequence GTCGACGGCC ACGATTTCTC CTTTATTCTC CATCAGCGCC GCAGCATGGA AGGTCTTGGA 60 GCCAGGAGCT GCCGCCAGAT CGAGCAACCT TTCACCAGCC TCTGGCTTTA AAACGTGGGC 120 GACGTAGGGC CTGGCCAAGT CTTGAATTAC AAATTTGCCT TCCTTGTACC AGCCAAGCCT 180 TGTAACCGGC GTTTTGTACT CAAGAACCTT TAAAACGTCT GGAACTGGAG TTAGTGCAGT 240 TCTAACGCCG TTCTCTTCAA GGTAGTCGCG GAGGGAATCA ACGTCTGTCT TTAGCGTGTT 300 GGCCCTCACG TAATACCTCT GGGGCCGGTT GTTGCTGAGA AGGAGCCTTA CGGCCTCGTC 360 GTAGCCGAAG AGGTTTATAA CGTACTCCAC ATACCAGCGG GGGTGGGAAA AGCGGACGGA 420 GAGCCACTCA ATTCTGTCCT TCTCTTTGAG TCTCTTTAAA GCCCTCTCTCTATA AG CCGT ACGAGTCGGG ACGTTCAGTCGA ACTCGATCGCGA ACCGCTGAGCGA GAAGGA GGTTCGCAAG ATAGGGGTCA AGGTCTTCAA CCTTTGAACC 660 CTTGAGGACT GAGTTTATTA TGAAGTCTAT CTTCGCGCGC CACTTCTCGA TCTCGAAGAC 720 GTAGGCATGC GCCAAACCGC GAGCCTTCTC GCGGTCTCTT CCGGCCACCC TCTTAAAGAC 780 CCTCTCAAGG GCGTGCTTCG ATGACAGCTC ACGCTCCTCC ACAAGCATGA GCGCATCGGC 840 CACAACTTCC TGAAAAGCTC ACGCGGTAAA ACAGCTCCAT GAGGGGGCCT TTGAAAATTG 900 ACTTAAAAAG GTGGCGGTGA ATTTATTAGC CCTTAACTGT AATTGATTCT AGGTGTCCCT 960 T ATG ATC CTC GAC ACT GAC TAC ATA ACT GAG AAT GGA AAG CCC GTC 1006 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val 1 5 10 15 ATC AGG ATT TTC AAG AAG GAA AAC GGC GAG TTC AAG ATC GAG TAC GAC 1054 Ile Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp 20 25 30 AGG ACT TTT GAG CCC TAT ATC TAC GCC CTC CTG AAG GAC GAT TCT GCA 1102 Arg Thr Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala 35 40 45 ATA GAG GAA GTC AAG AAG ATA ACT GCC GAG AGA CAC GGG ACG GTT GTA 1150 Ile Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val 50 55 60 ACT GTA AAG CGC GCC GAG AAA GTG GAG AAG AAG TTC CTC GGA AGG CCG 1198 Thr Val Lys Arg Ala Glu Lys Val Glu Lys Lys Phe Leu Gly Arg Pro 65 70 75 TTG GAG GTC TGG AAG CTC TAC TTT ACT CAC CCG CAG GAC GTT CCA GCG 1246 Leu Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala 80 85 90 95 ATA AGG GAC AAG ATA CGA GAG CAT CCA GCA GTT ATT GAC ATC TAC GAG 1294 Ile Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu 100 105 110 TAC GAC ATA CCC TTC GCC AAG CGC TAC CTC ATA GAT AAG GGC CTG ATC 1342 Tyr Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile 115 120 125 CCG ATG GAA GGC GAC GAG GAT CTG AAA ATG CTC GCC TTC GAC ATT GAA 1390 Pro Met Glu Gly Asp Glu Asp Leu Lys Met Leu Ala Phe Asp Ile Glu 130 135 140 ACG CTC TAC CAC GAG GGC GAG GAG TTT GCC GAG GGG CCA ATC CTT ATG 1438 Thr Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met 145 150 155 ATA AGC TAC GCC GAC GAA GAA GGA GCG AGG GTG ATA ACG TGG AAG AAC 1486 Ile Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn 160 165 170 175 ATA GAC CCC CCC TAC GTT GAC GTC GTC TCG ACG GAG AGG GAG ATG ATA 1534 Ile Asp Pro Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile 180 185 190 AAG CGC TTT CTC AGG ATT GTG AAG GAG AAA GAC CCG GAT GTA CTG ATA 1582 L ys Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile 195 200 205 ACG TAC AAC GGT GAC AAC TTT GAC TTT GCC TAC CTC AAG AAG CGC TGT 1630 Thr Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys 210 215 220 GAA AAG CTT GGA ATA AAC TTT GCC CTC GGA AGG GAC GGA AGC GAG CCT 1678 Glu Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro 225 230 235 AAG ATT CAG AGG ATG GGC GAT AGG TTT GCT GTC GAG GTT AAG GGC AGG 1726 Lys Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg 240 245 250 255 ATA CAC TTT GAT CTC TAC CCC GTG ATA AGA CGG ACG ATA AAC CTC CCC 1774 Ile His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro 260 265 270 ACA TAT ACG CTT GAG GCC GTC TAT GAA GCG ATC TTT GGA AAG CCA AAG 1822 Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys 275 280 285 GAG AAA GTC TAC GCC GAG GAG ATA GCC ACC GCT TGG GAG AGC GGG GAG 1870 Glu Lys Val Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Ser Gly Glu 290 295 300 GGG CTT GAG AGG GTA GCT AAA TAC TCT ATG GAG GAC GCC AAG GTT ACC 1918 Gly Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr 305 310 315 TAC GAG CTC GGA AAG GAA TTC TTC CCG ATG GAG GCC CAG CTT TCC CGC 1966 Tyr Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg 320 325 330 335 TTG ATC GGC CAG TCC CTC TGG GAC GTT TCC CGC TCC AGC ACT GGC AAC 2014 Leu Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn 340 345 350 CTC GTC