KR20100107873A - Dna polymerase derived from thermococcus celer and its use - Google Patents

Abstract

PURPOSE: A thermal resistant DNA polymerase which is derived from Thermococcus celer with DNA polymerization activity and proofreading activity is provided to widely use in various nucleic acid polymerization. CONSTITUTION: A thermal resistant Tce DNA polymerase isolated from Thermococcus celer contains an amino acid of seuqnce number 10. The Tce DNA polymerase has an optimal activity in 20-35 mM of (NH_4)_2SO_4 and 4-16 mM of Mg^2+ at pH 6.5 and 75°C. A nucleic acid molecule encoding the Tce DNA polymerase contains a nucleic acid sequence of sequence number 9. A pTCE vector contains a nucleic acid molecule encoding the Tce DNA polymerase. A method for preparing the Tce DNA polymerase comprises: a step of preparing a vector expressing the Tce DNA polymerase; a step of transforming the vector to the microorganism; a step of culturing the transformant; and a step of collecting protein from the transformant.

Description

Heat-resistant DNA polymerase derived from thermococcus sealer strain and use thereof {DNA POLYMERASE DERIVED FROM THERMOCOCCUS CELER AND ITS USE}

The present invention relates to a DNA polymerase derived from a hyperthermophilic archaeum Thermococcus celer strain and its use.

DNA polymerase (deoxyribonucleic acid polymerase, DNA polymerase, EC number 2.7.7.7) is an enzyme that synthesizes complementary DNA in the 5 '→ 3' direction depending on template DNA. enzymes that play the most important role in replication or DNA repair (Lehninger, AL et al ., 1993, Principles of biochemistry, 2nd ed ., Worth Publishers). DNA polymerase was first discovered in Escherichia coli by Kornberg et al in 1957 and is the current E. coli DNA polymerase I (Konberg, A. and Baker, T., 1992, DNA replication, 2nd ed ., Freeman). Company). This Escherichia coli DNA polymerase I ( E. coli DNA polymerase I) is a 5 ′ → 3 ′ nucleic acid terminal hydrolase activity (5 ′ → 3 ′ exonuclease activity, 3 ′ called proofreading activity). → 5 ′ nucleic acid end hydrolase activity (3 ′ → 5 ′ exonuclease activity) and DNA polymerization activity (DNA polymerization activity) 5 ′ → 3 ′ polymerase activity (5 '→ 3' polymerase activity) together. The heat-resistant DNA polymerase was first isolated from the thermophilic Thermos aquaticus YT-1 by Chien et al. In 1976, and then Thermus flavus belonging to the same genus ) And its properties have been isolated from Thermus thermophilus HB8 and the like, but have not received much attention (see Chien, A. et al. , 1976, J. Bacteriol. 127 , 1550-1557; Kaledin, AS et al ., 1981, Biokh imiya 46 , 1576-1584; Ruettimann, C. et al. , 1985, Eur. J. Biochem . 149 , 41-46). However, in 1988, Saiki et al . Developed a PCR technique using heat-resistant DNA polymerase (see Saiki, RK et al ., 1988, Science 239 , 487-491). as Thermococcus litoralis (Thermococcus litoralis), pie far Caucus furiosus (Pyrococcus furiosus), nanoah keum Iquique Tansu (Nanoarchaeum equitans), sseomeoseu knife filler's not K-24 (Thermus caldophilus GK24), sseomeoseu Filippo Miss (Thermus Heat-resistant DNA polymerase has been competitively researched and developed from several thermophile and hyperthermic archaea, such as filiformis ) and Pyrococcus woesei ( Hong , H. et al ., 1993, The Journal of Biological Chemistry 168 (3) 1965-1975; Lundberg, KS et al. , 1991, Gene 108 , 1-6; Choi, JJ et al ., 2008, Appl. Environ.Microbiol. 74 , 6563-6569; Park, JH et al. , 1993, Eur. J. Biochem. 214 , 135-140; Jung, SE et al. , 1997, Mol. Cells 7 , 769-776; Dabrowski, S. and Kur, J., 1998, Protein Expres. Purif. 14 , 131-138). In particular, DNA polymerases derived from S. aureus and Pyrococcus strains have been attracting more attention because they are heat-resistant DNA polymerases with corrective activity, unlike DNA polymerases derived from S. aureus, and PCR requiring high accuracy. It is used for.

PCR is a technique for mass amplification of trace amounts of template DNA using DNA polymerase and primers, DNA denaturation (94 ° C), primer annealing (40-65 ° C), and DNA synthesis (DNA). extension, 72 ° C.) is repeated 20-30 times in succession. Therefore, since high temperature is required due to the nature of the reaction, the development of heat-resistant DNA polymerase, which is the most important factor in PCR, is indispensable for the development and development of various PCR technologies (see Erlich HA et al. , 1989, Stockton Press 193). ? 208). Heat-resistant DNA polymerase is a very useful enzyme for identification and amplification of genes by PCR, DNA sequencing, clinical diagnosis, etc., including molecular biology and genetic engineering research, early diagnosis of viral and cancer genes, genetic diagnosis and forensic It is used in a wide range of fields from research to the demand is increasing day by day.

To date, there have been no studies on the use of this enzyme and the characteristics of the thermococcal sealer, which is a hyperthermic archaea, for the DNA sequence of the DNA polymerase gene and the heat-resistant DNA polymerase encoded by the electric gene. Therefore, the present inventors intended to isolate the heat-resistant DNA polymerase gene from the ultra-high temperature archaea thermococcus sealer, to express and purify the enzyme encoded by the electric gene in E. coli, and to identify the enzyme properties and use it for PCR. This will be of significant significance for the industrial use of heat-resistant DNA polymerases from the Thermococcus sealer, which has not been the case so far.

Accordingly, the present inventors have attempted to identify a heat-resistant DNA polymerase derived from Thermococcus celer , which is a kind of hyperthermophilic archaea. Thermococcus celer DNA polymerase, Tce DNA polymerase, hereinafter "Tce DNA polymerase, ") by selecting a gene and determining the nucleotide sequence and amino acid sequence deduced therefrom of the gene, Tce DNA polymerase is a conventional heat-resistant B type DNA It was confirmed that it is a new heat-resistant B series DNA polymerase having high amino acid sequence homology with family B type DNA polymerases. Next, an expression vector of the gene was prepared, cloned into Escherichia coli and expressed, and the protein was purified through heat treatment and column chromatography. Subsequently, various enzyme properties of the purified Tce DNA polymerase were examined to confirm the optimum activity condition, and it was confirmed that the Tce DNA polymerase had a proofreading activity that improved the accuracy during DNA polymerization. Finally, by performing (referred to as polymerase chain reaction, hereinafter 'PCR') polymerase chain reaction using the Tce DNA polymerase, it is intended to use the purified Tce DNA polymerase in PCR.

The present invention relates to a Tce DNA polymerase isolated from Thermococcus celer , which is a kind of hyperthermic archaea. Specifically, the present invention relates to a heat resistant Tce DNA polymerase isolated from a thermococcal sealer having the amino acid sequence of SEQ ID NO: 10.

The Tce DNA of the present invention is a heat-resistant B-type DNA polymerase, which is a DNA polymerization activity of 5 ′ → 3 ′ polymerase activity, as well as 5 ′ → 3 ′ polymerase activity. , 3 '→ 5' nucleic acid terminal hydrolase activity (proof'ing activity) is characterized in that it has an exonuclease activity (3 '→ 5').

Tce DNA polymerase of the present invention is composed of 2,325 bp, including stop codon, and consists of 774 amino acids.

