KR20110126011A - Dna polymerase t7 and genes thereof - Google Patents

Abstract

PURPOSE: A DNA polymerase T7 and genes thereof are provided to ensure processivity, fidelity, and elongation length and to be applied in various fields. CONSTITUTION: A DNA polymerase T7 has an amino acid sequence of sequence number 2. A DNA polymerase T7 gene encodes an amino acid sequence of sequence number 2. The DNA polymerase T7 gene contains a base sequence of sequence number 1. A recombinant vector contains the T7 gene. The polymerase T7 is prepared by culturing a host cells transformed by the recombinant vector, inducing the recombinant protein expression, and isolating polymerase protein. The recombinant vector contains the DNA polymerase T7 gene. The DNA polymerase T7 is derived from Thermococcus sp. NA1.

Description

DNA Polymerase T7 and Genes Thereof {DNA Polymerase T7 and Genes Thereof}

The present invention relates to a mutant DNA polymerase T7 and a gene sequence encoding the same, a preparation method, and a PCR polymerization using the same. More specifically, the present invention is derived from Thermococcus sp. NA1 and induces mutation at a specific site. The present invention relates to a DNA polymerase, an amino acid sequence thereof, a gene encoding the mutant DNA polymerase, and a method for producing a polymerase using the same.

Recent advances in genomic research have yielded enormous amounts of gene sequence information. With the general combination of conventional genetic engineering and genomic research techniques, the genomic sequences of some highly thermophilic microorganisms are of interest because they are characterized by heat-resistant enzymes in the field of biotechnology, and various thermally stable enzymes are used for biotechnological purposes. Is being developed.

Such thermostable thermophilic DNA polymerase and polymerase chain reaction (PCR) using the same have important contributions in protein and gene research, and are widely used in biological applications. In fact, more than 50 DNA polymerase genes have been cloned from various organisms, including thermophilic and archaea, and recently, highly thermophilic bacteria with higher fidelity in PCR based on proofreading activity than common Taq polymerase. Pyrococcus sp.) and the genus Thermococcus sp.) B family DNA polymerase was isolated and widely used. Nevertheless, high fidelity polymerases are low in DNA elongation ability and require many improvements despite the above advantages.

We have already isolated new highly thermophilic strains from the deep-sea hydrothermal spout area of PACMANUS field and analyzed the 16S rDNA sequence for thermococcus. Genus (Thermococcus sp.) strain, and then analyzed the entire genome sequence to find a heat stable enzyme from the strain. Analysis of the genomic information confirmed that the strain had a DNA polymerase of type B, cloned the gene corresponding to the DNA polymerase from the strain and expressed this gene in E. coli, and recombinant DNA polymerase There was a patent registration for a DNA polymerase having high-extension ability and high-fidelity by pure separation (see Korean Patent Registration No. 10-0777227).

Accordingly, the present inventors continued efforts to develop a DNA polymerase suitable for long PCR polymerization using wild-type TNA1_pol DNA polymerase of Korean Patent Registration No. 10-0777227, resulting in a thermocacus. Genus ( Thermococcus sp.) DNA polymerase isolated from the strain was mutated to select DNA polymerase T7, and confirmed that they are superior in elongation, rapidity and fidelity compared to the conventional DNA polymerase and completed the present invention.

The present invention aims to provide DNA polymerase T7 and genes thereof.

For the above object, the present invention is Rhodococcus genus Thermococcus (Thermococcus sp.) from the strain and mutated wild type TNA1_pol DNA polymerase of Korean Patent Registration No. 10-0777227 to provide DNA polymerase T7.

A first aspect of the invention provides a DNA polymerase T7 comprising the amino acid sequence of SEQ ID NO.

As used herein, "DNA polymerase" refers to DNA in the 5 '->3' direction from deoxynucleotide triphosphate by successively adding nucleotides to the growing 3'hydroxy group using complementary template DNA strands and primers. It is an enzyme that synthesizes. The template strand determines the order of nucleotides added by Watson-Crick base pairs.

Mutant DNA polymerases of the present invention may also include their "functional equivalents." Functional equivalents herein include amino acid sequence variants in which some or all of the amino acid sequences of the mutant DNA polymerase are substituted or a portion of the amino acid is deleted or added. At this time, the substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur containing amino acids (Cys, Met). In this case, the deletion of the amino acid is preferably located at a portion not directly involved in the activity of the DNA polymerase.

