JP3132624B2 - DNA polymerase gene derived from hyperthermophilic archaeon and uses thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel hyperthermophilic archaeon, KO
The present invention relates to a DNA polymerase derived from D1, a gene encoding the polymerase, and a method for producing a DNA polymerase using the gene.

[0002]

2. Description of the Related Art Many studies have been made on DNA polymerases derived from mesophilic bacteria such as Escherichia coli and phage-derived DNA polymerases that infect mesophilic bacteria. Recently, many studies have been made on thermostable DNA polymerases useful for recombinant DNA technology using nucleic acid amplification such as polymerase chain reaction (PCR). The thermostable DNA polymerase used for the PCR reaction is mainly Thermos thermophilus (T.
hermus thermophilus) (Tth polymerase) and Thermus aquaticus (Thermus aqucus).
aticus) derived DNA polymerase (Taq polymerase)
Etc. have been used.

[0003]

However, although the heat-resistant DNA polymerases known in the prior art have heat resistance, they still have some problems in their thermal stability and stability to organic solvents. . In addition, there is a lack of accuracy in the incorporation of nucleic acids, and problems to be solved remain in using these enzymes in DNA sequencing and polymerase chain reaction. Therefore, a novel thermostable DNA polymerase that solves these drawbacks has been long-awaited. Pyrococcus furiosas
osus) derived thermostable DNA polymerase (Pfu polymerase, WO92 / 09689, JP-A-5-328969), thermostable DNA polymerase derived from Thermococcus litoralis (Tli polymerase, JP-A-6-7160)
Is also known. However, although these thermostable DNA polymerases are more accurate in incorporation of nucleic acids than Tag DNA polymerase and The DNA polymerase, they are not perfect and new heat-resistant DNA polymerases have been desired.

[0004]

The present inventors have determined that the thermal stability D
We succeeded in obtaining one of the novel hyperthermophilic archaeon producing NA polymerase, elucidated its gene, and reached the present invention. That is, the present invention relates to a hyperthermophilic archaeon
1 derived DNA polymerase.

[0005] The present invention is also an isolated DNA encoding a DNA polymerase derived from the hyperthermophilic archaeon KOD1.

Further, the present invention is a recombinant DNA expression vector in which an isolated DNA encoding a DNA polymerase derived from the hyperthermophilic archaeon KOD1 is inserted into a vector.

[0007] The present invention is also a recombinant host cell transformed with a DNA recombinant expression vector in which an isolated DNA encoding a DNA polymerase derived from the hyperthermophilic archaeon KOD1 has been inserted into a vector.

[0008] The present invention relates to DN derived from the hyperthermophilic archaeon KOD1.
Culturing a recombinant host cell transformed with a DNA recombinant expression vector having the isolated DNA encoding A polymerase inserted into the vector, and collecting the DNA polymerase from the culture from the culture. This is a method for producing a DNA polymerase derived from the hyperthermophilic archaeon KOD1.

Further, the present invention provides a method for culturing a recombinant host cell transformed with a DNA recombinant expression vector in which an isolated DNA encoding a DNA polymerase derived from the hyperthermophilic archaeon KOD1 has been inserted into a vector. (A) after collecting the recombinant host cells, crushing to prepare a cell extract,
(B) DN derived from hyperthermophilic primordial bacterium KOD1, comprising a step of removing an impurity protein derived from a recombinant host cell.
This is a method for purifying A polymerase.

[0010] One of the hyperthermophilic archaeon used in the present invention
The species, KOD1, is a strain isolated from sulfur dioxide at Kohojima, Kagoshima Prefecture. The bacteriological properties of the strain are described below. Cell morphology Streptococcus, diplococcus, with flagella Growth temperature range 65-100 ° C Optimum growth temperature 95 ° C Growth pH range 5-9 Optimum pH 6 Optimum salt concentration 2-3% GC content of ether type DNA 38%

The hyperthermophilic archaeon KOD1 strain has a diameter of about 1 μm.
And had multiple polar flagella. This strain is a Pfu DNA polymerase producing strain (Pyroc
occus furiosus) and Tli (Vent) DNA polymerase-producing bacteria (Thermococcus litoralis).

The cloning of the thermostable DNA polymerase gene of the present invention is performed by the following method. The cloning method is based on the amino acid sequence of the conserved region of Pfu DNA polymerase (Nucleic Acids Research, 1993, vol. 21, No.
Based on 2, 259-265), primers are designed and synthesized.

First, the chromosome DN of the hyperthermophilic archaeon KOD1 strain
A as a template, the primer prepared above (eg, SEQ ID NO: 4)
And 5) to perform a PCR reaction to amplify a DNA fragment. DNA sequence of the amplified fragment (eg, SEQ ID NO: 6)
After confirming that the amino acid sequence encodes the initially set amino acid sequence, Southern hybridization is performed on the fragment of the chromosomal DNA by using the fragment as a probe. It is preferable to limit the approximate size of the fragment containing the target DNA polymerase gene to about 4 to 7 Kbp.

Further, a DNA fragment of about 4 to 7 Kbp is recovered from the gel, and using this, a DNA library is prepared in Escherichia coli, and the PCR-amplified DNA fragment described above (eg,
Colony hybridization is performed using SEQ ID NO: 6) as a probe to obtain a clone strain.

The DNA polymerase gene of the KOD1 strain cloned in the present invention is composed of 5010 bases (estimated 1670 amino acids) (SEQ ID NO: 1).
As compared with other DNA polymerases, the gene of the present invention has regions 1 to 5 which are conserved regions of eukaryotic α-DNA polymerase. In addition, EXO1,2,3 which is a 3 '→ 5' exonuclease motif is present on the N-terminal side of the gene. The conserved regions of the heat-resistant DNA polymerase gene derived from the hyperthermophilic archaeon KOD1 strain, Regions 1 and 2, each contain an intervening sequence and have an open reading frame (OR
F) in the preserved form.

The thermostable DNA polymerase gene of the hyperthermophilic archaebacterium KOD1 strain was replaced with a known enzyme, Pfu DNA polymerase gene derived from Pyrococcus furiosus (Japanese Patent Laid-Open No. 5-328969), and Thermococcus. Compared with the Tli (Vent) DNA polymerase gene derived from litoralis (Thermococcus litoralis) (JP-A-6-7160), the KOD1 of the present invention
Although there are intervening sequences in the strain gene, the above PfuDN
There is no intervening sequence in the A polymerase gene, and there are two intervening sequences in the Tli DNA polymerase gene, but their locations are in the conserved regions Regions 2 and 3, respectively. It differs greatly from the location of the intervening sequence in the thermostable DNA polymerase gene of the KOD1 strain (see FIG. 4).

The gene of the present invention is a DNA encoding a DNA polymerase derived from the hyperthermophilic archaeon KOD1. One example of the DNA contains a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2. Further, such DNA contains the base sequence described in SEQ ID NO: 1 or 3 or a part thereof. The hyperthermophilic archaeon K of the present invention
In order to express the heat-resistant DNA polymerase derived from the OD1 strain in Escherichia coli, 137 of the nucleotide sequence shown in SEQ ID NO: 1 was used.
An intervening sequence of 4 to 2453 bp and 2708 to 4316 bp was removed by a PCR gene fusion method to obtain a complete DN.
Construct the A polymerase gene. Specifically, a cloned gene containing an intervening sequence is subjected to a PCR reaction using a combination of three primers to amplify three fragments separated by the intervening sequence. When designing a primer to be used here, a part of a fragment to be bound to its end is included in its 5 'end. Next, a PCR reaction is carried out using the overlapping sequences of the ends of the fragments to be ligated, and the fragments are ligated. Further, a PCR reaction was carried out in the same manner using the two types of obtained fragments,
A complete DNA polymerase gene without the DNA polymerase gene from strain D1 is obtained.