Specifically, 50 mM Tris-HCl (pH 7.8) / 10 mM (NH 4) 2 SO 4/5 mM MgSO 4 / 0.1% Triton under a reaction buffer consisting of X-100 lambda phage to genomic DNA of (λ phage) PCR was performed using the Tce DNA polymerase of the present invention as a template, and the synthesis was possible up to 6 kb. In addition, as a result of PCR using Tce DNA polymerase under the buffer solution, 2 kb of DNA was amplified in a short time compared with Taq DNA polymerase and Pfu DNA polymerase. In addition, PCR showed high accuracy with a significantly lower error rate.

Tce DNA polymerase of the present invention exhibits physical and chemical properties that exhibit optimal activity at pH 6.5, temperature 75 ° C., 20-35 mM (NH ₄ ) ₂ SO ₄ concentration and 4-16 mM Mg ²⁺ concentration. Has characteristics. However, KCl inhibited the activity of Tce DNA polymerase.

The present invention provides protein variants having sequences that differ from the amino acid sequence of wild-type Tce DNA polymerase by deletion, insertion, non-conservative or conservative substitution, addition or combination of some sequences at one or more positions within the scope of the present invention. Include. In addition, the Tce DNA polymerase of the present invention includes the modified form of phosphorylation, sulfidation, acrylated, glycosylated, methylated, farnesylated and the like within the scope of the present invention.

The variant may be a functional equivalent having substantially the same activity as the wild type protein or a form with increased or decreased physicochemical properties. For example, variants with enhanced enzyme activity. Or physical factors such as temperature, moisture, pH, electrolyte, reducing sugar, pressurization, drying, freezing, interfacial tension, light, repetition of freezing and thawing, high concentration conditions, acid, alkali, neutral salt, organic solvent, metal ion, oxidation It is a variant with enhanced structural stability to the external environment of chemical factors such as reducing agents and proteases.

The present invention also relates to a nucleic acid molecule encoding the heat resistant Tce DNA polymerase.

The Tce DNA polymerase having an amino acid sequence of SEQ ID NO: 10 preferably has a nucleic acid sequence of SEQ ID NO: 9.

The present invention also encompasses nucleic acid molecules encoding the aforementioned Tce DNA polymerase variants.

Nucleic acid molecules encoding Tce DNA polymerase or variants thereof of the invention may be mutated by substitutions, deletions, insertions or combinations thereof at one or more positions. Such nucleic acid sequence variants are also within the scope of the present invention.

The present invention also relates to a vector comprising a nucleic acid molecule encoding the heat resistant Tce DNA polymerase.

In the present invention, a "vector" is a recombinant vector capable of expressing a protein of interest in a host cell, and is a gene construct containing essential regulatory elements operably linked to express a gene insert. As used herein, “operably linked” refers to a functional linkage between an expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Can be prepared using well-known genetic recombination techniques, and site-specific DNA cleavage and ligation can be facilitated using enzymes generally known in the art, etc. The vector of the present invention is a plasmid vector, course All common vectors, including mid vectors, bacteriophage vectors, viral vectors, etc. Preferably, they are plasmid vectors.

Suitable expression vectors can include expression control element sequences such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. The start codon and the stop codon must be functional in the subject when the gene construct is administered and must be in frame with the coding sequence. The promoter may be constitutive or inducible and may include the origin of replication in the case of a replicable expression vector. It may also include selectable markers that confer a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins for screening transformed microorganisms.

Specifically, the present invention provides a vector which is pTCE expressing Tce DNA polymerase having the amino acid sequence of SEQ ID NO: 10.

In addition, the present invention relates to a microorganism transformed with the vector.

Microorganisms usable as host cells that can be transformed are not particularly limited. For example, E. coli (Escherichia coli), Rhodococcus (rhodococcus), Pseudomonas (Pseudomonas), Streptomyces (Streptomyces), Staphylococcus ( Staphylococcus ), Syfolobus , Thermoplasma , Thermoproteus and the like. Preferably, as E. coli, E. coli XL1-blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH series, E. coli TOP10, E. coli HB101 and the like.

Specifically, the present invention provides Escherichia coli Rosetta (DE3) pLysS / pTCE, which is a microorganism transformed with a pTCE vector. Escherichia coli Rosetta (DE3) pLysS / pTCE was deposited with the accession number KACC95097P as of February 11, 2009 to KACC (Korean Agricultural Culture Collection, 88-20 Seodun-dong, Gwonseon-gu, Suwon-si).

Vector introduction into host cells can be carried out by known methods such as electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, PEG, textlan sulfate, lipofectamine, etc. .

The present invention also relates to a kit comprising a heat resistant Tce DNA polymerase isolated from a thermococcal sealer having the amino acid sequence of SEQ ID NO: 10.

Tce DNA polymerase of the present invention can be used as a kit component of a nucleic acid amplification reaction. Such nucleic acid amplification reactions include polymerase chain reation (PCR), ligand chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), strand displacement amplification (SDA), and branched DNA signal amplification. (branched DNA signal amplification), nested PCR (nested PCR), multiplex PCR (multiplex PCR) and the like.

In addition to the Tce DNA polymerase, the kit for nucleic acid amplification reaction of the present invention includes one or more other components, solutions or devices suitable for the analysis method. For example, the kit of the present invention may include one or more components selected from a container containing a detection primer, an amplification tube or container, reaction buffer, dNTPs, RNase, sterile water, and the like.

Kits containing the Tce DNA polymerase of the present invention can be usefully used in various fields such as genetic engineering and molecular biology experiments, clinical diagnosis, forensic research.

The present invention also relates to a method for producing Tce DNA polymerase.

The Tce DNA polymerase of the present invention can be prepared by a method of isolation from a source of natural origin, by chemical synthesis, or by introducing a vector encoding a Tce DNA polymerase into a suitable host, expressing it, and then separating it from the host.

Systems for preparing an expression vector capable of efficiently expressing Tce DNA polymerase and expressing it in a microorganism are well known to those skilled in the art, and can be implemented by various modifications / applications. One example of such an implementation is as follows.

Tce DNA polymerase comprises the steps of (i) preparing a vector expressing the Tce DNA polymerase of the present invention; (ii) transforming the vector with a microorganism; (iii) culturing the transformant; And (iv) may be prepared by a process comprising the step of recovering the protein from the transformant.

The type of vector in step (i), the method of transforming the vector in step (ii) and the type of microorganism used as a host cell are as described above.

The culturing process of step (iii) is carried out according to a method well known for the type of microorganism used as a transformant, and conditions such as a medium component, a culture temperature and a culture time can be appropriately adjusted. Specifically, the culture medium contains all the nutrients essential for the growth and survival of microorganisms such as carbon sources, nitrogen sources, trace element components and the like. The pH of the medium can be adjusted appropriately, and components such as antibiotics can be included.

In addition, the expression of the protein may be induced by treating an inducer such as isopropyl thiogalactopyranoside (hereinafter referred to as “IPTG”). The type of inducer to be treated may be determined according to the vector system, and conditions such as the induction agent administration time and dosage may be appropriately controlled.

In step (iv) the protein is recovered and purified in a conventional manner. For example, the cells recovered by the centrifugation method are crushed using a French press, an ultrasonic crusher or the like. If protein is secreted into the culture, the culture supernatant is collected. When aggregated by overexpression, it can be obtained by dissolving, denaturing and refolding the protein in the appropriate solution. Oxidation and reduction systems of glutathione, dithiothreitol, β-mercaptoethanol, β-mercaptomethanol, cystine and cystamine are used, and refolding agents such as urea, guanidine, arginine and the like are used. Some of the salts may be used with the refolding agent.

