JP2002051788A - Heat resistant enzyme having primase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a heat resistant enzyme having an excellent activity even under the condition of high temperature and a primase activity capable of synthesizing a nucleic acid primer. SOLUTION: A gene supposed encoding a protein exhibiting the primase activity is identified from a gene sequence of a super thermophilic archaebacterium growing at 90-100 deg.C, further the gene is incorporated into an Escherichia coli, an enzyme is produced by using the transformed Escherichia coli, and the enzyme is stable at high temperature (more than 80 deg.C) and is confirmed to exhibit the primase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a protein having a primase activity under high temperature conditions, and a gene encoding the protein.

[0002]

2. Description of the Related Art Primase, also called DNA primase, is an RNA polymerase that synthesizes a primer RNA for initiating DNA replication. That is, the primase synthesizes a short RNA primer of about 10 nucleotides at the origin of replication on the leading strand and the origin of synthesis of the Okazaki fragment on the lagging strand in the DNA replication machinery. Therefore, primase is DN
It is an essential component in the A duplication mechanism.

[0003] Primers known at present include:
DnaG gene product (60 kDa) in E. coli, T
7 phage gene 4 product (Gp4, 63 kDa), T
4 phage gene 41 product (54 kDa) and gene 61 product (40 kDa) and two subunits of eukaryotic DNA polymerase α (about 60 kDa and about 50 kDa).
kDa).

[0004] However, since these primases are derived from normal-temperature organisms, they are extremely unstable under temperature conditions of room temperature or higher. I will. For this reason, for example, PC which repeats the heat denaturation temperature of double-stranded DNA, rapid cooling, and enzymatic reaction
In the R reaction and the like, currently known primase cannot be used. If a primase having excellent heat resistance is found, a DNA amplification reaction by a PCR method and a primer synthesis reaction can be performed in the same tube. As a result, a novel gene labeling method and a gene mutation method can be developed. Therefore, primases exhibiting excellent activity even under high temperature conditions have been craved. However, no primase having excellent heat resistance has been known at present.

[0005]

Accordingly, an object of the present invention is to provide a thermostable enzyme having a primase activity capable of synthesizing a nucleic acid primer while exhibiting excellent activity even under high temperature conditions. And

[0006]

Means for Solving the Problems In order to achieve the above object, the present inventors focused on hyperthermophilic archaea that grow at 90 to 100 ° C. and encoded a protein exhibiting primase activity from its gene sequence. Then we found a putative gene. Furthermore, the gene was incorporated into Escherichia coli, and an enzyme was produced using the transformed Escherichia coli. It was confirmed that the enzyme was stably present at a high temperature (80 ° C. or higher) and exhibited primase activity. Thus, the present invention has been completed.

[0007] That is, the present invention relates to a primase which binds to a predetermined site in a base sequence and synthesizes a nucleic acid primer complementary to the base sequence.
Purified from heat-resistant archaea that can grow in the above environment,
A primase having an optimum temperature of 80 ° C. or higher.

Further, the present invention provides the following (a) or (b)
Is a protein. (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (B) a protein having an amino acid sequence in which at least one amino acid in the amino acid sequence represented by SEQ ID NO: 1 has been deleted, substituted or added, and has a subunit function of an enzyme having primase activity.

Here, in the protein of (a), SEQ ID NO: 1 is the amino acid sequence of the small subunit of primase collected from hyperthermophilic archaea. Therefore, the protein (a) has a function of becoming a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. In the protein (b), “having a subunit function of an enzyme having primase activity” means that the protein (b) becomes a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. It means to have.

Further, the present invention relates to the following protein (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2; (B) a protein having an amino acid sequence in which at least one amino acid in the amino acid sequence represented by SEQ ID NO: 2 has been deleted, substituted or added, and having a subunit function of an enzyme having primase activity.

Here, in the protein (a), SEQ ID NO: 2 is the amino acid sequence of the large subunit of primase collected from hyperthermophilic archaea. Therefore, the protein (a) has a function of becoming a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. Therefore, in the protein of (b), “having a subunit function of an enzyme having primase activity” means that the protein of (b) becomes a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. It means to have.

Furthermore, the present invention is a composite protein comprising a heterodimer formed by the protein of claim 1 and the protein of claim 2 and having a primase activity. Furthermore, the present invention is a gene encoding the primase according to claim 1. Furthermore, the present invention is a gene encoding the protein according to claim 2. Furthermore, the present invention is a gene encoding the protein according to claim 3.

Further, the present invention relates to a gene having the following DNA (a) or (b). (A) DN consisting of the base sequence represented by SEQ ID NO: 3
A. (B) a DNA consisting of a base sequence in which at least one base in the base sequence represented by SEQ ID NO: 3 has been deleted, substituted or added, and having a function of encoding a subunit of an enzyme having primase activity.

Here, in the DNA of (a), SEQ ID NO: 3 is a nucleotide sequence encoding a small subunit of primase collected from hyperthermophilic archaea. Therefore, the DNA of (a) has a function of encoding a small subunit which is a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. Also, D in (b)
In NA, “having the function of encoding a subunit of an enzyme having primase activity” means that a small subunit that is a component of a primase that synthesizes a nucleic acid primer under high-temperature conditions, It has the function of coding.

Further, the present invention is a gene having the following DNA (a) or (b). (A) DN consisting of the base sequence represented by SEQ ID NO: 4
A. (B) DNA comprising a base sequence in which at least one base in the base sequence represented by SEQ ID NO: 4 has been deleted, substituted or added, and having a function of encoding a subunit of an enzyme having primase activity.

Here, in the DNA of (a), SEQ ID NO: 4 is a nucleotide sequence encoding a large subunit of primase collected from hyperthermophilic archaea. Therefore, the DNA of (a) has a function of encoding a large subunit which is a component of a primase that synthesizes a nucleic acid primer under high temperature conditions. Also, D in (b)
In NA, “has a function of encoding a subunit of an enzyme having primase activity” means that a large subunit which is a component of a primase that synthesizes a nucleic acid primer under high-temperature conditions, It has the function of coding.

Furthermore, the present invention is a recombinant vector containing the gene according to claim 6 or 8. Furthermore, the present invention is a recombinant vector containing the gene according to claim 7 or claim 9. Furthermore, the present invention is a transformant comprising the recombinant vector according to claim 10 and / or the recombinant vector according to claim 11.
Furthermore, the present invention provides a method for culturing the transformant according to claim 12, collecting a small subunit and a large subunit from the obtained culture, and forming a heterodimer from the small subunit and the large subunit. This is a method for producing primase.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The primase of the present invention is purified from heat-resistant archaea that can grow under cyclization at 80 ° C or higher, and is characterized in that the optimum temperature is 80 ° C or higher. In other words, this primase can be used at temperatures
It binds to a predetermined site in the base sequence and has a primase activity for synthesizing a nucleic acid primer complementary to the base sequence.

Here, as the heat-resistant archaea capable of growing under cyclization at 80 ° C. or higher, hyperthermophilic bacteria can be used, preferably thermophilic archaea, and more preferably sulfur-metabolized thermophilic archaea. Bacteria can be used. Examples of the thermophilic archaea include, for example, Pyrococcus horikosi
i) Pyrococcus furios
us) and Pyrococcus woe
sei), Thermococcus litoralis, Thermococcus celer, Thermococcus profundus and Thermococcus peptonopylus
And the like, or the genus Thermococcus or the genus Methanobacterium.