The vector used in the present invention is KO
Any substance can be used as long as it allows cloning and expression of the thermostable DNA polymerase derived from D1, such as phage and plasmid.
Examples of the plasmid include a plasmid vector capable of inducible expression with a T7 promoter, such as pET-8c. As another example of a plasmid,
pUC19, pBR322, pBluescript,
pSP73, pGW7, pET3A, pET11C and the like. Examples of phage include λgt11, λD
ASH, λZapII and the like. The host cells used in the present invention include E. coli, yeast and the like. As Escherichia coli, for example, JM109, 101,
XL1, PR1, BL21 (DE3) plysS, and the like. In the present invention, the above-mentioned heat-resistant DN derived from KOD1 is used.
A gene encoding A polymerase is inserted into the above vector to form a recombinant expression vector, and a host cell is transformed with the recombinant expression vector.

In the production method of the present invention, the above-mentioned recombinant host cell is cultured to obtain a heat-resistant DNA polymer derived from the KOD1 strain.
The ze gene is induced and expressed. The medium and conditions used for culturing the recombinant host cells follow conventional methods. As a specific example, the complete form of D without the intervening sequence derived from the KOD1 strain
Escherichia coli transformed with the pET-8c plasmid containing the NA polymerase gene is cultured in, for example, a TB medium and subjected to induction treatment. Preferably, the T7 promoter is induced by adding isopropioti-β-D-galactoside.

In the purification method of the present invention, after culturing the recombinant host cells, (a) collecting and crushing the recombinant host cells;
Preparing a cell extract, and (b) removing host protein-derived impure proteins. The heat-resistant DNA polymerase produced from the recombinant host cells is obtained by culturing the host cells in a medium.
After the induction treatment, the cells are separated and collected from the culture solution by centrifugation or the like. After resuspending the cells in a buffer, the cells are disrupted by sonication, Dynomill French Press, or the like. Next, heat treatment is performed, and a heat-resistant DNA polymerase is recovered from the supernatant. As the method for crushing cells, ultrasonic treatment, Dynomill French Press method and the like are preferable. Heat treatment is preferred as one of the steps for removing host cell-derived impure proteins. Heat treatment condition is 70 ° C or more, preferably 90 ° C
That is all. As a method for removing other impurity proteins, various types of chromatography and the like are performed.

The hyperthermophilic archaeon KOD thus obtained
The molecular weight of a heat-resistant DNA polymerase derived from one strain is about 9
0KDa (see FIG. 2).

When a polymerase chain reaction is carried out using this heat-resistant DNA polymerase, sufficient DNA
Amplification of the A fragment is confirmed (see FIG. 3).

[0023]

The DNA polymerase derived from the hyperthermophilic archaeon obtained by the present invention is an enzyme having high thermostability and suitable for the polymerase chain reaction and the like.

[0024]

Next, the present invention will be described with reference to examples. Example 1 DNA polymerase gene derived from hyperthermophilic archaeon KOD1 strain
Of black - after culturing hyperthermophilic archaeon strain KOD1 isolated by training Kagoshima Kodakara Island at 95 ° C., the cells were collected. Chromosomal DNA of the hyperthermophilic archaeon KOD1 strain was prepared from the obtained cells according to a conventional method. Pyrococcus furiosus DNA polymerase
Two primers (5'-GGATTAGTATAGTGCCAATGGAA) were used based on the amino acid sequence of the conserved region of ze (Pfu polymerase).
GGCGAC-3 '(SEQ ID NO: 4), 5'-GAGGGCGAAGTTTATTCCGAGCTT
-3 '(SEQ ID NO: 5) was synthesized. Using these two primers, a PCR reaction was performed using the prepared chromosomal DNA as a template.

After the base sequence (SEQ ID NO: 6) of the PCR amplified DNA fragment was determined and the amino acid sequence (SEQ ID NO: 7) was determined, the amplified DNA fragment was used as a probe to obtain KOD1.
Southern hybridization was performed on the product of the strain chromosomal DNA restriction enzyme treatment, and the size of the fragment encoding the DNA polymerase was determined (about 4 to 7 Kbp). Further, a DNA fragment of this size was recovered from an agarose gel, inserted into a plasmid pBS (manufactured by Stratagene), and Escherichia coli (E. coli JM109) was transformed with the mixture to prepare a library. Colony hybridization was carried out using the probe (SEQ ID NO: 6) used for Southern hybridization, and a clone strain (E. coli JM109 /
pBSK0D1) was obtained.

Example 2 Determination of the base sequence of the cloned fragment The cloned strain obtained in Example 1, E. coli JM109 / pBSK0D1
The plasmid and BSKOD1 were collected from the
(SEQ ID NO: 1) was determined. Further, the amino acid sequence was deduced from the obtained base sequence. DNA from KOD1 strain
The polymerase gene consists of 5010 bases and 1670
Amino acids were encoded.

Example 3 Construction of Recombinant Expression Vector In order to construct a complete polymerase gene, two intervening sequence portions (1374-2453 bp, 2708-43) were used.
16 bp) was removed by the PCR fusion method. In the PCR fusion method, three sets of primers (SEQ ID NOs: 8 to 13) were combined using a plasmid recovered from the clone strain as a template, and PCR was performed to amplify three fragments excluding the intervening sequence. At this time, the primers used for PCR were designed such that the same sequence as the binding partner was located on the side that binds to other fragments. In addition, separate restriction enzyme sites (N-terminal: EcoRV, C-terminal: BamHI) were designed to be created at both ends. Next, in the PCR-amplified fragment, the fragment located in the center of the structure and the fragment located on the N-terminal side were mixed, and PCR was performed using each fragment as a primer. Similarly, the fragment located at the center and the fragment located at the C-terminal side were mixed in structure, and PCR was performed using each fragment as a primer. PCR was performed again using the two kinds of fragments thus obtained, the intervening sequence was removed, and EcoRV at the N-terminus and Kam having a BamHI site at the C-terminus.
A complete gene fragment encoding the DNA polymerase from the OD1 strain was obtained. Furthermore, an expression vector capable of inducing the same gene with the T7 promoter, pET-8c N
Using the coI / BamHI site and the restriction enzyme site created earlier, subcloning was performed to obtain a recombinant expression vector (pET-pol).

Example 4 Expression and Purification of KOD1-Derived DNA Polymerase The recombinant expression vector obtained in Example 3 (pET-po
l) was used to transform Escherichia coli (E. coli JM109), and the resulting transformant was transformed into TB medium (Molecular Cloning, pA2,
1989), and 1 hour before collection, the T7 promoter was used.
The induction of tar was carried out by adding isopropioti-β-D-galactoside. Cells were collected from the culture by centrifugation. After resuspension in a buffer, the cells were disrupted by sonication to obtain a cell extract. In order to further remove impurity proteins from the host cells, the cell lysate
C. for 20 minutes to insolubilize host cell-derived impure proteins. The insoluble fraction is removed by centrifugation and KOD
One strain of thermostable DNA polymerase was obtained.

