JP3689736B2 - Thermostable enzyme with primase activity - Google Patents

Description

【図面の簡単な説明】
【図１】耐熱性プライマーゼのヘテロダイマー構造を確認するために行なったSDS-PAGEの電気泳動写真である。
【図２】 (A)ヘテロダイマー構造を有する耐熱性プライマーゼのＲＮＡプライミング反応の結果を示す電気泳動写真である。(B)耐熱性プライマーゼのＤＮＡプライミング反応及びＲＮＡプライミング反応の結果を示す電気泳動写真である。
【図３】耐熱性プライマーゼにおける温度と比活性との関係を示す特性図である。
【図４】耐熱性プライマーゼにおけるｐＨと比活性との関係を示す特性図である。
【図５】耐熱性プライマーゼにおけるＭｇ²⁺と比活性との関係を示す特性図である。
【図６】耐熱性プライマーゼにおける塩濃度と比活性との関係を示す特性図である。
【図７】耐熱性プライマーゼにおける加熱時間と残存活性との関係を示す特性図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a protein having primase activity under high temperature conditions, and a gene encoding the protein.
[0002]
[Prior art]
The primase is also called a DNA primase and 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 in the leading strand and the origin of synthesis of the Okazaki fragment in the lagging strand in the DNA replication mechanism. Thus, primase is an essential component in the DNA replication mechanism.
[0003]
Currently known primases include the dnaG gene product in E. coli (60 kDa), the T4 phage gene 4 product (Gp4, 63 kDa), the T4 phage gene 41 product (54 kDa) and the gene 61 product (40 kDa) and eukaryotic. There are two subunits (about 60 kDa and about 50 kDa) of the DNA polymerase α of the organism.
[0004]
However, since these primases are derived from room temperature organisms, they are extremely unstable under temperature conditions above room temperature, and in particular, they are quickly heat-inactivated under conditions of double-stranded DNA heat denaturation. For this reason, currently known primase cannot be used in, for example, a PCR reaction in which double-stranded DNA heat denaturation temperature, rapid cooling, and enzymatic reaction are repeated. If a primer with excellent heat resistance is discovered, a DNA amplification reaction and a primer synthesis reaction by PCR can be performed in the same tube. As a result, a novel gene labeling method and gene mutation method can be developed.
Accordingly, there is a need for a primase that exhibits excellent activity even under high temperature conditions. However, at present, no primase with excellent heat resistance is known.
[0005]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a thermostable enzyme having a primase activity that exhibits excellent activity even under high temperature conditions and can synthesize a nucleic acid primer.
[0006]
[Means for Solving the Problems]
In order to achieve the above object, the present inventor has focused on a hyperthermophilic archaea that grows at 90 to 100 ° C., and has found a gene presumed to encode a protein exhibiting primase activity from its gene sequence. Furthermore, the gene was incorporated into E. coli, and an enzyme was produced using this transformed E. coli, and it was confirmed that this enzyme is stably present at high temperature (over 80 ° C.) and exhibits primase activity. Thus, the present invention has been completed.
[0007]
  That is, the present invention is a primase that binds to a predetermined site of a base sequence and synthesizes a nucleic acid primer complementary to the base sequence, purified from heat-resistant archaea that can grow in an environment of 80 ° C. or higher. Primase characterized in that the optimum temperature is 80 ° C. or higherThe present invention is as shown in the following (1) to (6)..
[0008]
(1) a) a primase having the amino acid sequence represented by SEQ ID NO: 2 or 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 Consisting of a large subunit and having the amino acid sequence represented by SEQ ID NO: 1, or b) 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, A subunit for forming a complex protein with the small subunit of primase.
(2) A large subprime of primase having the amino acid sequence represented by SEQ ID NO: 2 or 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 A small sub-primase having a unit and an amino acid sequence represented by SEQ ID NO: 1 or 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 A complex protein having a primase activity, wherein a unit forms a heterodimer.
(3) A large subprime of primase having the amino acid sequence represented by SEQ ID NO: 2 or 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 Primer having a DNA encoding the unit and the amino acid sequence represented by SEQ ID NO: 1, or 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 In combination with DNA encoding the small subunit of
(4) The DNA encoding the large subunit of primase has the base sequence represented by SEQ ID NO: 4, or at least one base in the base sequence represented by SEQ ID NO: 4 has been deleted, substituted or added The combination according to (3) above, which is a DNA having a different base sequence.
(5) DNA encoding the large subunit of primase and DNA encoding the small subunit of primase according to (3) above Recombinant vector combinations each containing
(6) A combination of transformants each containing the recombinant vector according to (5) above.
