JP2000308494A - Hyperthermostable dna ligase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the stable subject new enzyme of a hyperthermostable DNA ligase containing a specific amino acid sequence, useful in a recombinant DNA technique or the like, and suitably usable for a ligase chain reaction(LCR) or the like. SOLUTION: This hyperthermostable DNA ligase is the one containing an amino acid sequence of the 1- to 562-position of the formula, or the one of a modified body having an amino acid sequence having deletion, substitution or insertion of one to several amino acids in the amino acid sequence of the 1- to 562-position of the formula, and having a DNA ligase activities. The DNA ligase is an enzyme having activities for linking DNA chains, usable for a recombinant DNA technique or the like, and further usable for a ligase chain reaction or the like useful for the detection or the like of a target base sequence in a sample. The enzyme is obtained by separating chromosomal DNA from hyperthermostable archaebacteria KOD-1 strain, making a chromosomal library by using the chromosomal DNA, screening the library by using the partial sequence as a probe, incorporating the obtained gene into a vector, and expressing the gene in a host.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a hyperthermostable DNA ligase and a DNA encoding the same.

[0002]

2. Description of the Related Art DNA ligase is an enzyme having an activity of linking DNA strands, and is widely used in recombinant DNA technology (see "Molecular Cloning: A Laboratory Manual").
Second Edition ", edited by Sambrook, Fritsch and Maniatis, Cold Sp.
ring Harbor Laboratory Press, 1989)).

[0003] Furthermore, DNA ligase is used in a specific gene amplification method called "ligase chain reaction (LCR)" which is a method for detecting the presence of a nucleotide sequence of interest in a sample (Landegren, U.S.A.). . Et al., (1988)
Science, 241: 1077-1080; Barany, F. (1991) PCR Meth
ods and Applications 1: 5-16; Marsh, E. et al., (1992) St.
rategies 5: 73-76). Since this reaction is performed using cycling that includes a denaturation step at elevated temperatures, it is desirable to use DNA ligase that has high thermostability. Pyrococcu as a thermostable DNA ligase
s furiosus-derived Pfu DNA ligase and the like have been isolated (Takahashi, M., Yamaguchi, E., and Uchida, T.,
(1984) The Journal of Biological Chemistry, 259 (1
6): 10041-10047).

[0004] For such uses, a novel DNA ligase that is more structurally stable than conventionally known DNA ligases derived from normal-temperature organisms or thermophilic bacteria has been desired.

[0005] Because hyperthermophilic archaeons survive at high temperatures, the proteins (eg, enzymes) they produce are generally highly thermostable (ie, structurally stable). In addition, the evolution of these organisms is evident from the fact that it has been proposed that the archaeon to which hyperthermophilic archaea belong is different from the conventionally known prokaryotes and eukaryotes. And different. Therefore, even though they have similar functions to known enzymes derived from prokaryotes and eukaryotes, enzymes derived from hyperthermophilic archaea are structurally and enzymatically different from conventional enzymes. Is often different. For example, the hyperthermophilic archaeon KOD-1 strain (Morikawa, M. et al., Appl. E
nviron. Microbiol. 60 (12), 4559-4566 (1994)) was isolated from Escherichia coli GroE.
It has the same function as L. However, GroEL itself forms a 14-mer, and GroES forms a further 7-mer.
In contrast, chaperonins from the KOD-1 strain function alone (Yan, Z et al., App.
l. Environ. Microbiol. 63 : 785-789).

[0006]

SUMMARY OF THE INVENTION The present invention relates to a super heat-resistant DN
Providing A ligase and DNA encoding it;
It is an object of the present invention to provide a vector containing the DNA and a host cell containing the vector, and to provide a method for producing a hyperthermostable DNA ligase including a step of culturing the host cell.

[0007]

According to the present invention, a hyperthermostable DNA ligase comprising the amino acid sequence from position 1 or 4 to 562 of SEQ ID NO: 2, or from position 1 or 4 to 562 of SEQ ID NO: 2 The present invention provides a variant containing an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence at position 1, and having DNA ligase activity.

In one embodiment, the super heat resistant DN
A ligase or a variant thereof is derived from the hyperthermophilic archaeon KOD-1 strain.

According to the present invention, there is provided a DNA encoding the above-mentioned hyperthermostable DNA ligase or a variant thereof.

[0010] In one embodiment, the DNA comprises the nucleotide sequence of positions 1 or 10 to 1686 of SEQ ID NO: 1.

[0011] In one embodiment, the DNA is derived from the hyperthermophilic archaeon strain KOD-1.

According to the present invention, there is provided a vector containing the above DNA.

According to the present invention, there is provided a recombinant host cell containing the above-mentioned vector.

According to the present invention, there is provided a method for producing a hyperthermostable DNA ligase or a variant thereof, comprising a step of culturing the host cell.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method of producing a hyperthermostable DNA ligase produced by a hyperthermophilic archaeon, by culturing a hyperthermophilic archaeon that originally produces the enzyme, or a DNA encoding the enzyme. And constructing a vector containing the DNA, producing a host cell containing the vector, and culturing the host cell to produce a hyperthermostable DNA ligase.

[0016] DNA ligase is an enzyme having an activity of linking a 3'-OH group and a 5'-phosphate group of a DNA chain by a phosphodiester bond. DNA ligase is used in recombinant DNA technology.
Used to join chains ("Molecular Cloning:
A Laboratory Manual, 2nd ed., Edited by Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press,
1989)).

