KR0136446B1 - Method for mass producing novel heat-resistant top dna polymerase - Google Patents

Abstract

The present invention provides a novel heat resistant TOP DNA polymerase isolated from the thermophilic strain Thermusthermophilus HB27, the nucleotide sequence of its gene and the amino acid sequence encoded by it, an expression vector containing the gene and the expression vector. The present invention relates to a method for producing a novel heat resistant TOP DNA polymerase comprising culturing the transformed Escherichia coli and purifying the expressed polymerase.

Description

[Name of invention]

Mass production method of novel heat-resistant thiophyll DNA synthesis enzyme

[Brief Description of Drawings]

FIG. 1A shows the results of electrophoresis by cleaving chromosomal DNA of Thermos thermophilus HB27 with restriction enzymes of Bam HI, BamHI / HindIII, HindIII, HindIII / BamHI, PstI / HindIII, PstI / BamHI Will,

B of FIG. 1 shows the result of hybridization of the electrophoretic gel with a Tca DNA polymerase gene labeled with a radioisotope.

2 shows the position of the TOP DNA polymerase gene contained in the plasmids pTTB and pTTH together with the restriction map of the TOP DNA polymerase gene.

3 shows the nucleotide sequence of the TOP DNA polymerase gene and the amino acid sequence encoded by it,

4 shows the construction of a plasmid pGTOP containing a complete TOP DNA polymerase,

5 is a plasmid pTOP for expressing the TOP DNA polymerase in E. coli and a plasmid pTOPM for expressing the TOP DNA polymerase gene in which a part of the 5 'region is modified.

Figure 6 shows the result of the modified polyacrylamide gel electrophoresis of the expressed TOP DNA polymerase and the fraction in each purification step,

7 shows the results of electrophoresis on agarose gels after PCR using purified TOP DNA polymerase.

Detailed description of the invention

The present invention relates to a novel heat resistant TOP DNA polymerase, a gene thereof, an expression vector containing the same, and a method for mass production of TOP DNA polymerase by culturing E. coli transformed with the vector.

More specifically, the present invention provides a novel heat resistant TOP DNA polymerase isolated from the thermophilic strain Thermos thermophilus HB27, the nucleotide sequence of its gene and the amino acid sequence encoded by it, and an expression containing the gene thereof. The present invention relates to a method for producing a novel heat resistant TOP DNA polymerase comprising culturing a vector and E. coli transformed with the expression vector.

DNA polymerase is an enzyme that catalyzes the synthesis of double stranded DNA by complementary binding of nucleotide triphosphates to a single stranded DNA template. DNA polymerases having these characteristics have been studied in Escherichia coli, mesophilic bacteria, and among them, in particular, DNA nucleotide sequence and total amino acid sequence of DNA polymerase I have been determined, followed by 5 '' of this polymerase I. → The tertiary structure of the Klenow fragment, from which the amino terminal region with 3 'exonuclease activity was removed (Jocye, CM, Kelley, WS and Grindley, DF, J. Biol. Chem. 257, 1958-1964 (1982); Jocye, CM and Steitz, TA, Trends Biochem. Sci. 12, 288-292 (1987)). On the other hand, relatively few studies have been conducted on heat-resistant DNA polymerases produced from high-temperature strains. A typical study was Taq DNA polymerase of Thermos aquaticus YT-1 (Lawyer, FC et al. , J. Biol. Chem. 264, 6427-6437 (1989).

DNA polymerase is used for determining the sequencing of DNA and nick translation in the field of molecular biology using its properties. Recently, a technology called polymerase chain reaction (PCR) using DNA polymerase has been developed and widely used for diagnosis of various genetic diseases including DNA amplification.

(Erlich, H. A., PCR Technoloqy, Stockton Press, New York, 1989). This technique is characterized by three reactions of DNA denatured by heat treatment, primer annealing at 55-60 ° C. and polymerization by heat resistant DNA polymerase at 72 ° C., denaturation of template DNA at 94 ° C. The method of amplifying a desired DNA fragment by repeating it once or more requires a heat resistant DNA polymerase that is stable at high temperatures.

