Patent application title: NOVEL DNA POLYMERASE
Inventors:
Yoshizumi Ishino (Fukuoka-Shi, JP)
Takeshi Yamagami (Fukuoka-Shi, JP)
Hiroaki Matsukawa (Fukuoka-Shi, JP)
Assignees:
KYUSHU UNIVERSITY, NATIONAL UNIVERSITY CORPORATION
IPC8 Class: AC12N912FI
USPC Class:
435194
Class name: Enzyme (e.g., ligases (6. ), etc.), proenzyme; compositions thereof; process for preparing, activating, inhibiting, separating, or purifying enzymes transferase other than ribonuclease (2.) transferring phosphorus containing group (e.g., kineases, etc.(2.7))
Publication date: 2014-12-11
Patent application number: 20140363875
Abstract:
Provided are various novel DNA polymerases.
Provided are: a DNA polymerase comprising: an amino acid sequence
modified from the amino acid sequence of SEQ ID NO: 12, which has a
substitution of arginine at position 651 by an amino acid residue having
a negatively charged side chain, preferably by asparatic acid or glutamic
acid, more preferably by glutamic acid; and a DNA polymerase comprising
an amino acid sequence modified from the amino acid sequence of SEQ ID
NO: 14, which has a substitution of proline at position 653 by an amino
acid residue having a negatively charged side chain, preferably by
asparatic acid or glutamic acid, more preferably by glutamic acid.Claims:
1-25. (canceled)
26. A DNA polymerase having a reverse transcriptional activity which is any one of (a) to (c) mentioned below: (a) a DNA polymerase comprising: an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of the arginine residue at position 651 by an amino acid residue having a negatively charged side chain; or an amino acid sequence that is modified from an amino acid sequence of a Family A DNA polymerase derived from a thermophilic eubacterium, which has a substitution of an amino acid residue corresponding to position 651 in SEQ ID NO: 12 by an amino acid residue having a negatively charged side chain; (b) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of the DNA polymerase as recited in (a), which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues which exclude the amino acid residue substituted in (a); and (c) a DNA polymerase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the DNA polymerase as recited in (a), with the proviso that the amino acid residue substituted in (a) is the same as the amino acid residue in (a).
27. The DNA polymerase as recited in claim 26, wherein the Family A DNA polymerase derived from a thermophilic eubacterium is selected from the group consisting of DNA polymerases derived from Thermus aquaticus or DNA polymerases derived from Thermus thermophilus.
28. The DNA polymerase as recited in claim 26 or 27, wherein the DNA polymerase of (a) is: (a1) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of the arginine residue at position 651 by an amino acid residue having a negatively charged side chain.
29. The DNA polymerase as recited in claim 28, wherein the DNA polymerase of (a1) comprises the amino acid sequence of SEQ ID NO: 20.
30. The DNA polymerase as recited in claim 28, wherein the DNA polymerase of (b) or (c) has a reverse transcriptional activity of at least 16.0.times.103 U/mg.
31. A polynucleotide which is any one of (A1) to (D1) mentioned below: (A1) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19; (B1) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in claim 28; (C1) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 19, and which encodes a DNA polymerase; and (D1) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 19.
32. The polynucleotide as recited in claim 30, wherein the DNA polymerase in (C1) or (D1) has a reverse transcriptional activity of at least 16.0.times.103 U/mg.
33. The DNA polymerase as recited in claim 26 or 27, wherein the DNA polymerase of (a) is: (a2) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 14, which has a substitution of the proline residue at position 653 by an amino acid residue having a negatively charged side chain.
34. The DNA polymerase as recited in claim 33, wherein the DNA polymerase of (a2) comprises the amino acid sequence of SEQ ID NO: 22.
35. The DNA polymerase as recited in claim 33, wherein the DNA polymerase of (b) or (c) has a reverse transcriptional activity of at least 16.0.times.103 U/mg.
36. A polynucleotide which is any one of (A2) to (D2) mentioned below: (A2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 21; (B2) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in claim 33; (C2) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 21, and which encodes a DNA polymerase; and (D2) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 21.
37. The polynucleotide as recited in claim 36, wherein the DNA polymerase in (C2) or (D2) has a reverse transcriptional activity of at least 16.0.times.103 U/mg.
38. A DNA polymerase having a primer extension activity using DNA as a template which is any one of (a4) to (c4) mentioned below: (a4) a DNA polymerases comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of at least one selected from the glutamic acid residue at position 117, the asparatic acid residue at position 119, the asparatic acid residue at position 142, and the asparatic acid residue at position 144, as well as the glutamic acid residue at position 742 by an amino acid residue having a non-polar aliphatic side chain; (b4) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of the DNA polymerase as recited in (a4), which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues, with the proviso that amino acid residues corresponding to at least one selected from positions 117, 119, 142 and 144, as well as position 742, in the amino acid sequence of the DNA polymerase as recited in (a4) remain the same; and (c4) a DNA polymerase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the DNA polymerase as recited in (a4), with the proviso that amino acid residues corresponding to at least one selected from positions 117, 119, 142 and 144, as well as position 742, in the amino acid sequence of the DNA polymerase as recited in (a4) remain the same.
39. The DNA polymerase as recited in claim 38, wherein the DNA polymerase of (a4) comprises the amino acid sequence of SEQ ID NO: 18.
40. The DNA polymerase as recited in claim 38 or 39, wherein the DNA polymerase of (b4) or (c4) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin.
41. A polynucleotide which is any one of (A4) to (D4) mentioned below: (A3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17; (B4) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in claim 38; (C4) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 17, and which encodes a DNA polymerase; and (D4) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 17.
42. The polynucleotide as recited in claim 41, wherein the DNA polymerase of (C4) or (D4) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin.
43. A recombinant vector comprising the polynucleotide as recited in claim 32.
44. A transformant comprising the recombinant vector as recited in claim 43.
45. A process for preparing the DNA polymerase as recited in any one of claims 1 to 5, 8 to 10, and 18 to 20, the process comprising a step of culturing the transformant as recited in claim 24.
Description:
TECHNICAL FIELD
[0001] The present invention relates to novel DNA polymerases. The DNA polymerases of this invention are particularly useful for PCR.
BACKGROUND ART
[0002] DNA polymerases are enzymes that can synthesize new DNA strands along template DNA strands in vitro. DNA polymerases can synthesize new DNA strands from a template DNA, an oligonucleotide serving as a primer, and four types of deoxynucleotides (dATP, dGTP, dCTP, and dTTP). DNA polymerases are also used in many genetic engineering techniques, including nucleotide sequencing and PCR.
[0003] Thermostability of polymerases is essential for PCR, and the current protocol of nucleotide sequencing generally uses the cycle sequencing method using a thermostable DNA polymerase as a standard technique. In order to find a thermostable enzyme, one would usually search enzymes produced by thermophilic microorganisms. Among thermophilic bacteria, those which proliferate at an optimum growth temperature of at least 80° C. are particularly referred to as "hyperthermophilic bacteria" and serve as excellent resources for thermostable enzymes. Taq DNA polymerases (also referred to as "Taq polymerases") which are currently widely used in PCR were originally isolated from the thermophilic eubacterium Thermus aquaticus.
[0004] Based on the similarity in amino acid sequences, DNA polymerases are categorized into seven groups: Families A, B, C, D, E, X and Y. Enzymes belonging to the same family basically exhibit very similar properties. The enzymes that are in practical use are those belonging to Families A and B.
[0005] Family A enzymes have superior performance in recognizing dideoxynucleotides as substrates and are most appropriate for nucleotide sequencing. Thus, the enzymes contained in currently commercially available sequencing kits are all those which belong to Family A and are derived from thermophilic eubacteria. In PCR, Family A and B enzymes are selectively used depending on the purpose.
[0006] Family B enzymes are not suitable for nucleotide sequencing because of poor incorporation of dideoxynucleotides but have 3'-5' exonuclease activity which is involved in the accuracy in synthesizing DNA strands according to the sequences of template strands--during amplification, the enzymes of this family produce less errors than Family A enzymes such as Taq polymerases with no exonuclease activity. The Family B enzymes that are commercialized are those derived from hyperthermophilic archaea. In order to perform PCR more accurately, it is advisable to use Family B enzymes, whereas in order to amplify long-chain DNA, Family A enzymes can be selected due to their superior extensibility and superior DNA synthesis efficiency.
[0007] Comparison between the two DNA polymerases that are derived from bacteria belonging to the genus Thermus and which have been up to now widely used as PCR enzymes shows that Taq DNA polymerase only has weak reverse transcriptional activity, while Tth DNA polymerase ("Tth polymerase") derived from Thermus thermophilus has significantly strong reverse transcriptional activity. This property of Tth polymerase is utilized in a simple RT-PCR technology in which a single enzyme is used in a single reaction tube to synthesize cDNA from mRNA by reverse transcription and then amplify the synthesized cDNA. Since the optimum temperature of this enzyme is high, the enzyme makes it possible to perform a reverse transcription reaction at relatively high temperatures (around 60° C.) and is also effective for the reverse transcription of RNA which easily forms a three-dimensional structure, but the enzyme is not suitable for the synthesis of long cDNAs like those reaching as long as several kilo bases in length.
[0008] PCR is a gene analysis technology that is widely used throughout the world as a routinely utilized technique. Accordingly, there is a need for a DNA polymerase that is more convenient, easier-to-use, and more reliable, and it is also desired to provide various DNA polymerases that can amplify various DNAs appropriately depending on the template to be used as well as the purpose to be needed for PCR such as extensibility, rapidity, and accuracy.
[0009] As regards modification of Taq polymerases, there have been hitherto reports stating that primers were designed based on the segments of an amino acid sequence highly conserved in Family A DNA polymerases, each of which contains an active site, gene fragments were amplified by PCR using DNA samples derived from hot spring soil as templates, and the corresponding segments of a wild-type Taq polymerase gene were substituted by the amplified fragments, whereby obtained were chimeric DNA polymerases with higher extension activity than Taq polymerases (Patent Documents 1 and 2). Another report showed that on the basis of metagenomic analysis and the three-dimensional structure information of DNA polymerases, one or more mutations were introduced that produce an increased the total electric charges of glutamic acid at position 742 and alanine at position 743 in an amino acid sequence of a Taq polymerase, whereby obtained was a modified Taq polymerase that is superior to the Taq polymerase in at least one of: primer extension activity; binding activity on a primer annealed to a template DNA; and PCR performance (Patent Document 3).
CITATION LIST
Patent Documents
[0010] Patent Document 1: Japanese Patent Application Publication No. JP 2006-101791
[0011] Patent Document 2: Japanese Patent Application Publication No. JP 2006-204267
[0012] Patent Document 3: Japanese Patent No. JP 4193997
SUMMARY OF INVENTION
Technical Problem
[0013] The present inventors made detailed comparison between the amino acid sequences of Taq polymerases and Tth polymerases, focusing on the difference in the properties associated with their amino acid sequence identity (about 80%) and reverse transcriptional activity. However, based on this comparison alone, it was difficult to predict on what the difference in their properties depends.
[0014] On the other hand, the inventors have accumulated the results of the study in which the properties of DNA polymerases derived from a diverse range of organisms are reflected as those of chimeric Taq polymerases by the following method: hot spring soil samples are collected from various places, DNAs are directly extracted from the samples, fragments of DNA polymerase genes possessed by various kinds of organisms contained in the samples are amplified by PCR based on the obtained DNAs (metagenomes), and the resulting fragments are recombined in vitro with the homologous regions of Taq polymerase genes, whereby chimeric enzymes are constructed. It is, of course, ideal that the gene obtained from a metagenomic DNA contains a full-length sequence, but many metagenomic DNAs extracted from environmental samples are often fragmented or damaged; thus, it is a very difficult task to obtain a full-length gene directly. Therefore, we constructed the following study system; a gene segment encoding an active center that significantly affects the activity of a DNA polymerase and peripheral regions thereof is obtained from a metagenome, and the obtained segment is substituted with the homologous region of a Taq polymerase gene to create a chimeric enzyme gene, so that the obtained gene fragment could directly affect the basic properties of a DNA polymerase.
[0015] We created a phylogenetic tree (not shown in the present application) by checking the results of determining the activities of many chimeric Taq polymerases constructed in such a way as described above, against each other, and comparing the sequences of the gene fragments that are derived from metagenomic DNAs and which were introduced into wild-type Taq polymerases. As a result of predicting the factors that might change the activities of the chimeric Taq polymerases, we found the chimeric Taq polymerases 8-16, 18-7, 1-8, and 3-7 which have significantly strong reverse transcriptional activity as compared with the wild-type Taq polymerases.
[0016] First, we compared the sequences of 8-16, a Taq polymerase, and a Tth polymerase with each other and, as a result, did not find any amino acid residue that is common between 8-16 and the Tth polymerase but is different between 8-16 and the Taq polymerase. However, among ten amino acid residues that are different between the Taq and Tth polymerases, there were three amino acid residues that are completely different in nature between these three polymerases. Thus, we focused on these three amino acid residues and decided to introduce mutations to them.
[0017] Next, we compared the sequence of 18-7 with each of the sequences of a Taq polymerase and a Tth polymerase to thereby search for an amino acid residue that is common between 18-7 and the Tth polymerase which both have reverse transcriptional activity but not common between 18-7 and the Taq polymerase, and, as a result, four amino acid residues were found. Among them, there was one amino acid residue that is greatly different in nature between 18-7/Tth polymerase and the Taq polymerase; thus, we decided to introduce a mutation to this amino acid residue.
[0018] Further, as regards 1-8 and 3-7, these chimeric polymerases have strong primer extension activity and reverse transcriptional activity, whereas there was a chimeric enzyme (1-20) that has almost the same sequence as said polymerases but is extremely weak in activity. We compared the sequences of the three enzymes: 1-8, 3-7 as well as 1-20 mentioned above. As a result, we found two amino acids that are completely inconsistent between these sequences, and selected the one that is only inconsistent in 1-20, which is greatly different in activity, and decided to introduce a mutation to it.
[0019] We compared the mutant proteins of the site-specific mutated Taq polymerase and Tth polymerase constructed according to the above-noted strategy, with the wild-type enzymes. First, the mutant proteins were evaluated for nucleotide incorporation activity using an activated DNA as a template, whereupon the respective specific activities of the mutants which are relative to those of the wild-type Taq polymerase taken as 100% demonstrated that there is in principle no significant change in DNA polymerase activity. Next, these enzymes were determined for reverse transcriptional activity by a nucleotide incorporation assay using RNA as a template, whereupon there were found a mutant Taq polymerase (Taq R651E) and a mutant Tth polymerase (Tth P653E), which respectively have a reverse transcriptional activity 1.4 and 1.7 times greater than that of the wild-type Tth polymerase. Comparison of the sequences showed that the amino acid at position 653 in the Tth polymerase corresponds to that at position 651 in the Taq polymerase (refer to FIG. 1). The inventors thus created mutant DNA polymerases having presumably very useful properties, and completed the present invention.