GAG TGG TTC CTC CTC CGG AAG GCC TAT GAG GGG AAC GAG CTG 2062 Leu Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Gly Asn Glu Leu 355 360 365 GCC CCG AAC AAG CCC GAT GAA AAG GAG CTG GCC AGA AGA CGG CAG AGC 2110 Ala Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser 370 375 380 TAT GAA GGG GGC TAC GTA AAA GAG CCG GAG AGA GGT CTA TGG GAG AAC 2158 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn 385 390 395 ATA GTT TAT CTG GAT TTC CGT TCG CTG TAC CCC TCA ATC ATC ATC ACC 2206 I le Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 400 405 410 415 CAC AAC GTC TCG CCG GAT ACT CTC AAC AGG GAA GGA TGC AAG GAG TAT 2254 His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr 420 425 430 GAC ACC GCC CCC CAG GTT GGC CAC CGC TTC TGC AAG GAC TTC CCG GGC 2302 435 440 445 445 TTC ATT CCG AGC CTC CTT GGG GCC CTG CTT GAG GAG AGG CAG AAG ATA 2350 Phe Ile Pro Ser Leu Leu Gly Ala Leu Leu Glu Glu Arg Gln Lys Ile 450 455 460 AAG AAG AGA ATG AAG GCA ACG ATT GAT CCA CTT GAG AAG AAG CTC CTT 2398 Lys Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu 465 470 475 GAT TAC AGA CAG CGG GCT ATC AAG ATC CTA GCA AAC AGT TTT TAC GGC 2446 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ale Asn Ser Phe Tyr Gly 480 485 490 495 TAC TAC GGC TAT GCA AAG GCC CGC TGG TAC TGC AAG GAG TGT GCG GAG 2494 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 AGC GTC ACT GCC TGG GGA AGG GAG TAC ATA ACG ATG ACC ATC AGG GAG 2542 Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Arg Glu 515 520 525 ATA GAG GAA AAG TAT GGC TTT AAA GTT CTG TAC GCG GAC ACA GAC GGT 2590 Ile Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly 530 535 540 TTC TTC GCG ACA ATA CCT GGA GCT GAT GCC GAA ACC GTC AAA AAG AAA 2638 Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys 545 550 555 GCG ATG GAG TTC CTC AAG TAC ATC AAC GAT AAA CTT CCG GGC GCG CTT 2686 Ala Met Glu Phe Leu Lys Tyr Ile Asn Asp Lys Leu Pro Gly Ala Leu 560 565 570 575 GAG CTC GAG TAC GAG GGC TTC TAC AAG CGC GGC TTC TTC GTC ACT AAA 2734 Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys 580 585 590 AAG AAG TAC GCG GTG ATA GAC GAG GAA GGC AAG ATA ACA ACG CGC GGA 2782 Lys Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly 595 600 605 CTT GAG ATT GTG AGG CGC GAC TGG AGC GAG ATA GCT AAG GAG ACG CAG 2830 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 GCT AGA GTT CTT GAA GCT TTA CTA AAG GAC GGT GAC GTT GAA GAG GCG 2878 Ala Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Glu Ala 625 630 635 GTT AAA ATC GTT AAA GAA GTA ACG GAA AAG CTG AGC AAG TAC GAG GTT 2926 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val 640 645 650 655 CCG CCG GAG AAA CTG GTG ATC CAC GAG CAG ATA ACA AGG GAT CTG AGC 2974 Pro Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Ser 660 665 670 GAC TAC AAA GCG ACA GGA CCT CAC GTT GCC GTT GCT AAG AGA TTG GCC 3022 Asp Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 GCG AGG GGC GTC AAA ATA CGC CCT GGA ACG GTG ATA AGC TAC ATC GTG 3070 Ala Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val 690 695 700 CTA AAG GGC TCT GGA AGG ATT GGG GAC AGA GCT ATT CCA TTC GAC GAG 3118 Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu 705 710 715 TTT GAC CCG ACA AAG CAC AAG TAC GAC GCT GAG TAC TAC ATT GAA AAC 3166 Phe Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 720 725 730 735 CAG GTT CTG CCT GCC GTT GAG AGA ATC CTG AGG GCC TTT GGC TAC CGC 3214 Gln Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg 740 745 750 AAG GAA GAC CTG CGC TAC CAG AAG ACG AGA CAG GTT GGT TTG AGT GCT 3262 Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala 755 760 765 TGG CTG AAG CCG AAG GGA ACT TGACCTTTCC ATTTGTTTTC CAGCGGGGATAA 3313 Trp Leu LyTA TC CTT GCA AACTCTCCCT CAGAGGGAAG GCCATGAAAA TAGAAATCCG 3373 TCGAAGTCCC GTCAAGTATG CGAGGATAGA GGTGAAGCCG GACGGCAGGG TAGTCGTCAC 3433 CGCCCCCGAG GGGTTCAACG TTGAGGAGTT CATAGCAAAG AACGCCGCCT GGCTGGAGGG 3493 GAAGTTGGCC CAGATTGAAG ACCTGAAAGA GCTTGCAGAG TCGGGCTTCC CTCTGAACGG 3553 TGAGTTCTAC AAGGTCGTAC ATGGAAGGAG GGCTAAAGTC CATGACCGCT TTAAGACCGT 3613 TGTCCTTCCC CCTTATCCCG AAGACATGAG GGAAGAACTG AAAAGGCTTC TCCGACCTAA 3673 GTTAGTGGAG CTTATTGAGA AATACGCTGG AAAAATGGGT GTCTCCCCCA GTAAGATC 3731