Based on these facts, the present inventors conducted a study to develop a novel heat resistant DNA polymerase, and isolated the new heat resistant TOP DNA polymerase gene from the thermophilic thermos thermophilus HB27, and the nucleotide sequence thereof And revealed the amino acid sequence of the TOP DNA polymerase, after producing an expression vector containing the gene of the enzyme, by culturing the transformed Escherichia coli thereby producing a large amount of TOP DNA polymerase, and purified the present invention Completed.

It is an object of the present invention to provide a novel heat resistant DNA polymerase. Another object of the present invention is to provide an expression vector comprising the gene of the enzyme and E. coli transformed thereby.

Still another object of the present invention is to provide a method for mass-producing a novel heat-resistant DNA polymerase by culturing the transformed Escherichia coli and a method for effectively purifying the produced polymerase. The present invention is described in more detail as follows.

1. Heat resistant TOP DNA polymerase

The novel heat resistant TOP DNA polymerase of the present invention is isolated from Thermos Thermophilus HB27 and is a protein having a molecular weight of 93,000 to 95,000 consisting of 834 amino acids of the sequence as shown in FIG. TOP DNA polymerase has a homology of 87.1% at the amino acid level and 85.5% at the amino acid level with Taq DNA polymerase reported by Lawyer et al., And Tca DNA polymerase (Applicant's prior Korean Patent Application Publication No. 93-21789) and 97.6% and 98.3% homology, respectively.

In addition, E. coli DNA polymerase I has a homology of 37.6% at the amino acid level. The heat-resistant TOP DNA polymerase of the present invention is a protein that replaces or removes one or more amino acids of the amino acid sequence of FIG. 3 as long as the activity thereof is substantially maintained, in particular, a portion of the amino terminus is removed or one or more amino acids are substituted. Also included are proteins having an amino acid sequence to which the addition is made.

As described above, the heat resistant TOP DNA polymerase of the present invention expresses heat resistant TOP DNA polymerase by transforming a microorganism into a vector containing a gene or a modification of the heat resistant TOP DNA polymerase isolated from or from Thermos Thermophilus HB27. It can be prepared by the method of the present invention or by chemical amino acid synthesis.

2. DNA encoding heat resistant TOP DNA polymerase

DNA encoding the heat resistant TOP DNA polymerase preferably has, but is not limited to, a nucleotide sequence beginning with the start codon ATG (methionine) and ending with the stop codon TAA as shown in FIG. Some of the nucleotide sequences include substituted or deleted nucleotide sequences so long as they encode proteins having substantially the same activity, and are directly known by the method of cloning the thermos thermophilus HB27 (Takashi Matsumoto et al., J. Biochemistry 107). , 331-338 (1990) or can be chemically synthesized using a DNA synthesizer.The separation from Thermos Thermophilus HB27 is, for example, a hybrid using a gene of known heat resistant DNA polymerase as a probe. By the hybridization method (Sambrook, J., et al., Molecular Cloning, 2nd ed., 9.31-9.62 (1989)). After recovering the DNA fragments of the strongly hybridized region from the DNA of thermos thermophilus HB27 digested with restriction enzymes and cloning them, the deleted DNA fragments were synthesized by PCR using the chromosomal DNA of thermos thermophilus HB27 as a template. By obtaining a complete TOP DNA polymerase gene.

3. Expression Vector

DNA encoding the heat resistant TOP DNA polymerase of the present invention or modifications thereof can be expressed using any of the appropriate prokaryotic or eukaryotic expression systems well known in the art (Sambrook et al., Molecular Cloning, A Laboratory). Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, USA (1989).

Expression is Escherichia coli, eg, Escherichia coli MV1184, Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), Escherichia coli NM522, or yeast, for example, Saccharomyces cerevisiae, Saccharomyces diastas. It can be carried out in Taccus (Saccharomyces diastaticus) and the like, it is preferable to perform in E. coli MV1184.