At this time, a step of heat treatment for 10 to 60 minutes at 70 to 85 ℃ can be added.

Proteins obtained by culturing the transformant can be used without purification and are subjected to conventional biochemical separation techniques such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, chromatography Photography and the like can be used alone or in combination. Chromatography includes ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like.

The scale of the protein manufacturing process can be adjusted to suit the purpose. In addition, there may be additional and charging processes between each process.

Thus obtained protein can be converted into a salt by a well-known method when obtained as a free body, and when obtained into a salt, it can be converted into a free body or another salt by a well-known method.

In a specific embodiment of the present invention, the cells were harvested by centrifugation of the cultured cells, and then crushed by ultrasound and subjected to column chromatography using a HiTrap ^™ Heparin HP column (GE Healthcare).

Proteins obtained by this process exist in a "substantially pure" state that is sufficiently separated from other proteins in a physical environment that is distinct from the naturally occurring environment. The protein prepared by the above method can be used as a heat-resistant enzyme having excellent DNA polymerization activity in a wide range of fields from molecular biology and genetic engineering experiments to early diagnosis of viral and cancer genes, genetic disease diagnosis and forensic research.

Since the Tce DNA polymerase of the present invention has excellent DNA polymerization activity and high correction activity, it can be widely used for various nucleic acid polymerization reactions such as PCR.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Culture of Thermococcus Sealer Strains

Thermococcus celer strain (DSM 2476) was incubated in DifcoTM Marine Broth 2216 medium. DifcoTM Marine Broth 2216 medium (5.0 g of peptone per liter, 1.0 g of yeast extract, 1.0 g of Ferric Citrate, 19.45 g of NaCl ₂ , 5.9 g of MgCl ₂ , 3.24 g of MgSO ₄ , 1.8 g of CaCl ₂ , KCl 0.55 g, Na ₂ B ₄ O ₇ 0.16 g, KBr 0.08 g, SrCl ₂ 34.0 mg, H ₃ BO ₃ 22.0 mg, NA ₂ SIO ₃ 4.0 mg, NaF 2.5 mg, NH ₄ NO ₃ 1.6 mg, Na ₂ HPO ₄ 8.0 mg) was prepared, and then adjusted to pH 5.8 with 5 N sulfuric acid. At this time, peptone and yeast extract (prepared as 10% stock) and elemental sulphur are prepared separately without addition together. The solution was heated overnight (about 8 hours) in an incubator at 100 ° C. to sterilize and remove oxygen from the solution. Some of the precipitate formed after the heating was quickly filtered and then again heated for 3 hours in a 100 ℃ incubator. 0.5 g of Elemental sulphur was added to a 120 ml serum bottle (Wheaton Co., USA) and 100 ml of heated electric media solution was added. The gas inside the serum bottle containing the media solution was replaced with nitrogen gas for 30 minutes. Then, the bottle bottle was sealed with a rubber bottle cap and an aluminum ring, and then sterilized at 95 ° C. for 24 hours. After the sterilized peptone and yeast extract were added to the prepared medium by using a sterilized syringe, a small amount of 5% Na ₂ S? 9H ₂ O solution was injected, and the 1% inoculation of the thermococcal sealer strain was then used at 80 ° C. Incubated for 24 hours.

Example 2: Isolation of Genomic DNA

The cultures incubated under anaerobic conditions were filtered to remove sulfur, the cells were recovered by centrifugation, and the cells were suspended in 10 mM Tris-HCl (pH 8.0) buffer containing 20% sucrose. After freezing and thawing, the cell membrane was attenuated twice, and Triton X-100 was added to a final concentration of 0.3%, followed by reaction at room temperature for 1 hour. To this was added SET (150 mM NaCl / 1 mM EDTA / 20 mM Tris-HCl (pH 8.0)) buffer, SDS and RNase were added and reacted at 55 ° C for 1 hour, followed by proteinase K ( proteinase K) was added and reacted at 37 ° C for 1 hour and at 55 ° C for 2 hours. Subsequently, the same amount of chloroform / isoamylalcohol (24: 1) was added thereto, followed by shaking overnight, followed by 2 times volume of 100% ethanol and 1/10 volume of 3M sodium acetate ( Genomic DNA was precipitated by addition of sodium acetate (pH 5.2), washed with 70% ethanol and dried in vacuo and dissolved in TE (10 mM Tris-HCl (pH 7.6) / 1 mM EDTA) buffer. The isolated thermococcal sealer genomic DNA was quantified by measuring the absorbance at 260 nm wavelength.

Example 3: Selection of DNA Polymerase Genes

Thermococcus kodakaraensis KOD1, a previously reported thermococcus kodakaraensis KOD1 template, was used as a template for screening Tce DNA polymerase genes from thermococcal sealer genomic DNA (see Takagi M. et al.). , 1997, Appl.Environ.Microbiol . 63 (11), 4504-4510), Thermococcus sp. NA1 (Kim YJ et al ., 2007, J Microbiol Biotechnol. 17 (7) 1090-7), and well conserved sites in the DNA polymerase genes of Pyrococcus furiosus (Lundberg, KS et al ., 1991, Gene 108 (1), 1-6). The mixed primers of SEQ ID NOS: 1 and 2 were prepared with reference to two amino acid sequences. SEQ ID NO: 1 primer is the nucleotide sequence encoding the amino acid sequence EYDIPFA. SEQ ID NO: 2 primer is a complementary sequence of nucleotide sequence encoding amino acid sequence YYIENQV. PCR was performed using these primers as a template for thermococcal sealer genomic DNA. The PCR conditions were reaction for 3 minutes at 94 ° C, 1 minute at 94 ° C, 1 minute at 47 ° C and 6 minutes at 67 ° C. The reaction process was repeated 25 times for 6 minutes, and the reaction product was electrophoresed on 0.8% agarose gel for 10 minutes at 72 ° C. to confirm that the DNA fragment of about 1.8 kb was amplified. The PCR reaction products were also purified on the 0.8% agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). The purified DNA was commissioned by CoBio Systems Co., Ltd. to determine the nucleotide sequence, and compared with other DNA polymerase sequences of known species. Therefore, it was confirmed that the purified 1.8 kb DNA fragment was part of the Tce DNA polymerase gene of the Thermococcus sealer. This was used as the center for screening the Tce DNA polymerase gene of the Thermococcus sealer.

SEQ ID NO: 1 Primer (T-111N): 5'-GARTACGACATACCCTTYGC-3 '

SEQ ID NO: 2 Primer (T-CR): 5'-AACCTGGTTCTCRATKTAGTA-3 '

(Wherein R is an A or G base, Y is a C or T base, and K is a base of G or T.)

Example 4 Using Primer Walking Method Tce Search for unknown contiguous DNA sites (N and C termini) from the center of the DNA polymerase gene

Four internal primers were prepared based on the DNA base sequence of 1.8 kb of the Tce DNA polymerase gene already secured in Example 3 (SEQ ID NOs: 3, 4, 5, and 6 primers). The Walking SpeedUp ^™ Premix Kit (seegene, Korea) was used to find unknown contiguous sequences. In order to clone the gene region corresponding to the 5 'upstream (N-terminal region) of the Tce DNA polymerase gene, four random primers and a SEQ ID NO: 3 primer (Tce-N-1) in the Walking SpeedUp ^TM Premix Kit and The first PCR was performed using Thermococcus sealer genomic DNA. As a template, a second PCR was performed using a SEQ ID No. 4 primer (Tce-N-2) and a second PCR primer in the kit as a template to specifically amplify a DNA fragment of about 1,700 bp. The amplified DNA fragments were electrophoresed on an agarose gel, extracted and purified using a QIA quick Gel Extraction Kit (QIAGEN Gmbh, Germany), and commissioned to Cobiobiosystem to determine the base sequence. As a result of determining the base sequence of these fragments, the base sequence in the 5 'upstream (N-terminal part) including the start codon was obtained.