Further, DNA ligase is used in a specific gene amplification method called "ligase chain reaction (LCR)". The ligase chain reaction involves two adjacent oligonucleotides complementary to the nucleotide sequence of interest and an oligonucleotide complementary to each, and a DN
A ligase is performed by mixing with a sample containing the template DNA of interest and subjecting it to a cycling consisting of a denaturation step at high temperature and an annealing / ligation step at low temperature. When a target template DNA having a nucleotide sequence complementary to two adjacent oligonucleotides is present in a sample, a ligation reaction between the two oligonucleotides occurs. Confirmation of the reaction product is performed using electrophoresis or the like (Landegren, U. et al., (19)
88) Science, 241: 1077-1080; Barany, F. (1991) PCR
Methods and Applications 1: 5-16; Marsh, E. et al., (199
2) See Strategies 5: 73-76). Therefore, the DNA ligase used in the ligase chain reaction preferably has high heat resistance. Furthermore, in this case, it is preferable to have a higher activity for a template-dependent ligation reaction for linking between two annealed oligonucleotides as compared to ligation between single-stranded DNAs and ligation between double-stranded blunt-ended DNAs. .

The enzymatic activity of DNA ligase is determined, for example, by reacting a sample containing DNA ligase on HindIII digest of phage λ DNA and subjecting the reaction product to agarose gel electrophoresis.
Observing changes in the mobility of the A fragment, or
A sample containing DNA ligase is allowed to act on an oligo dT labeled with ³² P, unreacted ³² P is removed with alkaline phosphatase, and then radioactivity can be measured, for example, (Rossi, R et al., (1997) Nucleic Acids Research,
25 (11): 2106-2113; Odell, M. et al., (1996) Virology 22
1: 120-129; Sriskanda, V. et al., (1998) Nucleic Acids R
esearch, 26 (20): 4618-4625; Takahashi, M. et al., (1984)
The Journal of Biological Chemistry, 259 (16): 1004
1-10047)).

The hyperthermophilic archaeon used in the present invention is defined as a microorganism that grows at 90 ° C. or higher. Preferably, the hyperthermophilic archaeon is a thermostable thiol protease-producing bacterium strain KOD-1 (Morikawa, M. et al., Appl. Environ. Micr), which produces a hyperthermostable DNA ligase.
obiol. 60 (12), 4559-4566 (1994)). The KOD-1 strain has been deposited at the National Institute of Bioscience and Human Technology,
Its accession number is FERM P-15007. This KOD-
One strain was initially classified as belonging to the genus Pyrococcus, as described in the above literature. But DNASIS
GenBank R91.0 entered in (Hitachi Software Engineering) October, 1995 + Daily Update
Comparison of the 16S rRNA sequences using the registered data of the KOD-1 strain revealed that the Thermococ strain was rather the Pyrococcus genus.
It has been suggested to be closely related to the genus cus.

Culture of the hyperthermophilic archaeon producing the original hyperthermostable DNA ligase of the present invention can be performed, for example, by the method of Appl. Environ.
icrobiol. 60 (12), 4559-4566 (1994) (supra). The culture can be either static culture or aeration and agitation culture with nitrogen gas, and can be either continuous or batch.

The conditions for culturing the recombinant host cell are appropriately selected depending on the type of the host cell used. As the host cell, any host cell that can be used in recombinant DNA technology can be used. These include, for example, bacterial cells, yeast cells, animal cells, plant cells, insect cells, and the like. Preferred host cells are bacterial cells.

After culturing, the hyperthermostable DNA ligase of the present invention can be purified from the resulting culture by a method known in the art. If the expression product is secreted extracellularly, a supernatant is obtained, for example, by centrifuging or filtering the culture,
This is purified directly or concentrated by precipitation or ultrafiltration and then purified. If the expression product accumulates in the cells, the cells are disrupted using cell wall lytic enzymes, changes in osmotic pressure, glass beads, homogenizer or sonication to obtain a cell extract, which is purified.
Purification can be performed by a combination of methods known in the art, such as ion exchange chromatography, gel filtration, affinity chromatography, and electrophoresis.

The DNA encoding the hyperthermostable DNA ligase is
For example, it can be obtained by isolating chromosomal DNA from hyperthermophilic archaeon, preparing a library containing the chromosomal DNA, and screening this library.

The chromosomal DNA of the hyperthermophilic archaeon is obtained by lysing cultured bacterial cells using a surfactant (for example, N-lauryl sarcosine) and subjecting the lysate to an equilibrium density of cesium ethidium bromide. It can be obtained by fractionation by gradient ultracentrifugation or the like (for example, Imanaka et al.,
J. Bacteriol. 147 : 776-786 (1981)). The library is obtained by cleaving the obtained chromosomal DNA with various restriction enzymes, and then cutting the same restriction enzyme or a restriction enzyme giving a common cleavage end to a vector (such as a phage or plasmid), and then adding T4 DNA ligase or the like. And can be obtained by linking. Screening of the library is performed by using
This can be done by selecting a clone containing DNA encoding A ligase. The selection is performed using, for example, an oligonucleotide designed based on a predetermined partial amino acid sequence of the hyperthermostable DNA ligase, a cloned DNA that is presumed to have homology to the target DNA, or the like as a probe. obtain. Alternatively, selection can be performed by expressing the enzyme of interest. For example, expression can be detected by detecting the activity of an expression product against a substrate added to a plate when the activity of the enzyme of interest can be easily detected, or when an antibody against the enzyme of interest is available. Can be carried out by utilizing the reactivity between the expression product and the antibody.

Analysis of the resulting cloned DNA can be performed, for example, by isolating the selected DNA, creating a restriction map thereof, and determining the nucleotide sequence. Preparation of cloned DNA, restriction enzyme treatment,
Techniques such as subcloning and nucleotide sequence determination are well known in the art, and include, for example, “Molecular
Cloning: A Laboratory Manual Second Edition "(Sambrook, Fr
edited by itsch and Maniatis, Cold Spring Harbor Laborato
ry Press, 1989).