The term “nucleic acid primer” used herein means both a DNA primer and an RNA primer. That is, the primase of the present invention can synthesize both a DNA primer and an RNA primer. The primase of the present invention can be artificially obtained using recombinant DNA technology as long as it has the above properties.

In the following, as an example of the present invention, a thermostable primase using Pyrococcus horikosii will be described. It is possible to get zero.

1. Isolation of thermostable primase gene The thermostable primase gene is derived from Pyrococcus horikoshi, a sulfur-metabolizing thermophilic archaeon.
i, accession number JCM9974). That is, first, Pyrococcus horikoshi is cultured and chromosomal DNA is extracted. In culturing Pyrococcus horikoshi, a commonly used medium can be used.

Next, the cultured Pyrococcus horikoshi is collected, and chromosomal DNA is prepared therefrom. Chromosome D
In order to prepare NA, a commonly used technique can be applied. To prepare chromosomal DNA, a commercially available kit (for example, ISOPLANT (Nippon Gene))
Can also be used.

Next, the prepared chromosomal DNA is fragmented with a restriction enzyme, and the fragmented chromosomal DNA is ligated to a predetermined plasmid vector or phage vector to prepare a chromosomal DNA library. At this time, clones are selected so as to cover all the chromosomes of Pyrococcus horikoshi and the chromosomal DNA library is sorted.

Next, the entire base sequence of the aligned chromosomal DNA library is determined. Specifically, when determining the nucleotide sequence, about 500 so-called shotgun clones were prepared for one chromosomal DNA library clone, and the nucleotide sequences of these shotgun clones were sequentially determined. Then, by linking and editing the nucleotide sequence of the shotgun clone with commercially available software, the entire nucleotide sequence of the chromosomal DNA library can be determined.

Next, the homology between the determined base sequence and the known primase gene is analyzed by a large computer. As a result of the analysis, a nucleotide sequence having high homology to a known primase gene was identified, and the small subunit gene (SEQ ID NO: 3) and the large subunit gene (SEQ ID NO: 4) encoding the primase in Pyrococcus horikoshi were identified. Can be identified.

2. Construction of Expression Plasmid To construct an expression plasmid, first, a small subunit is subjected to a PCR reaction using primers designed from the nucleotide sequence of chromosomal DNA, the nucleotide sequence of the small subunit gene, and the nucleotide sequence of the large subunit gene. Amplify unit gene and large subunit gene.

The primers are preferably designed so that restriction enzyme sites for integration into an expression plasmid vector are added to the 3 'end and 5' end of the structural gene to be amplified, respectively. More preferably,
It is preferable to design primers so that different restriction enzyme sites are added to the 3 ′ end and the 5 ′ end, respectively. As a result, the amplified structural gene can be incorporated in the correct direction into the expression plasmid vector.

Furthermore, when constructing an expression vector for a small subunit, it is preferable to design a primer that adds a histidine tag (His-Tag) to the N-terminal of the expressed small subunit. Thereby, the obtained small subunit becomes a fusion protein having a histidine tag added to the N-terminus, and can be fractionated according to the presence or absence of the histidine tag.

Specifically, as an upstream primer for amplifying a small subunit gene, for example, 5'-CTTTAAGAAGGAGATATACATATGCTGCTGCGTGAGGTAACCCGTGA to which a NedI site is added
GGAAAGAAAGAACTTTTAC-3 ′ (SEQ ID NO: 5) can be used, and as a downstream primer, for example, 5′-TTTTGAGCTCTTTGGATCCTTAGGCCATCTTTTAAGTTCCGAGACTT with a BamHI site added
TC-3 '(SEQ ID NO: 6) can be used.

The upstream primer and the downstream primer for amplifying the small subunit gene are not limited to those having the above base sequences, but are designed based on the base sequence of the chromosomal DNA library, and Any nucleotide sequence may be used as long as it is designed to sandwich the entire gene.

Further, specifically, as an upstream primer for amplifying a large subunit gene, for example, NedI
5'-TTTTGTCGACTTACATATGGCGATCATGCTCGACCCA-3 '(SEQ ID NO: 7) to which a site has been added can be used. As a downstream primer, for example, 5'-TTTTAAGCTTTTTGGATCCTTATTCCATTCATTGGTGTAA-3' (SEQ ID NO: 8) to which a BamHI site has been added can be used. Can be used.

The upstream primer and the downstream primer for amplifying the large subunit gene are not limited to those having the above base sequences, but are designed from the base sequence of the chromosomal DNA library, and Any nucleotide sequence may be used as long as it is designed to sandwich the entire gene.

Next, an expression plasmid for the small subunit capable of expressing the small subunit by incorporating the small subunit gene and the large subunit gene amplified by these primers into a predetermined expression plasmid vector, respectively. And an expression vector for the large subunit capable of expressing the large subunit.

Here, as a plasmid vector for expression, a commonly used plasmid vector can be used.
Examples of usable expression plasmid vectors include pET.
11a or pET15b.

Specifically, when the above primers are used, first, the amplified small subunit gene and large subunit gene are digested with NedI and BamHI, and the expression plasmid vector is digested with NedI and BamHI. Thereafter, the small subunit gene or large subunit gene and the expression plasmid vector are ligated with a ligation enzyme such as T4 ligase.

Thereafter, a part of the reaction solution is introduced into competent cells to obtain a transformant colony. Here, the competent cells that can be used may be those that are usually used for transformation, and include, for example, Escherichia coli and Bacillus subtilis. Then, the colony of the transformant is pure-cultured, and an expression vector for the small subunit and an expression vector for the large subunit are obtained by a technique such as the so-called alkaline method.

3. Purification of thermostable primase To purify the thermostable primase, first, the expression vector for the small subunit and the expression vector for the large subunit are separately expressed, and the small subunit (SEQ ID NO: 1) and Large subunits (SEQ ID NO: 2) are obtained respectively. Thereafter, by forming a heterodimer from the obtained small subunit and large subunit, a thermostable primase can be obtained.

Specifically, when isolating and purifying the small subunit or the large subunit, the expressed cells are melted to prepare a suspension. By using a biochemical method such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography or the like alone or in an appropriate combination, the desired small subunit and large subunit can be isolated from the suspension. It can be purified.

4. Evaluation of heat-resistant primase The heat-resistant primase obtained as described above has an optimum temperature of around 80 ° C and has primase activity at around 80 ° C. For this reason, the above-mentioned heat-resistant primase can be used for the following uses. PCR method A DNA serving as a template, a primase of the present invention, a dNTP mix, a heat-resistant DNA polymerase, and the like are mixed in the same tube, and a heat denaturation step of the template DNA, a nucleic acid primer synthesis step, and a DNA chain extension step are performed. By repeating a plurality of times, a PCR reaction can be performed. That is, in this PCR reaction, a synthetic oligo primer is not required, and the primer can be synthesized in the tube. Therefore, by using the primer of the present invention, a PCR reaction can be performed quickly and at low cost.