Suitable vectors that can be used for expression in Escherichia coli and yeast are described in Sambrook et al. (Homologous) and in Fiers et al. (Proced. 8th Int. Biotechnology Symposium, Soc. Frac. De Microbiol., Paris, ( Durand et al., Eds.), Pp. 680-697 (1988)), in the present invention prepared by inserting the gene of TOP DNA polymerase into the plasmid pJR (see Korean Patent Application No. 92-6370) Plasmid pTOP prepared by inserting the gene of TOP DNA polymerase modified with one plasmid pTOP or 6 nucleotides of the 5'-region into the plasmid pJR is preferred for expression in E. coli, and plasmid pTOPM is particularly preferred.

4. Microbial Transformants

Transformation of host cells by the vector described above can be carried out by any of the conventional methods (Sambrook et al., Molecular Cloning, A Laboratory Manual (1989)). Competent cells can be prepared and then processed according to known CaCL _2. Methods Transformation can also be performed after forming the protoplasts of the host cells or as known in the art and as Sambrook. It can also be carried out by other methods as described in Hom. Et al., E. coli MV1184 (pTOP / Escherichia coli MV1184) transformed with the plasmid pTOP described above was established by the Institute of Genetic Engineering on November 29, 1993. It was deposited with the gene bank (KCTC) under the accession number KCTC 8542P.

In addition, Escherichia coli MV1184 (pTOPM / Escherichia coli MV1184) transformed with plasmid pTOPM was deposited with KCTC as accession number KCTC 8543P on November 29, 1993.

5. Culture of Microbial Transformants

In general, host microorganisms containing the desired expression vector are cultured under conditions that maximize expression of the gene to be expressed and at the same time maintain optimal growth of the microorganism. For example, E. coli MV1184 cells transformed with the vector pTOPM were cultured in an LB medium (1% bactotrypton, 0.5% yeast extract, 0.5% NaCl) containing ampicillin, followed by induction of expression by addition of IPTG. do.

6. Recovery and Purification of Heat-Resistant TOP DNA Polymerase from Cultures

Heat resistant TOP DNA polymerase expressed in transformed E. coli cells can be recovered from cell culture or after cell disruption and / or by extraction by any suitable method known in the protein chemistry. For example, the following method may be used: That is, the culture of E. coli cells is centrifuged to suspend the harvested cells in buffer and disrupted with a French Press. The cell lysate is centrifuged to remove the unbroken cells, the streptomycin sulfate is added, and then centrifuged. The supernatant is heat-treated and centrifuged to remove E. coli derived proteins. At this time, the heat treatment is carried out at 70 ℃ to 80 ℃, preferably 77 ℃ 15 minutes to 30 minutes, preferably 20 minutes.

The supernatant is dialyzed and purified by DEAE-Sephacel column chromatography. Hereinafter, the present invention will be described in more detail with reference to the following Examples and Examples. The following examples are merely to illustrate the invention and are not intended to limit the scope of the invention in any aspect.

Reference Example 1 Enzyme Activity Measurement and Protein Quantitation

The activity of the DNA polymerase was measured by slightly modifying a known method of adsorbing onto a DE-81 filter paper. 25 mM Tris-HCl (pH8.3), 1 mM β-mercaptoethanol, 2 mM MgCl ₂ , 50 mM KCl, 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 48 μ Ci / mM [methyl, 1 ′, 2 ′, ^-3 H] Enzyme solution was added to 50 μl of vitrified mixture consisting of TTP and 1.43 μg active calf thymus DNA, reacted at 70 ° C. for 30 minutes, cooled in ice for 5 minutes, and then stopped by adding 2 μl of 0.5 M EDTA. 50 μl of the reaction mixture was dropped in the center of the filter paper (2.3 cm) and then dried by heating. The dried filter paper was placed in sodium phosphate buffer (0.5M Na ₂ HPO ₄ , pH7.0) and washed with shaking for 10 minutes. After repeating once more, it was transferred to a container containing 30 ml of 70% ethanol and washed with shaking for 5 minutes.