SEQ ID NO: 2 Sequence length: 774 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 Val Lys Arg Ala Glu Lys Val Glu Lys Lys Phe Leu Gly Arg Pro Leu 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Asp Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Pro Pro Tyr Val A sp Val Val Ser Thr Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Gly Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 Glu Gly Gly Tyr Val LysGlu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Thr Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Ala Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Arg Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Lys Tyr Ile Asn Asp Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile AspGlu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Glu Ala Val 625 630 635 640 Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Ser Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp 755 760 765 Leu Lys Pro Lys Gly Thr 770

SEQ ID NO: 3 Sequence length: 3799 Sequence type: Nucleic acid (DNA) Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin: Thermococcus peptonophilus Strain name: SM-2 Sequence characteristics 1-3492 CDS 1621-2790 Intervening sequence Sequence ATG ATC CTC GAC ACT GAC TAC ATA ACT GAG AAT GGA AAG CCC GTC ATC 48 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 AGG ATT TTC AAG AAG GAA AAC GGC GAG TTC ACG ATC GAG TAC GAC AGG 96 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Glu Tyr Asp Arg 20 25 30 ACT TTT GAG CCC TAT ATC TAC GCC CTC CTG AAG GAC GAT TCT GCA ATA 144 Thr Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 GAG GAA GTC AAG AAG ATA ACT GCC GAG AGA CAC GGG ACG GTT GTA ACG 192 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 GTT AAG CGA GCT GAA AAG GTT CAG AAG AAG TTC CTC GGA AGA CCG TTG 240 Val Lys Arg Ala Glu Lys Val Gln Lys L ys Phe Leu Gly Arg Pro Leu 65 70 75 80 GAT GTC TGG AAG CTC TAC TTT ACT CAC CCG CAG GAC GTC CCA GCG ATA 288 Asp Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 AGG GAC AAG ATA CGA GAG CAT CCG GCA GTT ATT GAC ATC TAC GAG TAC 336 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 GAC ATA CCC TTC GCC AAG CGC TAT CTC ATT GAC AAG GGC CTA ATC CCG 384 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 ATG GAG GGC GAT GAA GAG CTG AAA ATG CTA GCC TTC GAC ATT GAA ACG 432 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 CTC TAC CAC GAG GGC GAG GAG TTT GCC GAG GGG CCA ATC CTT ATG ATA 480 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 AGC TAC GCC GAC GAA GAA GGA GCG AGG GTG ATA ACG TGG AAG AAC ATA 528 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 GAC CTC CCC TAC GTT GAC GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG 576 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys 180 185 190 CGC TTT CTC AGG ATT GTG AAG GAG AAA GAC CCG GAC GTA CTG ATA ACG 624 Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 TAC AAC GGT GAC AAC TTT GAC TTT GCC TAC CTC AAG AAG CGC TGT GAA 672 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 AAG CTT GGA ATA AAC TTT GCC CTC GGA AGG GAC GGA AGC GAG CCT AGA 720 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 ATT CAG AGG ATG GGC GAT AGG TTT GCT GTC GAG GTT AAG GGC AGG ATA 768 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 CAC TTT GAT CTC TAC CCC GTG ATA AGA CGG ACG ATA AAC CTC CCC ACA 816 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 TAT ACG CTT GAG GCT GTC TAT GAA GCG ATC TTT GGA AAG CCA AAG GAG 864 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 AAA GTC TAC GCC GAG GAG ATA GCC ACC GCT TGG GAG AGC GGG GAG GGG 912 Lys Val Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Ser Gly Glu Gly 290 295 300 CTT GAG AGG GTA GCT AAA TAC TCT ATG GAG GAC GCC AAG GTT ACC TAC 960 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 GAG CTC GGA AAG GAG TTC TTC CCG ATG GAG GCC CAG CTT TCC CGC TTG 1008 