In addition, four random primers and SEQ ID NO: 5 primers (Tce-C-) in the Walking SpeedUp ^TM Premix Kit were used to clone the gene region corresponding to the unknown 3 'downstream (C-terminal region) adjacent to the conserved nucleotide sequence. 1) and the first PCR was performed using Thermococcus sealer genomic DNA. The PCR product was specifically amplified about 700 bp of DNA fragment using the SEQ ID NO: 6 primer (Tce-C-2) and the second PCR primer in the kit. The amplified DNA fragments were electrophoresed on an agarose gel, extracted and purified using a QIA quick Gel Extraction Kit (QIAGEN Gmbh, Germany), and commissioned to Cobiobiosystem to determine the base sequence. As a result of determining the nucleotide sequence of these fragments, a nucleotide sequence in the 3 'upstream (terminal C) portion including the stop codon was obtained. Thus, the entire nucleotide sequence encoding the Tce DNA polymerase gene was determined (SEQ ID NO: 9).

Internal primer sequence

SEQ ID NO: 3 Primer (Tce-N-1): 5'-GGAGGTCGATCTTCTTCCAGGT-3 '

SEQ ID NO: 4 Primer (Tce-N-2): 5'-TATCATCTCCTTCTCGGTCGAGA-3 '

SEQ ID NO: 5 Primer (Tce-C-1): 5'-GTGATCCACGAGCAGATAACGA-3 '

SEQ ID NO: 6 Primer (Tce-C-2): 5'-CGAGGTTCCGCCGGAGAAA-3 '

Tce DNA polymerase gene sequences were analyzed using the DNASTAR (DNASTAR Inc., USA) program, and thermococcus fumicolans and thermococcus codacarensis using the NCBI BLAST program. The sequence and similarity of the DNA sequences of Thermococcus kodakaraensis KOD1 and Pyrococcus furiosus DNA polymerase were compared.

As a result, the entire nucleotide sequence of the DNA polymerase gene isolated from the Thermococcus celer strain consisted of a total of 2,325 bp including a start codon (ATG) and a stop codon (TGA), and a total of 774 amino acids. (SEQ ID NO: 10). From the amino acid sequence, it was estimated that the molecular weight of the DNA polymerase enzyme of the present invention was about 89,788.9 Da. In addition, as a result of comparing the amino acid sequence of the DNA polymerase of the present invention isolated from the Thermococcus celer strain with other amino acid sequences of the DNA polymerase, Thermococcus fumicolans (see Marie-Anne) Cambon-Bonavita et al ., 2000, Extremophiles, Aug., 1431-0651) showed 87.9% sequence homology with the DNA polymerase, and the Thermococcus kodakaraensis KOD1 (Takagi M. et. al ., 1997, Applied and Environmental Microbiology, Nov., 4504-4510), having 86.2% sequence homology with Pyrococcus furiosus (Lundberg, KS et al ., 1991, Gene 108 (1), 1-6) and 76.9% sequence homology with the DNA polymerase was confirmed (see Fig. 1).

Example 5: Tce  Construction of Expression Vector pTCE for Expression of DNA Polymerase Gene

A recombinant plasmid (pTCE) incorporating a Tce DNA polymerase gene into an expression vector was constructed as follows (see FIG. 2).

First, Tce DNA polymerase SEQ encoding the N- terminal amino acid sequence from the nucleotide sequence of the Tce DNA polymerase gene determined in Example 4 to obtain 7 gene primers (TceN) (5 'terminal primer) and the C- terminal amino acid sequence After preparing each SEQ ID No. 8 primer (TceC) (3 'terminal primer) complementary to the DNA base coding for, the Tce DNA polymerase gene was amplified by PCR. The primer of SEQ ID NO: 7 was synthesized to contain the restriction enzyme Nde I cleavage site containing the start codon (ATG) of the Tce DNA polymerase gene and the primer of SEQ ID NO: 8 to the stop codon and restriction enzyme Hind III cleavage site, respectively. Thermococcal sealer genomic DNA was subjected to PCR using SEQ ID NOs: 7 and 8 primers. PCR was carried out at 95 ° C using a mixed solution consisting of 0.2 μg thermococcal sealer genomic DNA, 20 pmole 5 ′ and 3 ′ primers, 200 μM dNTPs, 10X PyroAce DNA polymerase buffer and 2.5 U Super PyroAce DNA polymerase. After reacting for 3 minutes at 30 ℃, 30 seconds at 58 ℃, 30 seconds, and repeated 30 times at the conditions of 120 seconds at 72 ℃, and then reacted for 10 minutes at 72 ℃. Electrophoresis on 0.8% agarose gel with DNA size marker confirmed the presence of a band at about 2.3 kb. After completion of the PCR, the reaction mixture was electrophoresed on a 0.8% agarose gel, and about 2.3 kb of DNA product amplified by PCR was purified by using a QIAquick Gel Extraction Kit (Qiagen Gmbh, Germany). The recovered DNA was digested with restriction enzymes Nde I and Hin dIII, and then electrophoresed again on a 0.8% agarose gel, and a gel fragment corresponding to about 2.3 kb was purified from the Tce DNA polymerase gene fragment using an electric kit. Was recovered. The purified Tce DNA polymerase gene fragment was inserted into the expression vector pET-22b (+) (Novagen, USA) digested with the same restriction enzyme and ligated overnight at 16 ° C. using a T4 DNA ligase. E. coli Escherichia coli Rosetta (DE3) pLysS (Stratagene, USA) was transformed (Sambrook, J. et al ., 1989, Molecular cloning, a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press). Plasmid DNA was isolated from the transformants by alkaline lysis, and then digested with restriction enzymes Nde I and Hin dIII, followed by electrophoresis with DNA size markers on 0.8% agarose gel to express the Tce DNA polymerase gene. It was again confirmed that it was correctly inserted in the vector. The expression vector constructed for the expression of the Tce DNA polymerase gene constructed as described above was named pTCE (FIG. 2). The E. coli containing the expression vector and the expression vector containing the Tce DNA polymerase gene and the gene were deposited on February 11, 2009, at the Korea Institute of Agricultural Biotechnology, Rural Development Agency, Agricultural Science and Biotechnology Center, namely Escherichia coli Rosetta (DE3) pLysS / pTCE, Accession No. KACC95097P.