SEQ ID NO: 1 is the amino acid sequence of the small subunit of primase collected from a hyperthermophilic archaea. Accordingly, the protein (b) in (1) has a function of becoming a primase component that synthesizes nucleic acid primers under high temperature conditions. SEQ ID NO: 2 is the amino acid sequence of the large subunit of primase collected from a hyperthermophilic archaea. Therefore, the protein a) becomes a primase component that synthesizes a nucleic acid primer under high temperature conditions, and forms a complex protein with the small subunit.
SEQ ID NO: 4 is a base sequence encoding a large subunit of primase collected from a hyperthermophilic archaea, and the DNA having the base sequence is a component of primase that synthesizes a nucleic acid primer under high temperature conditions It has a function of encoding a large subunit.
The specific base sequence of the DNA encoding the small subunit of the primase of (3) is shown in SEQ ID NO: 3, and the DNA having the base sequence was collected from a hyperthermophilic archaea and subjected to high temperature conditions. It has a function of encoding a small subunit that is a constituent element of a primase that synthesizes a nucleic acid primer.
In addition, the transformant of (6) above is cultured, small subunits and large subunits are collected from the resulting culture, and heterodimers are obtained from these small subunits and large subunits. By forming a primase, primase can be produced.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The primase of the present invention is purified from a heat-resistant archaea that can grow under cyclization at 80 ° C. or higher, and is characterized by an optimum temperature of 80 ° C. or higher. In other words, this primase binds to a predetermined site of the base sequence at a temperature of 80 ° C. or higher and has a primase activity for synthesizing a nucleic acid primer complementary to the base sequence.
[0019]
Here, as the thermostable archaea that can grow under cyclization at 80 ° C. or higher, hyperthermophilic bacteria can be used, preferably thermophilic archaea can be used, more preferably sulfur metabolizing thermophilic archaea. it can. Examples of thermophilic archaea include Pyrococcus horikosii, Pyrococcus furiosus and Pyrococcus woesei, Thermococcus litoralis (Thermococ litorris) Examples include Thermococcus genus such as Thermococcus celer, Thermococcus profundus and Thermococcus peptonopjilus, and genus Metanobacterium.
[0020]
In addition, here, the nucleic acid primer means both a DNA primer and an RNA primer. That is, the primer of the present invention can synthesize both DNA primers and RNA primers.
The primase of the present invention can be artificially obtained by using recombinant DNA technology as long as it has the above properties.
[0021]
In the following, a heat-resistant primase using Pyrococcus horikosii will be described as an example of the present invention, but the primase of the present invention is obtained from the heat-resistant archaea as described above by the same technique. It is possible.
[0022]
1. Isolation of thermostable primase gene
The thermostable primase gene is isolated from the chromosomal DNA of Pyrococcus horikoshii (registration number JCM9974), which is a sulfur-metabolizing thermophilic archaea.
That is, first, Pyrococcus horikoshi is cultured, and chromosomal DNA is extracted. In culturing Pyrococcus horikoshi, a commonly used medium can be used.
[0023]
Next, the cultured Pyrococcus horikoshi is collected and chromosomal DNA is prepared therefrom. In order to prepare chromosomal DNA, a commonly used technique can be applied. In order to prepare chromosomal DNA, a commercially available kit (for example, ISOPLANT (manufactured by Nippon Gene)) can also be used.
[0024]
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 chromosomes of Pyrococcus horikoshi, and this chromosomal DNA library is aligned.
[0025]
Next, the entire nucleotide sequence of the aligned chromosomal DNA library is determined. Specifically, in determining the base sequence, about 500 so-called shotgun clones were prepared for one chromosomal DNA library clone, and the base sequences of these shotgun clones were sequentially determined. The entire base sequence of the chromosomal DNA library can be determined by linking and editing the base sequence of the shotgun clone with commercially available software.
[0026]
Next, the homology between the determined base sequence and a known primase gene is analyzed by a large computer. As a result of analysis, a nucleotide sequence having high homology with a known primase gene was identified, and a small subunit gene (SEQ ID NO: 3) and a large subunit gene (SEQ ID NO: 4) encoding primase in Pyrococcus horicosi Can be identified.
[0027]
2. Construction of expression plasmid
In order to construct an expression plasmid, first, a small subunit gene and a large subunit are synthesized by PCR using primers designed from the base sequence of the chromosomal DNA, the base sequence of the small subunit gene, and the base sequence of the large subunit gene. Amplify subunit genes.