Example 5 Purification of KOD1-derived heat-resistant DNA polymerase The molecular weight of the KOD1-derived heat-resistant DNA polymerase obtained in Example 4 was determined by SDS-PAGE to be about 86-92 kDa. (FIG. 2). When PCR was carried out using the heat-resistant DNA polymerase derived from KOD1 obtained in Example 4 and a known template / primer, Thermococcus litoralis (Thermococcus litoralis) was obtained.
As in the case of using a heat-resistant DNA polymerase derived from lis), a target DNA fragment was confirmed (FIG. 3), and a high thermostable DNA polymerase activity was confirmed.

COMPARATIVE EXAMPLE 1 The hyperthermophilic archaeon KOD1 of the present invention is considered to be a related bacterium.
Pyrococcus furiosus
Or Thermococcus litoraris
lis) -derived thermostable DNA polymerase gene DNA polymerase gene (SEQ ID NO: 3) derived from the hyperthermophilic archaeon KOD1 of the present invention, Pyrococcus furiosus (P
yrococcus furiosus), a thermostable DNA polymerase gene (JP-A-5-328969),
Heat resistance D from litoralis (Thermococcus litoralis)
D of NA polymerase gene (JP-A-6-7160)
The amino acid sequence was deduced from the NA sequence and compared. The DNA polymerase derived from KOD1 of the present invention is Reg which is a conserved region of eukaryotic α DNA polymerase.
ions 1 to 5 were present. In addition, 3 '→
EXO1,2,2,5 'exonuclease-momotif
There were three. However, the intervening sequences IVS-A and IVS-B were present in the α DNA polymerase conserved regions Region 1 and Region 2 (see FIG. 4). On the other hand, Pyrococcus furiosus (Pyrococcus fu
riosus), a thermostable DNA polymerase derived from Pfu
There were no intervening sequences in the polymerase. Vent polymerase, a thermostable DNA polymerase derived from Thermococcus litoralis, has an αDNA polymerase storage region Region.
2 and Region 3, the intervening sequences IVS1 and IVS
2 was observed (see FIG. 4).