Gene labeling method The primerase of the present invention and a known thermostable DNA polymerase are coupled in the presence of a template DNA and a labeled nucleotide to cause a DNA extension reaction.
It is possible to obtain a single-stranded DNA whose 5 ′ end is composed of an RNA molecule or a DNA molecule. The resulting single-stranded DNA is
It is copied from a random position in the template DNA, and is labeled. That is,
By using the primase of the present invention, a novel gene labeling method can be established.

The gene mutation method The single-stranded DNA synthesized by the gene labeling method is re-annealed with a template DNA plasmid, and the gap between the re-annealed single-stranded DNAs is filled with DNA polymerase and then ligated with ligase. Thereafter, the host is transformed and a repair system in the host cell is activated to introduce a mutation at the position of the labeled nucleotide.
Thus, a novel gene mutation method can be established by using the primase of the present invention.

[0043]

EXAMPLES Hereinafter, the present invention will be described with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example 1] (1) Pyrococcus horikoshi (JCM9974)
Culture of Pyrococcus horikoshi JCM9974 (obtained from RIKEN Microorganism System Preservation Facility) was cultured by the following method.
13.5 g salt, 4 g Na ₂ SO ₄ , 0.7 g KCl, 0.2 g Na
HCO ₃ , 0.1 g KBr, 30 mg H ₃ BO ₃ , 10 g MgCl ₂ .6H ₂ O,
Dissolve 1.5 g CaCl ₂ , 25 mg SrCl ₂ , 1.0 ml resasulin solution (0.2 g / L), 1.0 g yeast extract, 5 g bactopeptone in 1 L, adjust the pH of this solution to 6.8 and sterilize by pressure. did. Then, dry heat sterilized elemental sulfur was added to a concentration of 0.2%, the medium was saturated with argon to make it anaerobic, and JCM9974 was inoculated. Whether the medium became anaerobic was confirmed by adding a Na ₂ S solution and not coloring the pink color of the resasurin solution due to Na ₂ S in the culture solution.
This culture was cultured at 95 ° C. for 2 to 4 days, and then centrifuged to collect the bacteria.

(2). Preparation of chromosomal DNA The chromosomal DNA of JCM9974 was prepared by the following method.
After completion of the culture, 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 sealed in an InCert Agarose (manufactured by FMC) block. 1% N-lauroylsarcosine, 1mg / ml
Chromosomal DNA by treating in protease K solution
Was separately prepared in an agarose block.

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

(4). Determination of Nucleotide Sequence The nucleotide sequence of the aligned BAC or Fosmid clones was determined sequentially by the following method. The DNA of each BAC or Fosmid clone recovered from Escherichia coli was fragmented by sonication, and 1k was obtained by agarose gel electrophoresis.
b and 2 kb DNA fragments were recovered. This fragment was inserted into the HincII restriction site of plasmid vector pUC118.
00 clones were made. The nucleotide sequence of each shotgun clone was determined using an automatic nucleotide sequence reader 373 or 377 manufactured by Perkin Elmer, ABI. The base sequence obtained from each shotgun clone was linked and edited using the base sequence automatic linking software Sequencher, and each BAC or Fos
The entire nucleotide sequence of the mid clone was determined.

(5). Identification of the thermostable primase gene The homology between the nucleotide sequence determined in (4) and the known primase gene was analyzed using a large-scale computer.
The small subunit gene (SEQ ID NO: 3) and the large subunit gene (SEQ ID NO: 4) encoding the primase at 9974 were identified.

Embodiment 2 (1). Construction of expression plasmid expressing small subunit In addition to introducing restriction enzyme sites around the small subunit gene region and adding a histidine tag to the N-terminus of the expressed small subunit, an upstream primer Upri-S
(5'-CTTTAAGAAGGAGATATACATATGCTGCTGCGTGAGGTAACCCGT
GAGGAAAGAAAGAACTTTTAC-3 ′: SEQ ID NO: 5) and downstream primer Lpri-S (5′-TTTTGAGCTCTTTGGATCCTTAGGCCATCTTTT)
AAGTTCCGAGACTTTC-3 ′: SEQ ID NO: 6) was designed and synthesized. It should be noted that an NdeI site is introduced from the 19th base to the 24th base in the Up-S, counting from the 5 'end. Lpri-S
From base 14 to base 19 from the 5 'end
BamHI site has been introduced.

After performing a PCR reaction using these Upri-S and Lpri-S, they were completely digested with restriction enzymes (NdeI and BamHI) (at 37 ° C. for 2 hours), and then the structural gene was purified. pET15b (Nova
gen) with restriction enzymes NdeI and BamHI.
The above structural gene was ligated by reacting with T4 ligase at 16 ° C. for 2 hours. A part of the ligated DNA was transferred to E. coli XL1-Blu
e MRF 'was introduced into competent cells to obtain transformant colonies. Expression plasmid capable of expressing small subunits from the obtained colonies (hereinafter referred to as “pET15b / PrimS”)
Was purified by an alkaline method.

(2). Construction of an expression plasmid expressing the large subunit In order to introduce restriction enzyme sites before and after the large subunit gene region, the upstream primer Upi-L (5'-TTTTGTCGACT
TACATATGGCGATCATGCTCGACCCA-3 ': SEQ ID NO: 7) and downstream primer Lpri-L (5'-TTTTAAGCTTTTTGGATCCTTATTCCA
TTCATTGGTGTAA-3 ′: SEQ ID NO: 8) was designed and synthesized.
In addition, an NdeI site has been introduced into the Up-L from the 14th to the 29th bases counted from the 5 'end. Lpri-L contains Ba from the 14th base to the 19th base counted from the 5 'end.
mHI site has been introduced.

After a PCR reaction using these Upri-L and Lpri-L, the structural gene was purified after complete digestion with restriction enzymes (NdeI and BamHI) (at 37 ° C. for 2 hours). pET11a (Nova
gen) with restriction enzymes NdeI and BamHI.
The above structural gene was ligated by reacting with T4 ligase at 16 ° C. for 2 hours. A part of the ligated DNA was transferred to E. coli XL1-Blu
e MRF 'was introduced into competent cells to obtain transformant colonies. Expression plasmid capable of expressing the large subunit from the obtained colonies (hereinafter referred to as “pET11a / PrimL”)
Was purified by an alkaline method.

(3). Gene expression Melt competent cells of E. coli (E. coli BL21 (DE3), Novagen) and E. coli (E. coli BL21 (DE3) Codon Plus-RIL, Novagen), and add 0.1 to each of the Falcon tubes. Transfer by ml. PE in the former Falcon tube
Add 0.005 mL of T15b / PrimS solution, add 0.005 mL of pET11a / PrimL solution to the latter Falcon tube, and then
After leaving these two Falcon tubes on ice for 30 minutes, a heat shock was performed at 42 ° C. for 30 seconds, and the SOC medium
Add 0.9mL and shake culture at 37 ° C for 1 hour. Thereafter, appropriate amounts of the culture solutions in the two Falcon tubes were separately spread on each of two 2YT agar plates containing ampicillin, and cultured at 37 ° C overnight to obtain two types of transformants from the two plates. Was.