[0028]
The primer is preferably designed so that restriction enzyme sites for incorporation into the expression plasmid vector are respectively added to the 3 'end side and 5' end side of the structural gene to be amplified. More preferably, the primer is designed so that different restriction enzyme sites are added to the 3 'end and the 5' end, respectively. Thus, the amplified structural gene can be incorporated in the correct direction with respect to the expression plasmid vector.
[0029]
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-terminus of the expressed small subunit. Thereby, the obtained small subunit becomes a fusion protein with a histidine tag added to the N-terminus, and can be fractionated according to the presence or absence of the histidine tag.
[0030]
Specifically, as an upstream primer for amplifying a small subunit gene, for example, a NedI site was added.
5'-CTTTAAGAAGGAGATATACATATGCTGCTGCGTGAGGTAACCCGTGAGGAAAGAAAGAACTTTTAC-3 '
(SEQ ID NO: 5) can be used, and as a downstream primer, for example, a BamHI site was added.
5'-TTTTGAGCTCTTTGGATCCTTAGGCCATCTTTTAAGTTCCGAGACTTTC-3 '
(SEQ ID NO: 6) can be used.
[0031]
The upstream primer and the downstream primer for amplifying the small subunit gene are not limited to those having the above base sequence, and are designed from the base sequence of the chromosomal DNA library, and the entire small subunit gene Any base sequence may be used as long as it is designed to be sandwiched.
[0032]
Specifically, as an upstream primer for amplifying the large subunit gene, for example, a NedI site was added.
5'-TTTTGTCGACTTACATATGGCGATCATGCTCGACCCA-3 '
(SEQ ID NO: 7) can be used, and as a downstream primer, for example, a BamHI site was added.
5'-TTTTAAGCTTTTTGGATCCTTATTCCATTCATTGGTGTAA-3 '
(SEQ ID NO: 8) can be used.
[0033]
The upstream primer and the downstream primer for amplifying the large subunit gene are not limited to those having the above-mentioned base sequence, and are designed from the base sequence of the chromosomal DNA library, and the entire large subunit gene Any base sequence may be used as long as it is designed to be sandwiched.
[0034]
Next, by incorporating the small subunit gene and the large subunit gene amplified by these primers into a predetermined expression plasmid vector, respectively, an expression plasmid and a large subunit for expressing the small subunit can be expressed. An expression vector for a large subunit capable of expressing the unit can be constructed.
[0035]
Here, as a plasmid vector for expression, a commonly used plasmid vector can be used. Examples of expression plasmid vectors that can be used include pET11a and pET15b.
[0036]
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 plasmid vector for expression are ligated with a linking enzyme such as T4 ligase.
[0037]
Thereafter, a part of the reaction solution is introduced into a competent cell to obtain transformant colonies. Here, usable competent cells may be those that are usually used for transformation, and examples thereof include Escherichia coli and Bacillus subtilis.
Then, a colony of the transformant is purely cultured, and an expression vector for a small subunit and an expression vector for a large subunit are obtained by a technique such as a so-called alkaline method.
[0038]
3. Purification of heat-resistant primase
In order 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 the large subunit (SEQ ID NO: 1) are expressed. 2) is obtained respectively. Thereafter, a heat-stable primase can be obtained by forming a heterodimer from the obtained small subunit and large subunit.
[0039]
Specifically, when isolating and purifying small subunits or large subunits, a suspension is prepared by thawing the expressed bacterial cells. Using methods such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, the desired small subunit and large subunit are isolated and purified from the suspension. be able to.
[0040]
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 said heat-resistant primase can be utilized for the uses as shown below.
・ PCR method
The template DNA, the primase of the present invention, the dNTP mix, the heat-resistant DNA polymerase, etc. are mixed in the same tube, and the template DNA thermal denaturation step, nucleic acid primer synthesis step, and DNA strand extension step are repeated multiple times. Thus, 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, the PCR reaction can be performed rapidly and at a low cost.
[0041]
・ Gene labeling method
When the primase of the present invention and a known thermostable DNA polymerase are coupled in the presence of template DNA and labeled nucleotides to cause a DNA extension reaction, the 5 ′ end is composed of an RNA molecule or a DNA molecule. Strand DNA can be obtained. The obtained single-stranded DNA is replicated from a random position in the template DNA and is labeled. That is, a novel gene labeling method can be established by using the primase of the present invention.