[0020] The present invention provides the following:
[1] A DNA polymerase which is any one of (a) to (c) mentioned below: (a) a DNA polymerase comprising: an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of the arginine residue at position 651 by an amino acid residue having a negatively charged side chain; or an amino acid sequence that is modified from an amino acid sequence of a Family A DNA polymerase derived from a thermophilic eubacterium, which has a substitution of an amino acid residue corresponding to position 651 in SEQ ID NO: 12 by an amino acid residue having a negatively charged side chain; (b) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of the DNA polymerase as recited in (a), which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues which exclude the amino acid residue substituted in (a); and (c) a DNA polymerase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the DNA polymerase as recited in (a), with the proviso that the amino acid residue substituted in (a) is the same as the amino acid residue in (a); [2] The DNA polymerase as recited in [1], wherein the Family A DNA polymerase derived from a thermophilic eubacterium is selected from the group consisting of DNA polymerases derived from Thermus aquaticus or DNA polymerases derived from Thermus thermophilus; [3] The DNA polymerase as recited in [11] or [2], wherein the DNA polymerase of (a) is: (a1) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of the arginine residue at position 651 by an amino acid residue having a negatively charged side chain; [4] The DNA polymerase as recited in [3], wherein the DNA polymerase of (a1) comprises the amino acid sequence of SEQ ID NO: 20; [5] The DNA polymerase as recited in [3] or [4], wherein the DNA polymerase of (b) or (c) has a reverse transcriptional activity of at least 16.0×103 U/mg; [6]A polynucleotide which is any one of (A1) to (D1) mentioned below: (A1) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19; (B1) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in [3]; (C1) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 19, and which encodes a DNA polymerase; and (D1) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ FD NO: 19; [7] The polynucleotide as recited in [6], wherein the DNA polymerase in (C1) or (D1) has a reverse transcriptional activity of at least 16.0×103 U/mg; [8] The DNA polymerase as recited in [1] or [2], wherein the DNA polymerase of (a) is: (a2) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 14, which has a substitution of the proline residue at position 653 by an amino acid residue having a negatively charged side chain; [9] The DNA polymerase as recited in [8], wherein the DNA polymerase of (a2) comprises the amino acid sequence of SEQ ID NO: 22; [10] The DNA polymerase as recited in [8] or [9], wherein the DNA polymerase of (b) or (c) has a reverse transcriptional activity of at least 16.0×103 U/mg; [11] A polynucleotide which is any one of (A2) to (D2) mentioned below: (A2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 21; (B2) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in [8]; (C2) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 21, and which encodes a DNA polymerase; and (D2) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 21; [12] The polynucleotide as recited in [11], wherein the DNA polymerase in (C2) or (D2) has a reverse transcriptional activity of at least 16.0×10 U/mg; [13] A DNA polymerase which is any one of (a3) to (c3) mentioned below: (a3) a DNA polymerases comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of at least one selected from the glutamic acid residue at position 117, the asparatic acid residue at position 119, the asparatic acid residue at position 142, and the asparatic acid residue at position 144 by an amino acid residue having a non-polar aliphatic side chain; (b3) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of the DNA polymerase as recited in (a3), which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues, with the proviso that at least one amino acid residue corresponding to at least one selected from positions 117, 119, 142 and 144 in the amino acid sequence of the DNA polymerase as recited in (a3) remains the same; and (c3) a DNA polymerase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the DNA polymerase as recited in (a3), with the proviso that at least one amino acid residue corresponding to at least one selected from positions 117, 119, 142 and 144 in the amino acid sequence of the DNA polymerase as recited in (a3) remains the same; [14] The DNA polymerase as recited in [13], wherein the DNA polymerase of (a3) comprises the amino acid sequence of SEQ ID NO: 16; [15] The DNA polymerase as recited in 113) or [14], wherein the DNA polymerase of (b3) or (c3) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin. [16]A polynucleotide which is any one of (A3) to (D3) mentioned below: (A3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15; (B3) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in 1:13); (C3) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 15, and which encodes a DNA polymerase; and (D3) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 15; [17] The polynucleotide as recited in [16], wherein the DNA polymerase of (C3) or (D3) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin. [18]A DNA polymerase which is any one of (a4) to (c4) mentioned below: (a4) a DNA polymerases comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of at least one selected from the glutamic acid residue at position 117, the asparatic acid residue at position 119, the asparatic acid residue at position 142, and the asparatic acid residue at position 144, as well as the glutamic acid residue at position 742 by an amino acid residue having a non-polar aliphatic side chain; (b4) a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of the DNA polymerase as recited in (a4), which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues, with the proviso that amino acid residues corresponding to at least one selected from positions 117, 119, 142 and 144, as well as position 742, in the amino acid sequence of the DNA polymerase as recited in (a4) remain the same; and (c4) a DNA polymerase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the DNA polymerase as recited in (a4), with the proviso that amino acid residues corresponding to at least one selected from positions 117, 119, 142 and 144, as well as position 742, in the amino acid sequence of the DNA polymerase as recited in (a4) remain the same; [19] The DNA polymerase as recited in [18], wherein the DNA polymerase of (a4) comprises the amino acid sequence of SEQ ID NO: 18; [20] The DNA polymerase as recited in 1181 or [19], wherein the DNA polymerase of (b4) or (c4) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin. [21] A polynucleotide which is any one of (A4) to (D4) mentioned below: (A3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17; (B4) a polynucleotide comprising a nucleotide sequence encoding the DNA polymerase as recited in [18]; (C4) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 17, and which encodes a DNA polymerase; and (D4) a polynucleotide encoding a DNA polymerase comprising a sequence at least 95% identical to the nucleotide sequence of SEQ ID) NO: 17; [22] The polynucleotide as recited in [21], wherein the DNA polymerase of (C4) or (D4) has a primer extension activity using DNA as a template, of at least 4.00 kb/Umin. [23]A recombinant vector comprising the polynucleotide as recited in any one of [7], [12], [17] and [22]; [24]A transformant comprising the recombinant vector as recited in [23]; and [25]A process for preparing the DNA polymerase as recited in any one of [1] to [5], [8] to [10], [13] to [15], and [18] to [20], the process comprising a step of culturing the transformant as recited in [24].
BRIEF DESCRIPTION OF DRAWINGS
[0021] FIG. 1-1 shows a comparison and alignment of the amino acid sequences of Taq and Tth DNA polymerases. The mark "∇" indicates the correspondence between the amino acid at position 651 in the Taq DNA polymerase and the amino acid at position 653 in the Tth DNA polymerase.
[0022] FIG. 1-2 shows a comparison and alignment of the amino acid sequences of Taq DNA polymerase and other family A DNA polymerases from thermophilic bacteria. The mark "" indicates the amino acid at position 651 in the Taq DNA polymerase.
[0023] FIG. 1-3 shows a comparison and alignment of the amino acid sequences of Taq DNA polymerase and other family A DNA polymerases from thermophilic bacteria. The mark "" indicates the amino acid at position 651 in the Taq DNA polymerase.
[0024] FIG. 2 shows a photograph of the SDS-PAGE gels concerning purification of Taq DNA polymerase mutants. The purity of each of the prepared Taq DNA polymerase mutants was confirmed by SDS-PAGE analysis (refer to Example 1).
[0025] FIG. 3 shows a graph of an actual example of enzymatic activity determination by nucleotide incorporation assay (refer to Example 2). Under the conditions of this test, Taq showed an activity of 3.9×105 U/mg.
[0026] FIG. 4 shows an example of a graph for determining primer extension rate. With the amount of enzyme kept constant, a time course of primer extension reaction is taken and the extension rate can be determined by measuring the length of strand extended per unit time through alkaline agarose electrophoresis in a region where the plots lie on a straight line (refer to Example 2). Under the conditions of this test, Taq showed a rate of 4.67 kb/min/U, Taq' (Taq mutant) showed a rate of 6.67 kg/min/U, and Chimera A (the chimera created by recombining a segment of the Taq gene with a homologous region obtained from the metagenome) showed a rate of 11.20 kb/min/U.
[0027] FIG. 5 shows a photograph of the alkaline agarose electrophoresis gels for comparing primer extension rates.
[0028] FIG. 6 shows the Taq WT amino acid sequence of SEQ ID) NO: 12 and the Taq WT nucleotide sequence of SEQ ID NO: 11.
[0029] FIG. 7 shows the Tth WT amino acid sequence of SEQ ID NO: 14 and the Tth WT nucleotide sequence of SEQ ID NO: 13.
[0030] FIG. 8 shows the Taq Exo- amino acid sequence of SEQ ID NO: 16 and the Taq Exo- nucleotide sequence of SEQ ID NO: 15.
[0031] FIG. 9 shows the Taq Exo-+E742A amino acid sequence of SEQ ID NO: 18 and the Taq Exo-+E742A nucleotide sequence of SEQ ID NO: 17.
[0032] FIG. 10 shows the Taq R651E amino acid sequence of SEQ ID NO: 20 and the Taq R651E nucleotide sequence of SEQ ID NO: 19.
[0033] FIG. 11 shows the Tth P653B amino acid sequence of SEQ ID NO: 22 and the Tth P653E nucleotide sequence of SEQ ID NO: 21.
DESCRIPTION OF EMBODIMENTS
Definitions, etc.
[0034] The "DNA polymerase" as referred to in the present invention, unless otherwise specified, means a protein having the activity of extending a complementary DNA strand to a template nucleic acid (DNA or RNA) using deoxyribonucleoside triphosphate as a substrate. The "Taq polymerase" or "Taq DNA polymerase" as referred to in this invention, unless otherwise specified, means a DNA polymerase derived from Thermus aquaticus. The amino acid sequence and the nucleotide sequence are respectively shown in SEQ ID NOs: 12 and 11 in the Sequence Listing, which constitutes a part of the present specification. The "Tth polymerase" or "Tth DNA polymerase" as referred to in this invention means a DNA polymerase derived from Thermus thermophilus. The amino acid sequence and the nucleotide sequence are respectively shown in SEQ ID NOs: 14 and 13 in the Sequence Listing, which constitutes a part of the present specification.
[0035] DNA Polymerase Activity:
[0036] The "activity" as referred to in the present invention in connection with a DNA polymerase includes transcriptional activity and primer extension activity.
[0037] The transcriptional activity includes transcriptional activity using DNA as a template, and transcriptional activity using RNA as a template. As well known to those skilled in the art, the transcriptional activity can be determined as the activity of incorporating deoxyribonucleoside triphosphate (dNTP) as a substrate. More specifically, a calf thymus DNA, a salmon sperm DNA, or the like is partially digested with DNase 1 to provide a nicked or gapped double-strand DNA as a template, and a radioisotope-labeled dNTP is mixed with a substrate dNTP; then, the DNA polymerase of interest is caused to act, so that the amount of nucleotides incorporated into nicks by nick translation or into gaps by primer extension activity can be determined using radioactivity as an indicator. This determination method, which is called nucleotide incorporation assay, is a standard method for determining DNA polymerase activity.
[0038] The "transcriptional activity" or "basic DNA polymerase activity" as referred to in the present invention, unless otherwise specified, means the activity of incorporating dNTP when DNA strand is used as a template. When the "transcriptional activity" or "basic DNA polymerase activity" is represented by numerical value in this invention, unless otherwise specified, the amount of enzyme, i.e., DNA polymerase, required to incorporate 10 nmol of nucleotides at 72° C. for 30 minutes is defined as 1 unit (U), and such activity is expressed as a value for specific activity (activity per protein amount) in U/mg or the like. Unless otherwise specified, the conditions for this determination are set as disclosed in the Examples section of the present application.
[0039] The "reverse transcriptional activity" as referred to in the present invention, unless otherwise specified, means the activity of incorporating dNTP when RNA strand is used as a template. When the "reverse transcriptional activity" is represented by numerical value in this invention, unless otherwise specified, the amount of enzyme, i.e., DNA polymerase, required to incorporate 10 nmols of nucleotide at 72° C. for 30 minutes is defined as 1 unit (U), and such activity is expressed as a value for specific activity (activity per protein amount) in U/mg or the like. Unless otherwise specified, the conditions for this determination are set as disclosed in the Examples section of the present application.
[0040] The "primer extension activity" as referred to in the present invention, unless otherwise specified, means the length of strand extended per unit time when a DNA polymerase of interest is caused to act on a substrate dNTP using DNA or RNA as a template, and the length can be expressed in kb/U/min, bp/pmol/min, or the like. Unless otherwise specified, the conditions for this determination are set as disclosed in the Examples section of the present application.
[0041] DNA Polymerases of the Present Invention:
[0042] The present invention provides novel DNA polymerases, more specifically mutants of Family A DNA polymerases derived from thermophilic eubacteria, notably particular mutants of Thermus aquaticus polymerases (Taq polymerases) or mutants of homologues thereof.
[0043] The "thermophilic eubacteria" as referred to herein means eubacteria having an optimum growth temperature of at least 45° C. or at least 60° C. Examples include bacteria of the genus Thermus, such as Thermus aquaticus and Thermus thermophilus, those of the genus Thermotoga, such as Thermotoga maritima, those of the genus Aquifex, such as Aquifex aeolicus, and those of the genus Thermodesulfobacerium, such as Thermodesulfobacterium commune, with preference given to Thermus aquaticus and Thermus thermophilus.
[0044] Thus, examples of the DNA polymerase of the present invention that are preferably provided include not only mutants of Thermus aquaticus-derived DNA polymerases (Taq polymerases) or mutants of Thermus thermophilus-derived DNA polymerases (Tth polymerases), but also mutants of Family A DNA polymerases derived from other thermophilic eubacteria, in which an amino acid that is shown by sequence comparison to correspond to a mutation site of a Taq polymerase is mutated.
[0045] For example, it was found that when the amino acid sequences of a Thermus aquaticus-derived DNA polymerase (Taq polymerase) and a Thermus thermophilus-derived DNA polymerase (Tth polymerase) are compared and aligned, the amino acid at position 651 in the Taq polymerase and the amino acid at position 653 in the Tth polymerase correspond to each other (FIG. 1).
[0046] As a result of investigation about chimeric Taq polymerases, the present inventors found two chimeric enzymes having high primer extension activity and reverse transcriptional activity, and other chimeric enzymes which have almost the same sequences as said two enzymes but show extremely low activities. The inventors also selected from those sequences one residue that is only inconsistent in the low-activity enzymes, and introduced a mutation to this residue, whereby the inventors found a mutant Taq polymerase (Taq R651E) and a mutant Tth polymerase (Tth P653E), which respectively have a reverse transcriptional activity 1.4 and 1.7 times greater than that of the wild-type Tth polymerase.
[0047] Thus, in the first aspect, the present invention provides a DNA polymerase comprising an amino acid sequence modified from an amino acid sequence of a Family A DNA polymerase derived from a thermophilic eubacterium, which has a substitution of one corresponding amino acid residue by an amino acid residue having a negatively charged side chain (in respect of the first aspect, refer to [1] and [2] mentioned above). Specifically, this invention provides a DNA polymerase comprising: an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of the arginine residue at position 651 by an amino acid residue having a negatively charged side chain; or an amino acid sequence modified from an amino acid sequence of a Family A DNA polymerase derived from a thermophilic eubacterium, which has a substitution of an amino acid residue corresponding to position 651 in SEQ ID NO: 12 by an amino acid residue having a negatively charged side chain (in respect of the first aspect, refer to [1] and [2] mentioned above). More specifically, this invention provides a DNA polymerase comprising the mutant Taq R651E or a homologue thereof, or the mutant Tth P653E or a homologue thereof (in respect of the first aspect, refer to [3] to [7] mentioned above for the mutant Taq R651E, and [8] to [12] for mutant Tth P653E).
[0048] The amino acid sequence of Taq R651E and the nucleotide sequence encoding the same are respectively shown in SEQ ID NOs: 20 and 19 in the Sequence Listing which constitutes a part of the present specification. The amino acid sequence of Tth P653E and the nucleotide sequence encoding the same are respectively shown in SEQ ID) NOs: 22 and 21 in the Sequence Listing which constitutes a part of the present specification.
[0049] The DNA polymerase according the first aspect of the present invention is characterized by having improved reverse transcriptional activity over the wild-type DNA polymerase. To be specific, the mutant Taq R651E and the homologues thereof have a reverse transcriptional activity of at least 5.00×103 U/mg, preferably at least 10.0×103 U/mg, more preferably at least 1.5.0×103 U/mg, and still more preferably at least 20.0×103 (U/mg. The mutant Tth P653E and the homologues thereof have a reverse transcriptional activity of at least 16.0×103 U/mg, preferably at least 18.0×103 U/mg, and more preferably at least 20.0×103 U/mg.
[0050] The present inventors further made extensive studies for the purpose of creating a superior PCR enzyme by modifying a Taq polymerase, and as a result found that the mutant Taq Exo- (E117A, D119A, D142A, D144A), in which four amino acid residues presumably important for the 5'→3' exonuclease activity inherent in a Taq polymerase are converted at the same time, are superior to the wild-type Taq polymerase in terms of primer extension activity using DNA as a template. Thus, in the second aspect, this invention provides the mutant Taq Exo- and homologues thereof, polypeptides encoding the same and homologues thereof (in respect of the second aspect, refer to [13] to [17] mentioned above).
[0051] The amino acid sequence of Taq Exo- and the nucleotide sequence encoding the same are respectively shown in SEQ ID NOs: 16 and 15 in the Sequence Listing which constitutes a part of the present specification.
[0052] In Taq Exo-, all of the glutamic acid residue at position 117, the asparatic acid residue at position 119, the asparatic acid residue at position 142, and the asparatic acid residue at position 144 in the amino acid sequence of SEQ ID NO: 12 are substituted, but the homologues of said mutant, which have an amino acid sequence in which at least one, preferably two, more preferably three, selected from these residues are each independently substituted by an amino acid having a non-polar aliphatic side chain, preferably by an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, and more preferably by alanine, are also encompassed by the present invention.
[0053] The mutant Taq Exo- and the homologues thereof according to the second aspect of the present invention have a primer extension activity using DNA as a template, at least 4.00 kb/Umin, preferably at least 8.00 kb/Umin, more preferably 9.00 kb/Umin, and still more preferably 14.0 kb/Umin.