SEQ ID NO: 7 Primer (TceN):

5'-GTGGT CATATG ATCCTGGAC-3 '

Nde I

SEQ ID NO: 8 Primer (TceC):

5'-AAAT AAGCTT CACCCCTTCC -3 '

Hind III

Example 6: Expression and Purification of Recombinant DNA Polymerase Gene

The recombinant protein transformed into Escherichia coli by the method of Example 5 was expressed as Tce DNA polymerase protein from Escherichia coli Rosetta (DE3) pLysS / pTCE, and the expressed protein was purified as follows. E. coli Rosetta (DE3) pLysS containing the recombinant plasmid in Example 5 was inoculated in LB liquid medium added with 100 ug / ul ampicillin overnight at 37 ℃ and 5 ml inoculated in 500 ml of the same medium 37 Incubated at ℃. When the absorbance at 600 nm was about 0.6, IPTG (isopropyl β-D-galactopyranoside) was added to a final concentration of 0.1 mM and incubated overnight. Cells (2.1 g / wet weight) were recovered by centrifugation at 6,000 rpm for 15 minutes and then 25 ml of sonication buffer containing 20 mM Tris-HCl (pH 8.0), 0.5 mM NaCl containing 1 mM PMSF. Suspension) was disrupted by ultrasonication and centrifuged at 18,000 rpm for 15 minutes to remove E. coli cell debris. Next, after heat treatment at 80 ° C. for 30 minutes, most of the proteins derived from E. coli were removed by centrifugation to recover the supernatant. The supernatant was finally purified by column chromatography using HiTrap ^™ Heparin HP column (GE Healthcare). Tce DNA polymerase purified as described above was stored in storage buffer (20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.1% Tween 20, 0.5% Nonidet P40, 200 mM KCl, 50% Glycerol) It was used for diagnosing the enzymatic properties of Tce DNA polymerase and storing it at -20 ° C and performing PCR using Tce DNA polymerase. In purifying heat-resistant Tce DNA polymerase from E. coli Rosetta (DE3) pLysS having a recombinant plasmid pTCE, the purification step results are shown in Table 1 below.

Purification of Tce DNA Polymerase from E. Coli Rosetta (DE3) pLysS / pTCE Total protein (mg) Total activity (Unit) Special activity (U / mg) Recovery
(%) Sonication 1561.2 115832.48 74.19 100 Heat 274.64 99410.78 361.97 85.82 Heparin 71.82 65406.21 910.7 56.47

The amount of protein in purification steps was determined using Lowry Assay . See J. Biol. Chem. 193, 261 (1951). In addition, denatured gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) was performed to confirm the degree of purification (see Figure 3). The degree of purification according to the purification step of the E. coli Rosetta (DE3) pLysS / pTCE derived Tce DNA polymerase is shown in Figure 3 by SDS-PAGE electrophoresis picture. lane 1 is an ultrasonic disrupted sample of E. coli Rosetta (DE3) pLysS / pTCE cells cultured without IPTG induction, lane 2 is an ultrasonic crushed sample of E. coli Rosetta (DE3) pLysS / pTCE cells cultured by IPTG induction, Lane 3 is a sample after heat treatment at 80 ℃ for 30 minutes, lane 4 is a sample after HiTrap ^TM Heparin HP column chromatography. Through purification, the Tce DNA polymerase protein had a molecular weight of about 90,000 Da, which was similar to 89,788.9 Da, which was estimated from the amino acid sequence of Example 4 above. lane M represents a protein high molecular weight marker. The specific activity of the purified enzyme is 910.7 U / mg.

Example 7: Tce  Investigation of enzyme properties in DNA polymerization activity of DNA polymerase

The enzymatic properties of the DNA polymerization activity of Tce DNA polymerase were investigated based on the following DNA polymerization activity measurement method (see Choi, JJ et al. , 1999, Biotechnol. Appl. Biochem. 30 , 19- 25). Reaction mixture ( Tce DNA polymerase purified in Example 6, 1.25 μg activated calf thymus DNA, 50 mM Tris-HCl, pH 7.5), 80 mM KCl, 14 mM MgCl ₂ , 1 mM β-mercaptoethanol, 100 μM dATP , dCTP and dGTP, 10 μM dTTP, 0.5 μCi [ methyl - ³ H] thymidine 5′-triphosphate) were reacted at 75 ° C. for 10 minutes and then quenched on ice. The reaction solution was added dropwise to a DE-81 filter paper disk (23 mm, Whatman Co., USA) and dried at 65 ° C., followed by 0.5 M sodium phosphate (pH 7.0) buffer. After washing for 10 minutes in 70% ethanol in order for 5 minutes, it was dried again at 65 ℃. The DE-81 filter paper disk prepared as above was used to measure DNA polymerization activity using the LS 6500 scintillation system (Beckman Co., USA). One unit (U) of DNA polymerization activity was defined as the amount of DNA polymerase capable of incorporating 10 pmol [ ³ H] TTP at 75 ° C. for 10 minutes using the reaction mixture.

When examining the enzyme properties in the DNA polymerization activity, it was carried out in the above method by changing the pH of the reaction mixture or the concentration of each component or the reaction temperature.

In order to observe the effect of pH on the enzymatic activity of the Tce DNA polymerase purified in Example 6, 50 mM MOPS buffer was used in the pH 6.0-7.0 range, and 50 mM Tris-HCl buffer was used in the pH 7.0-9.0 range. The enzyme activity was examined. As a result, Tce DNA polymerase showed maximum DNA polymerization activity at pH 6.5 (see FIG. 4). Figure 4 is a graph showing the relative degree of DNA polymerization activity according to the pH of the Tce DNA polymerase, in Figure 4 ▲ shows the results using a 50 mM MOPS buffer, ● is the result using a 50 mM Tris-HCl buffer Indicates.

The effect of temperature on the DNA polymerization activity of the Tce DNA polymerase purified in Example 6 was examined at 55-90 ° C., showing the maximum DNA polymerization activity at 75 ° C. (see FIG. 5). 5 is a graph showing the relative degree of DNA polymerization activity according to the temperature of the Tce DNA polymerase.

After a while the Tce DNA polymerase stored respectively at 90 ℃ and 95 ℃ for 3.5 hours sampling every 30 minutes, examination, the thermal stability by measuring DNA polymerization activity as described above, Tce DNA polymerase is 90 ℃ 50% of the DNA polymerization activity was maintained for about 2 hours (see FIG. 6). FIG. 6 is a graph showing the thermosafety of the thermococcal sealer DNA polymerase. In FIGS. 6 and 6, the Tce DNA polymerase incubated at 90 ° C. and 95 ° C., respectively.

In the DNA polymerase activity of the Tce DNA polymerase 1 surveyed the effects of cationic (monovalent cation) results, KCl was shown to inhibit the DNA polymerase activity of the Tce DNA polymerase (see Fig. 7), the best (NH ₄ ) ₂ SO ₄ concentration was 20-35 mM (see Figure 8). 7 is Tce DNA and relative graphs showing the degree of DNA polymerase activity according to the KCl concentration of the polymerase, Figure 8 (NH _{4) 2} SO DNA polymerization activity level graphs relatively showing the according to the _fourth concentration of the Tce DNA polymerase to be.

As a result of examining the effect of Mg ²⁺ , a divalent cation, on the DNA polymerization activity of Tce DNA polymerase, the optimum Mg ²⁺ concentration was 4-16 mM (see FIG. 9). 9 is a graph showing the relative degree of DNA polymerization activity according to the concentration of Mg ²⁺ of Tce DNA polymerase.