[0031]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 5342 Sequence type: nucleic acid (DNA) Number of strands: double-stranded Tropoie: linear Sequence type: cDNA Origin: Hyperthermophilic primordial Strain name: KOD1 Sequence characteristics 156- 5165 P CDS 1374-2453 Intervening sequence 2708-4316 Intervening sequence Sequence GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TGCCGGTTTT 60 ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TACAATAAAG 120 CCTGGATGATC TAG ATTCGGATGATC TAGATTACGGATAG GAG GAT GGA AAG CCT GTC ATA AGA ATT TTC AAG AAG GAA 221 Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu 10 15 20 AAC GGC GAG TTT AAG ATT GAG TAC GAC CGG ACT TTT GAA CCC TAC TTC 269 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Thr Phe Glu Pro Tyr Phe 25 30 35 TAC GCC CTC CTG AAG GAC GAT TCT GCC ATT GAG GAA GTC AAG AAG ATA 317 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Glu Val Lys Lys Ile 40 45 50 ACC GCC GAG AGG CAC GGG ACG GTT GTA ACG GTT AAG CGG GTT GAA AAG 365 Thr Ala Glu Arg His Gly Thr Val Val Thr Val Lys Arg Val Glu Lys 55 60 65 70 GTT CAG AAG AAG TTC CTC GGG AGA CCA GTT GAG GTC TGG AAA CTC TAC 413 Val Gln Lys Lys Phe Leu Gly Arg Pro Val Glu Val Trp Lys Leu Tyr 75 80 85 TTT ACT CAT CAT CCG CAG GAC GTC CCA GCG ATA AGG GAC AAG ATA CGA GAG 461 Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu 90 95 100 CAT GGA GCA GTT ATT GAC ATC TAC GAG TAC GAC ATA CCC TTC GCC AAG 509 His Gly Ala Val Ile Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys 105 110 115 CGC TAC CTC ATA GAC AAG GGA TTA GTG CCA ATG GAA GGC GAC GAG GAG 557 Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro Met Glu Gly Asp Glu Glu 120 125 130 CTG AAA ATG CTC GCC TTC GAC ATT CAA ACT CTC TAC CAT GAG GGC GAG 605 Leu Lys Met Leu Ala Phe Asp Ile Gln Thr Leu Tyr His Glu Gly Glu 135 140 145 150 GAG TTC GCC GAG GGG CCA ATC CTT ATG ATA AGC TAC GCC GAC GAG GAA 653 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu 155 160 165 165 GGG GCC AGG GTG ATA ACT TGG AAG AAC GTG GAT CTC CCC TAC GTT GAC 701 Gly Ala Arg Val Ile Thr Trp Lys Asn Val Asp Leu Pro Tyr Val Asp 170 175 180 GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG CGC TTC CTC CGT GTT GTG 749 Val Val Ser Thr Glu Arg Glu Met Ile Lys Arg Phe Leu Arg Val Val 185 190 195 AAG GAG AAA GAC CCG GAC GTT CTC ATA ACC TAC AAC GGC GAC AAC TTC 797 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe 200 205 210 GAC TTC GCC TAT CTG AAA AAG CGC TGT GAA AAG CTC GGA ATA AAC TTC 845 Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe 215 220 225 230 GCC CTC GGA AGG GAT GGA AGC GAG CCG AAG ATT CAG AGG ATG GGC GAC 893 Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp 235 240 245 AGG TTT GCC GTC GAA GTG AAG GGA CGG ATA CAC TTC GAT CTC TAT CCT 941 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro 250 255 260 GTG ATA AGA CGG ACG ATA AAC CTG CCC ACA TAC ACG CTT GAG GCC GTT 989 Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val 265 270 275 275 TAT GAA GCC GTC TTC GGT CAG CCGAAG GAG AAG GTT TAC GCT GAG GAA 1037 Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu 280 285 290 ATA ATA ACA CCA GCC TGG GAA ACC GGC GAG AAC CTT GAG AGA GTC GCC CGC 1085 Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn Leu Glu Arg Val Ala Arg 295 300 305 310 TAC TCG ATG GAA GAT GCG AAG GTC ACA TAC GAG CTT GGG AAG GAG TTC 1133 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe 315 320 325 CTT CCG ATG GAG GCC CAG CTT TCT CGC TTA ATC GGC CAG TCC CTC TGG 1181 Leu Pro Met Glu Ala Gln Leu Ser Arg Leu Ile Gly Gln Ser Leu Trp 330 335 340 GAC GTC TCC CGC TCC AGC ACT GGC AAC CTC GTT GAG TGG TTC CTC CTC 1229 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu 345 350 355 AGG AAG GCC TAT GAG AGG AAT GAG CTG GCC CCG AAC AAG CCC GAT GAA 1277 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu 360 365 370 AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT GAA GGA GGC TAT GTA AAA 1325 Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr Glu Gly Gly Tyr Val Lys 375 380 385 390 390 GAG CCC G AG AGA GGG TTG TGG GAG AAC ATA GTG TAC CTA GAT TTT AGA 1373 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg 395 400 405 TGC CAT CCA GCC GAT ACG AAG GTT GTC GTC AAG GGG AAG GGG ATT ATA 1421 Cys His Pro Ala Asp Thr Lys Val Val Val Lys Gly Lys Gly Ile Ile 410 415 420 AAC ATC AGC GAG GTT CAG GAA GGT GAC TAT GTC CTT GGG ATT GAC GGC 1469 Asn Ile Ser Glu Val Gln Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 425 430 435 TGG CAG AGA GTT AGA AAA GTA TGG GAA TAC GAC TAC AAA GGG GAG CTT 1517 Trp Gln Arg Val Arg Lys Val Trp Glu Tyr Asp Tyr Lys Gly Glu Leu 440 445 450 GTA AAC ATA AAC GGG TTA AAG TGT ACG CCC AAT CAT AAG CTT CCC GTT 1565 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 455 460 465 470 GTT ACA AAG AAC GAA CGA CAA ACG AGA ATA AGA GAC AGT CTT GCT AAG 1613 Val Thr Lys Asn Glu Arg Gln Thr Arg Ile Arg Asp Ser Leu Ala Lys 475 480 485 TCT TTC CTT ACT AAA AAA GTT AAG GGC AAG ATA ATA ACC ACT CCC CTT 1661 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Ile Ile Thr Thr Pro Leu 49 0 495 500 TTC TAT GAA ATA GGC AGA GCG ACA AGT GAG AAT ATT CCA GAA GAA GAG 1709 Phe Tyr Glu Ile Gly Arg Ala Thr Ser Glu Asn Ile Pro Glu Glu Glu 505 510 515 GTT CTC AAG GGA GAG CTC GCT GGC ATA CTA TTG GCT GAA GGA ACG CTC 1757 Val Leu Lys Gly Glu Leu Ala Gly Ile Leu Leu Ala Glu Gly Thr Leu 520 525 530 TTG AGG AAA GAC GTT GAA TAC