[0054] Furthermore, the present inventors found that the mutant Taq Exo- to which the mutation of Patent Document 3 mentioned above is further introduced achieves further improvement in extension activity. Thus, in the third aspect, the present invention provides the mutant Taq Exo-+E742A and homologues thereof; polypeptides encoding the same and homologues thereof (in respect of the third aspect, refer to [18] to [22] mentioned above).
[0055] The amino acid sequence of Taq Exo-+E742A and the nucleotide sequence encoding the same are respectively shown in SEQ ID NOs: 18 and 17 in the Sequence Listing, which constitutes a part of the present specification.
[0056] In Taq Exo-+E742A, all of the glutamic acid residue at position 117, the asparatic acid residue at position 119, the asparatic acid residue at position 142, and the asparatic acid residue at position 144, as well as the glutamic acid residue at position 742, in the amino acid sequence of SEQ ID NO: 12 are substituted, but the homologues of said mutant, which have an amino acid sequence in which at least one, preferably two, more preferably three, still more preferably four, selected from these residues are each independently substituted by an amino acid having a non-polar aliphatic side chain, preferably by an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, and more preferably by alanine, are also encompassed by the present invention.
[0057] The mutant Taq Exo-+E742A and the homologues thereof according to the third aspect of the present invention have a primer extension activity using DNA as a template, at least 4.00 kb/Umin, preferably at least 8.00 kb/Umin, more preferably 9.00 kb/Umin, and still more preferably 14.0 kb/Umin.
[0058] Any of the above-mentioned mutants of the present invention encompasses their variants in which a segment having exonuclease activity and consisting of multiple consecutive amino acid residues starting from the N terminal side is deleted. The N-terminal segment that may be deleted can be designed as appropriate by those skilled in the art. In the case of mutant Taq polymerases, the segment typically consists of the residues at positions 1-233, positions 1-293, or positions 1-302, in the amino acid sequence of SEQ ID NO: 12. In the case of mutant Tth polymerases, the segment typically consists of the residues at positions 1-237 in the amino acid sequence of SEQ ID NO: 14, which correspond to positions 1-233 in the Taq sequence. In every case, the mutant DNA polymerase of this invention even encompasses those variants in which a segment of a shorter amino acid residue length than in the above-mentioned deletion variants is deleted as long as the length is within the segment ranges mentioned above.
[0059] When the phrase "which has a substitution, deletion, insertion and/or addition of one to nine amino acid residues" is used in the present invention, the number of amino acids to be substituted or otherwise modified is not particularly limited as long as the protein (DNA polymerase) having the modified amino acid sequence has a desired function, and 1-9 or about 1-4 amino acids may be substituted or otherwise modified, or even more amino acids may be substituted or otherwise modified if said substitution or the like is intended to encode the same or similar amino acid sequence. Means for obtaining a protein having such an amino acid sequence are well known to those skilled in the art.
[0060] The substitution or the like of an amino acid(s) may be such a substitution or the like that does not cause an electrostatic change, for example, a substitution by an amino acid(s) that is(are) similar in electric charge and/or polarity. Examples of such a substitution include: substitution between amino acids having such an aliphatic side chain that the side chain (also expressed as "R group") is non-polar around physiological pH (7.0) (e.g., glycine, alanine, valine, leucine, isoleucine, and proline); substitution between amino acids having a polar uncharged side chain (e.g., serine, threonine, cysteine, methionine, asparagine, and glutamine); substitution between amino acids having a side chain that is positively charged around physiological pH (e.g., lysine, arginine, and histidine); substitution between amino acids having a negatively charged side chain (e.g., asparatic acid, glutamic acid); substitution between polar amino acids; and substitution between non-polar amino acids.
[0061] Examples of DNA polymerases preferred in the present invention include not only the mutant DNA polymerases encompassed by the above-mentioned first to third aspects of the invention, but also variants of wild-type Taq polymerases (SEQ ID NO: 12) or wild-type Tth polymerases (SEQ ID NO: 14) which have such an amino acid substitution as characterized above at position 651, 664, 674 or the like. Specific examples include a mutant of a wild-type Taq polymerase (SEQ ID NO; 12), which has a substitution of an arginine residue at position 651 by a glutamic acid residue, a mutant of the same polymerase having a substitution of a threonine residue at position 664 by an arginine residue, and a mutant of the same polymerase having a substitution of a serine residue at position 674 by an arginine residue (refer to FIG. 5).
[0062] The "stringent conditions" as referred to in the present invention can be determined as appropriate by those skilled in the art on the basis of the reference data such as polynucleotide length. As to the stringent conditions, those skilled in the art can make reference to the conditions described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press, 2001). The stringent conditions refer to, for example, the hybridization conditions of about 50% formamide, 2×SSC to 6×SSC at about 40-50° C. (or those conditions for other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42° C.). The pre-washing conditions of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), and/or the washing conditions of 0.5×SSC, 0.1% SDS at about 60° C. may also be applied. The stringent conditions more preferably involve not only the hybridization conditions of about 50% formamide, 2×SSC to 6×SSC at about 40-50° C. (or those conditions for other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42° C.), but also the washing conditions of 0.2×SSC, 0.1% SDS at about 68° C.
[0063] The high identity as referred to in the present invention in connection with an amino acid sequence, unless otherwise specified, means a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 96%, and most preferably at least 97%. The high identity as referred to in the present invention in connection with a nucleotide sequence, unless otherwise specified, also means a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 96%, and most preferably 97%. Search and analysis of polynucleotide or amino acid sequence identity can be made using an algorithm or program well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When the program is used, parameters can be appropriately set by those skilled in the art, or the default parameters of each program may also be used. Specific procedures for such analyses are also well known to those skilled in the art.
[0064] Of both amino acid and nucleotide sequences, an important site for performance of an intended function is described in the present specification. Accordingly, those skilled in the art can design, prepare and use different mutants in which the sequences of other segments than such an important site are modified as appropriate. Such mutants can also fall within the scope of the present invention.
[0065] The DNA polymerases of the present invention can be prepared by a method well known to those skilled in the art. In order to construct a recombinant vector, a DNA fragment of an appropriate length, which contains the coding region of a protein of interest, is prepared as a first step. In the nucleotide sequence of the coding region of the protein of interest, nucleotides may be so substituted as to give optimal codons for expression in host cells. Next, the prepared DNA fragment is inserted downstream of a promoter in an appropriate expression vector to construct a recombinant vector. It is necessary that said DNA fragment be incorporated into the vector so as to perform its function. The vector may contain not only promoters but also cis elements (e.g., enhancers), splicing signals, polyadenylation signals, selective markers (e.g., dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene), ribosome binding sequences (SD sequences), and/or the like. A transformant capable of producing a protein of interest can be obtained by introducing a recombinant vector into an appropriate host cell.
[0066] The expression vector is not particularly limited as long as it is capable of autonomous replication in a host cell, and examples of the vector that can be used include plasmid vectors, phage vectors, and viral vectors. Examples of the plasmid vectors that can be used include E. coli-derived plasmids (e.g., pRSET, pBR322, pBR325, pUC118, pUC119, pUC18, and pUC19), Bacillus subtilis-derived plasmids (e.g., pUB110 and pTPS), and yeast-derived plasmids (e.g., YEp13, YEp24, and YCp50). Examples of the phage vectors that can be used include λ phages (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP). Examples of the viral vectors that can be used include animal viruses such as retroviruses and vaccinia viruses, and insect viruses such as baculoviruses.
[0067] As the host cell, there can be used any of prokatyocytes, yeasts, animal cells, insect cells, plant cells, and other cells, as long as the cell is capable of expressing a DNA encoding a protein of interest. Animal individuals, plant individuals, silkworms, and the like may also be used.
[0068] When a bacterium is used as a host cell, examples of the bacterium that can be used as a host cell include bacteria of the genus Escherichia, such as Escherichia coli, those of the genus Bacillus, such as Bacillus subtilis, those of the genus Pseudomonas, such as Pseudomonas putida, and those of the genus Rhizobium, such as Rhizobium meliloti. Specific examples of the bacterium that can be used as a host cell include Escherichia coli such as Escherichia coli BL21, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli K 12, Escherichia coli JM109, and Escherichia coli HB101, and Bacillus subtilis such as Bacillus subtilis MI 114, and Bacillus subtilis 207-21. The promoter used in this case is not particularly limited as long as it can be expressed in a bacterium such as E. coli, and examples of the promoter that can be use include those derived from E. coli, phages and the like, such as trp promoter, lac promoter, PL promoter, and PR promoter. Artificially designed and modified promoters such as tac promoter, lacT7 promoter, and let 1 promoter can also be used. When a yeast is used as a host cell, examples of the yeast that can be used as a host cell include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. The promoter used in this case is not particularly limited as long as it can be expressed in a yeast, and examples of the promoter that can be used include gall promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1 promoter. When an insect cell is used as a host, examples of the insect cell that can be used as a host cell include Spodoptera frugiperda ovarian cells, Trichoplusia ni ovarian cells, and cultured cells derived from silkworm ovaries. Examples of the Spodoptera frugiperda ovarian cells that can be used include Sf9) and Sf21; examples of the Trichoplusia ni ovarian cells that can be used include High 5 and BTI-TN-5B1-4 (Invitrogen); and examples of the cultured cells derived from silkworm ovaries that can be used include Bombyx mori N4.
[0069] The method for introducing a recombinant vector into a host is not particularly limited, as long as the method allows introduction of a DNA into the host, and examples of the method that can be used include a calcium ion method, electroporation, a spheroplast method, and a lithium acetate method. The method for introducing a recombinant vector into an insect cell is not particularly limited, as long as the method allows introduction of a INA into the insect cell, and examples of the method that can be used include a calcium phosphate method, lipofection, and electroporation.
[0070] A transformant having introduced therein a recombinant vector incorporating a DNA encoding a protein of interest is cultured. Culturing of the transformant can be carried out according to a conventional method used for culture of host cells.
[0071] The protein of interest can be obtained by collecting it from the culture of the transformant. The "culture" as referred to herein encompasses all of culture supernatants, cultured cells, cultured microorganisms, and disrupted products of cells or microorganisms. In the case where the protein of interest is accumulated in transformant cells, the culture is centrifuged to collect the cells from the culture, and the collected cells are washed and disrupted to extract the protein of interest. In the case where the protein of interest is excreted outside the transformant cells, the culture supernatant is used as it is, or cells or microorganisms are removed from the culture supernatant by centrifugation or the like. The extracted protein can be purified by solvent extraction, salting-out/desalting with ammonium sulfate or the like, precipitation with an organic solvent, diethylaminoethyl (DEAE)-sepharose, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, or the like.
[0072] The mutants of the present invention are useful for DNA amplification (particularly, PCR) and can be used as a component of a DNA amplification (particularly, PCR) kit. In addition to the inventive mutant Taq polymerases or fragments thereof, the DNA amplification (particularly, PCR) kit can contain reagents (e.g., four types of dNTPs, Mg2+, buffer, additives), a vessel, an apparatus, and the like which are necessary for DNA amplification (particularly, PCR). The inventive DNA amplification method and kit are suitable for a variety of applications, including DNA sequencing, gene diagnosis, individual identification in paternity test and criminal investigation, variety identification, SNP (single nucleotide polymorphism) analysis for constitutional study, and archaeological excavation.
EXAMPLES
Example 1
Construction of Mutants
1. Introduction of Mutations:
[0073] In order to construct each of amino acid substitution mutants of a Taq polymerase, one or more site-specific mutations were introduced in a primer dependent manner by PCR using one or more sets of primers having sequences designed to ensure that a mutation is applied to a position of interest, with an expression plasmid having a Taq polymerase gene inserted therein being used as a template. The introduction of one or more site-specific mutations into a Taq polymerase was performed using one or more sets of primers shown below. The amino acid substitution sites employed to construct the respective mutants are underlined.
TABLE-US-00001 [Formula 1] Exo Taq117119A-F (SEQ ID NO: 1) CTCGAGGTCCCGGGCTACGCGGCGGCCGACGTCCTGGCCAGCCTG Taq117119A-R (SEQ ID NO: 2) CACGCTGGCCAGGACGTCGGCCGCCGCGTAGCCGGGACCTCGAG Taq142144A-F (SEQ ID NO: 3) GTCCGCATCCTCACCGCCGCCAAAGCCCTTTACCAGCTCCTTTCC Taq142144A-R (SEQ ID NO: 4) GGAAAGGAGCTGGTAAAGGGCTTTGGCGGCGGTGAGGATGCGGAC [Formula 2] Exo + E742A Taq117119A-F (SEQ ID NO: 1) CTCGAGGTCCCGGGCTACGCGGCGGCCGACGTCCTGGCCAGCCTG Taq117119A-R (SEQ ID NO: 2) CAGGCTGGCCAGGACGTCGGCCGCCGCGTAGCCCGGGACCTCGAG Taq142144A-F (SEQ ID NO: 3) GTCCGCATCCTCACCGCCGCCAAAGCCCTTTACCAGCTCCTTTCC Taq142144A-R (SEQ ID NO: 4) GGAAAGGAGCTGGTAAAGGGCTTTGGCGGCGGTGAGGATGCGGAC E742A-F (SEQ ID NO: 5) GTGAAGAGCGTGCGGGCGGCGGCCGAGCGCATG E742A-R (SEQ ID NO: 6) CATGCGCTCGGCCGCCGCCCGCACGCTCTTCAC [Formula 3] TaqR651E R651E-F (SEQ ID NO: 7) ATGTTCGGCGTCCCCGAGGAGGCCGTGGAC R651E-R (SEQ ID NO: 8) GTCCACGGCCTCCTCGGGGACGCCGAACAT [Formula 4] TthP653E P653E-F (SEQ ID NO: 9) ATGTTCGGCGTGCCCGAGGAGGCCGTGGAC P653E-R (SEQ ID NO: 10) GTCCACGGCCTCCTCGGGGACGCCGAACAT
[0074] Fifty microliters of a PCR reaction mixture (20 ng of pTV-Taq plasmid DNA, 0.5 μM each primer set, 0.2 mM dNTP, and 1 U of Pyrobest DNA polymerase (Takara Bio)) was subjected to initial denaturation in a Pyrobest buffer at 98° C. for 10 seconds, which was followed by PCR under the conditions of 16 cycles (98° C. for 10 seconds, 55° C. for 30 seconds, and 72° C. for 8 minutes). 5 U of the restriction enzyme Dpn1 was added to the resulting PCR product, and the mixture was incubated at 37° C. for 2 hours. Then, the reaction mixture was introduced into the E. coli JM109 strain, and the resulting strain was cultured. By using the procedure described in the next section, a plasmid was extracted from the resulting transformant clone, and then a check was made to see that the mutation(s) was(were) introduced in the position(s) of interest.
2. Preparation of Plasmids and Confirmation of Nucleotide Sequences:
[0075] With a drug resistance gene in the plasmid being used as a marker, selection was made of an E. coli transformant that was seeded onto an LB plate medium containing 50 μg/mL of ampicillin and cultured at 37° C. for 15 hours. The colony that has grown was inoculated into 4 mL of an LB liquid medium containing 50 μg/mL of ampicillin and cultured at 37° C. for 15 hours. A plasmid was extracted from the harvested microorganisms using a QIAprep Spin Miniprep Kit (QIAGEN) according to the kit's protocol. With the DNA of the resulting plasmid being used as a template, dideoxy reaction was performed using a DTCS Quick Start Master Mix (Beckman Coulter), so that the nucleotide sequence was confirmed using a multi-capillary DNA analysis system CEQ2000XL (BECKMAN COULTER).
[0076] The novel DNA polymerases constructed in the present study are listed in the following table.