After drying the filter, a disposable solution containing 2 ml of cocktail solution (4 g PPO (2,5-diphenyloxazol), 0.05 g POPOP (1,4-bis [2- (5-phenylox azolyl)] benzene), toluene 1ι) Into the measuring vessel, the amount of radioactivity bound to the filter was measured using a radiometer (Beckman LS 6800 Scintillation counter). One unit of enzyme was defined as the amount of enzyme incorporating 10 nM of [ ³ H] TTP into acid-insoluble DNA synthesis for 30 minutes at 70 ° C. The amount of protein was determined using the Bradford method (Bradford MM, Anal. Biochem. 72, 248-254 (1976)) using bovine serum as the standard protein.

Reference Example 2 Polymerase Chain Reaction

On a reaction mixture containing 1 μg template DNA, 1 μM primer, 250 μM dNTP (dATP, dCTP, dGTP and dTTP), 500 μg / ml BSA, 50 mM KCl, 20 mM Tris-HCl (pH8.3), 1.5 mM MgCL ₂ Mineral oil was added and denatured at 100 ° C. for 5 minutes. Where about 2.5 units of Tca DNA polymerase were added and 60 ° C., 1 minute (primer annealing); 74 ° C., 2 minutes (enzyme polymerization); The cycle of 94 degreeC and 1 minute (modification) is repeated 32 times.

Example 1 Cloning of TOP DNA Polymerase Gene

(Step 1) Preparation of Radio-labeled Probes

Plasmid pTCA (KCTC 0039BP), completely digested with restriction enzymes EcoRI, HinFI and SalI, was electrophoresed on low melting agarose gel at 0.8% concentration, respectively, to cleave a gel section containing about 2.5 kb fragments. The gel pieces thus obtained were heated to 65 ° C. to be completely dissolved, and the same amount of TE (10 mM Tris-HCl, pH7.6, 1 mM EDTA) buffer was added, followed by heating for another 5 minutes. The DNA / agarose solution was extracted with the same amount of phenol and chloroform and precipitated with ethanol to recover the DNA fragments.

The recovered DNA fragment was labeled with [α- ³² P] dATP by Nick translation according to a known method, and then unreacted [α- ³² P] dATP was removed by gel filtration (Rigby, PW). , et al., J. Mol. Biol. 113, 237 (1977)).

(Step 2) Agarose Gel Electrophoresis and Hybridization

Chromosomal DNA of Thermos Thermophilus HB27 was isolated by Marmer's method (Marmur, J., J. Mol. Biol 3,208-218 (1961)). The DNA was digested with restriction enzymes BamHI, HindIII and PstI, electrophoresed on 0.7% agarose gel with ethidium bromide, and photographed under UV lamp. As a result, the result of FIG. 1A was obtained, and the first column in A of FIG. The chromosome DNA of Thermophilus HB27 was cut with BamHI, and the second column was T. a. The chromosomal DNA of Thermophilus HB27 was cut with BamHI and HindIII, and the third column was T. Chromosome DNA of Thermophilus HB27 was digested with HindIII and BamHI. The chromosomal DNA of Thermophilus HB27 was digested with PstI and HindIII, and the sixth column was T. a. It is a sample obtained by digesting chromosomal DNA of Thermophilus HB27 with PstI and BamHI.