Example 8: Tce  Measurement of 3 '→ 5' Nucleic Acid Terminal Hydrolase Activity of DNA Polymerase

The 3 '→ 5' nucleic acid terminal hydrolase activity of Tce DNA polymerase was measured as follows. First, as a substrate for measuring 3 ′ → 5 ′ exonuclease activity, a pBluescript SK-vector was cleaved with restriction enzyme Not I, followed by radioisotope [α- ³² P] dCTP and Klenow fragments were used to label the 3 ′ end of the pBluescript SK-vector digested with electrorestriction enzyme. Next, the reaction mixture ( Tce DNA polymerase, the radioisotope labeled substrate at the 3 'end, 50 mM Tris-HCl (pH 7.5), 1 mM 2-mercaptoethanol, 10 mM MgCl ₂ , 0.01% BSA), respectively Samples were taken at 10-minute intervals, stored at 75 ° C for 1 hour in the presence or absence of dNTP, quenched on ice, centrifuged with 5% trichloroacetic acid solution, and then the supernatant was recovered. The nucleic acid terminal hydrolase activity was measured using the LS 6500 scintillation system. As a result, it was confirmed that the Tce DNA polymerase had 3 '→ 5' nucleic acid terminal hydrolase activity (see FIG. 10). In particular, it can be seen that the enzyme activity is higher in the absence of dNTP. The 3 '→ 5' nucleic acid terminal hydrolase activity of Tce DNA polymerase was estimated by amino acid sequence comparison and was consistent with the experimental results. This 3 ′ → 5 ′ nucleic acid terminal hydrolase activity is also called corrective activity and plays a role of enhancing the accuracy during DNA polymerization. FIG. 10 is a graph showing 3 ′ → 5 ′ nucleic acid terminal hydrolase activity of thermococcal sealer DNA polymerase. In FIG. 10, 3 ′ → 5 ′ nucleic acid terminal hydrolase activity in the presence of dNTP and ○ in the absence of dNTP. The result of a measurement is shown, respectively.

Example 9: Tce  Determination of Optimum Conditions for PCR Using DNA Polymerase

To examine the availability of Tce DNA polymerase in PCR where heat-resistant DNA polymerase is most importantly used, and to determine the optimal composition of the reaction buffer for PCR using Tce DNA polymerase, PCR was performed as follows. Was performed. 23 ng lambda phage genomic DNA was used as a template, respectively, based on the optimal conditions determined by the 5 'and 3' terminal primers of 1 pmole, 250 μM dNTPs, and the characterization of the Tce DNA polymerase in Example 8 above. Repeat the procedure of 20 seconds at 94 ° C. and 1 minute at 72 ° C. for 25 times using 1 × Tce DNA polymerase reaction buffer and 0.1 unit of purified Tce DNA polymerase (see Example 6 above) as a reaction mixture. PCR results were confirmed by 0.8% agarose gel electrophoresis. In determining the optimal reaction buffer composition in PCR using Tce DNA polymerase, the pH of the reaction buffer solution or the concentration of each component were varied.

As a result of examining the effect of pH on PCR using Tce DNA polymerase, the optimum pH of the PCR reaction buffer solution was 7.8 (see FIG. 11). Figure 11 shows the results according to pH in the polymerase chain reaction using Tce DNA polymerase, in Figure 11 M represents a 1 kb DNA ladder (New England BioLabs), each lane represents a pH. PCR amplification size is 2 kb.

As a result of examining the effects of monovalent cations on PCR using Tce DNA polymerase, KCl showed a result of inhibiting PCR using Tce DNA polymerase (see FIG. 12). 12 shows the results according to KCl concentration in the polymerase chain reaction using Tce DNA polymerase. In FIG. 12, M represents a 1 kb DNA ladder (New England BioLabs), and each lane represents KCl concentration. .

As a result of investigating the effect of (NH ₄ ) ₂ SO ₄ on PCR using Tce DNA polymerase, the optimum concentration was 10 mM (see FIG. 13). FIG. 13 shows the results according to (NH ₄ ) ₂ SO ₄ concentration in the polymerase chain reaction using Tce DNA polymerase. In FIG. 13, M represents a 1 kb DNA ladder (New England BioLabs), and each lane Shows the (NH ₄ ) ₂ SO ₄ concentration.

As a result of examining the effect of divalent cation Mg ²⁺ on PCR using Tce DNA polymerase, the optimum concentration was 5 mM (see FIG. 14). Figure 14 shows the results according to the concentration of MgCl ₂ in the polymerase chain reaction using Tce DNA polymerase, in Figure 14 M represents a 1 kb DNA ladder (New England BioLabs), each lane represents the MgCl ₂ concentration It is shown.

The results showed that Tce DNA polymerase was available for PCR, and the optimal reaction buffer composition for PCR using Tce DNA polymerase contained 50 mM Tris-HCl (pH) containing Triton X-100 as a stabilizer. 7.8), 10 mM (NH ₄ ) ₂ SO _4, 5 mM MgSO _4, 0.1% Triton X-100.

Example 10: Tce  Determination of PCR Amplifiable Size Using DNA Polymerase

23 ng of lambda phage genomic DNA as a template, respectively, 1 pmole 5 'and 3' terminal primer, 250 μM dNTPs, 1X Tce DNA polymerase optimal reaction buffer determined in Example 9 and purified in Example 6 According to the length of the target amplification product expected to investigate the amplifiable length of the PCR using the Tce DNA polymerase, PCR was performed as follows. The reaction was carried out at 94 DEG C for 20 seconds, and then at 50 DEG C / kb depending on the expected size of the DNA fragment at 72 DEG C. After PCR reaction, 0.8% agarose gel electrophoresis confirmed the PCR results. As a result, Tce DNA polymerase can be synthesized up to 6 kb by PCR using genomic DNA of lambda phage under optimal reaction buffer solution. It could be confirmed (see Fig. 15). FIG. 15 shows a result of polymerase chain reaction using Tce DNA polymerase using lambda phage genomic DNA as a reaction mixture under an optimal reaction buffer solution. In FIG. Represents the 1 kb DNA ladder (New England BioLabs).

Example 11: Tce  PCR at the shortest amplification time using DNA polymerase

Using 23 ng lambda phage genomic DNA as a template, 1 pmole of 5 'and 3' terminal primers, 250 μM dNTPs, the optimal reaction buffer for each polymerase and the respective polymerase were Tce , Taq And to investigate the shortest amplification time of PCR using Pfu DNA polymerase PCR was performed as follows according to the amplification time. The reaction was carried out at 94 DEG C for 20 seconds and then at 72 DEG C for 5, 10, 20 and 40 seconds. After the PCR reaction, 0.8% agarose gel electrophoresis confirmed the PCR results. In the case of PCR using Taq and Pfu DNA polymerase, Tce DNA polymerase was able to synthesize 2 kb in 40 seconds. 2 kb synthesis was possible (see FIG. 16). FIG. 16 shows the results of polymerase chain reaction using a thermococcal sealer DNA polymerase under an optimal reaction buffer using lambda phage genomic DNA as a template. In FIG. 16, lane shows amplification time at 72 ° C. The first lane 5-40 is the PCR result using Pfu DNA polymerase, the middle lane 5-40 is the PCR result using Taq DNA polymerase, the last lane 5-40 is the PCR result using Tce DNA polymerase, M Represents the 1 kb DNA ladder (New England BioLabs).

Example 12: Tce  Investigation of accuracy in PCR using DNA polymerase

PCR fidelity (PCR fidelity) in PCR using Tce DNA polymerase was performed by slightly modifying the method of Lundberg as follows (Lundberg et al ., 1991, Gene 108 (1), 1). -6). In fact, the experiment was measured by the same method as used by Song et al . And Choi, and the same expression vector pJR- lacZ (5.7 Kb) and primers were used (Song JM et al ., 2007, Enzyme Microbe.Technol . 40 , 1475-1483; Choi JJ et al., 2008, Appl. Environ.Microbio l. 74, 6563-6569). First, the 835 bp fragment of the 5 'portion of the expression vector pJR- lacZ into which the lacZ gene was inserted was amplified by PCR using Tce DNA polymerase. At this time, PCR using the Tce DNA polymerase later by performing the same PCR using DNA polymerase already advanced to the goods in place Tce DNA polymerase was to compare the results. Next, each of the PCR amplification products was digested with restriction enzymes Bam HI and Cla I, and then reinserted into the expression vector digested with the same restriction enzymes, and then transformed into E. coli by electroshock gene transfer. Antibiotic ampicillin and IPTG and X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) was evenly plated in agar plate medium and incubated for 16 hours at 37 ℃. Subsequently, the agar plate was stored at 4 ° C. for 2 hours, and then the number of blue colonies and white colonies was counted, and mutation frequencies and error rates were calculated based on the numbers of blue colonies and white colonies. The results are shown in Table 2 below, and the PCR accuracy in PCR using Tce DNA polymerase was similar to that of Pfu DNA polymerase, but much higher than that of Taq DNA polymerase which is mainly used for general PCR. It was confirmed that Tce DNA polymerase can be used for PCR requiring a high degree of accuracy.

