CN113308451B - Engineered Cas effector proteins and methods of use thereof - Google Patents

Engineered Cas effector proteins and methods of use thereof Download PDF

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CN113308451B
CN113308451B CN202110591698.9A CN202110591698A CN113308451B CN 113308451 B CN113308451 B CN 113308451B CN 202110591698 A CN202110591698 A CN 202110591698A CN 113308451 B CN113308451 B CN 113308451B
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nuclease
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cas12b
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CN113308451A (en
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李伟
周琪
陈阳灿
胡艳萍
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Beijing Institute Of Stem Cell And Regenerative Medicine
Institute of Zoology of CAS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

Provided herein are methods for engineering an enzyme, such as a Cas nuclease, to increase its enzymatic activity. Engineered Cas effector proteins, including engineered Cas12b, cas12i, and Cas9 nucleases and derivatives thereof, and methods of use thereof are also provided.

Description

Engineered Cas effector proteins and methods of use thereof
The present application is a divisional application of a patent application with chinese application number 2020114143843, the invention name "engineered Cas effector protein and method of use" and application day of 2020, 12 months and 7 days.
Technical Field
The present application relates generally to the field of biotechnology. More specifically, the present application relates to methods and compositions of engineered Cas effector proteins with improved activity (e.g., gene editing activity).
Background
Genome editing is an important and useful technique in genome research and a variety of applications. There are a number of systems available for genome editing including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -Cas systems, transcription activator-like effector nuclease (TALEN) systems, and Zinc Finger Nuclease (ZFN) systems.
The CRISPR-Cas system is an efficient and cost-effective genome editing technology that can be widely used in a range of eukaryotic organisms from yeast, plants to zebra fish and humans (see reviews: van der Oost 2013,Science 339:768-770, and Charpentier and Doudna,2013, nature 495:50-51). CRISPR-Cas systems provide adaptive immunity in archaebacteria and bacteria by binding Cas effector proteins and CRISPR RNA (crrnas). CRISPR-Cas systems comprising two classes six (type I-VI) (class 1 and class 2) have been characterized so far based on the outstanding functional and evolutionary modularity of the system. In class 2 CRISPR-Cas systems, type II Cas9 systems and V-a/B/E/J Cas12a/Cas12B/Cas12E/Cas12J systems have been utilized for genome editing and offer broad prospects for biomedical research.
However, current CRISPR-Cas systems have a number of limitations, including limited gene editing efficiency. Thus, there is a need for improved methods and systems for efficient genome editing across multiple loci.
Summary of The Invention
The present invention relates to:
(1) An engineered Cas nuclease comprising one or more mutations in a flexible region that increase the flexibility of a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has increased activity compared to the reference Cas nuclease, the flexible region having a flexibility score S of the amino acid residue of highest flexibility when determined using the DynaMine program 2 Prediction Less than or equal to 0.8, wherein the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k, and CasX.
(2) The engineered Cas nuclease of (1), wherein the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility.
(3) The engineered Cas nuclease of (1), which is an engineered Cas12b nuclease, comprising one or more mutations that increase the flexibility of the flexible region corresponding to amino acid residues 835-839 in a reference Cas12b nuclease, wherein the amino acid residue numbering is based on SEQ ID NO:1, wherein the engineered Cas12b nuclease has increased activity compared to the reference Cas12b nuclease.
(4) The engineered Cas nuclease of (1), which is an engineered Cas12i nuclease, comprising one or more mutations that increase flexibility of a flexible region in a reference Cas12i nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779, and amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8, wherein the engineered Cas12i nuclease has increased activity compared to the reference Cas12i nuclease.
(5) The engineered Cas nuclease of (1), which is an engineered Cas9 nuclease, the flexible region is selected from the group of regions corresponding to: amino acid residues 39-43, amino acid residues 135-139, amino acid residues 176-180, amino acid residues 274-278, amino acid residues 351-355, amino acid residues 389-393, amino acid residues 521-525, amino acid residues 541-545, amino acid residues 755-759, amino acid residues 774-778, amino acid residues 786-790, amino acid residues 811-815, amino acid residues 848-852, amino acid residues 855-859, amino acid residues 874-878, amino acid residues 891-895, amino acid residues 1019-1023, and amino acid residues 1036-1040, wherein the amino acid residue numbering is based on SEQ ID NO:25, wherein the engineered Cas9 nuclease has enhanced activity compared to the reference Cas9 nuclease.
(6) The engineered Cas nuclease of (1), which is an engineered Cas9 nuclease, the flexible region is selected from the group of regions corresponding to: amino acid residues 45-49, amino acid residues 84-88, amino acid residues 116-120, amino acid residues 128-132, amino acid residues 216-220, amino acid residues 318-322, amino acid residues 387-391, amino acid residues 497-501, amino acid residues 583-587, amino acid residues 594-598, amino acid residues 614-618, amino acid residues 696-700, and amino acid residues 739-743, wherein the amino acid residue numbering is based on SEQ ID NO:53, wherein the engineered Cas9 nuclease has increased activity compared to the reference Cas9 nuclease.