TABLE-US-00002 TABLE 1 DNA number of Theoretical Polymerase amino acids pl Mw. characteristics Taq WT (SEQ ID NO: 12) 832 6.04 93910.1 Taq Pol wild type Taq8 E742A 832 6.06 93852.1 E742A Taq8 A743H 832 6.10 93976.2 A743H 294L WT 540 6.88 60914.1 Deletion (293 as of N-ter) 294L E742A 540 6.97 60856.1 Deletion (293 as of N-ter), E742A 303E WT 531 6.07 59896.9 Deletion (302 as of N-ter) 303E E742A 531 6.18 59838.9 Deletion (302 as of N-ter), E742A Exo WT (SEQ ID NO: 16) 832 6.31 93720.0 E117A, D119A, D142A, D144A Exo E742A (SEQ ID NO: 18) 832 6.31 93662.0 E117A, D119A, D142A, D144A, E742A Taq R651E (SEQ ID NO: 20) 832 6.92 94785.2 R651E Taq T664R 832 6.10 93965.2 T664R Taq G668R 832 6.10 94009.2 G668R Taq S674R 832 6.10 93979.2 S674R Tth WT (SEQ ID NO: 14) 834 6.30 94049.4 Tth Pol Wild type Tth P653E (SEQ ID NO: 22) 834 6.23 94081.4 Tth Pol P653E indicates data missing or illegible when filed
3. Expression and Purification of DNA Polymerases:
[0077] Production of each of wild-type and mutant Taq polymerases was performed under the following conditions: the JM109 strain was transformed by a standard method using a plasmid that incorporated the gene of each polymerase into the pTV-118N vector, and the resulting transformant was cultured at 37° C. for 24 hours in 500 mL of an LB liquid medium containing 50 μg/mL of ampicillin. Production of each of wild-type and mutant Tth polymerases was performed under the following conditions; the BL21 (DE3) CodonPlus-RIPL strain (Stratagene) was transformed by a standard method using pET-3C as an expression vector, and the resulting transformant was cultured at 37° C. for 24 hours in 500 mL of an LB liquid medium containing 50 μg/mL of ampicillin and 331 μg/mL of chloramphenicol. Thereafter, the culture was centrifuged at 6,000 rpm for 15 minutes to harvest microorganisms, and the harvested microorganisms were suspended in 25 mL of Buffer A (50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol) supplemented with 1 mM PMSF, and were subjected to ultrasonication and centrifuged at 14,500 rpm for 15 minutes to obtain a crude cell extract. The crude extract was left to stand at 80° C. for 20-30 minutes to denature a non-thermostable protein, and was centrifuged at 14,500 rpm for 15 minutes to obtain a thermostable fraction in the supernatant. Polyethyleneimine was added to the fraction on ice so as to give a concentration of 0.15%, and the precipitate (nucleic acid) was removed by centrifugation at 14,500 rpm for 15 minutes. Next, ammonium sulfate was added to the supernatant on ice so as to give 80% saturation, and the suspension was stirred for at least 1 hour to effect salting-out. The suspension was centrifuged at 14,500 rpm for 15 minutes to effect protein precipitation, and the precipitate was suspended in Buffer A supplemented with 0.8 M ammonium sulfate; thereafter, the suspension was subjected to chromatography by passing it through a Hi Trap Phenyl column (5 mL) using an ÅKTA Explorer (GE Healthcare). After passage of the sample, a gradient from 1 M to 0 M ammonium sulfate was created and ultrapure water was passed through the column to thereby elute an enzyme of interest. The fraction was recovered and passed through a Hi Trap Heparin column (1 mL). Elution was performed with a gradient from 0 M to 800 mM sodium chloride as dissolved in Buffer A. The enzymes of interest thus obtained were each analyzed by SDS-PAGE to confirm their purity.
Example 2
Evaluation of Mutants
1. Transcriptional Activity:
[0078] In order to determine the basic DNA polymerase activity of each of the purified enzymes, a world-wide standard method was used. More specifically, the intensity of the activity of incorporating deoxyribonucleotides on a DNA template strand was determined, and the activity per unit protein amount was calculated in units. To perform a nucleotide incorporation reaction, a reaction mixture was prepared by adding 0.2 mg/mL of activated DNA (obtained by treating a calf thymus DNA with DNase I to partially nick or gap a double-strand DNA), 0.2 mM dNTP, 440 nM [3H]-dTTP, 50 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% TritonX-100, 100 μg/mL of BSA, and 1 nM DNA polymerase, and the mixture was reacted at 72° C.; then, 10 μL of the mixture was spotted onto DE81 paper. After air-dried for 10 minutes, the paper was washed with an aqueous 5% disodium hydrogenphosphate solution to remove unreacted nucleotides. The washing was repeated three times each for 10 minutes. After the DE81 paper was dried, radiation was measured by a liquid scintillation counter, whereby the amount of (3H-dTMP incorporated in the activated DNA due to the DNA polymerase activity was calculated to determine enzyme activity (in unit). One unit is defined as the amount of enzyme, i.e., DNA polymerase, required to incorporate 10 nmol of nucleotides at 72° C. for 30 minutes. The specific activity was calculated for each enzyme.
2. Extension Activity (Extension Rate):
[0079] Primer extension activity per unit was determined based on each of the calculated specific activity values. The primer extension reaction was performed using a substrate (primed DNA) obtained by annealing a 32P-radiolabeled oligonucleotide to an M13 phage single-strand DNA (7 kb). After 10 μL of a reaction mixture (5 nM M13 primed DNA, 0.2 mM dNTP, 50 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% TritonX-100, and 100 mL, BSA) was reacted at 72° C. for 5 minutes, the reaction was terminated by adding 2.5 μL of 6×loading buffer (300 nM NaOH, 6 mM EDTA, 18% Ficol 400, 0.15% BCG, and 0.25% XC). The reaction product was separated by agarose gel electrophoresis (agarose gel was prepared at a concentration of 1% in 50 mM NaOH and 1 mM EDTA) under alkaline conditions, and after the electrophoresis, the product was detected by autoradiography (using an image analyzer (FLA-5000, Fujifilm)).
[0080] The strand length of the reaction product was determined from the obtained image by comparing it with a size marker, whereby the strand length of the synthetic product obtained per unit time (1 min) was calculated. As a result, it was shown that whereas the wild-type Taq polymerase had an extension rate of 3.89 kb/min, Taq E742A (refer to Patent Document 3: JP 4193997 given above) had an extension rate of 10.7 kb/min, the mutant Exo- whose 5'-3' exonuclease activity residues were substituted in a site-specific manner had an extension rate of 9.17 kb/min, and Exo-+E742A produced by crossing Taq B742A with Exohad a rate of 15.4 kb/min.
3. Reverse Transcriptional Activity:
[0081] The intensity of the activity of incorporating deoxyribonucleotides on a RNA template strand was determined for each of the purified DNA polymerases. In order to perform the reaction, the DNA polymerase was added to a solution containing 20 ng/μL of poly(rA)*p(dT), 10 μM dTTP, 440 nM [3H]-dTTP, 50 mM Tris-HCl (pH 8.0), 1 mM MnCl2, 50 mM KCl, 0.1% TritonX-100, and 100 μg/mL of BSA, and the mixture was reacted at 60° C. for 10 minutes; then, 10 μL of the mixture was spotted onto DE81 paper. After air-dried, the paper was washed with an aqueous 5% disodium hydrogenphosphate solution to remove unreacted nucleotides. The washing was repeated three times each for 10 minutes. After the DE81 paper was dried, radiation was measured by a liquid scintillation counter, whereby the amount of [3H]-dTMP incorporated in the poly(rA)*p(dT) due to the DNA polymerase activity was calculated to determine enzyme activity (in unit). One unit is defined as the amount of enzyme, i.e., DNA polymerase, required to incorporate 10 nmol of nucleotides at 72° C. for 30 minutes. The specific activity was calculated for each enzyme.
[0082] First, a Tth polymerase with strong reverse transcriptional activity was prepared for use as a positive control. It was revealed that the Tth polymerase purified by the present inventors showed a reverse transcriptional activity of 1.54×10 U/mg, while the wild-type Taq DNA polymerase which was also purified by the inventors showed a reverse transcriptional activity of 0.42×104 U/mg (corresponding to a relative activity of 27% with respect to the Tth polymerase in the present study).
[0083] The reverse transcriptional activity of each of the mutants constructed according to the present invention was determined using the same conditions on the basis of its nucleotide incorporation activity. As a result, it was shown that Taq R651E and Tth P653E showed a reverse transcriptional activity of 2.09×104 U/mg and 2.54×104 U/mg, respectively. These results mean that Taq R651E and Tth P653E showed activities 1.36 and 1.65 times, respectively, greater than the wild-type Tth polymerase, whose reverse transcriptional activity is taken as 1.
[0084] The properties of the mutant Taq polymerases are summarized in the following table.
TABLE-US-00003 TABLE 2 Purified Protein DNA-DNA RNA-DNA from 500 Specific Relative Specific Relative Extension Date DNA mL culture Activity activity Activity activity DNA RNA Polymerase (mg) (×105 U/mg) (%) (×103 U/mg) (%) (kb/U min) (bp/pmol min) Taq WT (SEQ ID NO: 12) 1.65 5.11 100 4.24 27.5 3.89 14.0 Taq E742A 0.67 5.00 97.7 10.7 Taq A743H 0.89 3.84 77.1 5.80 294L WT 0.48 6.67 131 0.11 294L E742A 0.56 12.7 249 0.29 303E WT 0.71 6.50 127 0.11 303E E742A 0.63 13.5 254 0.26 Exo WT (SEQ ID NO: 16) 0.54 3.79 74.2 9.17 Exo E742A (SEQ ID NO: 18) 0.27 3.90 76.3 15.4 Taq R651E (SEQ ID NO: 20) 1.12 3.44 67.3 20.9 136 4.36 45.4 Taq T664R 0.65 2.03 39.8 7.74 Taq G568R 0.21 2.98 58.2 2.03 Taq S674R 0.31 4.56 89.3 6.00 Tth WT (SEQ ID NO: 14) 0.68 3.66 71.5 15.4 100 14.6 117 Tth P653E (SEQ ID NO: 22) 0.66 3.37 66.0 25.4 165 13.3 181
SEQUENCE LISTING FREE TEXT
[0085] SEQ ID NO: 1 PCR primer Taq117119A-F
[0086] SEQ ID NO: 2 PCR primer Taq117119A-R
[0087] SEQ ID) NO: 3 PCR primer Taq142144A-F
[0088] SEQ ID NO: 4 PCR primer Taq 142144A-R
[0089] SEQ ID NO: 5 PCR primer E742A-F
[0090] SEQ ID 3 NO: 6 PCR primer E742A-R
[0091] SEQ ID NO: 7 PCR primer R651E-F
[0092] SEQ ID NO: 8 PCR primer R651E-R
[0093] SEQ ID NO: 9 PCR primer P653E-F
[0094] SEQ ID NO: 10 PCR primer P653E-R
[0095] SEQ ID NO: 11 Taq WT nucleotide sequence
[0096] SEQ ID NO: 12 Taq WT amino acid sequence
[0097] SEQ ID NO: 13 Tth WT nucleotide sequence
[0098] SEQ ID NO: 14 Tth WT amino acid sequence
[0099] SEQ ID NO: 15 Taq Exo- nucleotide sequence
[0100] SEQ ID) NO: 16 Taq Exo- amino acid sequence
[0101] SEQ ID NO: 17 Taq Exo-+E742A nucleotide sequence
[0102] SEQ ID NO: 18 Taq Exo-+E742A amino acid sequence
[0103] SEQ ID NO: 19 Taq R651E nucleotide sequence
[0104] SEQ ID NO: 20 Taq R651E amino acid sequence
[0105] SEQ ID NO: 21 Tth P653E nucleotide sequence
[0106] SEQ ID NO: 22 Tth P653E amino acid sequence
Sequence CWU
1
1
22145DNAArtificial sequenceTaq117119A-F primer 1ctcgaggtcc cgggctacgc
ggcggccgac gtcctggcca gcctg 45245DNAArtificial
sequenceTaq117119A-R primer 2caggctggcc aggacgtcgg ccgccgcgta gcccgggacc
tcgag 45345DNAArtificial sequenceTaq142144A-F primer
3gtccgcatcc tcaccgccgc caaagccctt taccagctcc tttcc
45445DNAArtificial sequenceTaq142144A-R primer 4ggaaaggagc tggtaaaggg
ctttggcggc ggtgaggatg cggac 45533DNAArtificial
SequenceE742A-F primer 5gtgaagagcg tgcgggcggc ggccgagcgc atg
33633DNAArtificial SequenceE742A-R primer 6catgcgctcg
gccgccgccc gcacgctctt cac
33730DNAArtificial SequenceR651E-F primer 7atgttcggcg tccccgagga
ggccgtggac 30830DNAArtificial
SequenceR651E-R primer 8gtccacggcc tcctcgggga cgccgaacat
30930DNAArtificial SequenceP653E-F primer 9atgttcggcg
tccccgagga ggccgtggac
301030DNAArtificial SequenceP653E-R primer 10gtccacggcc tcctcgggga
cgccgaacat 30112499DNAThermus
aquaticusCDS(1)..(2499) 11atg agg ggg atg ctg ccc ctc ttt gag ccc aag ggc
cgg gtc ctc ctg 48Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly
Arg Val Leu Leu 1 5 10
15 gtg gac ggc cac cac ctg gcc tac cgc acc ttc cac gcc ctg
aag ggc 96Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu
Lys Gly 20 25 30
ctc acc acc agc cgg ggg gag ccg gtg cag gcg gtc tac ggc ttc gcc
144Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
aag agc ctc ctc aag gcc ctc aag gag gac ggg gac gcg gtg atc gtg
192Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60 gtc
ttt gac gcc aag gcc ccc tcc ttc cgc cac gag gcc tac ggg ggg 240Val
Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65
70 75 80 tac aag gcg
ggc cgg gcc ccc acg ccg gag gac ttt ccc cgg caa ctc 288Tyr Lys Ala
Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95 gcc ctc atc aag gag
ctg gtg gac ctc ctg ggg ctg gcg cgc ctc gag 336Ala Leu Ile Lys Glu
Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100
105 110 gtc ccg ggc tac gag gcg gac
gac gtc ctg gcc agc ctg gcc aag aag 384Val Pro Gly Tyr Glu Ala Asp
Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120
125 gcg gaa aag gag ggc tac gag gtc cgc
atc ctc acc gcc gac aaa gac 432Ala Glu Lys Glu Gly Tyr Glu Val Arg
Ile Leu Thr Ala Asp Lys Asp 130 135
140 ctt tac cag ctc ctt tcc gac cgc atc cac gtc
ctc cac ccc gag ggg 480Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val
Leu His Pro Glu Gly 145 150 155
160 tac ctc atc acc ccg gcc tgg ctt tgg gaa aag tac ggc
ctg agg ccc 528Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly
Leu Arg Pro 165 170
175 gac cag tgg gcc gac tac cgg gcc ctg acc ggg gac gag tcc gac
aac 576Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp
Asn 180 185 190
ctt ccc ggg gtc aag ggc atc ggg gag aag acg gcg agg aag ctt ctg
624Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205 gag
gag tgg ggg agc ctg gaa gcc ctc ctc aag aac ctg gac cgg ctg 672Glu
Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210
215 220 aag ccc gcc
atc cgg gag aag atc ctg gcc cac atg gac gat ctg aag 720Lys Pro Ala
Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225
230 235 240 ctc tcc tgg gac ctg
gcc aag gtg cgc acc gac ctg ccc ctg gag gtg 768Leu Ser Trp Asp Leu
Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245
250 255 gac ttc gcc aaa agg cgg gag
ccc gac cgg gag agg ctt agg gcc ttt 816Asp Phe Ala Lys Arg Arg Glu
Pro Asp Arg Glu Arg Leu Arg Ala Phe 260
265 270 ctg gag agg ctt gag ttt ggc agc ctc
ctc cac gag ttc ggc ctt ctg 864Leu Glu Arg Leu Glu Phe Gly Ser Leu
Leu His Glu Phe Gly Leu Leu 275 280
285 gaa agc ccc aag gcc ctg gag gag gcc ccc tgg
ccc ccg ccg gaa ggg 912Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp
Pro Pro Pro Glu Gly 290 295 300
gcc ttc gtg ggc ttt gtg ctt tcc cgc aag gag ccc atg
tgg gcc gat 960Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met
Trp Ala Asp 305 310 315
320 ctt ctg gcc ctg gcc gcc gcc agg ggg ggc cgg gtc cac cgg gcc
ccc 1008Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala
Pro 325 330 335
gag cct tat aaa gcc ctc agg gac ctg aag gag gcg cgg ggg ctt ctc
1056Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350 gcc
aaa gac ctg agc gtt ctg gcc ctg agg gaa ggc ctt ggc ctc ccg 1104Ala
Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365 ccc ggc gac
gac ccc atg ctc ctc gcc tac ctc ctg gac cct tcc aac 1152Pro Gly Asp
Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370
375 380 acc acc ccc gag ggg
gtg gcc cgg cgc tac ggc ggg gag tgg acg gag 1200Thr Thr Pro Glu Gly
Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390
395 400 gag gcg ggg gag cgg gcc gcc
ctt tcc gag agg ctc ttc gcc aac ctg 1248Glu Ala Gly Glu Arg Ala Ala
Leu Ser Glu Arg Leu Phe Ala Asn Leu 405
410 415 tgg ggg agg ctt gag ggg gag gag agg
ctc ctt tgg ctt tac cgg gag 1296Trp Gly Arg Leu Glu Gly Glu Glu Arg
Leu Leu Trp Leu Tyr Arg Glu 420 425
430 gtg gag agg ccc ctt tcc gct gtc ctg gcc cac
atg gag gcc acg ggg 1344Val Glu Arg Pro Leu Ser Ala Val Leu Ala His
Met Glu Ala Thr Gly 435 440
445 gtg cgc ctg gac gtg gcc tat ctc agg gcc ttg tcc ctg
gag gtg gcc 1392Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu
Glu Val Ala 450 455 460
gag gag atc gcc cgc ctc gag gcc gag gtc ttc cgc ctg gcc ggc
cac 1440Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly
His 465 470 475 480
ccc ttc aac ctc aac tcc cgg gac cag ctg gaa agg gtc ctc ttt gac
1488Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495 gag
cta ggg ctt ccc gcc atc ggc aag acg gag aag acc ggc aag cgc 1536Glu
Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510 tcc acc agc
gcc gcc gtc ctg gag gcc ctc cgc gag gcc cac ccc atc 1584Ser Thr Ser
Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515
520 525 gtg gag aag atc ctg
cag tac cgg gag ctc acc aag ctg aag agc acc 1632Val Glu Lys Ile Leu
Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530
535 540 tac att gac ccc ttg ccg gac
ctc atc cac ccc agg acg ggc cgc ctc 1680Tyr Ile Asp Pro Leu Pro Asp
Leu Ile His Pro Arg Thr Gly Arg Leu 545 550
555 560 cac acc cgc ttc aac cag acg gcc acg
gcc acg ggc agg cta agt agc 1728His Thr Arg Phe Asn Gln Thr Ala Thr
Ala Thr Gly Arg Leu Ser Ser 565 570
575 tcc gat ccc aac ctc cag aac atc ccc gtc cgc
acc ccg ctt ggg cag 1776Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg
Thr Pro Leu Gly Gln 580 585
590 agg atc cgc cgg gcc ttc atc gcc gag gag ggg tgg cta ttg
gtg gcc 1824Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu
Val Ala 595 600 605
ctg gac tat agc cag ata gag ctc agg gtg ctg gcc cac ctc tcc ggc
1872Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620 gac
gag aac ctg atc cgg gtc ttc cag gag ggg cgg gac atc cac acg 1920Asp
Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625
630 635 640 gag acc gcc
agc tgg atg ttc ggc gtc ccc cgg gag gcc gtg gac ccc 1968Glu Thr Ala
Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655 ctg atg cgc cgg gcg
gcc aag acc atc aac ttc ggg gtc ctc tac ggc 2016Leu Met Arg Arg Ala
Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660
665 670 atg tcg gcc cac cgc ctc tcc
cag gag cta gcc atc cct tac gag gag 2064Met Ser Ala His Arg Leu Ser
Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680
685 gcc cag gcc ttc att gag cgc tac ttt
cag agc ttc ccc aag gtg cgg 2112Ala Gln Ala Phe Ile Glu Arg Tyr Phe
Gln Ser Phe Pro Lys Val Arg 690 695
700 gcc tgg att gag aag acc ctg gag gag ggc agg
agg cgg ggg tac gtg 2160Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg
Arg Arg Gly Tyr Val 705 710 715
720 gag acc ctc ttc ggc cgc cgc cgc tac gtg cca gac cta
gag gcc cgg 2208Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu
Glu Ala Arg 725 730
735 gtg aag agc gtg cgg gag gcg gcc gag cgc atg gcc ttc aac atg
ccc 2256Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met
Pro 740 745 750
gtc cag ggc acc gcc gcc gac ctc atg aag ctg gct atg gtg aag ctc
2304Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765 ttc
ccc agg ctg gag gaa atg ggg gcc agg atg ctc ctt cag gtc cac 2352Phe
Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770
775 780 gac gag ctg
gtc ctc gag gcc cca aaa gag agg gcg gag gcc gtg gcc 2400Asp Glu Leu
Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785
790 795 800 cgg ctg gcc aag gag gtc
atg gag ggg gtg tat ccc ctg gcc gtg ccc 2448Arg Leu Ala Lys Glu Val
Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805
810 815 ctg gag gtg gag gtg ggg ata ggg
gag gac tgg ctc tcc gcc aag gag 2496Leu Glu Val Glu Val Gly Ile Gly
Glu Asp Trp Leu Ser Ala Lys Glu 820 825
830 tga
249912832PRTThermus aquaticus 12Met Arg Gly Met
Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 Val Asp Gly His His Leu Ala Tyr Arg
Thr Phe His Ala Leu Lys Gly 20 25
30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly
Phe Ala 35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50
55 60 Val Phe Asp Ala Lys
Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70
75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu
Asp Phe Pro Arg Gln Leu 85 90
95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu
Glu 100 105 110 Val
Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115
120 125 Ala Glu Lys Glu Gly Tyr
Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135
140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val
Leu His Pro Glu Gly 145 150 155
160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175 Asp Gln
Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180
185 190 Leu Pro Gly Val Lys Gly Ile
Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200
205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn
Leu Asp Arg Leu 210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225
230 235 240 Leu Ser Trp
Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245
250 255 Asp Phe Ala Lys Arg Arg Glu Pro
Asp Arg Glu Arg Leu Arg Ala Phe 260 265
270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe
Gly Leu Leu 275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290
295 300 Ala Phe Val Gly
Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310
315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly
Gly Arg Val His Arg Ala Pro 325 330
335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly
Leu Leu 340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365 Pro Gly Asp Asp
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370
375 380 Thr Thr Pro Glu Gly Val Ala Arg
Arg Tyr Gly Gly Glu Trp Thr Glu 385 390
395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu
Phe Ala Asn Leu 405 410
415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430 Val Glu Arg
Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435
440 445 Val Arg Leu Asp Val Ala Tyr Leu
Arg Ala Leu Ser Leu Glu Val Ala 450 455
460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu
Ala Gly His 465 470 475
480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495 Glu Leu Gly Leu
Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500
505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala
Leu Arg Glu Ala His Pro Ile 515 520
525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys
Ser Thr 530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545
550 555 560 His Thr Arg Phe Asn
Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565
570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val
Arg Thr Pro Leu Gly Gln 580 585
590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val
Ala 595 600 605 Leu
Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610
615 620 Asp Glu Asn Leu Ile Arg
Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630
635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg
Glu Ala Val Asp Pro 645 650
655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670 Met Ser
Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675
680 685 Ala Gln Ala Phe Ile Glu Arg
Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695
700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg
Arg Gly Tyr Val 705 710 715
720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735 Val Lys Ser
Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740
745 750 Val Gln Gly Thr Ala Ala Asp Leu
Met Lys Leu Ala Met Val Lys Leu 755 760
765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu
Gln Val His 770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785
790 795 800 Arg Leu Ala Lys
Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805
810 815 Leu Glu Val Glu Val Gly Ile Gly Glu
Asp Trp Leu Ser Ala Lys Glu 820 825
830 132505DNAThermus thermophilusCDS(1)..(2505) 13atg gag
gcg atg ctt ccg ctc ttt gaa ccc aaa ggc cgg gtc ctc ctg 48Met Glu
Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1
5 10 15 gtg gac ggc cac
cac ctg gcc tac cgc acc ttc ttc gcc ctg aag ggc 96Val Asp Gly His
His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20
25 30 ctc acc acg agc cgg ggc
gaa ccg gtg cag gcg gtc tac ggc ttc gcc 144Leu Thr Thr Ser Arg Gly
Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35
40 45 aag agc ctc ctc aag gcc ctg aag
gag gac ggg tac aag gcc gtc ttc 192Lys Ser Leu Leu Lys Ala Leu Lys
Glu Asp Gly Tyr Lys Ala Val Phe 50 55
60 gtg gtc ttt gac gcc aag gcc ccc tcc ttc
cgc cac gag gcc tac gag 240Val Val Phe Asp Ala Lys Ala Pro Ser Phe
Arg His Glu Ala Tyr Glu 65 70 75
80 gcc tac aag gcg ggg agg gcc ccg acc ccc gag gac
ttc ccc cgg cag 288Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp
Phe Pro Arg Gln 85 90
95 ctc gcc ctc atc aag gag ctg gtg gac ctc ctg ggg ttt acc
cgc ctc 336Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr
Arg Leu 100 105 110
gag gtc ccc ggc tac gag gcg gac gac gtt ctc gcc acc ctg gcc aag
384Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys
115 120 125
aag gcg gaa aag gag ggg tac gag gtg cgc atc ctc acc gcc gac cgc
432Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg
130 135 140 gac
ctc tac caa ctc gtc tcc gac cgc gtc gcc gtc ctc cac ccc gag 480Asp
Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145
150 155 160 ggc cac ctc
atc acc ccg gag tgg ctt tgg gag aag tac ggc ctc agg 528Gly His Leu
Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg
165 170 175 ccg gag cag tgg gtg
gac ttc cgc gcc ctc gtg ggg gac ccc tcc gac 576Pro Glu Gln Trp Val
Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180
185 190 aac ctc ccc ggg gtc aag ggc
atc ggg gag aag acc gcc ctc aag ctc 624Asn Leu Pro Gly Val Lys Gly
Ile Gly Glu Lys Thr Ala Leu Lys Leu 195 200
205 ctc aag gag tgg gga agc ctg gaa aac
ctc ctc aag aac ctg gac cgg 672Leu Lys Glu Trp Gly Ser Leu Glu Asn
Leu Leu Lys Asn Leu Asp Arg 210 215
220 gta aag cca gaa aac gtc cgg gag aag atc aag
gcc cac ctg gaa gac 720Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys
Ala His Leu Glu Asp 225 230 235
240 ctc agg ctc tcc ttg gag ctc tcc cgg gtg cgc acc gac
ctc ccc ctg 768Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp
Leu Pro Leu 245 250
255 gag gtg gac ctc gcc cag ggg cgg gag ccc gac cgg gag ggg ctt
agg 816Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu
Arg 260 265 270
gcc ttc ctg gag agg ctg gag ttc ggc agc ctc ctc cac gag ttc ggc
864Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285 ctc
ctg gag gcc ccc gcc ccc ctg gag gag gcc ccc tgg ccc ccg ccg 912Leu
Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290
295 300 gaa ggg gcc
ttc gtg ggc ttc gtc ctc tcc cgc ccc gag ccc atg tgg 960Glu Gly Ala
Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305
310 315 320 gcg gag ctt aaa gcc
ctg gcc gcc tgc agg gac ggc cgg gtg cac cgg 1008Ala Glu Leu Lys Ala
Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg 325
330 335 gca gca gac ccc ttg gcg ggg
cta aag gac ctc aag gag gtc cgg ggc 1056Ala Ala Asp Pro Leu Ala Gly
Leu Lys Asp Leu Lys Glu Val Arg Gly 340
345 350 ctc ctc gcc aag gac ctc gcc gtc ttg
gcc tcg agg gag ggg cta gac 1104Leu Leu Ala Lys Asp Leu Ala Val Leu
Ala Ser Arg Glu Gly Leu Asp 355 360
365 ctc gtg ccc ggg gac gac ccc atg ctc ctc gcc
tac ctc ctg gac ccc 1152Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala
Tyr Leu Leu Asp Pro 370 375 380
tcc aac acc acc ccc gag ggg gtg gcg cgg cgc tac ggg
ggg gag tgg 1200Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly
Gly Glu Trp 385 390 395
400 acg gag gac gcc gcc cac cgg gcc ctc ctc tcg gag agg ctc cat cgg
1248Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg
405 410 415
aac ctc ctt aag cgc ctc gag ggg gag gag aag ctc ctt tgg ctc tac
1296Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr
420 425 430 cac
gag gtg gaa aag ccc ctc tcc cgg gtc ctg gcc cac atg gag gcc 1344His
Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala
435 440 445 acc ggg gta
cgg cgg gac gtg gcc tac ctt cag gcc ctt tcc ctg gag 1392Thr Gly Val
Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu 450
455 460 ctt gcg gag gag atc
cgc cgc ctc gag gag gag gtc ttc cgc ttg gcg 1440Leu Ala Glu Glu Ile
Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465 470
475 480 ggc cac ccc ttc aac ctc aac
tcc cgg gac cag ctg gaa agg gtg ctc 1488Gly His Pro Phe Asn Leu Asn
Ser Arg Asp Gln Leu Glu Arg Val Leu 485
490 495 ttt gac gag ctt agg ctt ccc gcc ttg
ggg aag acg caa aag aca ggc 1536Phe Asp Glu Leu Arg Leu Pro Ala Leu
Gly Lys Thr Gln Lys Thr Gly 500 505
510 aag cgc tcc acc agc gcc gcg gtg ctg gag gcc
cta cgg gag gcc cac 1584Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala
Leu Arg Glu Ala His 515 520
525 ccc atc gtg gag aag atc ctc cag cac cgg gag ctc acc
aag ctc aag 1632Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr
Lys Leu Lys 530 535 540
aac acc tac gtg gac ccc ctc cca agc ctc gtc cac ccg agg acg
ggc 1680Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr
Gly 545 550 555 560
cgc ctc cac acc cgc ttc aac cag acg gcc acg gcc acg ggg agg ctt
1728Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu
565 570 575 agt
agc tcc gac ccc aac ctg cag aac atc ccc gtc cgc acc ccc ttg 1776Ser
Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
580 585 590 ggc cag agg
atc cgc cgg gcc ttc gtg gcc gag gcg ggt tgg gcg ttg 1824Gly Gln Arg
Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595
600 605 gtg gcc ctg gac tat
agc cag ata gag ctc cgc gtc ctc gcc cac ctc 1872Val Ala Leu Asp Tyr
Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu 610
615 620 tcc ggg gac gaa aac ctg atc
agg gtc ttc cag gag ggg aag gac atc 1920Ser Gly Asp Glu Asn Leu Ile
Arg Val Phe Gln Glu Gly Lys Asp Ile 625 630
635 640 cac acc cag acc gca agc tgg atg ttc
ggc gtc ccc ccg gag gcc gtg 1968His Thr Gln Thr Ala Ser Trp Met Phe
Gly Val Pro Pro Glu Ala Val 645 650
655 gac ccc ctg atg cgc cgg gcg gcc aag acg gtg
aac ttc ggc gtc ctc 2016Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val
Asn Phe Gly Val Leu 660 665
670 tac ggc atg tcc gcc cat agg ctc tcc cag gag ctt gcc
atc ccc tac 2064Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala
Ile Pro Tyr 675 680 685
gag gag gcg gtg gcc ttt ata gag cgc tac ttc caa agc ttc ccc
aag 2112Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro
Lys 690 695 700
gtg cgg gcc tgg ata gaa aag acc ctg gag gag ggg agg aag cgg ggc
2160Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly
705 710 715 720 tac
gtg gaa acc ctc ttc gga aga agg cgc tac gtg ccc gac ctc aac 2208Tyr
Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn
725 730 735 gcc cgg gtg aag
agc gtc agg gag gcc gcg gag cgc atg gcc ttc aac 2256Ala Arg Val Lys
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 740
745 750 atg ccc gtc cag ggc acc
gcc gcc gac ctc atg aag ctc gcc atg gtg 2304Met Pro Val Gln Gly Thr
Ala Ala Asp Leu Met Lys Leu Ala Met Val 755
760 765 aag ctc ttc ccc cgc ctc cgg gag
atg ggg gcc cgc atg ctc ctc cag 2352Lys Leu Phe Pro Arg Leu Arg Glu
Met Gly Ala Arg Met Leu Leu Gln 770 775
780 gtc cac gac gag ctc ctc ctg gag gcc ccc caa
gcg cgg gcc gag gag 2400Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln
Ala Arg Ala Glu Glu 785 790 795
800 gtg gcg gct ttg gcc aag gag gcc atg gag aag gcc tat
ccc ctc gcc 2448Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr
Pro Leu Ala 805 810
815 gtg ccc ctg gag gtg gag gtg ggg atg ggg gag gac tgg ctt tcc
gcc 2496Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser
Ala 820 825 830
aag ggt tag
2505Lys Gly
14834PRTThermus thermophilus 14Met Glu Ala Met Leu Pro Leu Phe Glu Pro
Lys Gly Arg Val Leu Leu 1 5 10
15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys
Gly 20 25 30 Leu
Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35
40 45 Lys Ser Leu Leu Lys Ala
Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe 50 55
60 Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg
His Glu Ala Tyr Glu 65 70 75
80 Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln
85 90 95 Leu Ala
Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu 100
105 110 Glu Val Pro Gly Tyr Glu Ala
Asp Asp Val Leu Ala Thr Leu Ala Lys 115 120
125 Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu
Thr Ala Asp Arg 130 135 140
Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145
150 155 160 Gly His Leu
Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165
170 175 Pro Glu Gln Trp Val Asp Phe Arg
Ala Leu Val Gly Asp Pro Ser Asp 180 185
190 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala
Leu Lys Leu 195 200 205
Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg 210
215 220 Val Lys Pro Glu
Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp 225 230
235 240 Leu Arg Leu Ser Leu Glu Leu Ser Arg
Val Arg Thr Asp Leu Pro Leu 245 250
255 Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly
Leu Arg 260 265 270
Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285 Leu Leu Glu Ala
Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290
295 300 Glu Gly Ala Phe Val Gly Phe Val
Leu Ser Arg Pro Glu Pro Met Trp 305 310
315 320 Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly
Arg Val His Arg 325 330