The transformant using pET15b / PrimS was
Named E.coli BL21 (DE3) / pET15b / PrimS, pET11a / Pri
E. coli BL21 (DE3) Codon Plus-
It was named RIL / pET11a / PrimL. Also, E.coli BL21 (DE
3) / pET15b / PrimS has been deposited as FERM P-17883 with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology. In addition, E.co
li BL21 (DE3) Codon Plus-RIL / pET11a / PrimL has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-17884.

In order to express the small subunit gene and the large subunit gene, these transformants were transformed with a 2YT liquid medium (2 liters) containing ampicillin.
And then cultured separately until the absorbance at 600 nm reaches 1 with IPTG (Isopropyl-β-D-thiogalactopyranosisi).
de) was added and the cells were further cultured for 6 hours. After culture, centrifuge (6,
(000 rpm, 20 min).

Embodiment 3 (1). Purification of small subunit The collected cells (E. coli BL21 (DE3) pET15b / PrimS) were
Freeze and thaw at 0 ° C and double the volume of 50 mM Tris-HCl buffer (p
H7.5) and 0.5 mg of DNase were added to obtain a suspension. The resulting suspension was kept at 37 ° C. for 30 minutes, heated at 85 ° C. for 30 minutes, and centrifuged (11,000 rpm, 20 minutes) to obtain a supernatant. This was converted to a Ni-column (Novagen, His Bindmetal chelati
on resin & His. Bind buffer kit) for affinity chromatography. The 100 mM imidazole effluent fraction (20 ml) obtained here was concentrated to 2 ml with Centriprep 10 (Amicon). Further, this was adsorbed to a HiTrap SP (Pharmacia) column equilibrated with 50 mM Tris-HCl buffer (pH 7.0), and elution was performed with a NaCl concentration gradient using the same buffer. Next, SDS-electrophoresis of each fraction was performed, and the molecular weight of the contained protein was measured. Since the molecular weight of the small subunit was predicted to be 40,000 Da based on the gene sequence, fractions containing proteins of this molecular weight were collected to obtain a purified small subunit. This small subunit has the amino acid sequence shown in SEQ ID NO: 1 from the nucleotide sequence determined in Example 1 (4), and has a molecular weight of 40,358 Da.

(2). Purification of large subunit Collected cells (E. coli BL21 (DE3) Codon Plus-RIL / pET1
1a / PrimL) was freeze-thawed at −20 ° C., and twice the volume of 50 mM Tris-HCl buffer (pH 7.5) was added to obtain a suspension. The resulting suspension was heated at 85 ° C. for 10 minutes, centrifuged (11,000 rp
m, 20 minutes) to obtain a supernatant. The supernatant was adsorbed on a HiTrap SP (Pharmacia) column equilibrated with 50 mM phosphate buffer (pH 6.0), and eluted with a NaCl concentration gradient using the same phosphate buffer. Next, SDS-electrophoresis of each fraction was performed, and the molecular weight of the contained protein was measured. From the gene sequence, the molecular weight of the large subunit is 46,000 Da
Therefore, fractions containing proteins of this molecular weight were collected and diluted with three times the volume of distilled water. Increase this further by 50 mM
It was adsorbed to a HiTrap heparin (Pharmacia) column equilibrated with a Tris-HCl buffer (pH 8.0), and eluted with a NaCl concentration gradient using the same buffer to obtain a purified large subunit. Note that this large subunit is similar to that of the first embodiment.
It has the amino acid sequence shown in SEQ ID NO: 2 from the nucleotide sequence determined in (4), and has a molecular weight of 46,271 Da.

(3). Confirmation of heterodimer structure That the heat-resistant primase forms a heterodimer structure consisting of a small subunit and a large subunit,
It was confirmed as follows. First, 2 μg of the small subunit to which the histidine tag had been added and 3 μg of the large subunit were added to a final concentration of 20 mM Tris-HCl buffer (pH 7.9) to a total volume of 180 μl. This also includes 0.5M NaCl, 5mM imidazole, 1% Triton X-100, 0.04% bovine serum albumin. The solution was then kept on ice for 30 minutes.
As a result, a complex of the small subunit and the large subunit was formed.

Using this solution, a histidine tag attached to a small subunit was added to His-Bindresin containing nickel.
(Novagen), adsorbed to 16 μl, repeated centrifugation and suspension, and then binding buffer (Novagen, His.
ffer kit) three times.

This centrifuged sediment was suspended in 50 μl of SDS sample buffer, heated at 100 ° C. for 5 minutes, and centrifuged to obtain a supernatant. The obtained supernatant was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the type of subunit contained in this supernatant.

FIG. 1 shows the results of SDS-PAGE. In FIG. 1, lane 1 is a molecular weight marker, lane 2 is a sample obtained, and lane 3 is a sample prepared in the same manner as described above except that a small subunit is not added.
Lane 4 is a control, a sample obtained by adding the SDS sample buffer to the histidine-tagged small subunit and boiled, and Lane 5 is a sample obtained by adding the SDS sample buffer to the large subunit and boiled. Lane 6 is a control, which is a sample obtained by adding SDS sample buffer to bovine serum albumin and boiling the protein.

As can be seen from FIG. 1, bands of the small subunit and the large subunit can be detected in lane 2. In lane 3, a large subunit band cannot be detected. From this,
The thermostable primase was found to have a heterodimer structure consisting of a small subunit and a large subunit.

(4). Preparation of thermostable primase A thermostable primase was prepared according to the following procedure. First, 16 μg of the small subunit to which the histidine tag was added and 24 μg of the large subunit were mixed with 1% Triton X-100 and 0.01%
Was added to a solution containing bovine serum albumin to make a total volume of 200 μl. This solution was kept in ice for 10 minutes to form a heterodimer structure.

Thereafter, the solution was added to Microcon YM-10 and concentrated by ultra-concentration. 25% glycerol, 10 mM MgCl
_2. 500 mM Tris-HCl buffer (pH 7.5) containing 1 mM dithiothreitol (DTT) (hereinafter abbreviated as A buffer)
μl and repeat the ultraconcentration procedure three times to obtain A
200 μl of a thermostable primase enzyme solution completely replaced with a buffer solution was obtained. In this heat-resistant primase enzyme solution, the heat-resistant primase concentration was 2 μM.

(5). Evaluation of thermostable primase activity Using the thermostable primase enzyme solution prepared in (4),
The RNA priming reaction under high temperature conditions was performed according to the following procedure, and the activity of the thermostable primase was evaluated. First, in 20 μl of a thermostable primase enzyme solution, 12.5 mM Tris-HCl (pH 7.5), 6.25% glycerol, 0.5 μg of template single-stranded DNA (ssM13 DNA), 10 mM MgCl ₂ , 4 mM DTT, 250 mM
μM ATP, GTP, UTP, 20 μM CTP, 5 μCi [α- ³² P] CTP
Was added to prepare a reaction solution. In this reaction solution, 0.
5 μM thermostable primase is included.