(7) The engineered Cas nuclease of (1), wherein the one or more mutations comprises insertion of one or more G residues in the flexible region.
(8) The engineered Cas nuclease of (1), wherein the one or more mutations comprises a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W).
(9) The engineered Cas nuclease of (1), wherein the activity is a site-specific nuclease activity.
(10) The engineered Cas nuclease of (9), wherein the gene editing efficiency of the engineered Cas nuclease at one or more genomic sites in a eukaryotic cell is at least about 20% greater than the reference Cas nuclease.
(11) The engineered Cas nuclease of (10), wherein the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
(12) The engineered Cas nuclease of (1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 14, 18, 20, 27, 28, 33, 34, 41, 54, 58-59.
(13) An engineered Cas effector protein comprising the engineered Cas nuclease of any one of (1) to (12).
(14) The engineered Cas effector protein of (13), wherein the effector protein is capable of inducing a double-strand break or single-strand break in a DNA molecule.
(15) The engineered Cas effector protein of (13), further comprising a functional domain fused to the engineered Cas nuclease.
(16) The engineered Cas effector protein of (15), wherein the functional domain is selected from the group consisting of: translation initiation domains, transcription repression domains, transactivation domains, epigenetic modification domains, nucleobase editing domains, reverse transcriptase domains, reporter domains and nuclease domains.
(17) The engineered Cas effector protein of (13), comprising: a first polypeptide comprising an N-terminal portion of the engineered Cas nuclease and a second polypeptide comprising a C-terminal portion of the engineered Cas nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complex with a target nucleic acid comprising a target sequence complementary to the guide sequence.
(18) An engineered CRISPR-Cas system comprising:
(a) The engineered Cas effector protein of any one of (13) to (17); and
(b) A guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA,
wherein the engineered Cas effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence.
(19) A method of detecting a target nucleic acid in a sample, comprising:
(a) Contacting the sample with the engineered CRISPR-Cas system of (18) and a tagged detection nucleic acid that is single stranded and that does not hybridize to a guide sequence of the guide RNA; and
(b) Measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas effector protein, thereby detecting the target nucleic acid.
(20) A method of modifying a target nucleic acid comprising a target sequence, comprising contacting the target nucleic acid with the engineered CRISPR-Cas system of (18).
(21) The method of (20), wherein the target nucleic acid is cleaved, a target sequence in the target nucleic acid is altered, or expression of the target nucleic acid is altered by the engineered CRISPR-Cas system.
(22) An engineered cell comprising the target nucleic acid modified using the method as described in (20) or (21).
(23) The use of the engineered CRISPR-Cas system of (18) in the manufacture of a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual.
To meet the above and other needs, the present disclosure provides methods for engineering enzymes (such as Cas nucleases) to improve their enzymatic activity, engineered Cas effector proteins, and methods of using the engineered Cas effector proteins.
In one aspect, the present application provides a method of engineering an enzyme comprising: (a) Obtaining a plurality of engineered enzymes, each engineered enzyme comprising one or more mutations that increase the flexibility of a flexible region of a plurality of flexible regions of a reference enzyme; and (b) selecting one or more engineered enzymes from the plurality of engineered enzymes, wherein the one or more engineered enzymes have increased activity compared to the reference enzyme. In some embodiments, the method further comprises determining a plurality of flexible regions in the reference enzyme.
In some embodiments according to any of the methods above, the plurality of flexible regions is determined based on the amino acid sequence of the reference enzyme. In some embodiments, the plurality of flexible regions is determined without reference to the three-dimensional structure of the reference enzyme or equivalent thereof. In some embodiments, the plurality of flexible regions is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. In some embodiments, the plurality of flexible regions is determined using DynaMine.
In some embodiments according to any of the above methods, the method comprises: (i) Calculating a flexibility score for each amino acid residue of the reference enzyme, wherein a higher flexibility score indicates a lower conformational flexibility; (ii) Select at X i A plurality of peak amino acid residues at positions, wherein each peak amino acid residue has a flexibility score below a predetermined threshold and below that at X i -5 to X i -1 and X i +1 and X i A flexibility score for the amino acid residue at position +5; and (iii) defining the plurality of flexible regions as amino acid residues X i -2 to X i +2。
In some embodiments according to any of the above methods, the plurality of flexible regions are located at random convolutions.
In some embodiments according to any of the methods above, the one or more mutations comprise insertion of one or more glycine (G) residues in the flexible region. In some embodiments, the one or more mutations comprise insertion of two G residues in the flexible region. In some embodiments according to any of the methods above, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P). In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P.