Next, the obtained cloned DNA is operably inserted into an expression vector compatible with the host cell to be used, and the host cell is transformed with the expression vector. Super heat resistance by culturing
DNA ligase can be expressed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the invention is not limited by these examples.

[0028]

Examples (Example 1: Isolation of DNA encoding hyperthermostable DNA ligase) 1.1. Preparation of chromosomal DNA of KOD-1 strain KOD-1 strain was isolated from Appl. Environ. Microbiol. 60 (12), 4559-
4 × 2216 marine broth medium described in 4566 (1994) (221
6 Marine broth: 18.7 g / L, PIPES 3.48 g / L, CaCl ₂・ H ₂ O
0.725 g / L, 0.4 mL 0.2% resazurin, 475 mL artificial seawater (N
aCl 28.16 g / L, KCl 0.7 g / L, MgCl ₂・ 6H ₂ O 5.5 g / L, Mg
_{SO 4 · 7H 2 O 6.9 g} / L), distilled water 500 mL, was inoculated into pH 7.0) 1,000 ml, it was incubated with a 2-liter fermenter. During the culture, the inside of the fermenter was replaced with nitrogen gas, and the internal pressure was maintained at 0.1 kg / cm ² with the same gas. Culture is performed at a temperature of 85 ± 1 ℃
The cells were cultured for 4 hours. The cultivation was performed by stationary cultivation, and aeration and agitation of nitrogen gas were not performed during the culturing. After the completion of the culture, the culture was centrifuged at 10,000 rpm for 10 minutes to recover the cells.

1 g of the obtained cells was added to 10 ml of an A solution (50 mM Tr
is-HCl, 50 mM EDTA, pH 8.0) and centrifuged (8,00
After collecting cells by 0 rpm, 5 minutes, 4 ° C.), the cells were suspended in 3 ml of A solution containing 15% sucrose, incubated at 37 ° C. for 30 minutes, and then 1% N
3 ml of solution A containing lauryl sarcosine were added. To this solution, 5.4 g of cesium chloride and 300 μl of a 10 mg / ml ethidium bromide solution were added, and the mixture was added at 55,000 rpm for 16 hours.
Ultracentrifugation was performed at 8 ° C. to fractionate chromosomal DNA. After removing ethidium bromide from the obtained chromosomal DNA fraction by n-butanol extraction, a TE solution (10 mM Tris-HCl (pH 8.0),
(0.1 mM EDTA) overnight to obtain chromosomal DNA.

1.2. Screening of Chromosome Library The chromosomal DNA prepared in the above 1.1 was cut with the restriction enzyme SacI, and inserted into the SacI site of the plasmid vector pUC19. The nucleotide sequence of the fragment in the resulting plasmid was then sequenced randomly and a known DN
Clones encoding DNA ligase were identified based on homology to the nucleotide sequence of the gene encoding A ligase.

The chromosomal DNA prepared in the above 1.1 was partially digested with restriction enzyme EcoRI, and then ligated to phage vector λEMBL4 (Stratagene) using T4 DNA ligase.
This combined mixture is then converted to Max Plax ^™ Packaging Extract (EPI
(CENTER TECHNOLOGIES).

The phage library was 1.1 × 10 ⁹ pfu
To Escherichia coli XL1-Blue MRA (P2) strain,
The appeared plaque was screened by a conventional method using the fragment of the clone identified above as a probe to obtain a positive clone ("Molecular Clon").
ing: A Laboratory Manual Second Edition "Sambrook, Fritsch
And Maniatis, Cold Spring Harbor Laboratory Pr
ess, 1989).

1.3. Sequence Analysis Upon determining the nucleotide sequence of the fragment of the positive clone isolated in 1.2 above, a 1686 bp open sequence encoding a polypeptide having an amino acid sequence homologous to the amino acid sequence of a known DNA ligase was determined. A reading frame was found. This nucleotide sequence is shown in SEQ ID NO: 1.

This open reading frame has 562
It encodes a polypeptide consisting of amino acids (SEQ ID NO: 2), and the molecular weight of the gene product was estimated to be 64.079 kDa. However, if the second methionine codon in the open reading frame is the start codon, the length of the open reading frame, the number of encoded amino acids, and the estimated molecular weight of the gene product are 1677 bp, 559 amino acids, and 63.746 kDa, respectively. (Described later).

Next, this deduced amino acid sequence was converted to EMB using the software DNASIS, BLAST, and CLUSTALW.
Comparison was made with amino acid sequences of known proteins registered in databases such as L and PDB. As a result, high homology with amino acids of a known DNA ligase derived from an archaebacterium was found (FIG. 1). Next, a phylogenetic tree was created by the proximity junction method (NJ method). As a result, it was found that the DNA ligase encoded by the obtained clone belongs to ATP-dependent DNA ligase derived from eukaryotes and viruses.

(Example 2: Expression of DNA ligase gene)
In the vicinity of the N-terminal coding sequence of the open reading frame determined in Example 1, two methionine-encoding codons capable of acting as initiation codons were found (SEQ ID NO: 1, 1 to 3 and 10 to 10). 12th). The former codon usage is based on M. thermoformicicum and M. ther
It is suggested by homology with the amino acid sequence of DNA ligase from moautotrophicum, and the latter codon usage is P. h
The results were suggested by homology with the amino acid sequences of DNA ligases derived from orikoshii, M. jannaschii, and D. ambivalens. Therefore, it was expected that both of these methionine codons could act as initiation codons.