Next, the gel was denatured in 0.5N NaOH solution for 1 hour and then washed again in distilled water for 30 minutes. The gel was sandwiched between paper towels and compressed to remove moisture as much as possible. The gel, about 1 mm thick, was transferred onto Whatman paper (3MM) and then dried under reduced pressure at 60 ° C. for 30 minutes on a gel dryer to obtain a membrane. The membrane was prepared using a prehybridization solution (6 x SSC (SSC: 17.53 g of NaCl per liter, 8.82 OH, pH 7.0), 5 x Denhardt solution (Denhardt solution: Ficoll 0.2 g per liter, polyvinyl) Pyrrolidone 0.2h, BSA 0.2g), 50 mM sodium phosphate buffer (pH6.5), 250 μg / ml sonicated salmon testis DNA (Sigma, type III Na salt)) at 65 ° C. for 1 hour Then, in a hybridization solution (6 x SSC, 1 x Denharz's solution, 50 mM sodium phosphate buffer (pH6.5), 100 μg / ml sonicated salmon testis DNA) with a radioisotope labeled probe again The hybridization reaction was carried out at 60 ° C. for 16 hours. Gel washes were repeated three times for 10 min at 55 ° C. in a 2 × SSC solution. The water was removed, an X-ray film was placed, and the resultant was photosensitive overnight, followed by development to obtain the result of B in FIG. Columns 1 to 6 in FIG. 1B are the same samples as in A of FIG. 1, respectively.

(Step 3) Cloning of the Top DNA Polymerase Gene

In B of FIG. 1, about 2 kb of HindIII fragment and about 3 kb of BamHI fragment, which are suitable for cloning, especially among the regions strongly hybridizing to the probe, were recovered from the low melting agarose gel by the method described in step 1 above. . These were added to the fragments of the plasmid pUC18 purified by HindIII and added in equimolar amounts, respectively, and then reacted by binding in the presence of a DNA binding enzyme at 16 ° C. for 12 hours.

The bound plasmids were transformed into E. coli strain MV1184 (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, USA (1989)). A clone of the Top DNA polymerase gene was selected by colony hybridization method (Hanahan, D. and Meselson, M., Gene 10, 63 (1980)) using an element labeled probe. The plasmids that were strongly hybridized with the probes were selected and separated from them were named pTTH and pTTB, respectively, and these are shown in FIG.

(Step 4) Amplification of the 3'-terminal region of the top DNA polymerase

As can be seen in Figure 2, the plasmids pTTB and pTTH did not contain the complete DNA of the Top DNA polymerase, and the 0.31 kb gene fragment was missing at the 3'-end. In order to obtain the missing gene fragment, SalI sites were inserted immediately after the EcoRI and 3 'terminal stop codons, respectively, immediately before the ATG codon corresponding to about 1120 bp using a known PCR method. The terminal region was amplified. PCR was carried out as in Reference Example 2 using a chromosome isolated from Thermos Thermophilus HB27 as a template and using the primers KE2 and KS described below.

Primer KE2: 5'-GCGACGAATTCATGCTCCTCGCCTACCTCCRGGACC-3 '

EcoRI

Primer KS: 3'-CCCTCCTGACCGAGAGGCGGTTCCTCACTCAGCTGGGACG-5 '

SalI

After performing PCR, chloroform was treated to remove mineral oil. After treatment with phenol / chloroform, ethanol was precipitated and the amplified DNA was recovered and confirmed on agarose gel. The 3'-terminal portion of the Top DNA polymerase gene amplified by this PCR method was cloned into the EcoRI and SalI sites of pJR, an expression vector, which will be described later.

Example 2 Determination of Nucleotide Sequence of Top DNA Polymerase Gene

Top sequence in the plasmids pTTH and pTTB according to the sequencing method using dideoxynucleosides, which are doubled by F. Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 543 (1977)). The nucleotide sequence of the DNA polymerase gene was determined, and a 0.31 kb gene fragment amplified by PCR was used to determine the nucleotide sequence of fragments obtained from various transformants to correct the aberration of Tca DNA polymerase that may occur during PCR. Nucleotide and amino acid sequences of the complete Top DNA polymerase are shown in FIG.

Top DNA polymerases were identified as proteins having a molecular weight of 93,948 Daltons with 834 amino acids, beginning with initiation codon ATG (methionine) and ending with stop codon TAA. Top DNA polymerase has a homology of 87.1% at amino acid level and 85.5% at DNA level with Taq DNA polymerase reported by Lawyer et al., And 97.6% and 98.3% homology with Tca DNA polymerase, respectively. Has It was also found to have 37.6% homology with E. coli DNA polymerase I.