TTT GAT TCA TCC CGC AAA AAA CGG AGG 1805 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Lys Lys Arg Arg 535 540 545 550 550 ATT TCA CAC CAG TAT CGT GTT GAG ATA ACC ATT GGG AAA GAC GAG GAG 1853 Ile Ser His Gln Tyr Arg Val Glu Ile Thr Ile Gly Lys Asp Glu Glu 555 560 560 565 GAG TTT AGG GAT CGT ATC ACA TAC ATT TTT GAG CGT TTG TTT GGG ATT 1901 Glu Phe Arg Asp Arg Ile Thr Tyr Ile Phe Glu Arg Leu Phe Gly Ile 570 575 580 ACT CCA AGC ATC TCG GAG AAG AAA GGA ACT AAC GCA GTA ACA CTC AAA 1949 Thr Pro Ser Ile Ser Glu Lys Lys Gly Thr Asn Ala Val Thr Leu Lys 585 590 595 GTT GCG AAG AAG AAT GTT TAT CTT AAA GTC AAG GAA ATT ATG GAC AAC 1997 Val Ala Lys Lys Asn Val Tyr Leu Lys Val Lys Gl u Ile Met Asp Asn 600 605 610 ATA GAG TCC CTA CAT GCC CCC TCG GTT CTC AGG GGA TTC TTC GAA GGC 2045 Ile Glu Ser Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 615 620 625 630 GAC GGT TCA GTA AAC AGG GTT AGG AGG AGT ATT GTT GCA ACC CAG GGT 2093 Asp Gly Ser Val Asn Arg Val Arg Arg Ser Ile Val Ala Thr Gln Gly 635 640 645 ACA AAG AAC GAG TGG AAG ATT AAA CTG GTG TCA AAA CTG CTC TCC CAG 2141 Thr Lys Asn Glu Trp Lys Ile Lys Leu Val Ser Lys Leu Leu Ser Gln 650 655 660 CTT GGT ATC CCT CAT CAA ACG TAC ACG TAT CAG TAT CAG GAA AAT GGG 2189 Leu Gly Ile Pro His Gln Thr Tyr Thr Tyr Gln Tyr Gln Glu Asn Gly 665 670 675 AAA GAT CGG AGC AGG TAT ATA CTG GAG ATA ACT GGA AAG GAC GGA TTG 2237 Lys Asp Arg Ser Arg Tyr Ile Leu Glu Ile Thr Gly Lys Asp Gly Leu 680 685 690 ATA CTG TTC CAA ACA CTC ATT GGA TTC ATC AGT GAA AGA AAG AAC GCT 2285 Ile Leu Phe Gln Thr Leu Ile Gly Phe Ile Ser Glu Arg Lys Asn Ala 695 700 705 710 CTG CTT AAT AAG GCA ATA TCT CAG AGG GAA ATG AAC AAC TTG GAA AAC 2333 Leu Leu Asn Lys Ala Ile Ser Gln Arg Glu Met Asn Asn Leu Glu Asn 715 720 725 AAT GGA TTT TAC AGG CTC AGT GAA TTC AAT GTC AGC ACG GAA TAC TAT 2381 Asn Gly Phe Tyr Arg Leu Ser Glu Phe Asn Val Ser Thr Glu Tyr Tyr 730 735 740 740 GAG GGC AAG GTC TAT GAC TTA ACT CTT GAA GGA ACT CCC TAC TAC TTT 2429 Glu Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 745 750 755 GCC AAT GGC ATA TTG ACC CAT AAC TCC CTG TAC CCC TCA ATC ATC ATC 2477 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile 760 765 770 ACC CAC AAC GTC TCG CCG GAT ACG CTC AAC AGA GAA GGA TGC AAG GAA 2525 Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu 775 780 785 790 790 TAT GAC GTT GCC CCA CAG GTC GGC CAC CGC TTC TGC AAG GAC TTC CCA 2573 Tyr Asp Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro 795 800 805 GGA TTT ATC CCG AGC CTG CTT GGA GAC CTC CTA GAG GAG AGG CAG AAG 2621 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys 810 815 820 ATA AAG AAG AAG AAG ATG AAG GCC ACG ATT GAC CCG ATC GAG AGG AAG CTC 2669 Ile Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu 825 830 835 CTC GAT TAC AGG CAG AGG GCC ATC AAG ATC CTG GCA AAC AGC ATC CTA 2717 Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Ile Leu 840 845 850 CCC GAG GAA TGG CTT CCA GTC CTC GAG GAA GGG GAG GTT CAC TTC GTC 2765 Pro Glu Glu Trp Leu Pro Val Leu Glu Glu Gly Glu Val His Phe Val 855 860 865 870 870 AGG ATT GGA GAG CTC ATA GAC CGG ATG ATG GAG GAA AAT GCT GGG AAA 2813 Arg Ile Gly Glu Leu Ile Asp Arg Met Met Glu Glu Asn Ala Gly Lys 875 880 885 GTA AAG AGA GAG GGC GAG ACG GAA GTG CTT GAG GTC AGT GGG CTT GAA 2861 Val Lys Arg Glu Glu Glu Thr Glu Val Leu Glu Val Ser Gly Leu Glu 890 895 900 GTC CCG TCC TTT AAC AGG AGA ACT AAC AAG GCC GAG CTC AAG AGA GTA 2909 Val Pro Ser Phe Asn Arg Arg Thr Asn Lys Ala Glu Leu Lys Arg Val 905 910 915 AAG GCC CTG ATT AGG CAC GAT TAT TCT GGC AAG GTC TAC ACC ATC AGA 2957 Lys Ala Leu Ile Arg His Asp Tyr Ser Gly Lys Val Tyr Thr Ile Arg 920 925 930 CTG AAG TCG GGG AGG AGA ATA AAG ATA ACC TCT GGC CAC A GC CTC TTC 3005 Leu Lys Ser Gly Arg Arg Ile Lys Ile Thr Ser Gly His Ser Leu Phe 935 940 945 950 TCT GTG AGA AAC GGG GAG CTC GTT GAA GTT ACG GGC GAT GAA CTA AAG 3053 Ser Val Arg Asn Gly Glu Leu Val Glu Val Thr Gly Asp Glu Leu Lys 955 960 965 CCA GGT GAC CTC GTT GCA GTC CCG CGG AGA TTG GAG CTT CCT GAG AGA 3101 Pro Gly Asp Leu Val Ala Val Pro Arg Arg Leu Glu Leu Pro Glu Arg 970 975 980 980 AAC CAC GTG CTG AAC CTC GTT GAA CTG CTC CTT GGA ACG CCA GAA GAA 3149 Asn His Val Leu Asn Leu Val Glu Leu Leu Leu Gly Thr Pro Glu Glu 985 990 995 GAA ACT TTG GAC ATC GTC ATG ACG ATC CCA GTC AAG GGT AAG AAG AAC 3197 Glu Thr Leu Asp Ile Val Met Thr Ile Pro Val Lys Gly Lys Lys Asn 1000 1005 1010 TTC TTT AAA GGG ATG CTC AGG ACT TTG CGC TGG ATT TTC GGA GAG GAA 3245 Phe Phe Lys Gly Met Leu Arg Thr Leu Arg Trp Ile Phe Gly Glu Glu 1015 1020 1025 1030 AAG AGG CCC AGA ACC GCG AGA CGC TAT CTC AGG CAC CTT GAG GAT CTG 3293 Lys Arg Pro Arg Thr Ala Arg Arg Tyr Leu Arg His Leu Glu Asp Leu 1035 1040 1045 GGC TAT GTC CGG CTT AAG AAG ATC GGC TAC GAA GTC CTC GAC TGG GAC 3341 Gly Tyr Val Arg Leu Lys Lys Ile Gly Tyr Glu Val Leu Asp Trp Asp 1050 1055 1060 TCA CTT AAG AAC TAC AGA AGG CTC TAC GAG GCG CTT GTC GAG AAC GTC 3389 Ser Leu Lys Asn Tyr Arg Arg Leu Tyr Glu Ala Leu Val Glu Asn Val 1065 1070 1075 AGA TAC AAC GGC AAC AAG AGG GAG TAC CTC GTT GAA TTC AAT TCC ATC 3437 Arg Tyr Asn Gly Asn Lys Arg Glu Tyr Leu Val Glu Phe Asn Ser Ile 1080 1085 1090 CGG GAT GCA GTT GGC ATA ATG CCC CTA AAA GAG CTG AAG GAG TGG AAG 3485 Arg Asp Ala Val Gly Ile Met Pro Leu Lys Glu Leu Lys Glu Trp Lys 1095 1100 1105 1110 ATC GGC ACG CTG AAC GGC TTC AGA ATG AGA AAG CTC ATT GAA GTG GAC 3533 Ile Gly Thr Leu Asn Gly Phe