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 ATC GGC CAG TCC CTC TGG GAC GTT TCC CGC TCC AGC ACT GGC AAC CTC 1056 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 GTC GAG TGG TTC CTC CTC CGG AAG GCC TAT GAG AGG AAC GAG CTG GCC 1104 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 CCG AAC AAG CCC GAT GAA AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT 1152 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 GAA GGG GGC TAC GTA AAA GAG CCG GAG AGA GGT CTA TGG GAA AAC ATA 1200 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 GTT TAT CTG GAT TTC CGT TCG CTG TAC CCC TCA ATC ATC ATC ACC CA C 1248 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 AAC GTC TCG CCG GAT ACT CTT AAC AGG GAA GGA TGC AAG GAG TAT GAC 1296 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 ACC TCC CCC CAG GTT GGC CAC CGC TTC TGC AAG GAC TTC CCG GGC TTC 1344 Thr Ser Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 ATT CCG AGC CTC CTT GGG GCC CTG CTT GAG GAG AGG CAG AAG ATA AAG 1392 Ile Pro Ser Leu Leu Gly Ala Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 AAG AGA ATG AAG GCA ACG ATT GAT CCA CTT GAG AAG AAG CTC CTT GAT 1440 Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 TAC AGA CAG CGG GCT ATC AAG ATC CTA GCA AAC AGT TTT TAC GGC TAC 1488 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 TAC GGC TAT GCA AAG GCC CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC 1536 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 GTC ACT GCC TGG GGA AGG GAG TAC ATA ACG ATG ACC ATC AGG GAG ATA 1584 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Arg Glu Ile 515 520 525 GAG GAA AAG TAC GGC TTT AAA GTG CTG TAC GCT GAC AGC GTT ACG GGT 1632 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Ser Val Thr Gly 530 535 540 GAC TCT GAG GTC ATC ATA AGA AGA AAT GGC CGC ATT GAG TTC ATT CCT 1680 Asp Ser Glu Val Ile Ile Arg Arg Asn Gly Arg Ile Glu Phe Ile Pro 545 550 555 560 ATC GAA AAG CTC TTC GAA AGA GTG GAC TAC ACA GTT GGG GAG AAG GAG 1728 Ile Glu Lys Leu Phe Glu Arg Val Asp Tyr Thr Val Gly Glu Lys Glu 565 570 575 TAT CAT GTT CTA TCA TCA AAT GTT GAA GCT CTA ACA CTC GAC GAC AAC 1776 Tyr His Val Leu Ser Ser Asn Val Glu Ala Leu Thr Leu Asp Asp Asn 580 585 590 GGA AAG CTT ACG TGG AGA AAA GTC CCT TAC GTA ATG AGA CAT AAA ACG 1824 Gly Lys Leu Thr Trp Arg Lys Val Pro Tyr Val Met Arg His Lys Thr 595 600 605 GAG AAG AAG ATT TAC CGC GTC TGG CTG ACC AAC AGC TGG TAC CTT GAT 1872 Glu Lys Lys Ile Tyr Arg Val Trp Leu Thr Asn Ser Trp Tyr Leu Asp 610 615 620 GTC ACG GAG GAC CAT TCT CTT ATA GGG TAC CTA AAC ACA TCA AGA GTA 1920 Val Thr Glu Asp His Ser Leu Ile Gly Tyr Leu Asn Thr Ser Arg Val 625 630 635 640 AGG GCT GGA AAG CCT CTA AAA GAC CGC CTT TGT GAG GTT AAG CCA CTG 1968 Arg Ala Gly Lys Pro Leu Lys Asp Arg Leu Cys Glu Val Lys Pro Leu 645 650 655 GAA CTT GGT AAA TCT GTG AAG TCC CTC ATA ACG CCA AGA GCA CCT CTC 2016 Glu Leu Gly Lys Ser Val Lys Ser Leu Ile Thr Pro Arg Ala Pro Leu 660 665 670 TCT AGG GGC ATA AAA CCC AAT GAA ATA GCC CTG AAA TTC TGG GAG CTG 2064 Ser Arg Gly Ile Lys Pro Asn Glu Ile Ala Leu Lys Phe Trp Glu Leu 675 680 685 GTT GGG CTT CTC GTT GGC GAT GGC AAT TGG GGA GGT ACA TCA AAC TGG 2112 Val Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Thr Ser Asn Trp 690 695 700 GCG AAG TAC TAC GTG GGA CTT GCA TGT GGA GAA GAC AAA GAA GAA ATC 2160 Ala Lys Tyr Tyr Val Gly Leu Ala Cys Gly Glu Asp Lys Glu Glu Ile 705 710 715 720 GCC GAG AAG GTA CTT GAC CCG CTA AAA CGT GCA GGA GTC ATA TCC AAC 2208 Ala Glu Lys Val Leu Asp Pro Leu Lys Arg Ala Gly Val Ile Ser Asn 725 730 735 TAC TAC GAC AAG AGC AAG AAG GGA GAC GTT TCG ATC TTA TCG AAG GGT 2256 Tyr Tyr Asp Lys Ser Lys Lys Gly Asp Val Ser Ile Leu Ser Lys Gly 740 745 750 CTC GCA AAG CTC ATG GTT AGA TAC TTT AAG GAT GAG GAC GGG AAC AAA 2304 Leu Ala Lys Leu Met Val Arg Tyr Phe Lys Asp Glu Asp Gly Asn Lys 755 760 765 AAG ATT CCA GAG TTT ATG TTC AAC CTC CCA