Using a thermostable primase enzyme solution, a DNA priming reaction was carried out under high temperature conditions according to the following procedure. In 20 μl of heat-resistant primase enzyme solution, 12.5 mM Tris-HCl (pH 7.5), 6.25% glycerol, 0.5 μg of template single-stranded DNA (ssM13 DNA), 10 mM MgC
l _2, 4mM DTT, dCTP of 250μM, dGTP, dTTP, dAT of 20μM
P and 5 μCi of [α- ³² P] dATP were added to prepare a reaction solution. Note that this reaction solution contains 0.5 μM heat-resistant primase.

Next, these reaction solutions were heated at 80 ° C. for 30 minutes to progress the reaction, and the reaction was stopped by adding EDTA to a final concentration of 10 mM. The reaction product was subjected to ethanol precipitation to remove unreacted nucleotides, and the precipitate was dissolved in 10 μl of a formamide solution and analyzed by 15% PAGE containing 7 M urea. At this time, as a molecular weight marker, 10 mer, 26 mer,
50 mer synthetic DNA oligomers were used for each T4 polynucleotide
The 5 ′ end was labeled using kinase and [γ- ³² P] ATP before use. For comparison, a sample in which the enzyme in the reaction solution was only a small subunit and a sample in which the enzyme in the reaction solution was only a large subunit were prepared, and the activity was similarly evaluated for these samples. .

The electrophoresis pattern of 15% PAGE containing 7 M urea was subjected to autoradiography with a PhosphoImager (manufactured by Bio-Rad), and the length and amount of the synthesized reaction product were measured.
A and B show. FIG. 2A is an electrophoresis pattern showing an RNA priming synthesis reaction. In FIG. 2A, lane 1 is a molecular weight marker flowing a 10-mer synthetic DNA oligomer, lane 2 is a molecular weight marker flowing a 26-mer synthetic DNA oligomer, and lane 3 is a molecular weight marker flowing a 50-mer synthetic DNA oligomer. In addition, lane 4 is a sample using a heat-resistant primase forming a heterodimer, lane 5 is a sample using only a small subunit, and lane 6 is a sample using only a large subunit.

FIG. 2B is an electrophoresis pattern comparing the DNA primer synthesizing activity and the RNA primer synthesizing activity of the heterodimeric thermostable primase.
In FIG. 2B, lane 1 is a DNA priming synthesis reaction, lane 2 is an RNA priming synthesis reaction, and lane 3 is a reaction product when no template single-stranded DNA (ssM13 DNA) is added in the DNA priming reaction. Yes, lane 4 is the reaction product when no template single-stranded DNA (ssM13 DNA) was added in the RNA priming reaction,
Lane 5 is the reaction product when heat-resistant primase is not added in the DNA priming reaction, and lane 6 is the reaction product when heat-resistant primase is not added in the RNA priming reaction.

As can be seen from FIG. 2A, when the small subunit or the large subunit was used alone, the synthesized RNA oligomer could not be detected, and almost no RNA priming reaction occurred. On the other hand, when a compound having a heterodimer structure composed of a small subunit and a large subunit is used, an RNA oligomer having a length corresponding to the replication origin of the template DNA can be detected, and the RNA priming activity can be detected. Can be admitted.

As can be seen from FIG. 2B, the thermostable primase has an activity of synthesizing a DNA primer and an RNA primer. Further, as can be seen from FIG. 2B, the activity of the thermostable primase depends on the presence of the template DNA and the thermostable primase. Further, as shown in FIG.
The priming activity was found to be about 10 times higher than the RNA priming activity.

(6). Characteristic evaluation of heat-resistant primase ・ Optimum temperature
The evaluation was performed by quantifying the reaction product when the temperature in the NA priming reaction was changed from 50 ° C to 95 ° C. Specifically, the reaction product at each temperature is
Separation was performed by the same electrophoresis as described above, and the specific activity at a predetermined temperature was calculated by quantification using a PhosphoImager to determine the optimum temperature. The results are shown in FIG. From FIG. 3, it was found that the optimum temperature of the heat-resistant primase was 80 ° C.

Optimum pH The optimum pH of the thermostable primase was determined by the R
The evaluation was performed by quantifying the reaction product when the pH in the NA priming reaction was changed from 6.5 to 9.6. Specifically, a phosphate buffer having a final concentration of 20 mM (pH 6.
PH from 5 to 8.0) and Na ₂ B ₄ O ₇ -HCl buffer (pH 8.0 to 9.6), and the reaction products at each pH were separated by electrophoresis in the same manner as (5). By quantifying with a PhosphoImager, the specific activity at a predetermined pH was calculated, and the optimum pH was determined. FIG. 4 shows the results. This figure 4
From this, it was found that the optimum pH of the thermostable primase was 8.0.

[0073] in-optimal Mg ²⁺ concentration refractory primase the optimal Mg ²⁺ concentration, reaction products when changing up 0~30mM the MgSO ₄ concentration in the RNA primed reactions were performed in (5) Determination Was evaluated by: Specifically, the reaction product at each MgSO ₄ concentration was separated by electrophoresis as in (5),
By quantifying with PhosphoImager, the specified MgS
The specific activity at the O ₄ concentration was calculated to determine the optimal Mg ²⁺ concentration. FIG. 5 shows the results. From FIG. 5, it was found that the optimal Mg ²⁺ concentration of the thermostable primase was 10 mM.

Optimum salt concentration The optimum salt concentration of the thermostable primase was determined by the NaCl concentration or KC in the RNA priming reaction performed in (5).
Evaluation was performed by quantifying the reaction product when the 1 concentration was changed from 2.5 to 100 mM, respectively. Specifically, the reaction product at each salt concentration was separated by electrophoresis in the same manner as in (5), and quantified by PhosphoImager, thereby calculating the specific activity at a predetermined salt concentration to determine the optimum salt concentration. FIG. 6 shows the results. From FIG. 6, the optimum salt concentration of the thermostable primase is 2.5 mM in the case of NaCl.
It was found that both NaCl and KCl caused strong activity inhibition at 40 mM. Therefore,
It was found that the salt concentration was preferably 20 mM or less.

Heat Stability The heat stability of the heat-resistant primase prepared in (4) was measured by heating the enzyme solution of the heat-resistant primase prepared at (4) at 80 ° C. for 0 to 60 minutes. Perform an RNA priming reaction in the same manner as in (5) using
The reaction products were evaluated by quantification. Specifically, the reaction product at each heat treatment time was separated by electrophoresis in the same manner as in (5), and quantified by PhosphoImager to calculate the residual activity at the predetermined heat treatment time,
The thermal stability was evaluated. FIG. 7 shows the results. From FIG. 7, it was found that the half-life of the thermostable primase was 30 minutes at 80 ° C.

[0076]

As described in detail above, the present invention provides a novel thermostable primase. This thermostable primase can be used for a gene amplification reaction (PCR reaction) and the like because it can perform accurate nucleic acid priming under a high temperature environment. In addition, since this thermostable primase can be used for gene labeling and gene mutation, a new method for gene sequence analysis using the enzyme can be developed.