The following was performed to express DNA ligase having two types of initiation codons in Escherichia coli. Primers DL-L-5T (SEQ ID NO: 3) and DL-3T (SEQ ID NO: 4), or DL-S-5T (SEQ ID NO: 5) and DL-3T
Using (SEQ ID NO: 4), fragments of the two DNA ligase coding regions were obtained using the phage clone fragment as a template and inserted into the HincII site of pUC18. The primers DL-L-5T (SEQ ID NO: 3), DL-L-5T
3T (SEQ ID NO: 4) and DL-S-5T (SEQ ID NO: 5) correspond to nucleotides 1-28 and 1 of the ORF, respectively.
709 to 1736, and nucleotides 10 to 31. The nucleotide sequence of the inserted fragment was confirmed, this plasmid was digested with NdeI and BamHI, and the resulting fragment was inserted into plasmid pET21a (Novagen) to construct two types of plasmids (p.
ET-ligL and pET-ligS). Confirmation of expression and activity was performed as follows.

Using each of the above plasmids, Escher
The ichia coli BL21 (DE3) strain was transformed. Appearing ampicillin-resistant transformants were transformed into NZCYM medium (1% NZ amine, 0.5% NaCl, 0.5%) containing ampicillin (50 μg / ml).
Yeast extract, 0.1% casamino _{acid, 0.2% MgSO 4 · 7H 2} O
(PH 7)), and cultured at 37 ° C. until the OD ₆₆₀ becomes 1.0. Then, isopropyl-β-D-thiogalactopyranoside (IPTG, 0.1 mM) was added, and the cells were further cultured at 37 ° C. Continued. After completion of the culture, the cells were collected by centrifugation, disrupted by sonication, and centrifuged again to collect the soluble fraction, and then heat-treated at 80 ° C for 30 minutes. A sample was obtained by centrifuging the thermostable soluble fraction. This sample was further purified using anion exchange chromatography, cation chromatography, and gel filtration according to the standard methods described below.

Anion exchange chromatography: pH
7.5 of 50 mM Tris-HCl, 10 mM Mg
Amersham pha equilibrated with Cl ₂ buffer
Macia Biotech Resin Resource
This was performed using a column packed with Q (about 6 ml bed volume). The sample was applied to the column, and the contaminants were adsorbed on the resin. DNA ligase was collected as effluent through the column.

Cation exchange chromatography: DNA ligase partially purified by anion chromatography was purified from Amersham pharmacia B equilibrated with 50 mM Tris-HCl buffer, pH 7.5.
Adsorbed on ResinResourceS manufactured by iotech,
After washing the resin with the same buffer, 50 mM T
DNA ligase activity was eluted using ris-HCl, 100 mM MgCl ₂ buffer.

Gel filtration: 50 mM Tri at pH 7.5
Amersham Pharmacia Biote equilibrated with s-HCl, 100 mM MgCl ₂ buffer
ch Resin Superdex 200HR10 / 30
Was carried out using a column packed with (a bed capacity of about 24 ml). About 2 times the column volume of the same buffer was passed through the column, and DNA ligase was separated as a peak fraction.

FIG. 2 shows that the sample in each purification step was 1
The result of performing electrophoresis by applying to a 2.5% acrylamide gel is shown. 1 to 6 in FIG. 2 are respectively 1: a molecular weight marker, 2: supernatant liquid after sonication of transformant cells containing plasmid pET-ligL, 3: 3: 8
The lane in which the supernatant liquid after heat treatment at 0 ° C. for 30 minutes, 4: anion exchange chromatography effluent, 5: cation exchange chromatography adsorption fraction, and 6: gel filtration peak fraction are shown. As shown in FIG.
The ligase migrated as a band with a molecular weight of approximately 62.0 kDa (indicated by A in FIG. 2) and 56.2 kDa (indicated by B in FIG. 2). This is because both had the same DNA ligase, and bands having different three-dimensional structures appeared depending on the degree of denaturation. The band indicated by C in FIG. 2 is considered to be a degradation product of DNA ligase.

The enzyme activity after the action of the sample obtained in HindIII digest of λ phage DNA, the reaction was subjected to agarose gel electrophoresis, a method for observing the change in mobility of DNA fragments or ³² P, The obtained sample is allowed to act on the labeled oligo dT, unreacted ³² P is removed with alkaline phosphatase, and then measured by a method of measuring radioactivity (Rossi, R et al., (1997) Nucleic Acids Res.
earch, 25 (11): 2106-2113; Odell, M. et al., (1996) Virol
ogy 221: 120-129; Sriskanda, V. et al., (1998) Nucleic A
cids Research, 26 (20): 4618-4625; Takahashi, M. et al.
(1984) The Journal of Biological Chemistry, 259 (1
6): 10041-10047)). Here, the oligomers shown in FIG. 3 (30 mer, 40 mer and 80 mer,
SEQ ID NOs: 6, 7, and 8) were used as DNA substrates for measuring DNA ligase activity, and the activity of DNA ligase was measured. These were designed to have a GC content of around 50%, not to be a primer dimer, and to have no secondary structure. The following reaction system and reaction conditions were used for the measurement.

Composition of reaction system: 20 mM Bicine-
KOH, pH 8.0, 15 mM MgCl ₂ , 20 mM
KCl, 1 mM ATP, 10 μM 30 mer, 10
μM 40mer, 5μM 80mer, 270mg DNA
Ligase. Reaction conditions: 80 ° C., 15 minutes After completion of the reaction, the reaction solution is subjected to electrophoresis on a 6% amylylamide gel, stained with a 0.5 μg / ml ethidium bromide solution, and generated by ligation of a 30-mer with a 40-mer. Ligase activity was detected by the presence or absence of the 70-mer.
Note that the 80 mer functions as a template in the reaction system.