AAA GAA TAC TTG GAG GCG 2352 Lys Ile Pro Glu Phe Met Phe Asn Leu Pro Lys Glu Tyr Leu Glu Ala 770 775 780 TTT CTC AGG GGA TTG TTC AGT GCA GAT GGT ACT GTT AGC GTG AAG AGA 2400 Phe Leu Arg Gly Leu Phe Ser Ala Asp Gly Thr Val Ser Val Lys Arg 785 790 795 800 GGT GTA CCT GAA GTC AGG CTC ACG ACG ATT AGT GAT AGA TTA GCT AGT 2448 Gly Val Pro Glu Val Arg Leu Thr Thr Ile Ser Asp Arg Leu Ala Ser 805 810 815 GAC GTT AGA AAA CTG CTT TGG CTT GTG GGA ATT TCA AAT TCC ATC TTC 2496 Asp Val Arg Lys Leu Leu Trp Leu Val Gly Ile Ser Asn Ser Ile Phe 820 825 830 AGG GAG CAA AAT CCC AAC AGG TAC AAT GGA AAG TCG AGT GGC ACT TAC 2544 Arg Glu Gln Asn Pro Asn Arg Tyr Asn Gly Lys Ser Ser Gly T hr Tyr 835 840 845 TCC AAG CAC GTC CGC ATA AAG GAC AAA CTC CAG TTT GCG CAG AGA ATT 2592 Ser Lys His Val Arg Ile Lys Asp Lys Leu Gln Phe Ala Gln Arg Ile 850 855 860 AGA TTC ATA ATC AAC AGA AAA CAA GAA AAG CTC ATT AAA AAT CTA AAG 2640 Arg Phe Ile Ile Asn Arg Lys Gln Glu Lys Leu Ile Lys Asn Leu Lys 865 870 875 880 GAG TCA CAG TAC AAG AGA ACG ACG TTT AAG TAC GAG TTT GAC ATC ACT 2688 Glu Ser Gln Tyr Lys Arg Thr Thr Phe Lys Tyr Glu Phe Asp Ile Thr 885 890 895 CCC GTC AAG AAA GTT GAA GAG GTA ACC TAT AAT GGG TAC GTC TAC GAT 2736 Pro Val Lys Lys Val Glu Glu Val Thr Tyr Asn Gly Tyr Val Tyr Asp 900 905 910 ATC GAG GTT GAA GGC ACT CAT AGG TTC TTC GCT AAC GGA ATA CTA GTT 2784 Ile Glu Val Glu Gly Thr His Arg Phe Phe Ala Asn Gly Ile Leu Val 915 920 925 CAT AAT ACA GAC GGT TTC TTC GCG ACA ATA CCT GGA GCC GAT GCT GAA 2832 His Asn Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu 930 935 940 ACC GTC AAA AAG AAG GCA ATG GAG TTC ATC AAT TAC ATC AAC GAC AAG 2880 Thr Val Lys Lys Lys Ala Met Glu Phe I le Asn Tyr Ile Asn Asp Lys 945 950 955 960 CTT CCA GGT GCG CTT GAG CTC GAG TAT GAG GGT TTC TAC AAA CGC GGC 2928 Leu Pro Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly 965 970 975 TTC TTC GTC GTC ACG AAG AAG AAG TAC GCG GTG ATA GAC GAG GAA GGC AAG 2976 Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys 980 985 990 ATA ACA ACG CGC GGA CTT GAG ATT GTG AGG CGC GAC TGG AGC GAG ATA 30 Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile 995 1000 1005 GCG AAA GAG ACG CAG GCA AGG GTT CTT GAA GCT TTG CTA AAG GAC GGT 3072 Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Leu Leu Lys Asp Gly 1010 1015 1020 GAC GTC GAA AAG GCC GTG AGG ATA GTC AAG GAA GTC ACT GAA AAG CTG 3120 Asp Val Glu Lys Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys Leu 1025 1030 1035 1040 AGC AAG TAC GAG GTT TCG CCG GAG AAG CTG GTG ATC CAC GAG CAG ATA 3168 Ser Lys Tyr Glu Val Ser Pro Glu Lys Leu Val Ile His Glu Gln Ile 1045 1050 1055 ACG AGG GAT TTA AAG GAC TAC AAG GCG ACG GGA CCC CAC GTT GCC GTT 3216 Th r Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala Val 1060 1065 1070 GCC AAG AGG TTA GCC GCG AGA GGA GTC AAA ATA CGC CCT GGA ACG GTG 3264 Ala Lys Arg Leu Ala Ala Arg Gly Val Lys Ile Arg Pro Gly Thr Val 1075 1080 1085 ATA AGC TAC ATC GTG CTA AAG GGC TCT GGA AGG ATA GGC GAC AGG GCA 3312 Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala 1090 1095 1100 ATA CCG TTC GAC GAG TTC GAC CCG ACG AAG CAC AAG TAC GAC GCC GAG 3360 Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Lys Tyr Asp Ala Glu 1105 1110 1115 1120 TAC TAC ATT GAG AAC CAG GTT CTC CCA GCC GTT GAG AGA ATT CTG AGA 3408 Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu Arg Ile Leu Arg 1125 1130 1135 GCC TTC GGT TAT CGC AAG GAA GAC CTG CGC TAC CAG AAG ACG AGA CAG 3456 Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln 1140 1145 1150 GTT GGT TTG AGT GCT TGG CTG AAG CCG AAG GGA AAG TAGGGAATTA 3502 Val Gly Leu Ser Ala Trp Leu Lys Pro Lys Gly Lys 1155 1160 ACCCTTCCAG GTGAATGGAA TGGAACTAGA AATCCGTCGA AGTCCCGTCA A GTATGCGAG 3562 GATAGAAGTG AAGCCGGACG GCAGGGTAGT CGTCACCGCC CCCGAGGGGT TCAACGTTGA 3622 GGAGTTCATA GCAAAGAACG CCGCCTGGCT GGGACTCCTGACATGACTAG 682GAAAGAGCTT ATCCCGGCGCCAG