335 Ala Ala Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly
340 345 350 Leu Leu Ala
Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp 355
360 365 Leu Val Pro Gly Asp Asp Pro Met
Leu Leu Ala Tyr Leu Leu Asp Pro 370 375
380 Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly
Gly Glu Trp 385 390 395
400 Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg
405 410 415 Asn Leu Leu Lys
Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr 420
425 430 His Glu Val Glu Lys Pro Leu Ser Arg
Val Leu Ala His Met Glu Ala 435 440
445 Thr Gly Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser
Leu Glu 450 455 460
Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465
470 475 480 Gly His Pro Phe Asn
Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu 485
490 495 Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly
Lys Thr Gln Lys Thr Gly 500 505
510 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala
His 515 520 525 Pro
Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys 530
535 540 Asn Thr Tyr Val Asp Pro
Leu Pro Ser Leu Val His Pro Arg Thr Gly 545 550
555 560 Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr
Ala Thr Gly Arg Leu 565 570
575 Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
580 585 590 Gly Gln
Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595
600 605 Val Ala Leu Asp Tyr Ser Gln
Ile Glu Leu Arg Val Leu Ala His Leu 610 615
620 Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu
Gly Lys Asp Ile 625 630 635
640 His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val
645 650 655 Asp Pro Leu
Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660
665 670 Tyr Gly Met Ser Ala His Arg Leu
Ser Gln Glu Leu Ala Ile Pro Tyr 675 680
685 Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser
Phe Pro Lys 690 695 700
Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly 705
710 715 720 Tyr Val Glu Thr
Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn 725
730 735 Ala Arg Val Lys Ser Val Arg Glu Ala
Ala Glu Arg Met Ala Phe Asn 740 745
750 Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala
Met Val 755 760 765
Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln 770
775 780 Val His Asp Glu Leu
Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu 785 790
795 800 Val Ala Ala Leu Ala Lys Glu Ala Met Glu
Lys Ala Tyr Pro Leu Ala 805 810
815 Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser
Ala 820 825 830 Lys
Gly 152499DNAArtificial sequenceTaq Exo- 15atg agg ggg atg ctg ccc ctc
ttt gag ccc aag ggc cgg gtc ctc ctg 48Met Arg Gly Met Leu Pro Leu
Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 gtg gac ggc cac cac ctg gcc tac cgc
acc ttc cac gcc ctg aag ggc 96Val Asp Gly His His Leu Ala Tyr Arg
Thr Phe His Ala Leu Lys Gly 20 25
30 ctc acc acc agc cgg ggg gag ccg gtg cag gcg
gtc tac ggc ttc gcc 144Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala
Val Tyr Gly Phe Ala 35 40
45 aag agc ctc ctc aag gcc ctc aag gag gac ggg gac gcg
gtg atc gtg 192Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala
Val Ile Val 50 55 60
gtc ttt gac gcc aag gcc ccc tcc ttc cgc cac gag gcc tac ggg
ggg 240Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly
Gly 65 70 75 80
tac aag gcg ggc cgg gcc ccc acg ccg gag gac ttt ccc cgg caa ctc
288Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95 gcc
ctc atc aag gag ctg gtg gac ctc ctg ggg ctg gcg cgc ctc gag 336Ala
Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110 gtc ccg ggc
tac gcg gcg gcc gac gtc ctg gcc agc ctg gcc aag aag 384Val Pro Gly
Tyr Ala Ala Ala Asp Val Leu Ala Ser Leu Ala Lys Lys 115
120 125 gcg gaa aag gag ggc
tac gag gtc cgc atc ctc acc gcc gcc aaa gcc 432Ala Glu Lys Glu Gly
Tyr Glu Val Arg Ile Leu Thr Ala Ala Lys Ala 130
135 140 ctt tac cag ctc ctt tcc gac
cgc atc cac gtc ctc cac ccc gag ggg 480Leu Tyr Gln Leu Leu Ser Asp
Arg Ile His Val Leu His Pro Glu Gly 145 150
155 160 tac ctc atc acc ccg gcc tgg ctt tgg
gaa aag tac ggc ctg agg ccc 528Tyr Leu Ile Thr Pro Ala Trp Leu Trp
Glu Lys Tyr Gly Leu Arg Pro 165 170
175 gac cag tgg gcc gac tac cgg gcc ctg acc ggg
gac gag tcc gac aac 576Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly
Asp Glu Ser Asp Asn 180 185
190 ctt ccc ggg gtc aag ggc atc ggg gag aag acg gcg agg
aag ctt ctg 624Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg
Lys Leu Leu 195 200 205
gag gag tgg ggg agc ctg gaa gcc ctc ctc aag aac ctg gac cgg
ctg 672Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg
Leu 210 215 220
aag ccc gcc atc cgg gag aag atc ctg gcc cac atg gac gat ctg aag
720Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240 ctc
tcc tgg gac ctg gcc aag gtg cgc acc gac ctg ccc ctg gag gtg 768Leu
Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255 gac ttc gcc
aaa agg cgg gag ccc gac cgg gag agg ctt agg gcc ttt 816Asp Phe Ala
Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260
265 270 ctg gag agg ctt gag
ttt ggc agc ctc ctc cac gag ttc ggc ctt ctg 864Leu Glu Arg Leu Glu
Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275
280 285 gaa agc ccc aag gcc ctg gag
gag gcc ccc tgg ccc ccg ccg gaa ggg 912Glu Ser Pro Lys Ala Leu Glu
Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295
300 gcc ttc gtg ggc ttt gtg ctt tcc cgc
aag gag ccc atg tgg gcc gat 960Ala Phe Val Gly Phe Val Leu Ser Arg
Lys Glu Pro Met Trp Ala Asp 305 310
315 320 ctt ctg gcc ctg gcc gcc gcc agg ggg ggc cgg
gtc cac cgg gcc ccc 1008Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg
Val His Arg Ala Pro 325 330
335 gag cct tat aaa gcc ctc agg gac ctg aag gag gcg cgg
ggg ctt ctc 1056Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg
Gly Leu Leu 340 345 350
gcc aaa gac ctg agc gtt ctg gcc ctg agg gaa ggc ctt ggc ctc
ccg 1104Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu
Pro 355 360 365
ccc ggc gac gac ccc atg ctc ctc gcc tac ctc ctg gac cct tcc aac
1152Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380 acc
acc ccc gag ggg gtg gcc cgg cgc tac ggc ggg gag tgg acg gag 1200Thr
Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385
390 395 400 gag gcg ggg
gag cgg gcc gcc ctt tcc gag agg ctc ttc gcc aac ctg 1248Glu Ala Gly
Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415 tgg ggg agg ctt gag
ggg gag gag agg ctc ctt tgg ctt tac cgg gag 1296Trp Gly Arg Leu Glu
Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420
425 430 gtg gag agg ccc ctt tcc gct
gtc ctg gcc cac atg gag gcc acg ggg 1344Val Glu Arg Pro Leu Ser Ala
Val Leu Ala His Met Glu Ala Thr Gly 435 440
445 gtg cgc ctg gac gtg gcc tat ctc agg gcc
ttg tcc ctg gag gtg gcc 1392Val Arg Leu Asp Val Ala Tyr Leu Arg Ala
Leu Ser Leu Glu Val Ala 450 455
460 gag gag atc gcc cgc ctc gag gcc gag gtc ttc cgc
ctg gcc ggc cac 1440Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg
Leu Ala Gly His 465 470 475
480 ccc ttc aac ctc aac tcc cgg gac cag ctg gaa agg gtc ctc
ttt gac 1488Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu
Phe Asp 485 490 495
gag cta ggg ctt ccc gcc atc ggc aag acg gag aag acc ggc aag cgc
1536Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510 tcc
acc agc gcc gcc gtc ctg gag gcc ctc cgc gag gcc cac ccc atc 1584Ser
Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525 gtg gag aag
atc ctg cag tac cgg gag ctc acc aag ctg aag agc acc 1632Val Glu Lys
Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530
535 540 tac att gac ccc ttg
ccg gac ctc atc cac ccc agg acg ggc cgc ctc 1680Tyr Ile Asp Pro Leu
Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550
555 560 cac acc cgc ttc aac cag acg
gcc acg gcc acg ggc agg cta agt agc 1728His Thr Arg Phe Asn Gln Thr
Ala Thr Ala Thr Gly Arg Leu Ser Ser 565
570 575 tcc gat ccc aac ctc cag aac atc ccc
gtc cgc acc ccg ctt ggg cag 1776Ser Asp Pro Asn Leu Gln Asn Ile Pro
Val Arg Thr Pro Leu Gly Gln 580 585
590 agg atc cgc cgg gcc ttc atc gcc gag gag ggg
tgg cta ttg gtg gcc 1824Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly
Trp Leu Leu Val Ala 595 600
605 ctg gac tat agc cag ata gag ctc agg gtg ctg gcc cac
ctc tcc ggc 1872Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His
Leu Ser Gly 610 615 620
gac gag aac ctg atc cgg gtc ttc cag gag ggg cgg gac atc cac
acg 1920Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His
Thr 625 630 635 640
gag acc gcc agc tgg atg ttc ggc gtc ccc cgg gag gcc gtg gac ccc
1968Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655 ctg
atg cgc cgg gcg gcc aag acc atc aac ttc ggg gtc ctc tac ggc 2016Leu
Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670 atg tcg gcc
cac cgc ctc tcc cag gag cta gcc atc cct tac gag gag 2064Met Ser Ala
His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675
680 685 gcc cag gcc ttc att
gag cgc tac ttt cag agc ttc ccc aag gtg cgg 2112Ala Gln Ala Phe Ile
Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690
695 700 gcc tgg att gag aag acc ctg
gag gag ggc agg agg cgg ggg tac gtg 2160Ala Trp Ile Glu Lys Thr Leu
Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710
715 720 gag acc ctc ttc ggc cgc cgc cgc tac
gtg cca gac cta gag gcc cgg 2208Glu Thr Leu Phe Gly Arg Arg Arg Tyr
Val Pro Asp Leu Glu Ala Arg 725 730
735 gtg aag agc gtg cgg gag gcg gcc gag cgc atg gcc
ttc aac atg ccc 2256Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala
Phe Asn Met Pro 740 745
750 gtc cag ggc acc gcc gcc gac ctc atg aag ctg gct atg gtg
aag ctc 2304Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val
Lys Leu 755 760 765
ttc ccc agg ctg gag gaa atg ggg gcc agg atg ctc ctt cag gtc cac
2352Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780 gac
gag ctg gtc ctc gag gcc cca aaa gag agg gcg gag gcc gtg gcc 2400Asp
Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785
790 795 800 cgg ctg gcc
aag gag gtc atg gag ggg gtg tat ccc ctg gcc gtg ccc 2448Arg Leu Ala
Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815 ctg gag gtg gag gtg
ggg ata ggg gag gac tgg ctc tcc gcc aag gag 2496Leu Glu Val Glu Val
Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820
825 830 tga
249916832PRTArtificial
sequenceSynthetic Construct 16Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys
Gly Arg Val Leu Leu 1 5 10
15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30 Leu Thr
Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35
40 45 Lys Ser Leu Leu Lys Ala Leu
Lys Glu Asp Gly Asp Ala Val Ile Val 50 55
60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu
Ala Tyr Gly Gly 65 70 75
80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95 Ala Leu Ile
Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100
105 110 Val Pro Gly Tyr Ala Ala Ala Asp
Val Leu Ala Ser Leu Ala Lys Lys 115 120
125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala
Ala Lys Ala 130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145
150 155 160 Tyr Leu Ile Thr
Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165
170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu
Thr Gly Asp Glu Ser Asp Asn 180 185
190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys
Leu Leu 195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210
215 220 Lys Pro Ala Ile Arg
Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230
235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr
Asp Leu Pro Leu Glu Val 245 250
255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala
Phe 260 265 270 Leu
Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275
280 285 Glu Ser Pro Lys Ala Leu
Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295
300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu
Pro Met Trp Ala Asp 305 310 315
320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335 Glu Pro
Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340
345 350 Ala Lys Asp Leu Ser Val Leu
Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360
365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu
Asp Pro Ser Asn 370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385
390 395 400 Glu Ala Gly
Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405
410 415 Trp Gly Arg Leu Glu Gly Glu Glu
Arg Leu Leu Trp Leu Tyr Arg Glu 420 425
430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu
Ala Thr Gly 435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450
455 460 Glu Glu Ile Ala
Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470
475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln
Leu Glu Arg Val Leu Phe Asp 485 490
495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly
Lys Arg 500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525 Val Glu Lys Ile
Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530
535 540 Tyr Ile Asp Pro Leu Pro Asp Leu
Ile His Pro Arg Thr Gly Arg Leu 545 550
555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly
Arg Leu Ser Ser 565 570
575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590 Arg Ile Arg
Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595
600 605 Leu Asp Tyr Ser Gln Ile Glu Leu
Arg Val Leu Ala His Leu Ser Gly 610 615
620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp
Ile His Thr 625 630 635
640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655 Leu Met Arg Arg
Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660
665 670 Met Ser Ala His Arg Leu Ser Gln Glu
Leu Ala Ile Pro Tyr Glu Glu 675 680
685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys
Val Arg 690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705
710 715 720 Glu Thr Leu Phe Gly
Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725
730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg
Met Ala Phe Asn Met Pro 740 745
750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys
Leu 755 760 765 Phe
Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770
775 780 Asp Glu Leu Val Leu Glu
Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790
795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr
Pro Leu Ala Val Pro 805 810
815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
172499DNAArtificial SequenceTaq Exo- + E742A 17atg agg ggg atg ctg ccc
ctc ttt gag ccc aag ggc cgg gtc ctc ctg 48Met Arg Gly Met Leu Pro
Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 gtg gac ggc cac cac ctg gcc tac
cgc acc ttc cac gcc ctg aag ggc 96Val Asp Gly His His Leu Ala Tyr
Arg Thr Phe His Ala Leu Lys Gly 20 25
30 ctc acc acc agc cgg ggg gag ccg gtg cag
gcg gtc tac ggc ttc gcc 144Leu Thr Thr Ser Arg Gly Glu Pro Val Gln
Ala Val Tyr Gly Phe Ala 35 40
45 aag agc ctc ctc aag gcc ctc aag gag gac ggg gac
gcg gtg atc gtg 192Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp
Ala Val Ile Val 50 55 60
gtc ttt gac gcc aag gcc ccc tcc ttc cgc cac gag gcc tac
ggg ggg 240Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr
Gly Gly 65 70 75
80 tac aag gcg ggc cgg gcc ccc acg ccg gag gac ttt ccc cgg caa ctc
288Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
gcc ctc atc aag gag ctg gtg gac ctc ctg ggg ctg gcg cgc ctc gag
336Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110 gtc
ccg ggc tac gcg gcg gcc gac gtc ctg gcc agc ctg gcc aag aag 384Val
Pro Gly Tyr Ala Ala Ala Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125 gcg gaa aag
gag ggc tac gag gtc cgc atc ctc acc gcc gcc aaa gcc 432Ala Glu Lys
Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Ala Lys Ala 130
135 140 ctt tac cag ctc ctt
tcc gac cgc atc cac gtc ctc cac ccc gag ggg 480Leu Tyr Gln Leu Leu
Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150
155 160 tac ctc atc acc ccg gcc tgg
ctt tgg gaa aag tac ggc ctg agg ccc 528Tyr Leu Ile Thr Pro Ala Trp
Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165
170 175 gac cag tgg gcc gac tac cgg gcc ctg
acc ggg gac gag tcc gac aac 576Asp Gln Trp Ala Asp Tyr Arg Ala Leu
Thr Gly Asp Glu Ser Asp Asn 180 185
190 ctt ccc ggg gtc aag ggc atc ggg gag aag acg
gcg agg aag ctt ctg 624Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr
Ala Arg Lys Leu Leu 195 200
205 gag gag tgg ggg agc ctg gaa gcc ctc ctc aag aac ctg
gac cgg ctg 672Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu
Asp Arg Leu 210 215 220
aag ccc gcc atc cgg gag aag atc ctg gcc cac atg gac gat ctg
aag 720Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu
Lys 225 230 235 240
ctc tcc tgg gac ctg gcc aag gtg cgc acc gac ctg ccc ctg gag gtg
768Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255 gac