In some embodiments according to any of the methods above, the one or more mutations comprise replacing one or more non-G residues with one or more G residues. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W).
In some embodiments according to any of the above methods, the enzyme is a bacterial or archaeal enzyme. In some embodiments, the enzyme is a Cas nuclease. In some embodiments, the Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the plurality of flexible regions are in a domain of the reference Cas nuclease that interacts with DNA and/or RNA. In some embodiments, the activity described in step (b) is a site-specific nuclease activity. In some embodiments, the activity is a gene editing activity in a eukaryotic cell (e.g., a mammalian cell). In some embodiments, the activity is a gene editing activity in a human cell. In some embodiments, the gene editing efficiency of the selected engineered Cas nuclease in step (b) is at least about 20% (e.g., at least about 30%, 40%, 50%, 60%,70%, 80% or more) higher than the reference Cas nuclease at the genomic locus (genomic locus) in the cell. In some embodiments, the average gene editing efficiency of the engineered Cas nuclease selected in step (b) is at least about 20% (e.g., at least about 30%, 40%, 50%, 60%,70%, 80% or more) higher than the reference Cas nuclease at the plurality of genomic sites in the cell. In some embodiments, the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
Another aspect of the present application provides an engineered Cas nuclease obtained using any one of the above methods.
In some embodiments, the present application provides an engineered Cas nuclease comprising one or more mutations that areIncreased flexibility of a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has increased activity as compared to the reference Cas nuclease. In some embodiments, the flexible region is determined based on the amino acid sequence of the reference Cas nuclease. In some embodiments, the flexible region is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. In some embodiments, the flexible region is determined using DynaMine, and wherein the flexibility score S of the amino acid residue with the highest flexibility in the flexible region 2 Prediction Not greater than about 0.8.
In some embodiments of any of the engineered Cas nucleases according to the above, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments of any of the engineered Cas nucleases according to the above, the flexible region is located at a random coil.
In some embodiments of any of the above-described engineered Cas nucleases, the flexible region is in a domain of the reference Cas nuclease that interacts with DNA and/or RNA.
In some embodiments according to any of the engineered Cas nucleases above, the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX.
In some embodiments, the present application provides an engineered Cas12b nuclease comprising one or more mutations that increase flexibility of the flexible region corresponding to amino acid residues 835-839 in a reference Cas12b nuclease, wherein the amino acid residue numbering is based on SEQ ID NO:1, wherein the engineered Cas12b nuclease has increased activity compared to the reference Cas12b nuclease. In some embodiments, the engineered Cas12b nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID No. 2.
In some embodiments, the present application provides an engineered Cas12i nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas12i nuclease selected from the group of regions corresponding to: amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779, and amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8, wherein the engineered Cas12i nuclease has increased activity compared to the reference Cas12i nuclease. In some embodiments of any of the engineered Cas12i nucleases according to the above, the flexible region corresponds to amino acid residues 439-443 or amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8. In some embodiments, the engineered Cas12i nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:14, 18, and 20.
In some embodiments, the present application provides an engineered Cas9 nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas9 nuclease selected from the group of regions corresponding to: amino acid residues 39-43, amino acid residues 135-139, amino acid residues 176-180, amino acid residues 274-278, amino acid residues 351-355, amino acid residues 389-393, amino acid residues 521-525, amino acid residues 541-545, amino acid residues 755-759, amino acid residues 774-778, amino acid residues 786-790, amino acid residues 811-815, amino acid residues 848-852, amino acid residues 855-859, amino acid residues 874-878, amino acid residues 891-895, amino acid residues 1019-1023, and amino acid residues 1036-1040, wherein the amino acid residue numbering is based on SEQ ID NO:25, wherein the engineered Cas9 nuclease has enhanced activity compared to the reference Cas9 nuclease. In some embodiments according to any of the engineered Cas9 nucleases described above, the flexible region is selected from the group of regions corresponding to: amino acid residues 135-139, amino acid residues 176-180, amino acid residues 541-545, amino acid residues 755-759, and amino acid residues 811-815, wherein the amino acid residue numbering is based on SEQ ID NO. 25. In some embodiments of the engineered Cas9 nuclease according to any one of the above, the engineered nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 27, 28, 33, 34, and 41.
In some embodiments, the present application provides an engineered Cas9 nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas9 nuclease selected from the group of regions corresponding to: amino acid residues 45-49, amino acid residues 84-88, amino acid residues 116-120, amino acid residues 128-132, amino acid residues 216-220, amino acid residues 318-322, amino acid residues 387-391, amino acid residues 497-501, amino acid residues 583-587, amino acid residues 594-598, amino acid residues 614-618, amino acid residues 696-700, and amino acid residues 739-743, wherein the amino acid residue numbering is based on SEQ ID NO:53, wherein the engineered Cas9 nuclease has enhanced activity compared to the reference Cas9 nuclease. In some embodiments, the flexible region corresponds to amino acid residues 45-49 or amino acid residues 116-120, wherein the amino acid residue numbering is based on SEQ ID NO. 53. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:54 and 58-59.