Example 3 Preparation of Whole Gene of Top DNA Polymerase

First, the plasmid pTTB was digested with restriction enzyme EcoRI, and then the BamHI site at the N-terminus was removed with an Erase-a-base kit (Promega) using ExoIII and SI nucleic acid enzymes. The plasmid pTTB was completely digested with BamHI and HindIII, and then the large fragments from which the fragments which had been cut were removed by the method described in Step 1 of Example 1. In addition, the 3'-terminal portion of the Top DNA polymerase gene amplified by PCR method was completely digested with BamHI and HindIII, and a small fragment of 0.31 kb was recovered in the same manner. E. coli MV1184 was transformed after the small fragment of 0.31 kb was added to the large fragment of the purified plasmid pTTB in an equimolar amount and bound as in Step 3 of Example 1. The newly constructed plasmid was completely digested with HindIII, and then the linear plasmid was recovered by the method of Step 2 of Example 1, and plasmid pTTH was completely digested with HindIII, followed by the 2 kb small gene fragment by the method of Step 2 of Example 1. Recovered.

A homologous amount of a 2 kb gene fragment was added to the linear plasmid recovered as described above, followed by binding as in Step 3 of Example 1, and transformed into E. coli MV1184 to prepare a plasmid pGTOP containing a complete Top DNA polymerase gene. The construction of the plasmid pGTOP is shown in FIG.

Example 4 Preparation of Top DNA Polymerase Expression Vector

The expression vector is a higher expression vector than pKK223-3, which replaces the origin of the plasmid pKK223-3 with the origin of replication of the PUC and removes part of the tetracycline resistance gene including the restriction enzyme SalI site of pKK223-3. It was prepared using phosphorus plasmid pJR (see Applicant's previous Korean Patent Application No. 92-6370). First, we tried to insert the Top DNA polymerase gene into the EcoRI and SalI sites of the expression vector pJR, but the EcoRI and SalI sites were not present in the 5 'and 3' regions of the Top DNA polymerase gene. Using this method, the gene region of the Top DNA polymerase was amplified by inserting the EcoRI site immediately before the 5'-end ATG (Met) and the SalI site immediately after the 3'-end stop codon.

PCR reaction was carried out in the same manner as in Reference Example 2 using the primer of Top DNA polymerase as a template and using the primers KE3 and KG.

Primer KE3: 5'-CCCTGGGAATTCATGGAGGCGATGCTTCCGCTCTTTG-3 'EcoRI

Primer KG: 3'-CTCCTGACCGAAAGGCGGTTCCCAATCAGCTGGGGAC-5 '

SalI

The amplified gene was completely digested with EcoRI and SalI, and electrophoresed on a low melting agarose gel. Then, a band of about 2.5 kb corresponding to the gene of the Top DNA polymerase was recovered in the same manner as in Step 3 of Example 1 After insertion and binding to the EcoRI / SalI site of the expression vector pJR, E. coli MV1184 was transformed to obtain the plasmid pTOP shown in FIG.

E. coli MV1184 / pTOP transformed with this plasmid was deposited with the accession number KCTC 8542P to the Gene Bank on November 29, 1993.

Example 5 Expression of Top DNA Polymerase Gene

To express the cloned Top DNA polymerase gene, Escherichia coli MV1184 (KCTC 8542P) containing the plasmid pTOP was added to LB medium (10 g NaCl, 10 g Bactot tryptone, 5 g yeast extract per 50 ml of medium). / L). After incubation at 37 ° C. for about 2 hours, about 0.1-1.0 mM IPTG (isopropyl-thio-D-galacto-pyranoside) was added and culture was continued for about 7 hours to induce gene expression. The cells were recovered by centrifugation, and then buffer A (50 mM Tris-HCl, pH7.5, 1 mM β-mercaptoethanol, 0.5 mM EDTA, 0.04 M KCl, 1 mg / ml pepstatin P and 1 mM containing trypsin inhibitor After dissolving in phenyl methyl sulfonyl fluoride (PMSF), cells were completely disrupted at 14,000 psi by using a French press, and the DNA polymerase activity was measured in the same manner as in Reference Example 1 using this cell disruption solution. The measurement resulted in 54 units of activity per ml of culture.