A second aspect of the invention provides a DNA polymerase T7 gene encoding the amino acid sequence of SEQ ID NO. The DNA polymerase gene encoding the amino acid sequence of SEQ ID NO: 2 may have various types of sequences by degeneration of heredity. The gene sequence is preferably a DNA polymerase gene represented by SEQ ID NO: 1. Those skilled in the art can make functional equivalents to the DNA polymerase sequence of SEQ ID NO: 2, and the scope of the present invention includes gene sequences encoding the functional equivalents.

A third aspect of the invention provides a recombinant vector comprising the DNA polymerase T7 gene.

The term vector, as used in the present invention, refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto. The term expression vector includes plasmids, cosmids or phages capable of synthesizing the protein encoded by each recombinant gene carried by the vector. Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound. The vector may be a vector of plasmid, cosmid, phagemid, phage, viral form. Methods of making recombinant vectors comprising the DNA polymerase gene of the present invention using such vectors are known in the art.

A fourth aspect of the present invention provides a host cell transformed with the recombinant vector. The term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.

A fifth aspect of the present invention is characterized by culturing a host cell transformed with a recombinant vector comprising a DNA polymerase gene encoding the amino acid sequence of SEQ ID NO: 2 and inducing the expression of the recombinant protein to isolate the polymerase protein. It provides a method for producing a polymerase.

The mutant DNA polymerase of the present invention exhibits superior performance compared to the conventional DNA polymerase in the processability, fidelity, and elongation length of the polymerization activity compared to the wild type or the mutant type. As a result, it can be widely applied to various fields requiring PCR.

Figure 1 shows the cleavage map of the recombinant plasmid vector.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example  1. Wild type polymerase TNA1 _ pol  Gene Cloning  And primary sequence analysis

Thermococcus genus ( Thermococcus sp.) NA1 strain was isolated from deep-sea hydrothermal spouts in the Western Pacific region of Papua New Guinea. YPS Badges It was used to culture DNA of genus NA1 strain, Culture and strain maintenance of genus NA1 were performed by standard methods. Thermococcus In order to prepare genus NA1 seed culture, YPS medium in 25 ml serum container was inoculated with a single colony formed on a pattagel plate and incubated at 90 ° C. for 20 hours. Seed culture was used to inoculate 700 ml of YPS medium in an anaerobic jar and incubated at 90 ° C. for 20 hours.

Example  1. Wild type polymerase TNA1 _ pol  Gene production and its challenge Mutation

Thermococcus genus ( Thermococcus sp . ) The E. coli DH5α strain was used for plasmid proliferation and nucleic acid sequencing including the polymerase TNA1_pol gene of NA1 strain. E. coli BL21-Codonplus (DE3) -RIL cells (stratazine, Lazola, CA) and plasmid pET-24a (+) (Novagen, Madison, Wisconsin) were used for gene expression. E. coli strains were cultured using Luria-Bertani medium at 37 ° C. and added to the medium so that kanamycin was at a final concentration of 50 μg / ml.

In addition, DNA manipulations were done in standard ways as described by Sambrok and Russell. Genomic DNA in the thermococcus was isolated by standard methods. Restriction enzymes and other modified enzymes were purchased from Promega (Madison, Wisconsin). Small amounts of plasmid DNA from E. coli cells were made using the plasmid mini kit (Qiagen, Hilden, Germany). DNA sequencing was performed using an automated sequencer (AB3100) using the Big Die Terminator Kit (PE Applied Biosystems, Foster City, CA).

Analysis of the genomic sequence revealed an open reading frame (3,927 bp) encoding a protein consisting of 1,308 amino acids and showed very high similarity with the B family type DNA polymerase. The molecular weight of the protein obtained from the inferred amino acid sequence was 151.9 kDa, which was much larger than expected for the average thermostable DNA polymerase. In addition, sequence analysis revealed that the DNA polymerase gene was conserved between the putative 3'-5 'exonuclease domain, the α-like DNA polymerase domain, and the α-like DNA polymerase of eukaryotes and archaea. It was confirmed to include one intervening sequence of 1605 bp (535 amino acids) located in Pol III). Also, the amino acid sequence of inferred intein was very similar to the intein of other archaea polymerase, and pol_1 intein 1 (537 amino acids origin of DNA polymerase derived from Thermococcus sp. Strain GE8, AJ25033). At 81.0% homology, 69.0% homology to IVS-B (537 amino acids originating from KOD DNA polymerase, D29671) and 67.0% homology to intein (537 amino acids originating from deep vent DNA polymerase, U00707) I showed same sex.