ttc gcc aaa agg cgg gag ccc gac cgg gag agg ctt agg gcc ttt 816Asp
Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270 ctg gag agg
ctt gag ttt ggc agc ctc ctc cac gag ttc ggc ctt ctg 864Leu Glu Arg
Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275
280 285 gaa agc ccc aag gcc
ctg gag gag gcc ccc tgg ccc ccg ccg gaa ggg 912Glu Ser Pro Lys Ala
Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290
295 300 gcc ttc gtg ggc ttt gtg ctt
tcc cgc aag gag ccc atg tgg gcc gat 960Ala Phe Val Gly Phe Val Leu
Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310
315 320 ctt ctg gcc ctg gcc gcc gcc agg ggg
ggc cgg gtc cac cgg gcc ccc 1008Leu Leu Ala Leu Ala Ala Ala Arg Gly
Gly Arg Val His Arg Ala Pro 325 330
335 gag cct tat aaa gcc ctc agg gac ctg aag gag
gcg cgg ggg ctt ctc 1056Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu
Ala Arg Gly Leu Leu 340 345
350 gcc aaa gac ctg agc gtt ctg gcc ctg agg gaa ggc ctt
ggc ctc ccg 1104Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu
Gly Leu Pro 355 360 365
ccc ggc gac gac ccc atg ctc ctc gcc tac ctc ctg gac cct tcc
aac 1152Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser
Asn 370 375 380
acc acc ccc gag ggg gtg gcc cgg cgc tac ggc ggg gag tgg acg gag
1200Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400 gag
gcg ggg gag cgg gcc gcc ctt tcc gag agg ctc ttc gcc aac ctg 1248Glu
Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415 tgg ggg agg
ctt gag ggg gag gag agg ctc ctt tgg ctt tac cgg gag 1296Trp Gly Arg
Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420
425 430 gtg gag agg ccc ctt
tcc gct gtc ctg gcc cac atg gag gcc acg ggg 1344Val Glu Arg Pro Leu
Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435
440 445 gtg cgc ctg gac gtg gcc tat
ctc agg gcc ttg tcc ctg gag gtg gcc 1392Val Arg Leu Asp Val Ala Tyr
Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455
460 gag gag atc gcc cgc ctc gag gcc gag
gtc ttc cgc ctg gcc ggc cac 1440Glu Glu Ile Ala Arg Leu Glu Ala Glu
Val Phe Arg Leu Ala Gly His 465 470
475 480 ccc ttc aac ctc aac tcc cgg gac cag ctg gaa
agg gtc ctc ttt gac 1488Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu
Arg Val Leu Phe Asp 485 490
495 gag cta ggg ctt ccc gcc atc ggc aag acg gag aag acc
ggc aag cgc 1536Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr
Gly Lys Arg 500 505 510
tcc acc agc gcc gcc gtc ctg gag gcc ctc cgc gag gcc cac ccc
atc 1584Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro
Ile 515 520 525
gtg gag aag atc ctg cag tac cgg gag ctc acc aag ctg aag agc acc
1632Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540 tac
att gac ccc ttg ccg gac ctc atc cac ccc agg acg ggc cgc ctc 1680Tyr
Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545
550 555 560 cac acc cgc
ttc aac cag acg gcc acg gcc acg ggc agg cta agt agc 1728His Thr Arg
Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575 tcc gat ccc aac ctc
cag aac atc ccc gtc cgc acc ccg ctt ggg cag 1776Ser Asp Pro Asn Leu
Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580
585 590 agg atc cgc cgg gcc ttc atc
gcc gag gag ggg tgg cta ttg gtg gcc 1824Arg Ile Arg Arg Ala Phe Ile
Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600
605 ctg gac tat agc cag ata gag ctc agg
gtg ctg gcc cac ctc tcc ggc 1872Leu Asp Tyr Ser Gln Ile Glu Leu Arg
Val Leu Ala His Leu Ser Gly 610 615
620 gac gag aac ctg atc cgg gtc ttc cag gag ggg
cgg gac atc cac acg 1920Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly
Arg Asp Ile His Thr 625 630 635
640 gag acc gcc agc tgg atg ttc ggc gtc ccc cgg gag gcc
gtg gac ccc 1968Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala
Val Asp Pro 645 650
655 ctg atg cgc cgg gcg gcc aag acc atc aac ttc ggg gtc ctc tac
ggc 2016Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr
Gly 660 665 670
atg tcg gcc cac cgc ctc tcc cag gag cta gcc atc cct tac gag gag
2064Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685 gcc
cag gcc ttc att gag cgc tac ttt cag agc ttc ccc aag gtg cgg 2112Ala
Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690
695 700 gcc tgg att
gag aag acc ctg gag gag ggc agg agg cgg ggg tac gtg 2160Ala Trp Ile
Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705
710 715 720 gag acc ctc ttc ggc
cgc cgc cgc tac gtg cca gac cta gag gcc cgg 2208Glu Thr Leu Phe Gly
Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725
730 735 gtg aag agc gtg cgg gcg gcg
gcc gag cgc atg gcc ttc aac atg ccc 2256Val Lys Ser Val Arg Ala Ala
Ala Glu Arg Met Ala Phe Asn Met Pro 740
745 750 gtc cag ggc acc gcc gcc gac ctc atg
aag ctg gct atg gtg aag ctc 2304Val Gln Gly Thr Ala Ala Asp Leu Met
Lys Leu Ala Met Val Lys Leu 755 760
765 ttc ccc agg ctg gag gaa atg ggg gcc agg atg
ctc ctt cag gtc cac 2352Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met
Leu Leu Gln Val His 770 775 780
gac gag ctg gtc ctc gag gcc cca aaa gag agg gcg gag
gcc gtg gcc 2400Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu
Ala Val Ala 785 790 795
800 cgg ctg gcc aag gag gtc atg gag ggg gtg tat ccc ctg gcc gtg
ccc 2448Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val
Pro 805 810 815
ctg gag gtg gag gtg ggg ata ggg gag gac tgg ctc tcc gcc aag gag
2496Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830 tga
249918832PRTArtificial SequenceSynthetic Construct 18Met Arg Gly Met Leu
Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr
Phe His Ala Leu Lys Gly 20 25
30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala 35 40 45 Lys
Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50
55 60 Val Phe Asp Ala Lys Ala
Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70
75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp
Phe Pro Arg Gln Leu 85 90
95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110 Val Pro
Gly Tyr Ala Ala Ala Asp Val Leu Ala Ser Leu Ala Lys Lys 115
120 125 Ala Glu Lys Glu Gly Tyr Glu
Val Arg Ile Leu Thr Ala Ala Lys Ala 130 135
140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu
His Pro Glu Gly 145 150 155
160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175 Asp Gln Trp
Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180
185 190 Leu Pro Gly Val Lys Gly Ile Gly
Glu Lys Thr Ala Arg Lys Leu Leu 195 200
205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu
Asp Arg Leu 210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225
230 235 240 Leu Ser Trp Asp
Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245
250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp
Arg Glu Arg Leu Arg Ala Phe 260 265
270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
Leu Leu 275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290
295 300 Ala Phe Val Gly Phe
Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310
315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly
Arg Val His Arg Ala Pro 325 330
335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu
Leu 340 345 350 Ala
Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355
360 365 Pro Gly Asp Asp Pro Met
Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375
380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly
Gly Glu Trp Thr Glu 385 390 395
400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415 Trp Gly
Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420
425 430 Val Glu Arg Pro Leu Ser Ala
Val Leu Ala His Met Glu Ala Thr Gly 435 440
445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser
Leu Glu Val Ala 450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465
470 475 480 Pro Phe Asn
Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485
490 495 Glu Leu Gly Leu Pro Ala Ile Gly
Lys Thr Glu Lys Thr Gly Lys Arg 500 505
510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala
His Pro Ile 515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530
535 540 Tyr Ile Asp Pro
Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550
555 560 His Thr Arg Phe Asn Gln Thr Ala Thr
Ala Thr Gly Arg Leu Ser Ser 565 570
575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
Gly Gln 580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605 Leu Asp Tyr Ser
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610
615 620 Asp Glu Asn Leu Ile Arg Val Phe
Gln Glu Gly Arg Asp Ile His Thr 625 630
635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu
Ala Val Asp Pro 645 650
655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670 Met Ser Ala
His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675
680 685 Ala Gln Ala Phe Ile Glu Arg Tyr
Phe Gln Ser Phe Pro Lys Val Arg 690 695
700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg
Gly Tyr Val 705 710 715
720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735 Val Lys Ser Val
Arg Ala Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740
745 750 Val Gln Gly Thr Ala Ala Asp Leu Met
Lys Leu Ala Met Val Lys Leu 755 760
765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln
Val His 770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785
790 795 800 Arg Leu Ala Lys Glu
Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805
810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp
Trp Leu Ser Ala Lys Glu 820 825
830 192499DNAArtificial SequenceTaq R651E 19atg agg ggg atg ctg
ccc ctc ttt gag ccc aag ggc cgg gtc ctc ctg 48Met Arg Gly Met Leu
Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 gtg gac ggc cac cac ctg gcc
tac cgc acc ttc cac gcc ctg aag ggc 96Val Asp Gly His His Leu Ala
Tyr Arg Thr Phe His Ala Leu Lys Gly 20
25 30 ctc acc acc agc cgg ggg gag ccg gtg
cag gcg gtc tac ggc ttc gcc 144Leu Thr Thr Ser Arg Gly Glu Pro Val
Gln Ala Val Tyr Gly Phe Ala 35 40
45 aag agc ctc ctc aag gcc ctc aag gag gac ggg
gac gcg gtg atc gtg 192Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly
Asp Ala Val Ile Val 50 55 60
gtc ttt gac gcc aag gcc ccc tcc ttc cgc cac gag gcc
tac ggg ggg 240Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala
Tyr Gly Gly 65 70 75
80 tac aag gcg ggc cgg gcc ccc acg ccg gag gac ttt ccc cgg caa
ctc 288Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln
Leu 85 90 95
gcc ctc atc aag gag ctg gtg gac ctc ctg ggg ctg gcg cgc ctc gag
336Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110 gtc
ccg ggc tac gag gcg gac gac gtc ctg gcc agc ctg gcc aag aag 384Val
Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125 gcg gaa aag
gag ggc tac gag gtc cgc atc ctc acc gcc gac aaa gac 432Ala Glu Lys
Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130
135 140 ctt tac cag ctc ctt
tcc gac cgc atc cac gtc ctc cac ccc gag ggg 480Leu Tyr Gln Leu Leu
Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150
155 160 tac ctc atc acc ccg gcc tgg
ctt tgg gaa aag tac ggc ctg agg ccc 528Tyr Leu Ile Thr Pro Ala Trp
Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165
170 175 gac cag tgg gcc gac tac cgg gcc ctg
acc ggg gac gag tcc gac aac 576Asp Gln Trp Ala Asp Tyr Arg Ala Leu
Thr Gly Asp Glu Ser Asp Asn 180 185
190 ctt ccc ggg gtc aag ggc atc ggg gag aag acg
gcg agg aag ctt ctg 624Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr
Ala Arg Lys Leu Leu 195 200
205 gag gag tgg ggg agc ctg gaa gcc ctc ctc aag aac ctg
gac cgg ctg 672Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu
Asp Arg Leu 210 215 220
aag ccc gcc atc cgg gag aag atc ctg gcc cac atg gac gat ctg
aag 720Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu
Lys 225 230 235 240
ctc tcc tgg gac ctg gcc aag gtg cgc acc gac ctg ccc ctg gag gtg
768Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255 gac
ttc gcc aaa agg cgg gag ccc gac cgg gag agg ctt agg gcc ttt 816Asp
Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270 ctg gag agg
ctt gag ttt ggc agc ctc ctc cac gag ttc ggc ctt ctg 864Leu Glu Arg
Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275
280 285 gaa agc ccc aag gcc
ctg gag gag gcc ccc tgg ccc ccg ccg gaa ggg 912Glu Ser Pro Lys Ala
Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290
295 300 gcc ttc gtg ggc ttt gtg ctt
tcc cgc aag gag ccc atg tgg gcc gat 960Ala Phe Val Gly Phe Val Leu
Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310
315 320 ctt ctg gcc ctg gcc gcc gcc agg ggg
ggc cgg gtc cac cgg gcc ccc 1008Leu Leu Ala Leu Ala Ala Ala Arg Gly
Gly Arg Val His Arg Ala Pro 325 330
335 gag cct tat aaa gcc ctc agg gac ctg aag gag
gcg cgg ggg ctt ctc 1056Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu
Ala Arg Gly Leu Leu 340 345
350 gcc aaa gac ctg agc gtt ctg gcc ctg agg gaa ggc ctt
ggc ctc ccg 1104Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu
Gly Leu Pro 355 360 365
ccc ggc gac gac ccc atg ctc ctc gcc tac ctc ctg gac cct tcc
aac 1152Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser
Asn 370 375 380
acc acc ccc gag ggg gtg gcc cgg cgc tac ggc ggg gag tgg acg gag
1200Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400 gag
gcg ggg gag cgg gcc gcc ctt tcc gag agg ctc ttc gcc aac ctg 1248Glu
Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415 tgg ggg agg
ctt gag ggg gag gag agg ctc ctt tgg ctt tac cgg gag 1296Trp Gly Arg
Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420
425 430 gtg gag agg ccc ctt
tcc gct gtc ctg gcc cac atg gag gcc acg ggg 1344Val Glu Arg Pro Leu
Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435
440 445 gtg cgc ctg gac gtg gcc tat
ctc agg gcc ttg tcc ctg gag gtg gcc 1392Val Arg Leu Asp Val Ala Tyr
Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455
460 gag gag atc gcc cgc ctc gag gcc gag
gtc ttc cgc ctg gcc ggc cac 1440Glu Glu Ile Ala Arg Leu Glu Ala Glu
Val Phe Arg Leu Ala Gly His 465 470
475 480 ccc ttc aac ctc aac tcc cgg gac cag ctg gaa
agg gtc ctc ttt gac 1488Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu
Arg Val Leu Phe Asp 485 490
495 gag cta ggg ctt ccc gcc atc ggc aag acg gag aag acc
ggc aag cgc 1536Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr
Gly Lys Arg 500 505 510
tcc acc agc gcc gcc gtc ctg gag gcc ctc cgc gag gcc cac ccc
atc 1584Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro
Ile 515 520 525
gtg gag aag atc ctg cag tac cgg gag ctc acc aag ctg aag agc acc
1632Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540 tac
att gac ccc ttg ccg gac ctc atc cac ccc agg acg ggc cgc ctc 1680Tyr
Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545
550 555 560 cac acc cgc
ttc aac cag acg gcc acg gcc acg ggc agg cta agt agc 1728His Thr Arg
Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575 tcc gat ccc aac ctc
cag aac atc ccc gtc cgc acc ccg ctt ggg cag 1776Ser Asp Pro Asn Leu
Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580
585 590 agg atc cgc cgg gcc ttc atc
gcc gag gag ggg tgg cta ttg gtg gcc 1824Arg Ile Arg Arg Ala Phe Ile
Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600
605 ctg gac tat agc cag ata gag ctc agg
gtg ctg gcc cac ctc tcc ggc 1872Leu Asp Tyr Ser Gln Ile Glu Leu Arg
Val Leu Ala His Leu Ser Gly 610 615
620 gac gag aac ctg atc cgg gtc ttc cag gag ggg
cgg gac atc cac acg 1920Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly
Arg Asp Ile His Thr 625 630 635
640 gag acc gcc agc tgg atg ttc ggc gtc ccc gag gag gcc
gtg gac ccc 1968Glu Thr Ala Ser Trp Met Phe Gly Val Pro Glu Glu Ala
Val Asp Pro 645 650
655 ctg atg cgc cgg gcg gcc aag acc atc aac ttc ggg gtc ctc tac
ggc 2016Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr
Gly 660 665 670
atg tcg gcc cac cgc ctc tcc cag gag cta gcc atc cct tac gag gag
2064Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685 gcc
cag gcc ttc att gag cgc tac ttt cag agc ttc ccc aag gtg cgg 2112Ala
Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690
695 700 gcc tgg att
gag aag acc ctg gag gag ggc agg agg cgg ggg tac gtg 2160Ala Trp Ile
Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705
710 715 720 gag acc ctc ttc ggc
cgc cgc cgc tac gtg cca gac cta gag gcc cgg 2208Glu Thr Leu Phe Gly
Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725
730 735 gtg aag agc gtg cgg gag gcg
gcc gag cgc atg gcc ttc aac atg ccc 2256Val Lys Ser Val Arg Glu Ala
Ala Glu Arg Met Ala Phe Asn Met Pro 740
745 750 gtc cag ggc acc gcc gcc gac ctc atg
aag ctg gct atg gtg aag ctc 2304Val Gln Gly Thr Ala Ala Asp Leu Met
Lys Leu Ala Met Val Lys Leu 755 760
765 ttc ccc agg ctg gag gaa atg ggg gcc agg atg
ctc ctt cag gtc cac 2352Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met