[0042]
・ Gene mutation method
The single-stranded DNA synthesized by the gene labeling method is reannealed with the template DNA plasmid, and the gap between the reannealed single-stranded DNAs is filled with DNA polymerase and then ligated with ligase. The mutation is then introduced at the labeled nucleotide position by transforming the host and acting a repair system in the host cell. Thus, a novel gene mutation method can be established by using the primase of the present invention.
[0043]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, the technical scope of this invention is not limited to this Example.
[Example 1]
(1) Cultivation of Pyrococcus horikoshi (JCM9974)
Pyrococcus horikoshi JCM9974 (obtained from the microbial strain storage facility of RIKEN) was cultured by the following method. 13.5g salt, 4g Na₂SO_Four, 0.7 g KCl, 0.2 g NaHCO_Three, 0.1 g KBr, 30 mg H_ThreeBO_Three, 10g MgCl₂・ 6H₂O, 1.5g CaCl₂25 mg SrCl₂1.0 ml of resasulin solution (0.2 g / L), 1.0 g of yeast extract and 5 g of bactopeptone were dissolved in 1 L, and the pH of this solution was adjusted to 6.8 and pasteurized under pressure. Next, elemental sulfur sterilized by dry heat was added to 0.2%, and the medium was saturated with argon to make anaerobic, and then JCM9974 was inoculated. Whether the medium is anaerobic or not is Na₂Add S solution and add Na₂This was confirmed by the fact that the pink color of the resazurin solution by S was not colored. This culture solution was cultured at 95 ° C. for 2 to 4 days, and then centrifuged to collect bacteria.
[0044]
(2). Chromosomal DNA preparation
  The chromosomal DNA of JCM9974 was prepared by the following method. After culturing, the cells are collected by centrifugation at 5000 rpm for 10 minutes. The cells are washed twice with 10 mM Tris (pH 7.5) 1 mM EDTA solution and then enclosed in an InCert Agarose (FMC) block. Chromosomal DNA was separated and prepared in an agarose block by treating this block in 1% N-lauroylsarcosine, 1 mg / ml protease K solution.
[0045]
(3). Creation of library clones containing chromosomal DNA
The chromosomal DNA obtained in (2) was partially decomposed with the restriction enzyme HindIII, and then a fragment of about 40 kb was prepared by agarose gel electrophoresis. This DNA fragment was ligated with Bac vectors pBAC108L and pFOS1 that had been completely degraded by the restriction enzyme HindIII using T4 ligase. When the former vector was used, the DNA after completion of binding was immediately introduced into E. coli by electroporation. When the latter vector pFOS1 is used, the DNA after binding is packed into λ phage particles in a test tube using GIGA Pack Gold (manufactured by Stratagene). Introduced. The E. coli population resistant to the antibiotic chloramphenicol obtained by these methods was used as BAC and Fosmid libraries. A clone suitable for covering the chromosome of JCM9974 was selected from the library, and the clones were aligned.
[0046]
(4). Determination of nucleotide sequence
The nucleotide sequence of the aligned BAC or Fosmid clone was sequentially determined by the following method. Each BAC or Fosmid clone DNA recovered from E. coli was fragmented by sonication, and 1 kb and 2 kb long DNA fragments were recovered by agarose gel electrophoresis. 500 shotgun clones were prepared for each BAC or Fosmid clone in which this fragment was inserted into the HincII restriction enzyme site of plasmid vector pUC118. The base sequence of each shotgun clone was determined using an automatic base sequence reader 373 or 377 manufactured by PerkinElmer, ABI. The base sequence obtained from each shotgun clone was ligated and edited using the base sequence automatic linking software Sequencher, and the entire base sequence of each BAC or Fosmid clone was determined.
[0047]
(5). Identification of thermostable primase gene
As a result of analyzing the homology between the base sequence determined in (4) and a known primase gene using a large computer, a small subunit gene (SEQ ID NO: 3) and a large subunit gene encoding primase in JCM9974 (SEQ ID NO: 4) was identified.
[0048]
[Example 2]
(1). Construction of expression plasmid expressing small subunit
In order to introduce restriction enzyme sites before and after the small subunit gene region and to add a histidine tag to the N-terminus of the expressed small subunit, the upstream primer Upri-S (5'-CTTTAAGAAGGAGATATACATATGCTGCTGCGTGAGGTAACCCGTGAGGAAAGAAAGAACTTTTAC-3 ': SEQ ID NO: 5 ) And downstream primer Lpri-S (5′-TTTTGAGCTCTTTGGATCCTTAGGCCATCTTTTAAGTTCCGAGACTTTC-3 ′: SEQ ID NO: 6) were designed and synthesized. In Upri-S, NdeI sites are introduced from the 19th base to the 24th base from the 5 'end. In Lpri-S, a BamHI site is introduced from the 14th base to the 19th base from the 5 'end.