In addition, the splicing site of intein could be predicted by sequencing because His-Asn-Cys / Thr was well conserved at Cys or Ser and C-terminal splice junctions at the N-terminus of intein. Thus, a gene of mature form of polymerase TNA1_pol without intein could be expected, which was determined to be 2,322 bp encoding a protein consisting of 773 amino acid residues. The estimated sequence of polymerase TNA1_pol was compared with those of other DNA polymerases. In the pairwise alignment, the deduced mature polymerase TNA1_pol amino acid sequence was 91.0% homologous with KOD DNA polymerase (gi: 52696275), 82.0% homology with deep vent DNA polymerase (gi: 436495) and pfu DNA polymerization. It showed 79.0% homology with the enzyme (gi: 18892147). To confirm the performance of polymerase TNA1_pol in PCR amplification, TNA1_pol DNA was prepared by removing intein from the polymerase full length as mentioned above.

Mature forms of DNA polymerase containing no intein were prepared as follows. Using primers designed to include overlapping sequences, the N-terminal and C-terminal portions of TNA1-pol were amplified, respectively. The full length of the mature TNA1_pol gene flanked by restriction enzymes Nde I and Xho I sites was then amplified using a mixture of the N-terminal and C-terminal partially amplified PCR segments as two primers and templates. It became. The amplified sequences were digested with restriction enzymes Nde I and Xho I, was linked to the restriction enzyme Nde I, and the plasmid vector pET-24a digested with Xho I (+). The conjugate was transformed into E. coli DH5α. Candidates with the correct structure were identified by restriction enzymes, and the DNA sequences of the clones were analyzed to confirm that they contained the genes of the mature DNA polymerase. Thus, wild-type DNA polymerase TNA1_pol was prepared. In order to mutate specific sites of the TNA_pol, site-directed mutagenesis using polymerase chain reaction (PCR) using various synthetic primers corresponding to each specific site according to the existing protocol. Induced. By verifying the activity of the mutated DNA polymerase to search for DNA polymerase having high elongation, fidelity and rapidity, the invention invented DNA polymerase T7 of SEQ ID NO: 2.