Leu Leu Gln Val His 770 775 780
gac gag ctg gtc ctc gag gcc cca aaa gag agg gcg gag
gcc gtg gcc 2400Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu
Ala Val Ala 785 790 795
800 cgg ctg gcc aag gag gtc atg gag ggg gtg tat ccc ctg gcc gtg
ccc 2448Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val
Pro 805 810 815
ctg gag gtg gag gtg ggg ata ggg gag gac tgg ctc tcc gcc aag gag
2496Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830 tga
249920832PRTArtificial SequenceSynthetic Construct 20Met Arg Gly Met Leu
Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr
Phe His Ala Leu Lys Gly 20 25
30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala 35 40 45 Lys
Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50
55 60 Val Phe Asp Ala Lys Ala
Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70
75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp
Phe Pro Arg Gln Leu 85 90
95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110 Val Pro
Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115
120 125 Ala Glu Lys Glu Gly Tyr Glu
Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135
140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu
His Pro Glu Gly 145 150 155
160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175 Asp Gln Trp
Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180
185 190 Leu Pro Gly Val Lys Gly Ile Gly
Glu Lys Thr Ala Arg Lys Leu Leu 195 200
205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu
Asp Arg Leu 210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225
230 235 240 Leu Ser Trp Asp
Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245
250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp
Arg Glu Arg Leu Arg Ala Phe 260 265
270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
Leu Leu 275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290
295 300 Ala Phe Val Gly Phe
Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310
315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly
Arg Val His Arg Ala Pro 325 330
335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu
Leu 340 345 350 Ala
Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355
360 365 Pro Gly Asp Asp Pro Met
Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375
380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly
Gly Glu Trp Thr Glu 385 390 395
400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415 Trp Gly
Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420
425 430 Val Glu Arg Pro Leu Ser Ala
Val Leu Ala His Met Glu Ala Thr Gly 435 440
445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser
Leu Glu Val Ala 450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465
470 475 480 Pro Phe Asn
Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485
490 495 Glu Leu Gly Leu Pro Ala Ile Gly
Lys Thr Glu Lys Thr Gly Lys Arg 500 505
510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala
His Pro Ile 515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530
535 540 Tyr Ile Asp Pro
Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550
555 560 His Thr Arg Phe Asn Gln Thr Ala Thr
Ala Thr Gly Arg Leu Ser Ser 565 570
575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
Gly Gln 580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605 Leu Asp Tyr Ser
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610
615 620 Asp Glu Asn Leu Ile Arg Val Phe
Gln Glu Gly Arg Asp Ile His Thr 625 630
635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Glu Glu
Ala Val Asp Pro 645 650
655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670 Met Ser Ala
His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675
680 685 Ala Gln Ala Phe Ile Glu Arg Tyr
Phe Gln Ser Phe Pro Lys Val Arg 690 695
700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg
Gly Tyr Val 705 710 715
720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735 Val Lys Ser Val
Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740
745 750 Val Gln Gly Thr Ala Ala Asp Leu Met
Lys Leu Ala Met Val Lys Leu 755 760
765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln
Val His 770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785
790 795 800 Arg Leu Ala Lys Glu
Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805
810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp
Trp Leu Ser Ala Lys Glu 820 825
830 212505DNAArtificial SequenceTth P653E 21atg gag gcg atg ctt
ccg ctc ttt gaa ccc aaa ggc cgg gtc ctc ctg 48Met Glu Ala Met Leu
Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 gtg gac ggc cac cac ctg gcc
tac cgc acc ttc ttc gcc ctg aag ggc 96Val Asp Gly His His Leu Ala
Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20
25 30 ctc acc acg agc cgg ggc gaa ccg gtg
cag gcg gtc tac ggc ttc gcc 144Leu Thr Thr Ser Arg Gly Glu Pro Val
Gln Ala Val Tyr Gly Phe Ala 35 40
45 aag agc ctc ctc aag gcc ctg aag gag gac
ggg tac aag gcc gtc ttc 192Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp
Gly Tyr Lys Ala Val Phe 50 55
60 gtg gtc ttt gac gcc aag gcc ccc tcc ttc cgc
cac gag gcc tac gag 240Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg
His Glu Ala Tyr Glu 65 70 75
80 gcc tac aag gcg ggg agg gcc ccg acc ccc gag gac ttc
ccc cgg cag 288Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe
Pro Arg Gln 85 90
95 ctc gcc ctc atc aag gag ctg gtg gac ctc ctg ggg ttt acc cgc
ctc 336Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg
Leu 100 105 110
gag gtc ccc ggc tac gag gcg gac gac gtt ctc gcc acc ctg gcc aag
384Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys
115 120 125 aag
gcg gaa aag gag ggg tac gag gtg cgc atc ctc acc gcc gac cgc 432Lys
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg 130
135 140 gac ctc tac
caa ctc gtc tcc gac cgc gtc gcc gtc ctc cac ccc gag 480Asp Leu Tyr
Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145
150 155 160 ggc cac ctc atc acc
ccg gag tgg ctt tgg gag aag tac ggc ctc agg 528Gly His Leu Ile Thr
Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165
170 175 ccg gag cag tgg gtg gac ttc
cgc gcc ctc gtg ggg gac ccc tcc gac 576Pro Glu Gln Trp Val Asp Phe
Arg Ala Leu Val Gly Asp Pro Ser Asp 180
185 190 aac ctc ccc ggg gtc aag ggc atc ggg
gag aag acc gcc ctc aag ctc 624Asn Leu Pro Gly Val Lys Gly Ile Gly
Glu Lys Thr Ala Leu Lys Leu 195 200
205 ctc aag gag tgg gga agc ctg gaa aac ctc ctc
aag aac ctg gac cgg 672Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu
Lys Asn Leu Asp Arg 210 215 220
gta aag cca gaa aac gtc cgg gag aag atc aag gcc cac
ctg gaa gac 720Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His
Leu Glu Asp 225 230 235
240 ctc agg ctc tcc ttg gag ctc tcc cgg gtg cgc acc gac ctc ccc
ctg 768Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro
Leu 245 250 255
gag gtg gac ctc gcc cag ggg cgg gag ccc gac cgg gag ggg ctt agg
816Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg
260 265 270 gcc
ttc ctg gag agg ctg gag ttc ggc agc ctc ctc cac gag ttc ggc 864Ala
Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285 ctc ctg
gag gcc ccc gcc ccc ctg gag gag gcc ccc tgg ccc ccg ccg 912Leu Leu
Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290
295 300 gaa ggg gcc ttc
gtg ggc ttc gtc ctc tcc cgc ccc gag ccc atg tgg 960Glu Gly Ala Phe
Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305
310 315 320 gcg gag ctt aaa gcc ctg
gcc gcc tgc agg gac ggc cgg gtg cac cgg 1008Ala Glu Leu Lys Ala Leu
Ala Ala Cys Arg Asp Gly Arg Val His Arg 325
330 335 gca gca gac ccc ttg gcg ggg cta
aag gac ctc aag gag gtc cgg ggc 1056Ala Ala Asp Pro Leu Ala Gly Leu
Lys Asp Leu Lys Glu Val Arg Gly 340 345
350 ctc ctc gcc aag gac ctc gcc gtc ttg gcc
tcg agg gag ggg cta gac 1104Leu Leu Ala Lys Asp Leu Ala Val Leu Ala
Ser Arg Glu Gly Leu Asp 355 360
365 ctc gtg ccc ggg gac gac ccc atg ctc ctc gcc tac
ctc ctg gac ccc 1152Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr
Leu Leu Asp Pro 370 375 380
tcc aac acc acc ccc gag ggg gtg gcg cgg cgc tac ggg ggg
gag tgg 1200Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly
Glu Trp 385 390 395
400 acg gag gac gcc gcc cac cgg gcc ctc ctc tcg gag agg ctc cat cgg
1248Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg
405 410 415 aac
ctc ctt aag cgc ctc gag ggg gag gag aag ctc ctt tgg ctc tac 1296Asn
Leu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr
420 425 430 cac gag gtg
gaa aag ccc ctc tcc cgg gtc ctg gcc cac atg gag gcc 1344His Glu Val
Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435
440 445 acc ggg gta cgg cgg
gac gtg gcc tac ctt cag gcc ctt tcc ctg gag 1392Thr Gly Val Arg Arg
Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu 450
455 460 ctt gcg gag gag atc cgc cgc
ctc gag gag gag gtc ttc cgc ttg gcg 1440Leu Ala Glu Glu Ile Arg Arg
Leu Glu Glu Glu Val Phe Arg Leu Ala 465 470
475 480 ggc cac ccc ttc aac ctc aac tcc cgg
gac cag ctg gaa agg gtg ctc 1488Gly His Pro Phe Asn Leu Asn Ser Arg
Asp Gln Leu Glu Arg Val Leu 485 490
495 ttt gac gag ctt agg ctt ccc gcc ttg ggg aag
acg caa aag aca ggc 1536Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys
Thr Gln Lys Thr Gly 500 505
510 aag cgc tcc acc agc gcc gcg gtg ctg gag gcc cta cgg
gag gcc cac 1584Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg
Glu Ala His 515 520 525
ccc atc gtg gag aag atc ctc cag cac cgg gag ctc acc aag ctc
aag 1632Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu
Lys 530 535 540
aac acc tac gtg gac ccc ctc cca agc ctc gtc cac ccg agg acg ggc
1680Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly
545 550 555 560 cgc
ctc cac acc cgc ttc aac cag acg gcc acg gcc acg ggg agg ctt 1728Arg
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu
565 570 575 agt agc tcc gac
ccc aac ctg cag aac atc ccc gtc cgc acc ccc ttg 1776Ser Ser Ser Asp
Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu 580
585 590 ggc cag agg atc cgc cgg
gcc ttc gtg gcc gag gcg ggt tgg gcg ttg 1824Gly Gln Arg Ile Arg Arg
Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595
600 605 gtg gcc ctg gac tat agc cag ata
gag ctc cgc gtc ctc gcc cac ctc 1872Val Ala Leu Asp Tyr Ser Gln Ile
Glu Leu Arg Val Leu Ala His Leu 610 615
620 tcc ggg gac gaa aac ctg atc agg gtc ttc
cag gag ggg aag gac atc 1920Ser Gly Asp Glu Asn Leu Ile Arg Val Phe
Gln Glu Gly Lys Asp Ile 625 630 635
640 cac acc cag acc gca agc tgg atg ttc ggc gtc ccc
gag gag gcc gtg 1968His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Pro
Glu Glu Ala Val 645 650
655 gac ccc ctg atg cgc cgg gcg gcc aag acg gtg aac ttc ggc
gtc ctc 2016Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly
Val Leu 660 665 670
tac ggc atg tcc gcc cat agg ctc tcc cag gag ctt gcc atc ccc tac
2064Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr
675 680 685 gag
gag gcg gtg gcc ttt ata gag cgc tac ttc caa agc ttc ccc aag 2112Glu
Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys 690
695 700 gtg cgg
gcc tgg ata gaa aag acc ctg gag gag ggg agg aag cgg ggc 2160Val Arg
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly 705
710 715 720 tac gtg gaa acc
ctc ttc gga aga agg cgc tac gtg ccc gac ctc aac 2208Tyr Val Glu Thr
Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn 725
730 735 gcc cgg gtg aag agc gtc
agg gag gcc gcg gag cgc atg gcc ttc aac 2256Ala Arg Val Lys Ser Val
Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 740
745 750 atg ccc gtc cag ggc acc gcc gcc
gac ctc atg aag ctc gcc atg gtg 2304Met Pro Val Gln Gly Thr Ala Ala
Asp Leu Met Lys Leu Ala Met Val 755 760
765 aag ctc ttc ccc cgc ctc cgg gag atg ggg
gcc cgc atg ctc ctc cag 2352Lys Leu Phe Pro Arg Leu Arg Glu Met Gly
Ala Arg Met Leu Leu Gln 770 775
780 gtc cac gac gag ctc ctc ctg gag gcc ccc caa gcg
cgg gcc gag gag 2400Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala
Arg Ala Glu Glu 785 790 795
800 gtg gcg gct ttg gcc aag gag gcc atg gag aag gcc tat ccc
ctc gcc 2448Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro
Leu Ala 805 810 815
gtg ccc ctg gag gtg gag gtg ggg atg ggg gag gac tgg ctt tcc gcc
2496Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala
820 825 830 aag
ggt tag 2505Lys
Gly
22834PRTArtificial SequenceSynthetic Construct 22Met Glu Ala Met Leu Pro
Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5
10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe
Phe Ala Leu Lys Gly 20 25
30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala 35 40 45 Lys
Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe 50
55 60 Val Val Phe Asp Ala Lys
Ala Pro Ser Phe Arg His Glu Ala Tyr Glu 65 70
75 80 Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu
Asp Phe Pro Arg Gln 85 90
95 Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu
100 105 110 Glu Val
Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys 115
120 125 Lys Ala Glu Lys Glu Gly Tyr
Glu Val Arg Ile Leu Thr Ala Asp Arg 130 135
140 Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val
Leu His Pro Glu 145 150 155
160 Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg
165 170 175 Pro Glu Gln
Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180
185 190 Asn Leu Pro Gly Val Lys Gly Ile
Gly Glu Lys Thr Ala Leu Lys Leu 195 200
205 Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn
Leu Asp Arg 210 215 220
Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp 225
230 235 240 Leu Arg Leu Ser
Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu 245
250 255 Glu Val Asp Leu Ala Gln Gly Arg Glu
Pro Asp Arg Glu Gly Leu Arg 260 265
270 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu
Phe Gly 275 280 285
Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290
295 300 Glu Gly Ala Phe Val
Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305 310
315 320 Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg
Asp Gly Arg Val His Arg 325 330
335 Ala Ala Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg
Gly 340 345 350 Leu
Leu Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp 355
360 365 Leu Val Pro Gly Asp Asp
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375
380 Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg
Tyr Gly Gly Glu Trp 385 390 395
400 Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg
405 410 415 Asn Leu
Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr 420
425 430 His Glu Val Glu Lys Pro Leu
Ser Arg Val Leu Ala His Met Glu Ala 435 440
445 Thr Gly Val Arg Arg Asp Val Ala Tyr Leu Gln Ala
Leu Ser Leu Glu 450 455 460
Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465
470 475 480 Gly His Pro
Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu 485
490 495 Phe Asp Glu Leu Arg Leu Pro Ala
Leu Gly Lys Thr Gln Lys Thr Gly 500 505
510 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg
Glu Ala His 515 520 525
Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys 530
535 540 Asn Thr Tyr Val
Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly 545 550
555 560 Arg Leu His Thr Arg Phe Asn Gln Thr
Ala Thr Ala Thr Gly Arg Leu 565 570
575 Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr
Pro Leu 580 585 590
Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu
595 600 605 Val Ala Leu Asp
Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu 610
615 620 Ser Gly Asp Glu Asn Leu Ile Arg
Val Phe Gln Glu Gly Lys Asp Ile 625 630
635 640 His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Pro
Glu Glu Ala Val 645 650
655 Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu
660 665 670 Tyr Gly Met
Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr 675
680 685 Glu Glu Ala Val Ala Phe Ile Glu
Arg Tyr Phe Gln Ser Phe Pro Lys 690 695
700 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg
Lys Arg Gly 705 710 715
720 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn
725 730 735 Ala Arg Val Lys
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 740
745 750 Met Pro Val Gln Gly Thr Ala Ala Asp
Leu Met Lys Leu Ala Met Val 755 760
765 Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu
Leu Gln 770 775 780
Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu 785
790 795 800 Val Ala Ala Leu Ala
Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala 805
810 815 Val Pro Leu Glu Val Glu Val Gly Met Gly
Glu Asp Trp Leu Ser Ala 820 825
830 Lys Gly
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