[0049]
After PCR reaction using these Upri-S and Lpri-S, complete structural degradation (37 hours at 37 ° C.) with restriction enzymes (NdeI and BamHI) was followed by purification of the structural gene. pET15b (manufactured by Novagen) was cleaved and purified with restriction enzymes NdeI and BamHI, and then reacted with the above structural gene and T4 ligase at 16 ° C. for 2 hours for ligation. A part of the ligated DNA was introduced into competent cells of E. coli XL1-Blue MRF ′ to obtain transformant colonies. An expression plasmid (hereinafter referred to as “pET15b / PrimS”) capable of expressing a small subunit from the obtained colony was purified by an alkaline method.
[0050]
(2). Construction of expression plasmid expressing large subunit
In order to introduce restriction enzyme sites before and after the large subunit gene region, upstream primer Upri-L (5'-TTTTGTCGACTTACATATGGCGATCATGCTCGACCCA-3 ': SEQ ID NO: 7) and downstream primer Lpri-L (5'-TTTTAAGCTTTTTGGATCCTTATTCCATTCATTGGTGTAA-3': SEQ ID NO: 8) was designed and synthesized. In Upri-L, NdeI sites are introduced from the 14th base to the 29th base from the 5 'end. In Lpri-L, a BamHI site is introduced from the 14th base to the 19th base from the 5 'end.
[0051]
After PCR reaction using these Upri-L and Lpri-L, complete digestion with restriction enzymes (NdeI and BamHI) (2 hours at 37 ° C.) was followed by purification of the structural gene. pET11a (Novagen) was cleaved and purified with restriction enzymes NdeI and BamHI, and then reacted with the above structural gene and T4 ligase at 16 ° C. for 2 hours for ligation. A part of the ligated DNA was introduced into competent cells of E. coli XL1-Blue MRF ′ to obtain transformant colonies. An expression plasmid (hereinafter referred to as “pET11a / PrimL”) capable of expressing a large subunit from the obtained colony was purified by an alkaline method.
[0052]
(3). Gene expression
Thaw E. coli BL21 (DE3), Novagen and E. coli BL21 (DE3) Codon Plus-RIL, Novagen competent cells and transfer 0.1 mL each to the Falcon tube. . Add 0.005 mL of pET15b / PrimS solution to the former Falcon tube, add 0.005 mL of pET11a / PrimL solution to the latter Falcon tube, and then leave these two Falcon tubes in ice for 30 minutes, then heat shock at 42 degrees For 30 seconds, add 0.9 mL of SOC medium, and incubate with shaking at 37 degrees for 1 hour. Thereafter, the culture solution in these two Falcon tubes is separately spread on each of two 2YT agar plates containing ampicillin and cultured overnight at 37 degrees to obtain two types of transformants from the two plates. It was.
[0053]
The transformant using pET15b / PrimS was named E. coli BL21 (DE3) / pET15b / PrimS, and the transformant using pET11a / PrimL was named E. coli BL21 (DE3) Codon Plus-RIL / pET11a Named / PrimL. E. coli BL21 (DE3) / pET15b / PrimS is deposited as FERM P-17883 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. Furthermore, E. coli BL21 (DE3) Codon Plus-RIL / pET11a / PrimL is deposited as FERM P-17884 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
[0054]
In order to express a small subunit gene and a large subunit gene, these transformants were separately cultured in a 2YT liquid medium (2 liters) containing ampicillin until absorption at 600 nm reached 1, respectively, and then IPTG ( Isopropyl-β-D-thiogalactopyranoside) was added and further cultured for 6 hours. After incubation, the cells were collected by centrifugation (6,000 rpm, 20 min).
[0055]
[Example 3]
(1). Purification of small subunits
The collected cells (E. coli BL21 (DE3) pET15b / PrimS) are frozen and thawed at -20 ° C and suspended in 2 volumes of 50 mM Tris-HCl buffer (pH 7.5) and 0.5 mg of DNase. A liquid was obtained. The obtained suspension was kept at 37 ° C. for 30 minutes, then heat-treated at 85 ° C. for 30 minutes, and then centrifuged (11,000 rpm, 20 minutes) to obtain a supernatant. This was applied to a Ni-column (Novagen, His / Bind metal chelation resin & His / Bind buffer kit), and affinity chromatography was performed. 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 from the gene sequence, fractions containing the protein 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 base sequence determined in Example 1 (4) and has a molecular weight of 40,358 Da.