Example 6 Mass Expression of Top DNA Polymerase Gene by Gene Manipulation

The expression vector pJR used to prepare the expression vector of the Top DNA polymerase gene in Example 4 is a high expression vector which was prepared in our laboratory and confirmed through various experiments. However, as shown in Example 5, the plasmid pTOP in which the Top DNA polymerase gene was inserted into the EcoRI and SalI sites of the expression vector pJR was not so high.

On the other hand, plasmid pKTPOL10 (Kwon, ST et al., Mol. Cells. 1, 369-375 (1991)) in which Taq DNA polymerase gene was inserted at the EcoRI and SalI sites of the expression vector pKCI, which is almost similar to the expression vector pJR. Has been previously reported (Kwon, ST et al., Mol. Cells. 1, 369-375 (1991)). In addition, when Taq DNA polymerase gene is inserted into and expressed in EcoRI and SalI sites of the expression vector pJR, the expression of Taq DNA polymerase gene is further increased and the tag DNA is polymerized by modifying five nucleotides of the DNA sequence in the 5 'region of the Tca DNA polymerase gene. It was found that when expressed identically to the nucleotide sequence of the 5'-region of the enzyme gene, it is expressed in large quantities (see Korean Patent Application No. 92-6370).

Therefore, as in the case of the Tca DNA polymerase, the PCR method was used to modify six nucleotides in the 5 'region of the Top DNA polymerase gene so as to be identical to the nucleotide sequence of the 5' region of the Taq DNA polymerase gene. . PCR was used in Example 4 and primer N1 having the following sequence identical to the nucleotide sequence of the 5'-region of the Taq DNA polymerase gene, which was used to amplify the Taq DNA polymerase gene by PCR and clone it into the expression vector pKCI or pJR. The primer KG was used in the same manner as in Reference Example 2.

The asterisk (*) in the above is different from the nucleotide sequence of the Top DNA polymerase gene.

The DNA of the Top DNA polymerase modified with the 5'-region nucleotide sequence obtained above was completely digested with EcoRI and sall and electrophoresed on low melting agarose gel. The amplified 2.5 kb region was cleaved and purified as in Step 2 of Example 1, bound to the EcoRI / sall site of the expression vector pJR, and then transformed into E. coli MV1184. . The transformed Escherichia coli MV1184 / pTOPM was deposited on November 29, 1993 with the accession number KCTC 8543P to the Gene Bank of the Genetic Engineering Center. E. coli MV1184 / pTOPM was cultured in the same manner as in Example 5 to induce the expression of the Top DNA polymerase gene, and the enzyme activity was measured.

Example 7 Purification of Top DNA Polymerase

Escherichia coli containing the plasmid pTOPM prepared in Example 6 was incubated as in Example 5, followed by centrifugation of 10 L to recover 120 g of cells. The cells were suspended in 250 ml of buffer A as in Example 5, and then the cells were disrupted using a press. The suspension was centrifuged at 5,000 rpm for 1 hour, after which the unbroken cells were removed. In order to remove the nucleic acid, the streptomycin sulfate (STREPTOMYCIN SULFATE) of 5% or less in final concentration was slowly added at 4 ° C, left for about 30 minutes, and then centrifuged again. The supernatant was heat-treated at 77 ° C. for 20 minutes to precipitate E. coli-derived protein, and then the denatured protein was removed by centrifugation at 5,000 rpm for 1 hour, and the supernatant was buffer B (20 mM Tris-C1 (pH 8.0). ), 1 mM EDTA, 1 μM PMSF). By the heat treatment, only a few E. coli-derived proteins exist, and Top DNA polymerase was almost purified.