Arg Met Arg Lys Leu Ile Glu Val Asp 1115 1120 1125 GAG TCG TTA GCA AAG CTC CTC GGC TAC TAC GTG AGC GAG GGC TAT GCA 3581 Glu Ser Leu Ala Lys Leu Leu Gly Tyr Tyr Val Ser Glu Gly Tyr Ala 1130 1135 1140 AGA AAG CAG AGG AAT CCC AAA AAC GGC TGG AGC TAC AGC GTG AAG CTC 3629 Arg Lys Gln Arg Asn Pro Lys Asn Gly Trp Ser Tyr Ser Val Ly s Leu 1145 1150 1155 TAC AAC GAA GAC CCT GAA GTG CTG GAC GAT ATG GAG AGA CTC GCC AGC 3677 Tyr Asn Glu Asp Pro Glu Val Leu Asp Asp Met Glu Arg Leu Ala Ser 1160 1165 1170 AGG TTT TTC GGG AAG GTG AGG GGGGC AGG AAC TAC GTT GAG ATA CCG 3725 Arg Phe Phe Gly Lys Val Arg Arg Gly Arg Asn Tyr Val Glu Ile Pro 1175 1180 1185 1190 AAG AAG ATC GGC TAC CTG CTC TTT GAG AAC ATG TGC GGT GTC CTA GCG 3773 Lys Lys Ile Gly Tyr Leu Leu Phe Glu Asn Met Cys Gly Val Leu Ala 1195 1200 1205 GAG AAC AAG AGG ATT CCC GAG TTC GTC TTC ACG TCC CCG AAA GGG GTT 3821 Glu Asn Lys Arg Ile Pro Glu Phe Val Phe Thr Ser Pro Lys Gly Val 1210 1215 1220 CGG CTG GCC TTC CTT GAG GGG TAC TCA TCG GCG ATG GCG ACG TCC ACC 3869 Arg Leu Ala Phe Leu Glu Gly Tyr Ser Ser Ala Met Ala Thr Ser Thr 1225 1230 1235 GAA CAA GAG ACT CAG GCT CTC AAC GAA AAG CGA GCT TTA GCG AAC CAG 3917 Glu Gln Glu Thr Gln Ala Leu Asn Glu Lys Arg Ala Leu Ala Asn Gln 1240 1245 1250 CTC GTC CTC CTC TTG AAC TCG GTG GGG GTC TCT GCT GTA AAA CTT GGG 3965 Leu Val Leu Leu L eu Asn Ser Val Gly Val Ser Ala Val Lys Leu Gly 1255 1260 1265 1270 CAC GAC AGC GGC GTT TAC AGG GTC TAT ATA AAC GAG GAG CTC CCG TTC 4013 His Asp Ser Gly Val Tyr Arg Val Tyr Ile Asn Glu Glu Leu Pro Phe 1275 1280 1285 GTA AAG CTG GAC AAG AAA AAG AAC GCC TAC TAC TCA CAC GTG ATC CCC 4061 Val Lys Leu Asp Lys Lys Lys Asn Ala Tyr Tyr Ser His Val Ile Pro 1290 1295 1300 AAG GAA GTC CTG AGC GAG GTC TTT GGG AAG GTT TTC CAG AAA AAC GTC 4109 Lys Glu Val Leu Ser Glu Val Phe Gly Lys Val Phe Gln Lys Asn Val 1305 1310 1315 AGT CCT CAG ACC TTC AGG AAG ATG GTC GAG GAC GGA AGA CTC GAT CCC 4157 Ser Pro Gln Thr Phe Arg Lys Met Val Glu Asp Gly Arg Leu Asp Pro 1320 1325 1330 GAA AAG GCC CAG AGG CTC TCC TGG CTC ATT GAG GGG GAC GTA GTG CTC 4205 Glu Lys Ala Gln Arg Leu Ser Trp Leu Ile Glu Gly Asp Val Val Leu 1335 1340 1345 1350 GAC CGC GTT GAG TCC GTT GAT GTG GAA GAC TAC GAT GGT TAT GTC TAT 4253 Asp Arg Val Glu Ser Val Asp Val Glu Asp Tyr Asp Gly Tyr Val Tyr 1355 1360 1365 GAC CTG AGC GTC GAG GAC ATC GAG AAC TTC CTC GTT GGC TTT GGG TTG 4301 Asp Leu Ser Val Glu Asp Asn Glu Asn Phe Leu Val Gly Phe Gly Leu 1370 1375 1380 GTC TAT GCT CAC AAC AGC TAC TAC GGT TAC TAC GGC TAT GCA AGG GCG 4349 Val Tyr Ala His Asn Ser Tyr Tyr Gly Tyr Tyr Gly Tyr Ala Arg Ala 1385 1390 1395 CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC GTA ACG GCC TGG GGA AGG 4397 Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser Val Thr Ala Trp Gly Arg 1400 1405 1410 GAGTAC ATA ACG ATG ACC ATC AAG GAG ATA GAG GAA AAG TAC GGC TTT 4445 Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile Glu Glu Lys Tyr Gly Phe 1415 1420 1425 1430 AAG GTA ATC TAC AGC GAC ACC GAC GGA TTT TTT GCC ACA ATACT GGA 4493 Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly 1435 1440 1445 GCC GAT GCT GAA ACC GTC AAA AAG AAG GCT ATG GAG TTC CTC AAC TAT 4541 Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Met Glu Phe Leu Asn Tyr 1450 1455 1460 ATC AAC GCC AAA CTT CCG GGC GCG CTT GAG CTC GAG TAC GAG GGC TTC 4589 Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe 1465 1470 147 5 TAC AAA CGC GGC TTC TTC GTC ACG AAT ACG AAG AAG TAT GCG GTG ATA GAC 4637 Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp 1480 1485 1490 GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT GAG ATT GTG AGG CGT GAC 4685 Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp 1495 1500 1505 1510 TGG AGC GAG ATA GCG AAA GAG ACG CAG GCG AGG GTT CTT GAA GCT TTG 4733 Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Leu 1515 1520 1525 CTA AAG GAC GGT GAC GTC GAG AAG GCC GTG AGG ATA GTC AAA GAA GTT 4781 Leu Lys Asp Gly Asp Val Glu Lys Ala Val Arg Ile Val Lys Glu Val 1530 1535 1540 ACC GAA AAG CTG AGC AAG TAC GAG GTT CCG CCG GAG AAG CTG GTG ATC 4829 Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile 1545 1550 1555 CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC TAC AAG GCA ACC GGT CCC 4877 His Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro 1560 1565 1570 CAC GTT GCC GTT GCC AAG AGG TTG GCC GCG AGA GGA GTC AAA ATA CGC 4925 His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Val Lys Ile Arg 1575 1580 1585 1590 CCT GGA ACG GTG ATA AGC TAC ATC GTG CTC AAG GGC TCT GGG AGG ATA 4973 Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile 1595 1600 1605 GGC GAC AGG GCG ATA CCG TTC GAC GAG TTC GAC CCG ACG AAG CAC AAG 5021 Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Lys 1610 1615 1620 TAC GAC GCC GAG TAC TAC ATT ATT GAG AAC CAG GTT CTC CCA GCC GTT GAG 5069 Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu 1625 1630 1635 AGA ATT CTG AGA GCC TTC GGT TAC CGC AAG GAA GAC CTG CGC TAC CAG 5117 Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln 1640 1645 1650 AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG CTG AAG CCG AAG GGA ACT 5165 Lys Thr Arg Gln Val Gly Leu Ser Ala Trp Leu Lys Pro Lys Gly Thr 1655 1660 1665 1670 TGACCTTTCC ATTTGTTTTC CAGCGGTCATCTCTCTCGAGCTAGATCATC CCATGAAGAT AGAAATCCGG CGGCGCCCGG TTAAATACGC TAGGATAGA 5285 GTGAAGCCAG ACGGCAGGGT AGTCGTCACT GCCCCGAGGG TTCAACGTTG AGAAGTT 534 Two