[0056]
(2). Purification of large subunits
The collected cells (E.coli BL21 (DE3) Codon Plus-RIL / pET11a / PrimL) are frozen and thawed at -20 ° C, and 2 volumes of 50 mM Tris-HCl buffer (pH 7.5) are added to the suspension. Got. The obtained suspension was heat-treated at 85 ° C. for 10 minutes, and a supernatant was obtained by centrifugation (11,000 rpm, 20 minutes). 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 phosphate 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 large subunit was predicted to be 46,000 Da from the gene sequence, fractions containing this molecular weight protein were collected and diluted with three times the amount of distilled water. This was further adsorbed on a HiTrap heparin (Pharmacia) column equilibrated with 50 mM Tris-HCl buffer (pH 8.0), and eluted with a NaCl concentration gradient using the same buffer to obtain a purified large subunit.
This large subunit has the amino acid sequence shown in SEQ ID NO: 2 from the base sequence determined in Example 1 (4) and has a molecular weight of 46,271 Da.
[0057]
(3). Confirmation of heterodimer structure
It was confirmed as follows that the heat-resistant primase formed a heterodimer structure composed of a small subunit and a large subunit.
First, 2 μg of a small subunit added with a histidine tag and 3 μg of a large subunit were added to a final concentration of 20 mM Tris-HCl buffer (pH 7.9) to make a total volume of 180 μl. This includes 0.5 M NaCl, 5 mM imidazole, 1% Triton X-100, 0.04% bovine serum albumin. The solution was then kept in ice for 30 minutes. As a result, a complex of a small subunit and a large subunit was formed.
[0058]
Using this solution, the histidine tag added to the small subunit is adsorbed to His / Bindresin (Novagen) containing nickel, 16 μl, and centrifuged and suspended repeatedly, and then binding buffer (Novagen, His • Bind Washed 3 times with buffer kit).
[0059]
This centrifugal sediment was suspended in 50 μl of SDS sample buffer, heated at 100 ° C. for 5 minutes, and a supernatant was obtained by centrifugation. In order to confirm the types of subunits contained in the supernatant, the obtained supernatant was analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE).
[0060]
The results of SDS-PAGE are shown in FIG. In FIG. 1, lane 1 is a molecular weight marker, lane 2 is a sample obtained, lane 3 is a sample prepared in the same manner as above except that a small subunit is not added, lane 4 is a control, Sample sample with boiled SDS sample buffer added to small subunit with histidine tag, lane 5 is a control, sample boiled with SDS sample buffer added to large subunit, lane 6 is control The protein is a sample obtained by adding SDS sample buffer to bovine serum albumin and boiling.
[0061]
As can be seen from FIG. 1, bands of small subunits and large subunits can be detected in lane 2. In lane 3, a large subunit band cannot be detected. From this, it was found that the heat-resistant primase has a heterodimer structure composed of a small subunit and a large subunit.
[0062]
(4). Adjustment of heat-resistant primase
A thermostable primase was prepared according to the following procedure. First, 16 μg of a small subunit to which a histidine tag was added and 24 μg of a large subunit were added to a solution containing 1% Triton X-100 and 0.01% bovine serum albumin to make the total volume of the solution 200 μl. This solution was kept in ice for 10 minutes to form a heterodimer structure.
[0063]
Thereafter, the solution was added to Microcon YM-10 and subjected to ultraconcentration. 25% glycerol, 10 mM MgCl₂Add 500 μl of 50 mM Tris-HCl buffer (pH 7.5) (hereinafter abbreviated as A buffer) containing 1 mM dithiothreitol (DTT), and repeat the ultraconcentration operation three times to A completely substituted 200 μl heat-resistant primase enzyme solution was obtained. In this thermostable primase enzyme solution, the thermostable primase concentration was 2 μM.
[0064]
(5). Activity evaluation of heat-resistant primase
Using the heat-resistant primase enzyme solution prepared in (4), an RNA priming reaction under high temperature conditions was performed according to the following procedure, and the activity of the heat-resistant primase was evaluated.
First, in 20 μl heat-resistant primase enzyme solution, 12.5 mM Tris-HCl (pH 7.5), 6.25% glycerol, 0.5 μg template single-stranded DNA (ssM13 DNA), 10 mM MgCl₂, 4 mM DTT, 250 μM ATP, GTP, UTP, 20 μM CTP, 5 μCi [α-³²P] CTP was added to prepare a reaction solution. This reaction solution contains 0.5 μM heat-resistant primase.