[0077]

[Sequence List] SEQUENCE LISTING <110> Secretary of Agency of Industrial Science and Technology <120> Heat-resistant Enzyme having Primerse Activity <130> 11900397 <150> JP 2000-165875 <151> 2000-06-02 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 346 <212> PRT <213> Pyrococcus horikoshii <400> 1 Met Leu Leu Arg Glu Val Thr Arg Glu Glu Arg Lys Asn Phe Tyr Thr 1 5 10 15 Asn Glu Trp Lys Val Lys Asp Ile Pro Asp Phe Ile Val Lys Thr Leu 20 25 30 Glu Leu Arg Glu Phe Gly Phe Asp His Ser Gly Glu Gly Pro Ser Asp 35 40 45 Arg Lys Asn Gln Tyr Thr Asp Ile Arg Asp Leu Glu Asp Tyr Ile Arg 50 55 60 Ala Thr Ala Pro Tyr Ala Val Tyr Ser Ser Val Ala Leu Tyr Glu Lys 65 70 75 80 Pro Gln Glu Met Glu Gly Trp Leu Gly Thr Glu Leu Val Phe Asp Ile 85 90 95 Asp Ala Lys Asp Leu Pro Leu Arg Arg Cys Glu His Glu Pro Gly Thr 100 105 110 Val Cys Pro Ile Cys Leu Asn Asp Ala Lys Glu Ile Val Arg Asp Thr 115 120 125 Val Ile Ile Leu Arg Glu Glu Leu Gly Phe Asn Asp Ile His Ile Ile 130 135 140 Tyr Ser Gly Arg Gly Tyr His Ile Arg Val Leu Asp Glu Trp Ala Leu 145 150 155 160 Lys Leu Asp Ser Lys Ser Arg Glu Arg Ile Leu Ser Phe Val Ser Ala 165 170 175 Ser Glu Ile Glu Asp Val Glu Glu Phe Arg Lys Leu Leu Leu Asn Lys 180 185 190 Arg Gly Trp Phe Val Leu Asn His Gly Tyr Pro Arg Ala Phe Arg Leu 195 200 205 Arg Phe Gly Tyr Phe Ile Leu Arg Ile Lys Leu Pro His Leu Ile Asn 210 215 220 Ala Gly Ile Arg Lys Ser Ile Ala Lys Ser Ile Leu Lys Ser Lys Glu 225 230 235 240 Glu Ile Tyr Glu Glu Phe Val Arg Lys Ala Ile Leu Ala Ala Phe Pro 245 250 255 Gln Gly Val Gly Ile Glu Ser Leu Ala Lys Leu Phe Ala Leu Ser Thr 260 265 270 Arg Phe Ser Lys Ser Tyr Phe Asp Gly Arg Val Thr Val Asp Leu Lys 275 280 285 Arg Ile Leu Arg Leu Pro Ser Thr Leu His Ser Lys Val Gly Leu Ile 290 295 300 Ala Lys Tyr Val Gly Thr Asn Glu Arg Asp Val Met Arg Phe Asn Pro 305 310 315 320 Phe Lys His Ala Val Pro Lys Phe Arg Lys Glu Glu Val Lys Val Glu 325 330 335 Tyr Lys Lys Phe Leu Glu Ser Leu Gly Thr 340 345 <210> 2 <211> 397 <212> PRT <213> Pyrococcus horikoshii <40 0> 2 Met Met Ile Met Leu Asp Pro Phe Ser Glu Lys Ala Lys Glu Leu Leu 1 5 10 15 Lys Gly Phe Gly Ser Ile Asn Asp Phe Met Asp Ala Ile Pro Lys Ile 20 25 30 Val Ser Val Asp Asp Val Ile Glu Arg Ile Arg Val Val Lys Asn Glu 35 40 45 Lys Leu Ile Asp Lys Phe Leu Asp Gln Asp Asn Val Met Asp Leu Ala 50 55 60 Gln Phe Tyr Ala Leu Leu Gly Ala Leu Ser Tyr Ser Pro Tyr Gly Ile 65 70 75 80 Glu Leu Glu Leu Val Lys Lys Ala Asn Leu Ile Ile Tyr Ser Glu Arg 85 90 95 Leu Lys Arg Lys Lys Glu Ile Lys Pro Glu Glu Ile Ser Ile Asp Val 100 105 110 Ser Thr Ala Ile Glu Phe Pro Thr Glu Asp Val Arg Lys Ile Glu Arg 115 120 125 Val Tyr Gly Lys Ile Pro Glu Tyr Thr Met Lys Ile Ser Asp Phe Leu 130 135 140 Asp Leu Val Pro Asp Glu Lys Leu Ala Asn Tyr Tyr Ile Tyr Glu Gly 145 150 155 160 Arg Val Tyr Leu Lys Arg Glu Asp Leu Ile Arg Ile Trp Ser Lys Ala 165 170 175 Phe Glu Arg Asn Val Glu Arg Gly Val Asn Met Leu Tyr Glu Ile Arg 180 185 190 Asp Glu Leu Pro Glu Phe Tyr Arg Lys Val Leu Gly Glu Ile Gln Ala 195 200 205 Phe Ala Glu Glu G lu Phe Gly Arg Lys Phe Gly Glu Ile Gln Gly Gly 210 215 220 Lys Leu Arg Pro Glu Phe Phe Pro Pro Cys Ile Lys Asn Ala Leu Lys 225 230 235 240 Gly Val Pro Gln Gly Ile Arg Asn Tyr Ala Ile Thr Val Leu Leu Thr 245 250 255 Ser Phe Leu Ser Tyr Ala Arg Ile Cys Pro Asn Pro Pro Arg Arg Asn 260 265 270 Val Arg Val Lys Asp Cys Ile Lys Asp Ile Arg Val Ile Thr Glu Glu 275 280 285 285 Ile Leu Pro Ile Ile Ile Glu Ala Ala Asn Arg Cys Ser Pro Pro Leu 290 295 300 Phe Glu Asp Gln Pro Asn Glu Ile Lys Asn Ile Trp Tyr His Leu Gly 305 310 315 320 Phe Gly Tyr Thr Ala Asn Pro Ser Leu Glu Asp Ser Gly Asn Ser Thr 325 330 335 Trp Tyr Phe Pro Pro Asn Cys Glu Lys Ile Arg Ala Asn Ala Pro Gln 340 345 350 Leu Cys Thr Pro Asp Lys His Cys Lys Tyr Ile Arg Asn Pro Leu Thr 355 360 365 Tyr Tyr Tyr Leu Arg Arg Leu Tyr Leu Glu Gly Arg Arg Asn Ala Pro Lys 370 375 380 Arg Gly Asn Lys Arg Gly Lys Lys Glu Leu Leu His Gln 385 390 395 <210> 3 <211> 1041 <212> DNA <213> Pyrococcus horikoshii <220> <221> CDS <222> (1) .. (1038) <400> 3 atg ctc c ta aga gag gta aca aga gag gaa aga aag aac ttt tac acc 48 Met Leu Leu Arg Glu Val Thr Arg Glu Glu Arg Lys Asn Phe Tyr Thr 1 5 10 15 aat gaa tgg aag gtt aag gac ata cca gat ttc att gtg aag aca tta 96 Asn Glu Trp Lys Val Lys Asp Ile Pro Asp Phe Ile Val Lys Thr Leu 20 25 30 gag tta agg gaa ttt gga ttc gat cat tcg gga gag ggg cct agt gac 144 Glu Leu Arg Glu Phe Gly Phe Asp His Ser Gly Glu Gly Pro Ser Asp 35 40 45 agg aaa aac caa tat act gac att agg gat ctc gaa gat tac ata agg 192 Arg Lys Asn Gln Tyr Thr Asp Ile Arg Asp Leu Glu Asp Tyr Ile Arg 50 55 60 gcc acg gcc ccc tat gcc gtt tat tcc agc gtt gca ctt tat gag aag 240 Ala Thr Ala Pro Tyr Ala Val Tyr Ser Ser Val Ala Leu Tyr Glu Lys 65 70 75 80 ccc cag gaa atg gag gga tgg tta ggg act gaa ctc gtc ttt gac ata 288 Pro Gln Glu Met Glu Gly Trp Leu Gly Thr Glu Leu Val Phe Asp Ile 85 90 95 gat gct aag gac tta ccg tta aga agg tgc gag cat gag cca gga aca 336 Asp Ala Lys Asp Leu Pro Leu Arg Arg Cys Glu His Glu Pro Gly Thr 100 105 110 gta tgt cca att t gc tta aac gac gct aag gag ata gtg agg gat acc 384 Val Cys Pro Ile Cys Leu Asn Asp Ala Lys Glu Ile Val Arg Asp Thr 115 120 125 gta ata ata cta agg gag gag tta gga ttc aat gac ata cat atc ata 432 Val Ile Ile Leu Arg Glu Glu Leu Gly Phe Asn Asp Ile His Ile Ile 130 135 140 tat tct gga agg ggt tac cat att aga gtt ttg gat gaa tgg gcc ctc 480 Tyr Ser Gly Arg Gly Tyr His Ile Arg Val Leu Asp Glu Trp Ala Leu 145 150 155 160 aag cta gat tcg aag tcc agg gaa aga att ttg tca ttt gtt tct gct 528 Lys Leu Asp Ser Lys Ser Arg Glu Arg Ile Leu Ser Phe Val Ser Ala 165 170 175 agt gaa atc gag gat gttc gaa gagtt aga aaa ctt ctc ctg aat aag 576 Ser Glu Ile Glu Asp Val Glu Glu Phe Arg Lys Leu Leu Leu Asn Lys 180 185 190 agg gga tgg ttc