SEQ ID NO: 2 Sequence length: 774 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe 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 Thr Val Val Thr 50 55 60 Val Lys Arg Val Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Val 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Gly Ala 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 Gln Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Val 165 170 175 Asp Leu Pro Tyr Val A sp Val Val Ser Thr Glu Arg 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 Asn Phe Ala 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 Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Leu 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 Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 Glu Gly Gly Tly Val Lys G lu Pro Glu Arg Gly Leu Trp Glu 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 Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg 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 Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Asn Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 585 590 Lys Tyr Ala Val Ile Asp G lu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val 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 705 710 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp 755 760 765 Leu Lys Pro Lys Gly Thr 770

SEQ ID NO: 3 Sequence length: 5342 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Tropoie: linear Sequence type: cDNA Origin: Hyperthermophilic primordial Strain name: KOD1 sequence GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TGCCGGTTTT 60 ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TACAATAAAG 120 CCTGGATTGT TCTACAAGAT TATGGGGGAT GAAAGATGAT CCTCGACACT GACTACATAA 180 CCGAGGATGG AAAGCCTGTC ATAAGAATTT TCAAGAAGGA AAACGGCGAG TTTAAGATTG 240 AGTACGACCG GACTTTTGAA CCCTACTTCT ACGCCCTCCT GAAGGACGAT TCTGCCATTG 300 AGGAAGTCAA GAAGATAACC GCCGAGAGGC ACGGGACGGT TGTAACGGTT AAGCGGGTTG 360 AAAAGGTTCA GAAGAAGTTC CTCGGGAGAC CAGTTGAGGT CTGGAAACTC TACTTTACTC 420 ATCCGCAGGA CGTCCCAGCG ATAAGGGACA AGATACGAGA GCATGGAGCA GTTATTGACA 480 TCTACGAGTA CGACATACCC TTCGCCAAGC GCTACCTCAT AGACAAGGGA TTAGTGCCAA 540 TGGAAGGCGA CGAGGAGCTG AAAATGCTCG CCTTCGAGACAT TCAAACTCTC TACCATGAGG 600 GCGAGGTCGACCGATCGAGGAG GG GGGTGATAAC TTGGAAGAAC GTGGATCTCC CCTACGTTGA CGTCGTCTCG ACGGAGAGGG 720 AGATGATAAA GCGCTTCCTC CGTGTTGTGA AGGAGAAAGA CCCGGACGTT CTCATAACCT 780 ACAACGGCGA CAACTTCGAC TTCGCCTATC TGAAAAAGCG CTGTGAAAAG CTCGGAATAA 840 ACTTCGCCCT CGGAAGGGAT GGAAGCGAGC CGAAGATTCA GAGGATGGGC GACAGGTTTG 900 CCGTCGAAGT GAAGGGACGG ATACACTTCG ATCTCTATCC TGTGATAAGA CGGACGATAA 960 ACCTGCCCAC ATACACGCTT GAGGCCGTTT ATGAAGCCGT CTTCGGTCAG CCGAAGGAGA 1020 AGGTTTACGC TGAGGAAATA ACACCAGCCT GGGAAACCGG CGAGAACCTT GAGAGAGTCG 1080 CCCGCTACTC GATGGAAGAT GCGAAGGTCA CATACGAGCT TGGGAAGGAG TTCCTTCCGA 1140 TGGAGGCCCA GCTTTCTCGC TTAATCGGCC AGTCCCTCTG GGACGTCTCC CGCTCCAGCA 1200 CTGGCAACCT CGTTGAGTGG TTCCTCCTCA GGAAGGCCCT ATGAGAGGAA TGAGCTGGCC 1260 CCGAACAAGC CCGATGAAAA GGAGCTGGCC AGAAGACGGC AGAGCTATGA AGGAGGCTAT 1320 GTAAAAGAGC CCGAGAGAGG GTTGTGGGAG AACATAGTGT ACCTAGATTT TAGATGCCAT 1380 CCAGCCGATA CGAAGGTTGT CGTCAAGGGG AAGGGGATTA TAAACATCAG CGAGGTTCAG 1440 GAAGGTGACT ATGTCCTTGG GATTGACGGC TGGCAGAGAG TTAGAAAAGT ATGGGAATAC 1500 GACTACAAAG GGGAGCTTGT AAACATAAAC GGGTTAAAGT GTACGCCCAA TCATAAGCTT 1560 CCCGTTGTTA CAAAGAACGA ACGACAAACG AGAATAAGAG ACAGTCTTGC TAAGTCTTTC 1620 CTTACTAAAA AAGTTAAGGG CAAGATAATA ACCACTCCCC TTTTCTATGA AATAGGCAGA, 1680 GCGACAAGTG AGAATATTCC AGAAGAAGAG GTTCTCAAGG GAGAGCTCGC TGGCATAGTA, 1740 TTGGCTGAAG GAACGCTCTT GAGGAAAGAC GTTGAATACT TTGATTCATC CCGCAAAAAA 1800 CGGAGGATTT CACACCAGTA TCGTGTTGAG ATAACCATTG GGAAAGACGA GGAGGAGTTT 1860 AGGGATCGTA TCACATACAT TTTTGAGCGT TTGTTTGGGA TTACTCCAAG CATCTCGGAG 1920 AAGAAAGGAA CTAACGCAGT AACACTCAAA GTTGCGAAGA AGAATGTTTA TCTTAAAGTC 1980 AAGGAAATTA TGGACAACAT AGAGTCCCTA CATGCCCCCT CGGTTCTCAG GGGATTCTTC 2040 GAAGGCGACG GTTCAGTAAA CAGGTTAGGA GGAGTATTGT TGCAACCCAG GGTACAAAGA 2100 ACGAGTGGAA GATTAAACTG GTGTCAAAAC TGCTCTCCCA GCTTGGTATC CCTCATCAAA 2160 CGTACACGTA TCAGTATCAG GAAAATGGGA AAGATCGGAG CAGGTATATA CTGGAGATAA 2220 CTGGAAAGGA CGGATTGATA CTGTTCCAAA CACTCATTGG ATTCATCAGT GAAAGAAAGA 2280 ACGCTCTGCT TAATAAGGCA ATATCTCAGA GGGAAATGAA CAACTTGGAA AACAATGGAT 2340 TTTACAGGCT CAGTG AATTC AATGTCAGCA CGGAATACTA TGAGGGCAAG GTCTATGACT 2400 TAACTCTTGA AGGAACTCCC TACTTTGCCA ATGGCATATT GACCCATAAC TCCCTGTACC 2460 CCTCAATCAT CATCACCCAC AACGTCTCGC CGGATACGCT CAACAGAGAA GGATGCAAGG 2520 AATATGACGT TGCCCCACAG GTCGGCCACC GCTTCTGCAA GGACTTCCCA GGATTTATCC 2580 CGAGCCTGCT TGGAGACCTC CTAGAGGAGA GGCAGAAGAT AAAGAAGAAG ATGAAGGCCA 2640 CGATTGACCC GATCGAGAGG AAGCTCCTCG ATTACAGGCA GAGGGCCATC AAGATCCTGG 2700 CAAACAGCAT CCTACCCGAG GAATGGCTTC CAGTCCTCGA GGAAGGGGAG GTTCACTTCG 2760 TCAGGATTGG AGAGCTCATA GACCGGATGA TGGAGGAAAA TGCTGGGAAA GTAAAGAGAG 2820 AGGGCGAGAC GGAAGTGCTT GAGGTCAGTG GGCTTGAAGT CCCGTCCTTT AACAGGAGAA 2880 CTAACAAGGC CGAGCTCAAG AGAGTAAAGG CCCTGATTAG GCACGATTAT TCTGGCAAGG 2940 TCTACACCAT CAGACTGAAG TCGGGGAGGA GAATAAAGAT AACCTCTGGC CACAGCCTCT 3000 TCTCTGTGAG AAACGGGGAG CTCGTTGAAG TTACGGGCGA TGAACTAAAG CCAGGTGACC 3060 TCGTTGCAGT CCCGCGGAGA TTGGAGCTTC CTGAGAGAAA CCACGTGCTG AACCTCGTTG 3120 AACTGCTCCT TGGAACGCCA GAAGAAGAAA CTTTGGACAT CGTCATGACG ATCCCAGTCA 3180 AGGGTAAGAA GAACTTCTTT AAAGGGATGC TCAGGACTTT GCGCTGGATT TTCGGAGAGG 3240 AAAAGAGGCC CAGAACCGCG AGACGCTATC TCAGGCACCT TGAGGATCTG GGCTATGTCC 3300 GGCTTAAGAA GATCGGCTAC GAAGTCCTCG ACTGGGACTC ACTTAAGAAC TACAGAAGGC 3360 TCTACGAGGC GCTTGTCGAG AACGTCAGAT ACAACGGCAA CAAGAGGGAG TACCTCGTTG 3420 AATTCAATTC CATCCGGGAT GCAGTTGGCA TAATGCCCCT AAAAGAGCTG AAGGAGTGGA 3480 AGATCGGCAC GCTGAACGGC TTCAGAATGA GAAAGCTCAT TGAAGTGGAC GAGTCGTTAG 3540 CAAAGCTCCT CGGCTACTAC GTGAGCGAGG GCTATGCAAG AAAGCAGAGG AATCCCAAAA 3600 ACGGCTGGAG CTACAGCGTG AAGCTCTACA ACGAAGACCC TGAAGTGCTG GACGATATGG 3660 AGAGACTCGC CAGCAGGTTT TTCGGGAAGG TGAGGCGGGG CAGGAACTAC GTTGAGATAC 3720 CGAAGAAGAT CGGCTACCTG CTCTTTGAGA ACATGTGCGG TGTCCTAGCG GAGAACAAGA 3780 GGATTCCCGA GTTCGTCTTC ACGTCCCCGA AAGGGGTTCG, GCTGGCCTTC CTTGAGGGGT 3840 ACTCATCGGC GATGGCGACG TCCACCGAAC AAGAGACTCA GGCTCTCAAC GAAAAGCGAG 3900 CTTTAGCGAA CCAGCTCGTC CTCCTCTTGA ACTCGGTGGG GGTCTCTGCT GTAAAACTTG 3960 GGCACGACAG CGGCGTTTAC AGGGTCTATA TAAACGAGGA GCTCCCGTTC GTAAAGCTGG 4020 ACAAGAAAAA GAACGCCTAC TACTCA CACG TGATCCCCAA GGAAGTCCTG AGCGAGGTCT 4080 TTGGGAAGGT TTTCCAGAAA AACGTCAGTC CTCAGACCTT CAGGAAGATG GTCGAGGACG 4140 GAAGACTCGA TCCCGAAAAG GCCCAGAGGC TCTCCTGGCT CATTGAGGGG GACGTAGTGC 4200 TCGACCGCGT TGAGTCCGTT GATGTGGAAG ACTACGATGG TTATGTCTAT GACCTGAGCG 4260 TCGAGGACAA CGAGAACTTC CTCGTTGGCT TTGGGTTGGT CTATGCTCAC AACAGCTACT 4320 ACGGTTACTA CGGCTATGCA AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA 4380 CGGCCTGGGG AAGGGAGTAC ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT 4440 TTAAGGTAAT CTACAGCGAC ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG 4500 AAACCGTCAA AAAGAAGGCT ATGGAGTTCC TCAACTATAT CAACGCCAAA CTTCCGGGCG 4560 CGCTTGAGCT CGAGTACGAG GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT 4620 ATGCGGTGAT AGACGAGGAA GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG 4680 ACTGGAGCGA GATAGCGAAA GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG 4740 GTGACGTCGA GAAGGCCGTG AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG 4800 AGGTTCCGCC GGAGAAGCTG GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA 4860 AGGCAACCGG TCCCCACGTT GCCGTTGCCA A GAGGTTGGC CGCGAGAGGA GTCAAAATAC 4920 GCCCTGGAAC GGTGATAAGC TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG 4980 CGATACCGTT CGACGAGTTC GACCCGACGA AGCACAAGTA CGATGCCGAG TACTACATTG 5040 AGAACCAGGT TCTCCCAGCC GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG 5100 ACCTGCGCTA CCAGAAGACG AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA 5160 CTTGACCTTT CCATTTGTTT TCCAGCGGAT AACCCTTTAA CTTCCCTTTC AAAAACTCCC 5220 TTTAGGGAAA GACCATGAAG ATAGAAATCC GGCGGCGCCC GGTTAAATAC GCTAGGATAG 5280 AAGTGAAGCC AGACGGCAGG GTAGTCGTCA CTGCCCCGAG GGTTCAACGT TGAGAAGTT 5339