FIG. 4 shows the results. In FIG. 4, 1 to 5 are 1: samples before the reaction, 2 to 5: samples after the reaction, 2 and 5 as cofactors, 2 and 5 after the reaction including ATP, and 4 after the reaction including NAD ^+. 3 shows the results of electrophoresis of the sample of No. 3, the reaction system containing no cofactor, and
Indicates the result of electrophoresis of the sample after the reaction which does not contain the 80 mer of the template. As shown in FIG.
NA ligase did not link the 30-mer and the 40-mer in the reaction system without ATP (lane 3), and also in the reaction system containing NAD ⁺ as a cofactor.
It was shown to link the mers, and was therefore shown to require ATP for their active expression and that NAD ⁺ also acts as a cofactor.

Example 3 Enzymatic Properties of DNA Ligase The following enzymatic properties of the obtained DNA ligase were examined.

1. Reaction optimum pH Reaction system: 10 mM MgCl ₂ , 50 mM KCl, 1
A reaction system containing mM ATP, 10 μM 30 mer, 10 μM 40 mer, 5 μM 80 mer, and 270 mg DNA ligase was used. The pH of the reaction system is adjusted to 2
The test was performed with the following buffer at a concentration of 0 mM. Citrate-
KOH (pH 4.0, 4.5, 5.0, 5.5), ME
S-KOH (pH 5.5, 6.0, 6.5, 7.0),
HEPES-KOH (pH 7.0, 7.5, 8.0),
Bicine-KOH (pH 8.0, 8.5, 9.
0), CHES-KOH (pH 9.0, 9.5, 10.
0). Reaction conditions: Performed at 80 ° C. for 15 minutes.

FIG. 5 shows the results. The vertical axis in FIG.
Is the relative value of the ligase activity, where the activity obtained in step 1 is taken as 100. As is clear from FIG. 5, the optimal pH of the obtained DNA ligase was around pH 8.0.

2. Effect on activity of Mg ²⁺ ion Reaction system: 20 mM Bicine-KOH, pH8.
0, 50 mM KCl, 1 mM ATP, 10 μM 3
0 mer, 10 μM 40 mer, 5 μM 80 mer, 27
A reaction system containing 0 mg DNA ligase is mixed with 0 to 50 mM.
The reaction system to which MgCl ₂ was added was used. Reaction conditions: Performed at 80 ° C. for 15 minutes.

FIG. 6 shows the results. The vertical axis in FIG.
This is a relative value of ligase activity, where the activity obtained in the presence of mM Mg ²⁺ is defined as 100. As shown in FIG.
No ligase activity was observed in the absence of ²⁺ ions,
The g ²⁺ ion was shown to be a cation essential for ligase activity. The optimal concentration of Mg2 ⁺ was about 14-18 mM.

3. Effect on K ⁺ ion activity Reaction system: 20 mM Bicine-KOH, pH8.
0, 15 mM MgCl ₂ , 1 mM ATP, 10 μM
30 mer, 10 μM 40 mer, 5 μM 80 mer,
The reaction system containing 270 mg DNA ligase was
A reaction system to which 0 mM KCl was added was used. Reaction conditions: Performed at 80 ° C. for 15 minutes.

FIG. 7 shows the results. The vertical axis of FIG.
It is a relative value of ligase activity, where the activity obtained in the presence of mM K ⁺ is defined as 100. K ⁺ had a ligase activity promoting effect, and its optimal concentration was in the range of about 10 to 30 mM.

The enzymatic properties of above 1 to 3 are based on KOD-1
It was consistent with the enzymatic properties of the DNA ligase obtained by culturing the strain.

4. Temperature characteristics The temperature characteristics of the obtained DNA ligase were determined based on the high concentration state (210 nM) and low concentration state (21 nM)
M).

Reaction system: 20 mM Bicine-KO
H, pH 8.0, 15 mM MgCl _Two, 20 mM K
Cl, 1 mM ATP, 10 μM 30 mer, 10 μM
 In a reaction system containing a 40 mer, 5 μM 80 mer, DNA
The reaction was performed by adding ligase at the above concentration. Reaction conditions: Performed at 20-100 ° C for 15 minutes.

FIGS. 8 and 9 show the temperature characteristics of the ligase activity when the DNA ligase of the present invention is used at a high concentration (including 210 nM DNA ligase in the reaction solution). FIG. 9 shows the results of electrophoresis of the DNA ligase reaction solution at each temperature shown in FIG. 8 using a 6% acrylamide gel. The reaction solution loaded in each lane corresponds to each of the reaction solutions shown in FIG. The vertical axis of FIG.
It is a relative value of ligase activity when the activity at 0 ° C. is set to 100. As shown in FIG. 8, when the DNA ligase of the present invention was used at a high concentration, its activity was maximum at about 80 ° C., and the activity was still observed at 100 ° C.

FIG. 10 shows temperature characteristics when the DNA ligase of the present invention was used at a low concentration (including 21 nM DNA ligase in the reaction solution). The vertical axis in FIG. 10 is a relative value of the ligase activity when the activity at 65 ° C. is set to 100.
As shown in FIG. 10, when the DNA ligase of the present invention was used at a low concentration, the maximum activity was observed at about 65 ° C.

The Tm value of the DNA substrate used in the reaction is 66 ° C., 73 ° C., 79 ° C. and 81 ° C. for the 30 mer, 40 mer, 70 mer and 80 mer, respectively. The ligase reaction should not normally occur theoretically above the 80-mer Tm value of the template,
The DNA ligase activity at 100 ° C., which was observed when the DNA ligase of the present invention was used at a high concentration, is considered that the DNA ligase binds to the substrate DNA and is involved in maintaining the double-stranded structure.

[0059]

According to the present invention, a hyperthermostable DNA ligase and a DNA encoding the same are provided. The present invention also provides a vector containing the DNA and a host cell containing the vector. Further, the present invention provides a method for producing a hyperthermostable DNA ligase, which comprises a step of culturing the host cell.