SEQ ID NO: 4 Sequence length: 2376 Sequence type: Nucleic acid (DNA) Topology: Linear Sequence type: Synthetic DNA Origin: Thermococcus peptonophilus Strain name: SM-2 Sequence characteristics 1-2322 CDS ATG ATC CTC GAC ACT GAC TAC ATA ACT GAG AAT GGA AAG CCC GTC ATC 48 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 AGG ATT TTC AAG AAG GAA AAC GGC GAG TTC ACG ATC GAG TAC GAC AGG 96 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Glu Tyr Asp Arg 20 25 30 ACT TTT GAG CCC TAT ATC TAC GCC CTC CTG AAG GAC GAT TCT GCA ATA 144 Thr Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 GAG GAA GTC AAG AAG ATA ACT GCC GAG AGA CAC GGG ACG GTT GTA ACG 192 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 GTT AAG CGA GCT GAA AAG GTT CAG AAG AAG TTC CTC GGA AGA CCG TTG 240 Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Leu 65 70 75 80 GAT GTC TGG AAG CTC TAC TT T ACT CAC CCG CAG GAC GTC CCA GCG ATA 288 Asp Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 AGG GAC AAG ATA CGA GAG CAT CCG GCA GTT ATT GAC ATC TAC GAG TAC 336 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 GAC ATA CCC TTC GCC AAG CGC TAT CTC ATT GAC AAG GGC CTA ATC CCG 384 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 ATG GAG GGC GAT GAA GAG CTG AAA ATG CTA GCC TTC GAC ATT GAA ACG 432 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 CTC TAC CAC GAG GGC GAG GAG TTT GCC GAG GGG CCA ATC CTT ATG ATA 480 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 AGC TAC GCC GAC GAA GAA GGA GCG AGG GTG ATA ACG TGG AAG AAC ATA 528 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 GAC CTC CCC TAC GTT GAC GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG 576 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys 180 185 190 CGC TTT CTC AGG A TT GTG AAG GAG AAA GAC CCG GAC GTA CTG ATA ACG 624 Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 TAC AAC GGT GAC AAC TTT GAC TTT GCC TAC CTC AAG AAG CGC TGT GAA 672 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 AAG CTT GGA ATA AAC TTT GCC CTC GGA AGG GAC GGA AGC GAG CCT AAG 720 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 ATT CAG AGG ATG GGC GAT AGG TTT GCT GTC GAG GTT AAG GGC AGG ATA 768 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 CAC TTT GAT CTC TAC CCC GTG ATA AGA CGG ACG ATA AAC CTC CCC ACA 816 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 TAT ACG CTT GAG GCT GTC TAT GAA GCG ATC TTT GGA AAG CCA AAG GAG 864 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 AAA GTC TAC GCC GAG GAG ATA GCC ACC GCT TGG GAG AGC GGG GAG GGG 912 Lys Val Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Ser Gly Glu Gly 290 295 300 CTT G AG AGG GTA GCT AAA TAC TCT ATG GAG GAC GCC AAG GTT ACC TAC 960 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 GAG CTC GGA AAG GAG TTC TTC CCG ATG GAG GCC CAG CTT TCC CGC TTG 1008 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 ATC GGC CAG TCC CTC TGG GAC GTT TCC CGC TCC AGC ACT GGC AAC CTC 1056 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 GTC GAG TGG TTC CTC CTC CGG AAG GCC TAT GAG AGG AAC GAG CTG GCC 1104 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 CCG AAC AAG CCC GAT GAA AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT 1152 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 GAA GGG GGC TAC GTA AAA GAG CCG GAG AGA GGT CTA TGG GAA AAC ATA 1200 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 GTT TAT CTG GAT TTC CGT TCG CTG TAC CCC TCA ATC ATC ATC ACC CAC 1248 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 AAC GTC TCG CCG GAT ACT CTT AAC AGG GAA GGA TGC AAG GAG TAT GAC 1296 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 ACC TCC CCC CAG GTT GGC CAC CGC TTC TGC AAG GAC TTC CCG GGC TTC 1344 Thr Ser Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 ATT CCG AGC CTC CTT GGG GCC CTG CTT GAG GAG AGG CAG AAG ATA AAG 1392 Ile Pro Ser Leu Leu Gly Ala Leu Leu Glu Arg Gln Lys Ile Lys 450 455 460 AAG AGA ATG AAG GCA ACG ATT GAT CCA CTT GAG AAG AAG CTC CTT GAT 1440 Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 TAC AGA CAG CGG GCT ATC AAG ATC CTA GCA AAC AGT TTT TAC GGC TAC 1488 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 TAC GGC TAT GCA AAG GCC CGC TGG TAC TGC AAG GAG TGT GCA GAG TGT GCA AGC 1536 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 GTC ACT GCC TGG GGA AGG GAG TAC ATA ACG ATG ACC ATC AGG GAG ATA 1584 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Arg Glu Ile 515 520 525 GAG GAA AAG TAC GGC TTT AAA GTG CTG TAC GCT GAT ACA GAC GGT TTC 1632 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 TTC GCG ACA ATA CCT GGA GCC GAT GCT GAA ACC GTC AAA AAG AAG GCA 1680 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 ATG GAG TTC ATC AAT TAC ATC AAC GAC AAG CTT CCA GGT GCG CTT GAG 1728 Met Glu Phe Ile Asn Tyr Ile Asn Asp Lys Leu Pro Gly Ala Leu Glu 565 570 575 CTC GAG TAT GAG GGT TTC TAC AAA CGC GGC TTC TTC GTC ACG AAG AAG 1776 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 AAG TAC GCG GTG ATA GAC GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT 1824 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 GAG ATT GTG AGG CGC GAC TGG AGC GAG ATA GCG AAA GAG ACG CAG GCA 1872 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 AGG GTT CTT GAA GCT TTG CTA AAG GAC GGT GAC GTC GAA AAG GCC GTG 1920 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 AGG ATA GTC AAG GAA GTC ACT GAA AAG CTG AGC AAG TAC GAG GTT TCG 1968 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Ser 645 650 655 CCG GAG AAG CTG GTG ATC CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC 2016 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670 TAC AAG GCG ACG GGA CCC CAC GTT GCC GTT GCC AAG AGG TTA GCC GCG 2064 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 AGA GGA GTC AAA ATA CGC CCT GGA ACG GTG ATA AGC TAC ATC GTG CTA 2112 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 AAG GGC TCT GGA AGG ATA GGC GAC AGG GCA ATA CCG TTC GAC GAG TTC 2160 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 GAC CCG ACG AAG CAC AAG TAC GAC GCC GAG TAC TAC ATT GAG AAC CAG 2208 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 GTT CTC CCA GCC GTT GAG AGA ATT CTG AGA GCC TTC GGT TAT CGC AAG 22 56 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 GAA GAC CTG CGC TAC CAG AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG 2304 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp 755 760 765 CTG AAG CCG AAG GGA AAG TAGGGAATTA ACCCTTCCAG GTGAATGGAA 2352 Leu Lys Pro Lys Gly Lys 770 TGGAACTAGA AATCCGTCGT CGAC 2376