gtt cta aat cat gga tat cca agg gct ttt agg ctc 624 Arg Gly Trp Phe Val Asn His Gly Tyr Pro Arg Ala Phe Arg Leu 195 200 205 aga ttt ggc tat ttt ata ctg agg att aag ctt ccc cac ctc ata aat 672 Arg Phe Gly Tyr Phe Ile Leu Arg Ile Lys Leu Pro His Leu Ile Asn 210 215 220 gct g ga ata agg aag agc att gcc aag agc atc ctt aaa tca aaa gag 720 Ala Gly Ile Arg Lys Ser Ile Ala Lys Ser Ile Leu Lys Ser Lys Glu 225 230 235 240 gaa att tac gaa gaa ttc gta agg aaa gct att ctt gca ttt ccc 768 Glu Ile Tyr Glu Glu Phe Val Arg Lys Ala Ile Leu Ala Ala Phe Pro 245 250 255 cag gga gtt gga atc gaa agc cta gcg aag ttg ttc gca ctc tcc acc 816 Gln Gly Val Gly Ile Glu Ser Leu Ala Lys Phe Ala Leu Ser Thr 260 265 270 agg ttc tca aag tca tac ttc gat ggg agg gta acc gtt gat cta aag 864 Arg Phe Ser Lys Ser Tyr Phe Asp Gly Arg Val Thr Val Asp Leu Lys 275 280 285 agg att cta aga ctc cct tca acg ttg cac tct aag gta gga cta ata 912 Arg Ile Leu Arg Leu Pro Ser Thr Leu His Ser Lys Val Gly Leu Ile 290 295 300 gca aag tac gta gga act aac gag agg gat gtt atg aga ttt aac ccc 960 Ala Lys Tyr Val Gly Thr Asn Glu Arg Asp Val Met Arg Phe Asn Pro 305 310 315 320 ttc aaa cat gcg gta cca aag ttc aga aaa gag gag gta aag gtg gaa 1008 Phe Lys His Ala Val Pro Lys Phe Arg Lys Glu Glu Val Lys Val Glu325 330 335 tac aaa aaa ttc ctg gaa agt ctc gga act taa 1041 Tyr Lys Lys Phe Leu Glu Ser Leu Gly Thr 340 345 <210> 4 <211> 1194 <212> DNA <213> Pyrococcus horikoshii <220> <221> CDS <222> (1) .. (1191) <400> 4 gtg atg atc atg ctc gac cca ttt agt gaa aaa gct aaa gaa cta cta 48 Met Met Ile Met Leu Asp Pro Phe Ser Glu Lys Ala Lys Glu Leu Leu 1 5 10 15 aaa gga ttc ggg tca att aat gat ttt atg gac gca att cca aaa ata 96 Lys Gly Phe Gly Ser Ile Asn Asp Phe Met Asp Ala Ile Pro Lys Ile 20 25 30 gtt agc gtt gac gat gtg ata gaa agg att agg gta gta aaa aat gag 144 Val Ser Val Asp Asp Val Ile Glu Arg Ile Arg Val Val Lys Asn Glu 35 40 45 aaa ctg ata gac aag ttt tta gat caa gat aac gta atg gat cta gct 192 Lys Leu Ile Asp Lys Phe Leu Asp Gln Asp Asn Val Met Asp Leu Ala 50 55 60 caa ttc tat gcc cta ttg ggg gcc cta tcc tac tca ccc tat ggt ata 240 Gln Phe Tyr Ala Leu Leu Gly Ala Leu Ser Tyr Ser Pro Tyr Gly Ile 65 70 75 80 gaa ctc gaa ctt gtg aaa aag gcg aac tta att att tat tca gag aga 288 Glu Leu Glu Leu Val Lys Lys Ala Asn Leu Ile Ile Tyr Ser Glu Arg 85 90 95 cta aaa aga aaa aag gag atc aag ccg gag gag ata agc att gat gtg 336 Leu Lys Arg Lys Lys Glu Ile Lys Pro Glu Glu Ile Ser Ile Asp Val 100 105 110 agc acc gcc ata gaa ttt cca aca gag gat gta agg aaa att gaa aga 384 Ser Thr Ala Ile Glu Phe Pro Thr Glu Asp Val Arg Lys Ile Glu Arg 115 120 125 gtt tac ggt aaa att cca gag tac aca atg aag atc tct gac ttc cta 432 Val Tyr Gly Lys Ile Pro Glu Tyr Thr Met Lys Ile Ser Asp Phe Leu 130 135 140 gat cta gtt ccc gat gaa aag tta gcg aac tat tac ata tac gaa ggg 480 Asp Leu Val Pro Asp Glu Lys Leu Ala Asn Tyr Tyr Ile Tyr Glu Gly 145 150 155 160 cgt gtg tac ctc aag agg gaa gat cta ata agg ata tgg agc aaa gcc 528 Arg Val Tyr Leu Lys Arg Glu Asp Leu Ile Arg Ile Trp Ser Lys Ala 165 170 175 ttt gag agg gtg gaa agg ggc gta aac atg ctt tac gaa att aga 576 Phe Glu Arg Asn Val Glu Arg Gly Val Asn Met Leu Tyr Glu Ile Arg 180 185 190 gat gaa ctc cca gaa ttc tac agg aaa gtt ctg ggt gaac Glu Leu Pro Glu Phe Tyr Arg Lys Val Leu Gly Glu Ile Gln Ala 195 200 205 ttt gcg gag gaa gag ttt gga aga aaa ttc gga gag att caa gga gga 672 Phe Ala Glu Glu Glu Phe Gly Arg Lys Phe Gly Glu Ile Gln Gly 210 215 220 aaa cta agg ccg gag ttc ttt ccc cct tgt att aag aac gcc cta aag 720 Lys Leu Arg Pro Glu Phe Phe Pro Pro Cys Ile Lys Asn Ala Leu Lys 225 230 235 240 ggt gtt cct cag gga att agg aac tat gca att acc gta tta ttg acg 768 Gly Val Pro Gln Gly Ile Arg Asn Tyr Ala Ile Thr Val Leu Leu Thr 245 250 255 agc ttt ttg agc tat gca aga atc tgt cca aac cca cca agg agg aac 816 Ser Phe Leu Ser Tyr Ala Arg Ile Cys Pro Asn Pro Pro Arg Arg Asn 260 265 270 270 gtt agg gtt aaa gac tgt ata aag gac att aga gta ata act gag gaa 864 Val Arg Val Lys Asp Cys Ile Lys Asp Ile Arg Val Ile Thr Glu Glu 275 280 285 att tta ccg ata ata att ata gaa gcc gca aat aga tgc tca cct cca tta 912 Ile Leu Pro Ile Ile Ile Glu Ala Ala Asn Arg Cys Ser Pro Pro Leu 290 295 300 ttt gag gat caa ccc aat gag atc aag aat atc tgg tac cac ctg 960 Phe Glu Asp Gln Pro Asn Glu Ile Lys Asn Ile Trp Tyr His Leu Gly 305 310 315 320 ttt gga tat aca gca aat cct agc ctc gaa gac agc gga aat tcc acc 1008 Phe Gly Tyr Thr Ala Asn Pro Ser Leu Glu Asp Ser Gly Asn Ser Thr 325 330 335 tgg tac ttt ccc cca aac tgt gaa aag ata agg gct aac gcc cca caa 1056 Trp Tyr Phe Pro Pro Asn Cys Glu Lys Ile Arg Ala Asn Ala Pro Gln 340 345 350 ttg tgc act ccc gat aag cac tgt aaa tac atc aga aat cca ttg acg 1104 Leu Cys Thr Pro Asp Lys His Cys Lys Tyr Ile Arg Asn Pro Leu Thr 355 360 365 tat tat tat atta agg agg tta tac ctg gag gga aga agg aat gct cct aag 1152 Tyr Tyr Leu Arg Arg Leu Tyr Leu Glu Gly Arg Arg Asn Ala Pro Lys 370 375 380 aga ggt aac aag aga gga aag aaa gaa ctt tta cac caa tga 1194 Arg Gly Asn Lys Arg Gly Lys Lys Glu Leu Leu His Gln 385 390 395 <210> 5 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An upper primer designed to create the NdeI site.Primer <400> 5 ctttaagaag gagatataca tatgctgctg cgtgaggtaa cc cgtgagga aagaaagaac 60 ttttac 66 <210> 6 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A lower primer designed to create the BamHI site.Primer <400> 6 ttttgagctc tttggatcct taggccatct tttaagttcc gagactttc 49 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An upper primer designed to create the NdeI site.Primer <400> 7 ttttgtcgac ttacatatgg cgatcatgct cgaccca 37 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A lower primer designed to create the BamHI site.Primer <400> 8 ttttaagctt tttggatcct tattccattc attggtgtaa 40