[0060]

[Sequence List] SEQUENCE LISTING <110> Imanaka, Tadayuki <120> Highly heat-stable DNA ligase <130> J100432093 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1689 <212> DNA <213 > KOD-1 <220> <221> CDS <222> (1) .. (1686) <400> 1 atg agc gat atg cgc tac tct gaa ctg gcc gat ctc tat aga cgg ctc 48 Met Ser Asp Met Arg Tyr Ser Glu Leu Ala Asp Leu Tyr Arg Arg Leu 1 5 10 15 gaa aag acc acg ctc aag acc ctg aag aca aag ttc gtt gcc gat ttt 96 Glu Lys Thr Thr Leu Lys Thr Leu Lys Thr Lys Phe Val Ala Asp Phe 20 25 30 tta aaa aag act cca gac gag ctc ctt gag ata gtt cca tac cta atc 144 Leu Lys Lys Thr Pro Asp Glu Leu Leu Glu Ile Val Pro Tyr Leu Ile 35 40 45 ctc gga aag gtc ttt ccc gat tgg gac gag agg gag cta ggc gtg gga 192 Leu Gly Lys Val Phe Pro Asp Trp Asp Glu Arg Glu Leu Gly Val Gly 50 55 60 gag aag ctc cta ata aaa gct gtc tct atg gca acg gga gtc ccc gaa 240 Glu Lys Leu Leu Ile Lys Ala Val Ser Met Ala Thr Gly Val Pro Glu 65 70 75 80 aag gag ata gaa gac tcc gtt agg gac act ggc gac ctg gga gag agc 288 Lys Glu Ile Glu Asp Ser Val Arg Asp Thr Gly Asp Leu Gly Glu Ser 85 90 95 gtt gct ttg gcc ata aag aaa aag aag cag aag agc ttc ttc tca cag 336 Val Ala Leu Ala Ile Lys Lys Lys Lys Gln Lys Ser Phe Phe Ser Gln 100 105 110 ccg ctc acg ata aag cgt gtt tac gac acc ttt gtc aag ata gcg gag 384 Pro Leu Thr Ile Lys Arg Val Tyr Asp Thr Phe Val Lys Ile Ala Glu 115 120 125 gcc caa ggc gaa gga agc cag gac agg aag atg aag tac ctc gcc aac 432 Ala Gln Gly Glu Gly Ser Gln Asp Arg Lys Met Lys Tyr Leu Ala Asn 130 135 140 ctc ttc atg gac gct gaa ccg gag gaa ggc aag tac ctg gcg agg acg 480 Leu Phe Pro Glu Glu Gly Lys Tyr Leu Ala Arg Thr 145 150 155 160 gtt ttg ggg acg atg cgc act gga gtc gct gag gga att ctt agg gat 528 Val Leu Gly Thr Met Arg Thr Gly Val Ala Glu Gly Ile Leu Arg Asp 165 170 175 gca ata gct gaa gcc ttc agg gtg aag cca gag ctc gtt gag agg gct 576 Ala Ile Ala Glu Ala Phe Arg Val Lys Pro Glu Leu Val Glu Arg Ala 180 185 190 tac atg ctg acg agc gac ttc gga tac gtt gcc ag aag ctc 624 Tyr Met Leu Thr Ser Asp Phe Gly Tyr Val Ala Lys Ile Ala Lys Leu 195 200 205 gag ggc aac gag ggg ctc tca aag gtc agg ata cag ata ggc aag ccg 672 Glu Gly Asn Glu Gly Leu Ser Lys Val Arg Ile Ile Gly Lys Pro 210 215 220 atc agg ccg atg ctt gcc cag aac gcg gcc agt gtt aaa gat gcc ctc 720 Ile Arg Pro Met Leu Ala Gln Asn Ala Ala Ser Val Lys Asp Ala Leu 225 230 235 240 atc gag atg ggt ggg gaa gcg gcc ttc gag ata aag tac gat ggc gcg 768 Ile Glu Met Gly Gly Glu Ala Ala Phe Glu Ile Lys Tyr Asp Gly Ala 245 250 255 aga gta cag gtg cac aag gac ggg gac aaa gtc atc gtg Gt tat tccaga Val His Lys Asp Gly Asp Lys Val Ile Val Tyr Ser Arg 260 265 270 agg ctt gag aac gtc acg aga tca atc cca gaa gtc att gag gcc ata 864 Arg Leu Glu Asn Val Thr Arg Ser Ile Pro Glu Val Ile Glu Ala Ile 275 280 285 aaa gcg gcc ctg aag cca gag aaa gcc ata gtc gag gga gaa ctt gtc 912 Lys Ala Ala Leu Lys Pro Glu Lys Ala Ile Val Glu Gly Glu Leu Val 290 295 300 gca gtt gga gaa aat gga cgg cca agg ccc tac gtg ctt agg 960 Ala Val Gly Glu Asn Gly Arg Pro Arg Pro Phe Gln Tyr Val Leu Arg 305 310 315 320 cgc ttc agg agg aag tac aac atc gac gag atg ata gag aag ata ccg 1008 Arg Phe Arg Arg Lys Tyr Asn Ile Asp Glu Met Ile Glu Lys Ile Pro 325 330 335 ctc gaa ctg aat ctc ttc gac gtc atg ttc gtt gac ggc gag agc ctg 1056 Leu Glu Leu Asn Leu Phe Asp Val Met Phe Val Asp Gly Glu Ser at Ru 340 act aag ttc atc gac agg agg aat aag ctt gaa gaa ata gtg 1104 Ile Glu Thr Lys Phe Ile Asp Arg Arg Asn Lys Leu Glu Glu Ile Val 355 360 365 aag gag agc gag aag ata aag ctc gcc gaa cag ctt ataa aca Lys Glu Ser Glu Lys Ile Lys Leu Ala Glu Gln Leu Ile Thr Lys Lys 370 375 380 gtt gag gaa gcg gag gcc ttc tac agg aga gca ctt gag ctc ggc cat 1200 Val Glu Glu Ala Glu Ala Phe Tyr Arg Arg Ala Leu Glu Gly His 385 390 395 400 gag ggg ctt atg gca aag agg ctt gat tcc atc tac gaa ccg ggg aac 1248 Glu Gly Leu Met Ala Lys Arg Leu Asp Ser Ile Tyr Glu Pro Gly Asn 405 410 415 aga ggc aag aag tgg ctc a ag att aag ccc acg atg gag aac ctc gat 1296 Arg Gly Lys Lys Trp Leu Lys Ile Lys Pro Thr Met Glu Asn Leu Asp 420 425 430 ctc gtc atc atc ggt gca gag tgg gga gag gga agg agg gcc cat ctg 1344 Leu Val Ile Gly Ala Glu Trp Gly Glu Gly Arg Arg Ala His Leu 435 440 445 ctt ggt tcg ttc ctc gtt gct gcc tac gac ccg cac agc ggc gag ttc 1392 Leu Gly Ser Phe Leu Val Ala Ala Tyr Asp