[0065]
In addition, a DNA priming reaction under high temperature conditions was performed using the thermostable primase enzyme solution according to the following procedure. In 20 μl of thermostable primase enzyme solution, 12.5 mM Tris-HCl (pH 7.5), 6.25% glycerol, 0.5 μg template single-stranded DNA (ssM13 DNA), 10 mM MgCl₂, 4 mM DTT, 250 μM dCTP, dGTP, dTTP, 20 μM dATP, 5 μCi [α-³²P] dATP was added to prepare a reaction solution. This reaction solution contains 0.5 μM heat-resistant primase.
[0066]
Next, these reaction solutions were heated at 80 ° C. for 30 minutes to advance the reaction, and the reaction was stopped by adding EDTA to a final concentration of 10 mM.
Unreacted nucleotides were removed from the reaction product by ethanol precipitation, and the precipitate was dissolved in 10 μl of formamide solution and analyzed by 15% PAGE containing 7M urea. At this time, 10-mer, 26-mer, and 50-mer synthetic DNA oligomers were used as molecular weight markers, T4 polynucleotide kinase and [γ-³²P] ATP was used to label the 5 ′ end. In addition, 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. .
[0067]
FIGS. 2A and 2B show the results of autoradiography of a 15% PAGE electrophoresis pattern containing 7M urea by PhosphoImager (manufactured by Bio-Rad), and measuring the length and amount of the synthetic reaction product.
FIG. 2A is an electrophoresis pattern showing the RNA priming synthesis reaction product. In FIG. 2A, lane 1 is a molecular weight marker in which a 10-mer synthetic DNA oligomer is flown, lane 2 is a molecular weight marker in which a 26-mer synthetic DNA oligomer is flowed, and lane 3 is a molecular weight marker in which a 50-mer synthetic DNA oligomer is flowed. Yes, lane 4 is a sample using a heat-resistant primase in which a heterodimer is formed, lane 5 is a sample using only a small subunit, and lane 6 is a sample using only a large subunit.
[0068]
FIG. 2B is an electrophoresis pattern comparing the DNA primer synthesis activity and the RNA primer synthesis activity of the heterodimer heat-resistant primase. In FIG. 2B, lane 1 is a DNA priming synthesis reaction product, lane 2 is an RNA priming synthesis reaction product, 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 a reaction product when no template single-stranded DNA (ssM13 DNA) is added in the RNA priming reaction, and lane 5 is a reaction product when no heat-resistant primase is added in the DNA priming reaction. 6 is a reaction product when no heat-resistant primase is added in the RNA priming reaction.
[0069]
As can be seen from FIG. 2A, when the small subunit or the large subunit is used alone, the synthesized RNA oligomer cannot be detected, and the RNA priming reaction hardly occurs. In contrast, when using a heterodimer structure consisting of a small subunit and a large subunit, an RNA oligomer having a length corresponding to the replication origin of the template DNA can be detected, and RNA priming activity can be detected. Can be recognized.
[0070]
Further, as can be seen from FIG. 2B, the heat-resistant primase has an activity of synthesizing a DNA primer and an RNA primer. As can be seen from FIG. 2B, the activity of the heat-resistant primase depends on the presence of the template DNA and the heat-resistant primase. Furthermore, as shown in FIG. 2B, it was found that the DNA priming activity of the thermostable primase was about 10 times higher than the RNA priming activity.
[0071]
(6). Characterization of heat-resistant primase
・ Optimum temperature
The optimum temperature of the heat-resistant primase was evaluated by quantifying the reaction product when the temperature in the RNA priming reaction performed in (5) was changed from 50 ° C to 95 ° C. Specifically, the reaction products at each temperature were separated by electrophoresis similar to (5), and the specific activity at a predetermined temperature was calculated by quantifying with a PhosphoImager, and the optimum temperature was determined. The results are shown in FIG. From FIG. 3, it was found that the optimum temperature for the heat-resistant primase was 80 ° C.
[0072]
・ Optimum pH
The optimum pH of the heat-resistant primase was evaluated by quantifying the reaction product when the pH in the RNA priming reaction performed in (5) was changed from 6.5 to 9.6. Specifically, a phosphate buffer (pH 6.5 to 8.0) with a final concentration of 20 mM and Na₂B_FourO₇By changing the pH using an HCl buffer (pH 8.0 to 9.6), separating the reaction products at each pH by electrophoresis similar to (5), and quantifying with PhosphoImager, the ratio at a given pH The activity was calculated and the optimum pH was determined. The results are shown in FIG. From FIG. 4, it was found that the optimum pH of the heat-resistant primase was 8.0.