Error Rate Comparison of PCR Products with Tce DNA Polymerase and Taq and Pfu DNA Polymerase Target (ng) Template doubling Mutant frequencies Error rate (10-5) Tce 58.85 6.12 0.023 0.48 Taq 58.85 5.84 0.086 1.77 Pfu 58.85 4.43 0.016 0.43

The heat-resistant polymerase of the present invention is expected to be applied in a wide range of fields such as genetic engineering research, early diagnosis of viral and cancer genes, genetic disease diagnosis, GMO and forensic research.

1 shows the amino acid sequence of Tce DNA polymerase according to the conventional heat-resistant B-based DNA polymerases ( Tce , Thermococcus sealer; Tfu , Thermococcus fumicolance ; Tko , Thermococcus codcarensis code source; Pfu , Pyrococcus) Compared to the amino acid sequence of Puriosus).

Figure 2 shows the construction of the recombinant plasmid pTCE for the expression of the Tce DNA polymerase gene.

Figure 3 shows the results of SDS modified gel electrophoresis according to the purification step (heat treatment, column chromatography) of Tce DNA polymerase expressed in E. coli.

4 is a graph showing the relative degree of DNA polymerization activity according to pH (pH 6.0-9.0) of Tce DNA polymerase. Optimal activity was shown in 50 mM MOPS buffer at pH 6.5.

5 is a graph showing the relative degree of DNA polymerization activity according to the temperature of the Tce DNA polymerase (55-90 ℃). Optimum activity was shown at 75 ° C.

Figure 6 is a graph showing the thermal stability of the Tce DNA polymerase relative to 90 ℃ and 95 ℃, respectively. 50% DNA polymerization activity was maintained at 90 ° C. for about 2 hours and at 95 ° C. for about 30 minutes.

7 is a graph showing the relative degree of DNA polymerization activity according to the KCl concentration (0-100 mM) of Tce DNA polymerase. KCl has been shown to inhibit the DNA polymerization activity of Tce DNA polymerase.

8 is a graph showing the relative degree of DNA polymerization activity according to (NH ₄ ) ₂ SO ₄ concentration (0-35 mM) of Tce DNA polymerase. Optimal (NH ₄ ) ₂ SO ₄ concentrations were 20-35 mM.

9 is a graph showing the relative degree of DNA polymerization activity according to MgCl ₂ concentration (0-20 mM) of Tce DNA polymerase. The optimal MgCl ₂ concentration was 4-16 mM.

10 is a graph showing the 3'-5 'nucleic acid terminal hydrolase activity of Tce DNA polymerase in the presence or absence of dNPT. It was confirmed that Tce DNA polymerase had 3 '→ 5' nucleic acid terminal hydrolase activity.

Figure 11 shows the results according to pH (pH 7.0-9.0) in the polymerase chain reaction (PCR) using Tce DNA polymerase. Optimal activity was shown in 50 mM Tris-HCl buffer at pH 7.8.

Figure 12 shows the results according to the KCl concentration (0-100 mM) in the polymerase chain reaction using Tce DNA polymerase. KCl has been shown to inhibit the DNA polymerization activity of Tce DNA polymerase.

Figure 13 shows the results according to (NH ₄ ) ₂ SO ₄ concentration (0-20 mM) in the polymerase chain reaction using Tce DNA polymerase. The optimal (NH ₄ ) ₂ SO ₄ concentration was 10 mM.

Figure 14 shows the results according to the concentration of MgCl ₂ (0-10 mM) in the polymerase chain reaction using Tce DNA polymerase. The optimal MgCl ₂ concentration was 5 mM.

Figure 15 shows the results of the polymerase chain reaction with amplification time of 50 seconds per kb using the Tce DNA polymerase under the optimal reaction buffer using lambda phage genomic DNA template. 2, 4 and 6 kb of DNA were amplified.