Pro His Ser Gly Glu 455 460 ctg cct gtg ggt aaa gtc ggg agc ggc ttc aca gac gag gac ttg gtc 1440 Leu Pro Val Gly Lys Val Gly Ser Gly Phe Thr Asp Glu Asp Leu Val 465 470 475 480 gag ttc acg aag atg ctc aagca cgc cag gaa ggc aag 1488 Glu Phe Thr Lys Met Leu Lys Pro Tyr Ile Val Arg Gln Glu Gly Lys 485 490 495 ttc gtc gag ata gag ccg aag ttc gtc att gag gtc acc tac cag gag 1536 Phe Val Glu Ile Glu Val Ile Glu Val Thr Tyr Gln Glu 500 505 510 ata cag aag agc ccg aag tac aag agc ggc ttc gcc ctg aga ttc ccg 1584 Ile Gln Lys Ser Pro Lys Tyr Lys Ser Gly Phe Ala Leu Arg Phe Pro 515 520 525 cgta c gtc gcc ctt agg gaa gac aaa agc cca gaa gag gcc gac aca 1632 Arg Tyr Val Ala Leu Arg Glu Asp Lys Ser Pro Glu Glu Ala Asp Thr 530 535 540 att gaa agg gtg gcg gag ctc tac gag agc cag agag gcg 1680 Ile Glu Arg Val Ala Glu Leu Tyr Glu Leu Gln Glu Arg Phe Lys Ala 545 550 555 560 aag aag tga 1689 Lys Lys <210> 2 <211> 562 <212> PRT <213> KOD-1 <400> 2 Met Ser Asp Met Arg Tyr Ser Glu Leu Ala Asp Leu Tyr Arg Arg Leu 1 5 10 15 Glu Lys Thr Thr Leu Lys Thr Leu Lys Thr Lys Phe Val Ala Asp Phe 20 25 30 Leu Lys Lys Thr Pro Asp Glu Leu Leu Glu Ile Val Pro Tyr Leu Ile 35 40 45 Leu Gly Lys Val Phe Pro Asp Trp Asp Glu Arg Glu Leu Gly Val Gly 50 55 60 Glu Lys Leu Leu Ile Lys Ala Val Ser Met Ala Thr Gly Val Pro Glu 65 70 75 80 Lys Glu Ile Glu Asp Ser Val Arg Asp Thr Gly Asp Leu Gly Glu Ser 85 90 95 Val Ala Leu Ala Ile Lys Lys Lys Lys Gln Lys Ser Phe Phe Ser Gln 100 105 110 Pro Leu Thr Ile Lys Arg Val Tyr Asp Thr Phe Val Lys Ile Ala Glu 115 120 125 Ala Gln Gly Glu Gly Ser Gln Asp Arg Lys Met L ys Tyr Leu Ala Asn 130 135 140 Leu Phe Met Asp Ala Glu Pro Glu Glu Gly Lys Tyr Leu Ala Arg Thr 145 150 155 160 Val Leu Gly Thr Met Arg Thr Gly Val Ala Glu Gly Ile Leu Arg Asp 165 170 175 Ala Ile Ala Glu Ala Phe Arg Val Lys Pro Glu Leu Val Glu Arg Ala 180 185 190 Tyr Met Leu Thr Ser Asp Phe Gly Tyr Val Ala Lys Ile Ala Lys Leu 195 200 205 Glu Gly Asn Glu Gly Leu Ser Lys Val Arg Ile Gln Ile Gly Lys Pro 210 215 220 Ile Arg Pro Met Leu Ala Gln Asn Ala Ala Ser Val Lys Asp Ala Leu 225 230 235 240 Ile Glu Met Gly Gly Glu Ala Ala Phe Glu Ile Lys Tyr Asp Gly Ala 245 250 255 255 Arg Val Gln Val His Lys Asp Gly Asp Lys Val Ile Val Tyr Ser Arg 260 265 270 270 Arg Leu Glu Asn Val Thr Arg Ser Ile Pro Glu Val Ile Glu Ala Ile 275 280 285 Lys Ala Ala Leu Lys Pro Glu Lys Ala Ile Val Glu Gly Glu Leu Val 290 295 300 Ala Val Gly Glu Asn Gly Arg Pro Arg Pro Phe Gln Tyr Val Leu Arg 305 310 315 320 Arg Phe Arg Arg Lys Tyr Asn Ile Asp Glu Met Ile Glu Lys Ile Pro 325 330 335 Leu Glu Leu Asn Leu Phe Asp Val Met Phe Val A sp Gly Glu Ser Leu 340 345 350 350 Ile Glu Thr Lys Phe Ile Asp Arg Arg Asn Lys Leu Glu Glu Ile Val 355 360 365 Lys Glu Ser Glu Lys Ile Lys Leu Ala Glu Gln Leu Ile Thr Lys Lys Lys 370 375 380 Val Glu Glu Alu Glu Ala Phe Tyr Arg Arg Ala Leu Glu Leu Gly His 385 390 395 400 Glu Gly Leu Met Ala Lys Arg Leu Asp Ser Ile Tyr Glu Pro Gly Asn 405 410 415 Arg Gly Lys Lys Trp Leu Lys Ile Lys Pro Thr Met Glu Asn Leu Asp 420 425 430 Leu Val Ile Ile Gly Ala Glu Trp Gly Glu Gly Arg Arg Ala His Leu 435 440 445 Leu Gly Ser Phe Leu Val Ala Ala Tla Asp Pro His Ser Gly Glu Phe 450 455 460 Leu Pro Val Gly Lys Val Gly Ser Gly Phe Thr Asp Glu Asp Leu Val 465 470 475 480 480 Glu Phe Thr Lys Met Leu Lys Pro Tyr Ile Val Arg Gln Glu Gly Lys 485 490 495 Phe Val Glu Ile Glu Pro Lys Phe Val Ile Glu Val Thr Tyr Gln Glu 500 505 510 Ile Gln Lys Ser Pro Lys Tyr Lys Ser Gly Phe Ala Leu Arg Phe Pro 515 520 525 Arg Tyr Val Ala Leu Arg Glu Asp Lys Ser Pro Glu Glu Ala Asp Thr 530 535 540 Ile Glu Arg Val Ala Glu Leu Tyr Glu Leu Gln Glu A rg Phe Lys Ala 545 550 555 560 Lys Lys <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <400> 3 cggtggtgca tatgagcgat atgcgctact ctgaactgg 39 <210> 4 <211> 38 <212> DNA <213 > Artificial Sequence <400> 4 ctcgggatcc ctgggaggga aaaggaatct cactcgcc 38 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <400> 5 gagaatgagc catatgcgct actctgaact ggccg 35 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 cagaggattg ttgaccggcc cgtttgtcag 30 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <400> 7 cgcaccgtga cgccaagctt gcattcctac aggtcgactc 40 <210> 8 <211> 80 <212> DNA <213> Artificial Sequence <400> 8 caaccgcgtg gcactgcggt tcgaacgtaa ggatgtccag ctgagtctc ctaacaactg gccgggc aaa cagtcgttgc 80