[Brief description of the drawings]

FIG. 1 is an SDS-PAGE electrophoresis photograph performed to confirm the heterodimer structure of a thermostable primase.

FIG. 2 (A) is an electrophoresis photograph showing the result of an RNA priming reaction of a thermostable primase having a heterodimer structure. (B) is an electrophoretic photograph showing the results of a DNA priming reaction and an RNA priming reaction of a thermostable primase.

FIG. 3 is a characteristic diagram showing the relationship between temperature and specific activity in a thermostable primase.

FIG. 4 is a characteristic diagram showing the relationship between pH and specific activity of a thermostable primase.

FIG. 5 is a characteristic diagram showing the relationship between Mg ²⁺ and specific activity in a thermostable primase.

FIG. 6 is a characteristic diagram showing the relationship between salt concentration and specific activity in thermostable primase.

FIG. 7 is a characteristic diagram showing a relationship between a heating time and a residual activity in a thermostable primase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/12 C12R 1:19) // (C12N 1/21 C12N 15/00 ZNAA C12R 1:19) 5 / 00 A F term (reference) 4B024 AA20 BA10 CA04 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL05 4B065 AA01Y AA26X AB01 BA02 CA29 CA46

Claims (13)

[Claims] 1. A primase that binds to a predetermined site in a base sequence and synthesizes a nucleic acid primer complementary to the base sequence, wherein the primase is purified from heat-resistant archaea that can grow in an environment of 80 ° C. or higher. A primase having an optimum temperature of 80 ° C. or higher. 2. A protein of the following (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (B) a protein having an amino acid sequence in which at least one amino acid in the amino acid sequence represented by SEQ ID NO: 1 has been deleted, substituted or added, and has a subunit function of an enzyme having primase activity. 3. A protein of the following (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2; (B) a protein having an amino acid sequence in which at least one amino acid in the amino acid sequence represented by SEQ ID NO: 2 has been deleted, substituted or added, and having a subunit function of an enzyme having primase activity. 4. A composite protein comprising a heterodimer formed by the protein according to claim 2 and the protein according to claim 3, and having a primase activity. A gene encoding the primase according to claim 1. 6. A gene encoding the protein according to claim 2. 7. A gene encoding the protein according to claim 3. 8. A gene having the following DNA (a) or (b): (A) DNA comprising the base sequence represented by SEQ ID NO: 3. (B) a DNA consisting of a base sequence in which at least one base in the base sequence represented by SEQ ID NO: 3 has been deleted, substituted or added, and having a function of encoding a subunit of an enzyme having primase activity. 9. A gene having the following DNA (a) or (b): (A) DNA comprising the base sequence represented by SEQ ID NO: 4. (B) a DNA comprising a base sequence in which at least one base in the base sequence represented by SEQ ID NO: 4 has been deleted, substituted or added, and having a function of encoding a subunit of an enzyme having primase activity. 10. A recombinant vector containing the gene according to claim 6 or 8. 11. A recombinant vector containing the gene according to claim 7 or 9. 12. A transformant comprising the recombinant vector according to claim 10 and / or the recombinant vector according to claim 11. 13. A transformant according to claim 12, which is cultured, a small subunit and a large subunit are collected from the obtained culture, and a primer that forms a heterodimer from the small subunit and the large subunit. Method of producing ze.