<110> Korea Ocean Research & Development Institute <120> DNA Polymerase T7 and Genes Thereof <130> PN100015 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 2322 <212> DNA <213> Thermococcus sp. <400> 1 atgatcctcg acgtcgatta catcacagag gacggaaagc ctgtcatcag gatcttcaag 60 aaggagaagg gtgagttcaa gattgaatac gacagagact tcgagcctta catctatgca 120 ctcctcaagg acgattctgc catcgaggaa gtgaagaaga taaccgcgga gcgccatgga 180 aaggttgtca aggtcaagcg tgccgagaag gtgaataaga agttcctcgg ccgaccggtt 240 gaggtatgga agctctactt cgagcacccg caggacgtgc ccgcaatccg cgacaagata 300 agggctcacc cgggggttat cgacatttac gagtacgaca tacccttcgc caagcgctac 360 ctcatagaca agggcctcgt ccccatggag ggcgatgaag aactgaagat gctcgccttt 420 gacatcgaga cgctctacca cgagggcgag gagttcggaa ccgggcccat actcatgata 480 agctacgcgg atgagaacga ggcgagggtt ataacctgga aaaagataga cctgccctac 540 gttgacgtcg tctcaaccga gaaggagatg ataaagcgct ttttgagggt tgttaaggag 600 aaggatcctg atgttctcat tacctacaac ggcgacaact ttgactttgc ttacctcaaa 660 aaacgttgcg aaaagcttgg gataagcttt accctcgggc gggacggaag cgagccgaag 720 atacaccgca tgggcgaccg cttcgccgtg gaggttaagg ggaggattca ttttgatctc 780 tatccggtca taaggcgtac catcaacctg ccgacctaca cccttgaggt tgtttatgag 840 gcggtctttg gcaaacccaa ggagaaggta tacgcggagg agataaccct tgcctgggag 900 agcggcgagg ggcttgagcg cgttgcgcgc tactctatgg aagatgcaaa ggcaacctat 960 gagctcggaa gagagttctt cccgatggag gcccagcttt cgaggctgat aggccagagc 1020 ctctgggacg tgtcgcgttc cagcaccggc aacctcgtgg agtggtttct cctgcggaag 1080 gcctacgaga ggaacgaact tgcccccaac aagccagacg agggggagtt agcgaggaga 1140 aggaacagtt accgcggcgg ctacgttaag gaaccagaac ggggattatg ggacaatatt 1200 gtgtatttag attttcgctc tctttacccc tcgatcataa tcacccacaa cgtctcgccg 1260 gatactctca acagagaggg ctgcaaggaa tatgacgtcg cccctcaggt cggtcacaag 1320 ttctgcaagg acttccccgg cttcattccg agccttctcg ggaacctgct cgaagagagg 1380 cagaagataa agaggaagat gaaggctaca atagatcccc tggagaagaa gctcctcgac 1440 tacaggcagc gggcaataaa aatcttagcg aacagctatt acgggtacta cggctatccc 1500 agggcaaggt ggtactgcaa ggagtgcgct gagagcgtta ccgcctgggg cagggaatac 1560 attgagatga cgataaggga aattgaggag aaatatggct ttaaagtgct gtatgcggac 1620 accgatggct tttatgctac aataccggga gcggacgctg aaactgtcaa aaagaaggct 1680 aaagagttcc ttaaatacat aaatgccaag ctgcctggat tacttgagct tgagtacgag 1740 ggcttctaca agcgcggctt cttcgtcacc aaaaagaagt acgctgttat cgacgaggag 1800 ggcaagatcg taactcgtgg gctggagata gtcaggcgtg attggagtga tatagctaaa 1860 gaaacacagg ctagggttct tgaggctctc ctgaaggacg gtaatgtaga gaaggccgtt 1920 aagatagtca aggagataac cgagaagctg agcaagtacg aaatcccgcc ggagaagctc 1980 gtcatccacg agcagataac ccgcgagctg aaggactaca aagcaacggg cccgcacgta 2040 gcgatagcaa agcgtttggc ggcgagggga ataaaagttc gccccggcac gataatcagc 2100 tacatcgtcc ttaagggaag tggaaggata ggtgacaggg cgataccctt cgacgagttc 2160 gacccaacga agcacaagta cgacgctgac tactacatcg agaaccaggt tctcccggct 2220 gtgatgagga ttttggaggc gtttgggtat aagaaagagg atttaagata ccagaagacg 2280 aggcaggttg gattgggggc ttggttgaag ccgaagaaat ga 2322 <210> 2 <211> 773 <212> PRT <213> Thermococcus sp. <400> 2 Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile   1 5 10 15 Arg Ile Phe Lys Lys Glu Lys Gly Glu Phe Lys Ile Glu 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 Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Lys      50 55 60 Val Lys Arg Ala Glu Lys Val Asn Lys Lys Phe Leu Gly Arg Pro Val  65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile                  85 90 95 Arg Asp Lys Ile Arg Ala His Pro Gly Val Ile 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 Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr     130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn Glu 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 Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr         195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu     210 215 220 Lys Leu Gly Ile Ser Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile His 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 Ile Asn Leu Pro Thr             260 265 270 Tyr Thr Leu Glu Val Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu         275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Leu Ala Trp Glu Ser Gly Glu Gly     290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr 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 Ser 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 Asp Glu Gly Glu Leu Ala Arg Arg Arg Asn Ser Tyr     370 375 380 Arg Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His                 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp             420 425 430 Val Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe         435 440 445 Ile Pro Ser Leu Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys     450 455 460 Arg Lys Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr                 485 490 495 Tyr Gly Tyr Pro Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser             500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Thr Ile Arg Glu Ile         515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe     530 535 540 Tyr Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu                 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys             580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly Leu         595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Asp Ile Ala Lys Glu Thr Gln Ala     610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asn Val Glu Lys Ala Val 625 630 635 640 Lys Ile Val Lys Glu Ile Thr Glu Lys Leu Ser Lys Tyr Glu Ile Pro                 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp             660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala         675 680 685 Arg Gly Ile Lys Val Arg Pro Gly Thr Ile Ile Ser Tyr Ile Val Leu     690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Asp Tyr Tyr Ile Glu Asn Gln                 725 730 735 Val Leu Pro Ala Val Met Arg Ile Leu Glu Ala Phe Gly Tyr Lys Lys             740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp         755 760 765 Leu Lys Pro Lys Lys     770

Claims (6)

DNA polymerase T7 comprising the amino acid sequence of SEQ ID NO: 2. DNA polymerase T7 gene encoding amino acid sequence of SEQ ID NO. The DNA polymerase gene of claim 2, wherein the DNA polymerase T7 gene is SEQ ID NO: 1. Recombinant vector comprising a DNA polymerase T7 gene of claim 2. A host cell transformed with the recombinant vector of claim 4. A method for producing polymerase T7, comprising culturing the host cell of claim 5 and inducing expression of the recombinant protein to separate the polymerase protein.

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