SEQ ID NO: 5 Sequence length: 774 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Leu 65 70 75 80 Asp Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 Glu Gly Gly Tyr Va l Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tlu Asp 420 425 430 Thr Ser Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Ala Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Arg Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Ile Asn Tyr Ile Asn A Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Ser 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp 755 760 765 Leu Lys Pro Lys Gly Lys 770

SEQ ID NO: 6 Sequence length: 23 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence features Sequence CCITTCGCIA AGCGITA (CT) CT CAT 23

SEQ ID NO: 7 Sequence length: 23 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence AG (CT) TACCACT CIACIAGGTT ICC 23

SEQ ID NO: 8 Sequence length: 26 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence GTIAGGCGIG ACTGGAG (CT) GA (AG) ATIGC 26

SEQ ID NO: 9 Sequence length: 23 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence ACIGCIGGCA IAAC (CT) TGGTT (CT) TC 23

SEQ ID NO: 10 Sequence length: 23 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence GGTGTCCCAT ATGATCCTCG ACA 23

SEQ ID NO: 11 Sequence length: 24 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence CGTAACGATA TCAGCGTACA GCAG 24

SEQ ID NO: 12 Sequence length: 26 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence ACAGACGGTT TCTTCGCGAC AATACC 26

SEQ ID NO: 13 Sequence length: 24 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence GGGTCGTCGA CGGATTTCTA GTTC 24

SEQ ID NO: 14 Sequence length: 24 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA Sequence CGCCAGGGTT TTCCCAGTCA CGAC 24

[Brief description of drawings]

FIG. 1 is a construction diagram of pOGSB.