Top DNA polymerase solution equilibrated with buffer B was adsorbed onto a DEAE-sephacel column (5 × 20 cm, Pharmacia), and then buffer B with KCI (4, 60, 90, 130, 180, 500 mM) was added. Were each passed at a rate of 60 ml per 1 liter hour.

The protein solution recovered according to the KCI concentration was used to investigate the activity of the polymerase as in Reference Example 1. Since a large amount of DNA polymerase activity was observed in the protein solution recovered from 180 mM KCI, it was concentrated in amicon and then buffer c (500 mM Tris-c1 (pH 8.0), 0.5 mM EDTA, 4 mM KCI, 2 mM GCl2, 1 mM). Equilibrate with dithiothreitol (DTT), 0.5 mg / ml BSA, 50% glycerol, 1 μM PMSF). It was passed through a DEAE-Sephacel column and electrophoresed on an agarose gel to confirm that the nucleic acid was completely removed. Using the KCI concentration gradient, a large amount of protein was easily and quickly obtained. FIG. 6 shows the results of electrophoresis on modified polyacrylamide gels for each step. In FIG. 6, column 1 is a standard molecular weight protein, column 2 is a whole protein of E. coli without plasmid. Row 3 is the total protein that appears when E. coli with the plasmid pTOPM is induced, row 4 is the polymerase after streptomycin sulfide treatment, row 5 is the polymerase after heat treatment, and row 6 is the DEAE. -Polymerase after purification with Sephacel. Table 1 below shows the purification degree of Top DNA polymerase produced in E. coli MV1184 / pTOPM by gene recombination.

Table 1. Purification of Top DNA Polymerase (Culturing Solution 10ℓ)

* One unit corresponds to the amount of polymerase that incorporates ¹⁰ mM [ ³ H) TTP into DNA at 70 ° C. for 30 minutes.

Glycerol was added to the purified enzyme solution at a concentration of 50% and stored at -70 ° C.

Example 8 Confirmation of the Efficacy of Top DNA Polymerase

PCR was carried out in the same manner as in Reference Example 2 using a reaction mixture of 50 μl to which primers KE3 and KG were added, using the Top DNA polymerase solution purified in Example 7 instead of Tca DNA polymerase as a template and pTOP plasmid DNA as a template. .

The PCR reaction product was confirmed by electrophoresis on 0.8% agarose gel. In FIG. 7, column 1 is a 1 kb standard molecular weight marker, and columns 2 to 5 are added to the 50 μl PCR reaction mixture by adding 3.2, 1.6, 0.8, and 0.2 units of the purified Top DNA polymerase of the present invention, respectively. DNA amplified by the PCR method. The results of FIG. 7 indicate that the Top DNA polymerase of the present invention, which has partially modified the nucleotide sequence of the 5'-region, is a heat resistant DNA polymerase useful for PCR reaction.

Claims (10)

A heat resistant Top DNA polymerase having an amino acid sequence of FIG. 3 derived from Thermos thermophilus HB27. The Top DNA polymerase according to claim 1, which has an amino acid sequence obtained by removing or modifying a part of the amino terminus of the amino acid sequence of FIG. DNA having a nucleotide sequence encoding the DNA polymerase of claim 1. 4. The DNA of claim 3 comprising a nucleotide sequence encoding the amino acid sequence of FIG. 4. The DNA of claim 3 comprising a portion or modification of the nucleotide sequence encoding the amino acid sequence of FIG. An expression vector comprising the DNA of any one of claims 3 to 5. The expression vector pTOP or pTOPM according to claim 6. E. coli transformed with the expression vector of claim 6. The method of claim 8, wherein pTOP / E. Coli MV1184 (KCTC 8542P) or pTOPM / E. Coli MV1184 (KCTC 8543P) A method for producing a DNA polymerase derived from Thermos thermophilus HB27, comprising culturing Escherichia coli according to claim 8 or 9.