SEQ ID NO: 4 Sequence length: 24 Sequence type: nucleic acid Topology: linear Sequence type: synthetic DNA sequence GGATTAGTGC CAATGGAAGG CGAC 24

SEQ ID NO: 5 Sequence length: 24 Sequence type: Nucleic acid Topology: Linear Sequence type: Synthetic DNA sequence GAGGGCGAAG TTTATTCCGA GCTT 24

SEQ ID NO: 6 Sequence length: 324 Sequence type: Nucleic acid (DNA) Number of chains: double-stranded Troposi: Linear Sequence type: cDNA sequence GGATTAGTGC CAATGGAAGG CGACGAGGAG CTGAAAATGC TCGCCTTCGA CATTCAAACT 60 CTCTACCATG AGGGCGAGGA GTTCGCAGAGGGGCAGAT CTACGCCGAC 120 GAGGAAGGGG CCAGGGTGAT AACTTGGAAG AACGTGGATC TCCCCTACGT TGACGTCGTC 180 TCGACGGAGA GGGAGATGAT AAAGCGCTTC CTCCGTGTTG TGAAGGAGAA AGACCCGGAC 240 GTTCTCATAA CCTACAACGG CGACAACTTC GACTTCGCAG AGCTGCTGATC AGCTGCTAGCTAGCTAGCTAGCTGACTGCCTATCTGATCAGCTGTCGACTGCCTATCTGAGAGCTCATGCGATCGCTCATCTGATCGCTCATCTGATCGATCAAGCGG

SEQ ID NO: 7 Sequence length: 108 Sequence type: amino acid Topology: linear Sequence type: protein sequence Gly Leu Val Pro Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe 1 5 10 15 Asp Ile Gln Thr Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro 20 25 30 Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr 35 40 45 Trp Lys Asn Val Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg 50 55 60 Glu Met Ile Lys Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp 65 70 75 80 Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys 85 90 95 Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe Ala Leu 100 105

SEQ ID NO: 8 Sequence length: 42 Sequence type: nucleic acid (DNA) Number of strands: single strand Sequence type: synthetic DNA sequence GCCATCAAGA TCCTGGCAAA CAGCTACTAC GGTTACTACG GC 42

SEQ ID NO: 9 Sequence length: 32 Sequence type: nucleic acid (DNA) Number of strands: single strand Sequence type: synthetic DNA sequence GATGGATCCA ACTTCTCAAC GTTGAACCCT CG 32

SEQ ID NO: 10 Sequence length: 46 Sequence type: nucleic acid (DNA) Number of strands: single strand Sequence type: synthetic DNA sequence GAACATAGTG TACCTAGATT TTAGATCCCT GTACCCCTCA ATCATC 46

SEQ ID NO: 11 Sequence length: 42 Sequence type: nucleic acid (DNA) Number of strands: single strand Sequence type: synthetic DNA sequence GCCGTAGTAA CCGTAGTAGC TGTTTGCCAG GATCTTGATG GC 42

SEQ ID NO: 12 Sequence length: 33 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Sequence type: Synthetic DNA sequence ATCGATATCC TCGACACTGA CTACATAACC GAG 33

SEQ ID NO: 13 Sequence length: 46 Sequence type: nucleic acid (DNA) Number of strands: single strand Sequence type: synthetic DNA sequence GATGATTGAG GGGTACAGGG ATCTAAAATC TAGGTACACT ATGTTC 46

[Brief description of the drawings]

FIG. 1 shows a construction diagram of a recombinant expression vector.

FIG. 2 is a photograph of electrophoresis showing the results of measuring the molecular weight of KOD1-derived heat-resistant DNA polymerase.

FIG. 3 is a photograph of electrophoresis showing a result of PCR using KOD1-derived heat-resistant DNA polymerase.

FIG. 4 shows a DNA polymerase gene derived from the hyperthermophilic archaeon KOD1 and a thermostable DNA polymerase gene derived from Pyrococcus furiosus which is considered to be a related bacterium, and Thermococc.
2 shows a comparison with a thermostable DNA polymerase gene derived from U. litoralis.

Continuation of the front page (51) Int.Cl. ⁷ identification symbol FI (C12N 9/12 C12R 1:01) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 9/12 C12N 1/21 C12N 15/54 BIOSIS (DIALOG) GenBank / EMBL / DDBJ / PIR / SwisProt / Genese q JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (16)

(57) [Claims] 1. A DNA polymerase comprising the amino acid sequence of SEQ ID NO: 2. 2. The method according to claim 2, which is derived from the hyperthermophilic archaeon KOD1.
2. The DNA polymerase according to 1 . 3. The DNA polymerase according to claim 1, which has a molecular weight of about 86 to 92 Kda. 4. The DNA polymerase according to claim 1, wherein the DNA polymerase is produced using a recombinant host cell. 5. A DNA polymerase comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2,
Isolated DNA characterized in that the DNA is 6. It contains the nucleotide sequence of SEQ ID NO: 3.
And an isolated DNA encoding a DNA polymerase . 7. A DNA polymerase derived from the hyperthermophilic archaeon KOD1.
6. The method according to claim 5, wherein the coding for the merase is carried out.
7. The isolated DNA according to 6. 8. The DNA according to any one of claims 5 to 7,
A recombinant DNA expression vector in which is inserted into a vector. 9. The recombinant DNA expression vector (pET-pol) according to claim 8, wherein the vector is a vector derived from pET-8c. 10. A recombinant host cell transformed with the DNA recombinant expression DNA vector according to claim 8. 11. The recombinant host cell according to claim 8, wherein the host cell is Escherichia coli. 12. A method for producing a DNA polymerase derived from the hyperthermophilic archaeon KOD1, comprising culturing the recombinant host cell according to claim 10 and collecting a DNA polymerase from the culture. 13. The recombinant host cell according to claim 10, which is cultured, (a) collecting the recombinant host cell, followed by crushing to prepare a cell extract, and (b) the recombinant host cell. A method for purifying a DNA polymerase derived from the hyperthermophilic archaeon KOD1, which comprises a step of removing an impurity protein derived from the protein. 14. The method for purifying a DNA polymerase derived from the hyperthermophilic archaeon KOD1 according to claim 13, wherein the method for disrupting the recombinant host cell is sonication. 15. The method for purifying a DNA polymerase derived from the hyperthermophilic archaebacterium KOD1 according to claim 13, wherein the step of removing the impurity protein derived from the recombinant host cell is a high-temperature heat treatment. 16. The method for purifying a DNA polymerase derived from the hyperthermophilic archaebacterium KOD1 according to claim 15, wherein the high-temperature heat treatment condition is 70 ° C. or more, preferably 90 ° C. or more.