FIG. 2 is a construction diagram of pSpolMT.

[Figure 3] Thermococcus peptonophilus (Thermoco
ccus peptonophilus) DNA polymerase, TliDN
100 of A polymerase and Taq DNA polymerase
It is a graph which shows heat stability in ° C.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/12 C12R 1:19) (72) Inventor Yoshihisa Kawamura 10-24 Toyo-cho, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Institute (72) Inventor Hiroki Horikoshi 4-39-8 Sakuradai, Nerima-ku, Tokyo

Claims (21)

[Claims] 1. A Thermococcus peptonophilus (The
rmococcus peptonophilus) thermostable DNA polymerase. 2. The thermostable DNA polymerase according to claim 1, which has the following physicochemical properties. Action: (1) Catalyze a polymerase reaction that forms a nucleic acid strand complementary to a nucleic acid template strand from nucleoside triphosphate. (2) It has a 3′-5 ′ exonuclease activity. Thermal stability: Residual activity of about 60% after treatment at 100 ° C. for 30 minutes. Molecular weight: about 86 to 92 KDa 3. Thermococcus peptonophilus (The
rmococcus peptonophilus) is Thermococcus peptonophilus OG-1 or Thermococcus peptonophilus (Thermococcus
peptonophilus) SM-2, The thermostable DNA polymerase according to claim 1. 4. Thermococcus peptonophylla (The
The thermostable DNA polymerase according to claim 1, wherein the rmococcus peptonophilus is Thermococcus peptonophilus OG-1. 5. The thermostable DNA polymerase according to claim 4, which has the following physicochemical properties. Action: (1) Catalyze a polymerase reaction that forms a nucleic acid strand complementary to a nucleic acid template strand from nucleoside triphosphate. (2) It has a 3′-5 ′ exonuclease activity. Thermal stability: Residual activity of about 60% after treatment at 100 ° C. for 30 minutes. Molecular weight: about 86 to 92 KDa Optimum pH: about 6.5 to 9 Optimum temperature: about 70 to 80 ° C. DNA synthesis rate: about 60 bases / second or more. 6. Thermococcus peptonophylla (The
The DNA polymerase according to claim 1, wherein the rmococcus peptonophilus) is Thermococcus peptonophilus SM-2. 7. The thermostable DNA polymerase according to claim 6, which has the following physicochemical properties. Action: (1) Catalyze a polymerase reaction that forms a nucleic acid strand complementary to a nucleic acid template strand from nucleoside triphosphate. (2) It has a 3′-5 ′ exonuclease activity. Thermal stability: Residual activity of about 60% after treatment at 100 ° C. for 30 minutes. Molecular weight: about 86 to 92 KDa Optimum pH: about 6.5 to 9 Optimum temperature: about 70 to 80 ° C. DNA synthesis rate: about 40 bases / second or more. 8. The thermostable DNA polymerase according to claim 1, which is produced using a recombinant host cell. 9. The thermostable DNA polymerase according to claim 1, which contains the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 10. The thermostable DNA polymerase according to claim 1, which comprises the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing. 11. Thermococcus peptonophyllus (T
isolated DNA encoding a DNA polymerase from hermococcus peptonophilus). 12. The isolated DNA according to claim 11, which contains a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 13. The isolated DNA according to claim 11, which contains a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing. 14. Thermococcus peptonophyllus (T
D with the isolated DNA encoding a DNA polymerase from hermococcus peptonophilus) inserted into the vector
NA recombinant expression vector. 15. The vector is pLED-M1 or pLED.
15. A vector derived from Bluescript.
The described DNA recombinant expression vector. 16. Thermococcus peptonophyllus (T
D with the isolated DNA encoding a DNA polymerase from hermococcus peptonophilus) inserted into the vector
A recombinant host cell transformed with the NA recombinant vector. 17. The host cell is Escherichia coli E. coli. The recombinant host cell according to claim 16, which is E. coli. 18. Thermococcus peptonophyllus (T
D with the isolated DNA encoding a DNA polymerase from hermococcus peptonophilus) inserted into the vector
Recombinant host cells transformed with the NA recombinant vector are cultured in a nutrient medium, and the thermostable DNA is obtained from the culture.
Thermostable DN characterized by harvesting polymerase
Method for producing A polymerase. 19. Thermococcus peptonophyllus (T
D with the isolated DNA encoding a DNA polymerase from hermococcus peptonophilus) inserted into the vector
Recombinant host cells transformed with the NA recombinant vector are cultured in a nutrient medium, (a) the recombinant host cells are collected and then crushed to prepare a cell extract, and (b) recombinant A method for producing a thermostable DNA polymerase, which comprises a step of removing an impure protein derived from a host cell. 20. The method for producing a thermostable DNA polymerase according to claim 19, wherein the step (b) of removing the impure protein derived from the recombinant host cell is a high temperature heat treatment. 21. The thermostable DN according to claim 19, wherein the high temperature heat treatment condition is 70 ° C. or higher, preferably 85 ° C. or higher.
A method for producing A polymerase.