[Brief description of the drawings]

FIG. 1A shows the alignment of DNA ligase.

FIG. 1B shows an alignment of DNA ligase.

FIG. 2 is a photograph showing a gel obtained by electrophoresing purified DNA ligase.

FIG. 3 is a diagram showing the structure of an oligonucleotide used for measuring the activity of DNA ligase.

FIG. 4 is a photograph showing a gel obtained by electrophoresis of a DNA ligase reaction solution.

FIG. 5 is a view showing a reaction optimum pH of DNA ligase.

FIG. 6 is a graph showing the effect of Mg ⁺ ions on DNA ligase activity.

FIG. 7 shows the effect of K ⁺ ions on DNA ligase activity.

FIG. 8 is a diagram showing temperature characteristics of a reaction of DNA ligase in a high concentration state.

FIG. 9 is a photograph showing a gel obtained by electrophoresis of a DNA ligase reaction solution reacted at different temperatures.

FIG. 10 is a diagram showing temperature characteristics of a reaction of DNA ligase in a low concentration state.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12N 9/00 C12R 1:19) (72) Inventor Satoshi Ezaki Ikebatamachi, Kamigamo, Kita-ku, Kyoto-shi, Kyoto 64 Floral Kitayama II-108 (72) Inventor Toshiaki Fukui 8-11 Shugakuin Inuzukacho, Sakyo-ku, Kyoto, Kyoto Prefecture 102 Petit-Dell Shugakuin 102 (72) Inventor Masaru Nakatani, Yoshiya-cho, Kamigyo-ku, Kyoto, Kyoto Pref. Town 360 Miyazaki Student 302

Claims (10)

[Claims] 1. A hyperthermostable DNA ligase comprising the amino acid sequence of positions 1 to 562 of SEQ ID NO: 2, or 1 of SEQ ID NO: 2
A variant thereof comprising an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence at positions 562 to 562 and having DNA ligase activity. 2. A hyperthermostable DNA ligase comprising the amino acid sequence of positions 4 to 562 of SEQ ID NO: 2, or 4 of SEQ ID NO: 2.
A variant thereof comprising an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence at positions 562 to 562 and having DNA ligase activity. 3. The hyperthermostable DNA ligase according to claim 1 or 2, which is derived from the hyperthermophilic archaeon KOD-1 strain. 4. A DNA encoding the hyperthermostable DNA ligase according to claim 1 or a variant thereof. 5. The DNA according to claim 4, comprising the nucleotide sequence at positions 1 to 1686 of SEQ ID NO: 1. 6. The DNA according to claim 4, comprising the nucleotide sequence of positions 10 to 1686 of SEQ ID NO: 1. 7. The DNA according to claim 4, which is derived from the hyperthermophilic archaeon KOD-1 strain. A vector comprising the DNA according to any one of claims 4 to 7. 9. A recombinant host cell comprising the vector according to claim 8. 10. A method for producing a hyperthermostable DNA ligase or a variant thereof, comprising the step of culturing the host cell according to claim 9.