[0073]
・ Optimum Mg²⁺concentration
Optimal Mg for heat-resistant primase²⁺Concentration was adjusted to MgSO in the RNA priming reaction performed in (5)._FourThe reaction product was evaluated by quantifying the concentration when the concentration was changed from 0 to 30 mM. Specifically, each MgSO_FourThe reaction product at a concentration is separated by electrophoresis similar to (5), and quantified with a PhosphoImager to obtain a predetermined MgSO._FourCalculate the specific activity at the concentration and optimize Mg²⁺The concentration was determined. The results are shown in FIG. From FIG. 5, the optimum Mg for heat-resistant primase²⁺The concentration was found to be 10 mM.
[0074]
・ Optimum salt concentration
The optimum salt concentration of the thermostable primase was evaluated by quantifying the reaction product when the NaCl concentration or KCl concentration in the RNA priming reaction performed in (5) was changed from 2.5 to 100 mM, respectively. Specifically, the reaction products at each salt concentration were separated by electrophoresis similar to (5), and quantified with PhosphoImager to calculate the specific activity at a predetermined salt concentration, and to determine the optimum salt concentration. The results are shown in FIG. From FIG. 6, it was found that the optimum salt concentration of the heat-resistant primase was 2.5 mM in the case of NaCl, and both NaCl and KCl caused strong activity inhibition at 40 mM. Therefore, it was found that the salt concentration is preferably 20 mM or less.
[0075]
・ Thermal stability
The heat stability of the heat-resistant primase was determined by heating the heat-resistant primase enzyme solution prepared in (4) at 80 ° C. for 0 to 60 minutes, and using heat-resistant primases with different heat treatment times (5) The RNA priming reaction was performed in the same manner as described above, and the reaction products were evaluated by quantification. Specifically, the reaction products in each heat treatment time were separated by electrophoresis similar to (5), and quantified with PhosphoImager to calculate the residual activity in a predetermined heat treatment time, and the thermal stability was evaluated. . The results are shown in FIG. From FIG. 7, it was found that the half-life of the heat-resistant primase was 30 minutes at 80 ° C.
[0076]
【The invention's effect】
As described above in detail, the present invention provides a novel heat-resistant primase. This thermostable primase is useful for gene amplification reaction (PCR reaction) and the like because it can perform accurate nucleic acid priming in a high temperature environment. In addition, since this heat-resistant primase can be used for gene labeling and gene mutation methods, it becomes possible to develop a new method for gene sequence analysis using the enzyme.
[0077]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an electrophoresis photograph of SDS-PAGE performed to confirm the heterodimer structure of heat-resistant primase.
FIG. 2 (A) is an electrophoresis photograph showing the results of RNA priming reaction of a thermostable primase having a heterodimer structure. (B) Electrophoresis photograph showing results of DNA priming reaction and RNA priming reaction of thermostable primase.
FIG. 3 is a characteristic diagram showing the relationship between temperature and specific activity in heat-resistant primase.
FIG. 4 is a characteristic diagram showing the relationship between pH and specific activity in heat-resistant primase.
FIG. 5: Mg in heat-resistant primase²⁺It is a characteristic view which shows the relationship between and specific activity.
FIG. 6 is a characteristic diagram showing the relationship between salt concentration and specific activity in heat-resistant primase.
FIG. 7 is a characteristic diagram showing the relationship between heating time and residual activity in heat-resistant primase.

Claims (6)

It consists of a large primase subunit having the amino acid sequence represented by SEQ ID NO: 2 or 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. A small subunit of primase having the amino acid sequence represented by SEQ ID NO: 1 or 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; Subunit for forming complex proteins. A large primase subunit having the amino acid sequence represented by SEQ ID NO: 2 or 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; A small subunit of primase having the amino acid sequence represented by SEQ ID NO: 1 or 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, A complex protein that forms a heterodimer and has primase activity. It encodes the large subunit of primase having the amino acid sequence represented by SEQ ID NO: 2 or 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 A primase having the amino acid sequence represented by SEQ ID NO: 1 or 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 Combination with DNA encoding subunit. The DNA encoding the large subunit of primase has the base sequence represented by SEQ ID NO: 4, or the base sequence in which at least one base in the base sequence represented by SEQ ID NO: 4 has been deleted, substituted or added The combination according to claim 3, which is DNA having 4. A combination of recombinant vectors each comprising DNA encoding the large subunit of primase and DNA encoding the small subunit of primase. A combination of transformants each comprising the recombinant vector according to claim 5.

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