16 shows various amplification times using Tce DNA polymerase, Taq DNA polymerase and Pfu DNA polymerase under the optimum reaction buffer using lambda phage genomic DNA as a template (5, 10, 20 and 40 seconds). It shows a comparison result by performing a polymerase chain reaction at.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> DNA polymerase derived from Thermococcus celer and its use <130> P09-058 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer T-111N <400> 1 gartacgaca tacccttygc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer T-CR <400> 2 aacctggttc tcratktagt a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer Tce-N-1 <400> 3 ggaggtcgat cttcttccag gt 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer Tce-N-2 <400> 4 tatcatctcc ttctcggtcg aga 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer Tce-C-1 <400> 5 gtgatccacg agcagataac ga 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer Tce-C-2 <400> 6 cgaggttccg ccggagaaa 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer TceN <400> 7 gtggtcatat gatcctggac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer TecC <400> 8 aaataagctt caccccttcc 20 <210> 9 <211> 2325 <212> DNA <213> Thermococcus celer <220> <221> CDS (222) (1) .. (1200) <400> 9 atg atc ctg gac gct gac tac atc acc gaa gat ggg aag ccc gtc gtg 48 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Val   1 5 10 15 agg ata ttc agg aag gag aag ggc gag ttc aga atc gac tac gac agg 96 Arg Ile Phe Arg Lys Glu Lys Gly Glu Phe Arg Ile Asp Tyr Asp Arg              20 25 30 gac ttc gag ccc tac atc tac gcc ctc ctg aag gac gat tcg gcc atc 144 Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile          35 40 45 gag gag gtg aag agg ata acc gtt gag cgc cac ggg aag gcc gtc agg 192 Glu Glu Val Lys Arg Ile Thr Val Glu Arg His Gly Lys Ala Val Arg      50 55 60 gtt aag cgg gtg gag aag gtc gaa aag aag ttc ctc aac agg ccg ata 240 Val Lys Arg Val Glu Lys Val Glu Lys Lys Phe Leu Asn Arg Pro Ile  65 70 75 80 gag gtc tgg aag ctc tac ttc aat cac ccg cag gac gtt ccg gcg ata 288 Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp Val Pro Ala Ile                  85 90 95 agg gac gag ata agg aag cat ccg gcc gtc gtt gat atc tac gag tac 336 Arg Asp Glu Ile Arg Lys His Pro Ala Val Val Asp Ile Tyr Glu Tyr             100 105 110 gac atc ccc ttc gcc aag cgc tac ctc atc gat aag ggg ctc gtc ccg 384 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro         115 120 125 atg gag ggg gag gag gag ctc aaa ctg atg gcc ttc gac atc gag acc 432 Met Glu Gly Glu Glu Glu Leu Lys Leu Met Ala Phe Asp Ile Glu Thr     130 135 140 ctc tac cac gag gga gac gag ttc ggg gag ggg ccg atc ctg atg ata 480 Leu Tyr His Glu Gly Asp Glu Phe Gly Glu Gly Pro Ile Leu Met Ile 145 150 155 160 agc tac gcc gac ggg gac ggg gcg agg gtc ata acc tgg aag aag atc 528 Ser Tyr Ala Asp Gly Asp Gly Ala Arg Val Ile Thr Trp Lys Lys Ile                 165 170 175 gac ctc ccc tac gtc gac gtc gtc tcg acc gag aag gag atg ata aag 576 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys             180 185 190 cgc ttc ctc cag gtg gtg aag gag aag gac ccg gac gtg ctc gta act 624 Arg Phe Leu Gln Val Val Lys Glu Lys Asp Pro Asp Val Leu Val Thr         195 200 205 tac aac ggc gac aac ttc gac ttc gcc tac ctg aag aga cgc tcc gag 672 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Arg Arg Ser Glu     210 215 220 gag ctt gga ttg aag ttc atc ctc ggg agg gac ggg agc gag ccc aag 720 Glu Leu Gly Leu Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 atc cag cgc atg ggc gac cgc ttc gcc gtc gag gtg aag ggg agg ata 768 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile                 245 250 255 cac ttc gac ctc tac ccg gtg ata agg cgc acc gtg aac ctg ccg acc 816 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Val Asn Leu Pro Thr             260 265 270 tac acg ctc gag gcg gtc tac gag gcc atc ttc ggg agg cca aag gag 864 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Arg Pro Lys Glu         275 280 285 aag gtc tac gcc ggg gag ata gtg gag gcc tgg gaa acc ggc gag ggt 912 Lys Val Tyr Ala Gly Glu Ile Val Glu Ala Trp Glu Thr Gly Glu Gly     290 295 300 ctt gag agg gtt gcc cgc tac tcc atg gag gac gca aag gtt acc ttc 960 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe 305 310 315 320 gag ctc ggg agg gag ttc ttc ccg atg gag gcc cag ctc tcg agg ctc 1008 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu                 325 330 335 atc ggc cag ggt ctc tgg gac gtc tcc cgc tcg agc acc ggc aac ctg 1056 Ile Gly Gln Gly Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu             340 345 350 gtc gag tgg ttc ctc ctg agg aag gcc tac gag agg aac gaa ctg gcc 1104 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala         355 360 365 ccg aac aag ccg agc ggc cgg gaa gtg gag atc agg agg cgt ggc tac 1152 Pro Asn Lys Pro Ser Gly Arg Glu Val Glu Ile Arg Arg Arg Gly Tyr     370 375 380 gcc ggt ggt tac gtt aag gag ccg gag agg ggt tta tgg gag aac atc 1200 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 gtgtacctcg actttcgctc tctttacccc tccatcatca taacccacaa cgtctcgccc 1260 gataccctaa acagggaggg ctgtgagaac tacgacgtcg ccccccaggt ggggcataag 1320 ttctgcaaag attttccggg cttcatcccg agcctgctcg gaggcctgct tgaggagagg 1380 cagaagataa agcggaggat gaaggcctct gtggatcccg ttgagcggaa gctcctcgat 1440 tacaggcaga gggccatcaa gatactggcc aacagcttct acggatacta cggctacgcg 1500 agggcgaggt ggtactgcag ggagtgcgcg gagagcgtta ccgcctgggg cagggagtac 1560 atcgataggg tcatcaggga gctcgaggag aagttcggct tcaaggtgct ctacgcggac 1620 acggacggac tgcacgccac gatccccggg gcggacgccg ggaccgtcaa ggagagggcg 1680 agggggttcc tgagatacat caaccccaag ctccccggcc tcctggagct cgagtacgag 1740 gggttctacc tgaggggttt cttcgtgacg aagaagaagt acgcggtcat agacgaggag 1800 ggcaagataa ccacgcgcgg cctcgagata gtcaggcggg actggagcga ggtggccaag 1860 gagacgcagg cgagggtcct ggaggcgata ctgaggcacg gtgacgtcga ggaggccgtt 1920 agaatcgtca gggaggtaac cgaaaagctg agcaagtacg aggttccgcc ggagaaactg 1980 gtgatccacg agcagataac gagggatttg agggactaca aagccacggg accgcacgtg 2040 gcggtggcga agcgcctggc cgggaggggg gtaaggatac gccccgggac ggtgataagc 2100 tacatcgtcc tcaagggctc cggaaggata ggggacaggg cgattccctt cgacgagttc 2160 gacccgacta agcacaggta cgacgccgac tactacatcg agaaccaggt tctgccagcc 2220 gtcgagagga tcctgaaggc cttcggctac cgcaaggagg acctgaaata ccagaagacg 2280 aggcaggtgg gcctgggtgc gtggctcaac gcggggaagg ggtga 2325 <210> 10 <211> 400 <212> PRT <213> Thermococcus celer <400> 10 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Val   1 5 10 15 Arg Ile Phe Arg Lys Glu Lys Gly Glu Phe Arg Ile Asp Tyr Asp Arg              20 25 30 Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile          35 40 45 Glu Glu Val Lys Arg Ile Thr Val Glu Arg His Gly Lys Ala Val Arg      50 55 60 Val Lys Arg Val Glu Lys Val Glu Lys Lys Phe Leu Asn Arg Pro Ile  65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp Val Pro Ala Ile                  85 90 95 Arg Asp Glu Ile Arg Lys His Pro Ala Val Val Asp Ile Tyr Glu Tyr             100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro         115 120 125 Met Glu Gly Glu Glu Glu Leu Lys Leu Met Ala Phe Asp Ile Glu Thr     130 135 140 Leu Tyr His Glu Gly Asp Glu Phe Gly Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Gly Asp Gly Ala Arg Val Ile Thr Trp Lys Lys Ile                 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys             180 185 190 Arg Phe Leu Gln Val Val Lys Glu Lys Asp Pro Asp Val Leu Val Thr         195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Arg Arg Ser Glu     210 215 220 Glu Leu Gly Leu Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile                 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Val Asn Leu Pro Thr             260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Arg Pro Lys Glu         275 280 285 Lys Val Tyr Ala Gly Glu Ile Val Glu Ala Trp Glu Thr Gly Glu Gly     290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu                 325 330 335 Ile Gly Gln Gly Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu             340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala         355 360 365 Pro Asn Lys Pro Ser Gly Arg Glu Val Glu Ile Arg Arg Arg Gly Tyr     370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400  

Claims (10)

A heat resistant Tce DNA polymerase isolated from a thermococcal sealer having the amino acid sequence of SEQ ID NO: 10. The Tce of claim 1, wherein the Tce has optimal activity at a pH of pH 6.5, at a temperature of 75 ° C., at a concentration of (NH ₄ ) ₂ SO ₄ at 20-35 mM and at a concentration of Mg ²⁺ of 4-16 mM. DNA polymerase. A nucleic acid molecule encoding the heat resistant Tce DNA polymerase of claim 1. 4. The nucleic acid molecule of claim 3 having the nucleic acid sequence of SEQ ID NO. A vector comprising a nucleic acid molecule encoding the heat resistant Tce DNA polymerase of claim 1. 6. The vector and microorganism transformed with the vector according to claim 5, wherein the vector is pTCE. Microorganisms transformed with the vector of claim 6. 8. The microorganism according to claim 7, wherein the microorganism is Escherichia coli Rosetta (DE3) pLysS / pTCE (accession number KACC95097P) transformed with a pTCE vector. A kit comprising the Tce DNA polymerase of claim 1. (i) preparing a vector expressing the Tce DNA polymerase of claim 1; (ii) transforming the vector with a microorganism; (iii) culturing the transformant; And (iv) recovering the protein from the transformant; It includes, Tce DNA polymerase production method.

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