In some embodiments of any of the engineered Cas nucleases according to the above, the one or more mutations comprise insertion of one or more G residues in the flexible region. In some embodiments, the one or more mutations comprise insertion of two G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P), wherein the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P.
In some embodiments of any of the engineered Cas nucleases according to the above, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W).
In some embodiments according to any of the engineered Cas nucleases described above, the engineered Cas nuclease comprises one or more mutations that increase the flexibility of two or more flexible regions in the reference Cas nuclease.
In some embodiments according to any of the engineered Cas nucleases described above, the increased activity of the Cas nuclease is a site-specific nuclease activity.
In some embodiments according to any of the engineered Cas nucleases described above, the increased activity of the Cas nuclease is gene editing activity in a eukaryotic cell (e.g., a mammalian cell). In some embodiments, the increased activity of the Cas nuclease is a gene editing activity in a human cell. In some embodiments, the gene editing efficiency of the engineered Cas nuclease is at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80% or more) higher than the reference Cas nuclease at the genomic site in the cell. In some embodiments, the average gene editing efficiency of the engineered Cas nuclease is at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80% or more) higher than the reference Cas nuclease at the plurality of genomic sites in the cell. In some embodiments, the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
In some embodiments, the present application provides Cas nucleases comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOs.2, 14, 18, 20, 27, 28, 33, 34, 41, 54 and 58-59.
In some embodiments, the present application provides an engineered Cas effector protein comprising any one of the above-described engineered Cas nucleases or functional derivatives thereof. In some embodiments, the engineered Cas nuclease or the functional derivative thereof has enzymatic activity. In some embodiments, the effector protein is capable of inducing a double strand break in a DNA molecule. In some embodiments, the engineered Cas effector protein is capable of inducing single strand breaks in a DNA molecule. In some embodiments, the effector protein comprises an enzyme-inactivating mutant of the engineered Cas nuclease.
In some embodiments according to any of the engineered Cas effect proteins above, the engineered Cas effect protein further comprises a functional domain fused to the engineered Cas nuclease or functional derivative thereof. In some embodiments, the functional domain is selected from the group consisting of: a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain.
In some embodiments according to any of the engineered Cas effector proteins described above, the engineered Cas effector protein comprises: a first polypeptide comprising an N-terminal portion of the engineered Cas nuclease or a functional derivative thereof and a second polypeptide comprising a C-terminal portion of the engineered Cas nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex with a target nucleic acid comprising a target sequence complementary to the guide sequence. In some embodiments, the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first dimerization domain and the second dimerization domain are associated with each other in the presence of an inducer. In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain.
Another aspect of the present application provides an engineered CRISPR-Cas system comprising: (a) any of the above engineered Cas effector proteins; (b) A guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence. In some embodiments, the guide RNA is crRNA comprising the guide sequence. In some embodiments, the system includes a precursor guide RNA array (array) encoding a plurality of crrnas. In some embodiments, the guide RNA comprises crRNA and tracrRNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the guide RNA comprises crRNA and scoutna. In some embodiments, wherein the engineered Cas effector protein is the main editor and the guide RNA is the pegRNA.
In some embodiments according to any of the above CRISPR-Cas systems, the system comprises one or more vectors encoding the engineered Cas effect protein. In some embodiments, the one or more carriers are selected from the group consisting of: retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors. In some embodiments, the one or more vectors are adeno-associated virus (AAV) vectors. In some embodiments, the AAV vector further encodes the guide RNA (e.g., crRNA, sgRNA, or precursor guide RNA array).
Another aspect of the present application provides an engineered CRISPR-Cas system comprising: (a) A Cas12i effector protein comprising a Cas12i nuclease (e.g., cas12i 2) or a functional derivative thereof; (b) crRNA that replaces one or more uridine (U) residues with one non-U residue in a repeat sequence comprising at least four U residues and a guide sequence complementary to a target sequence; wherein the Cas12i effector protein and the crRNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising a target sequence.
In another aspect of the present application, there is provided a method of detecting a target nucleic acid in a sample, the method comprising: (a) Contacting a sample with any of the above-described engineered CRISPR-Cas systems and a tagged detection nucleic acid that is single-stranded and that does not hybridize to a guide sequence of the guide RNA; and (b) measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas effector protein, thereby detecting the target nucleic acid.
Another aspect of the present application provides a method of modifying a target nucleic acid comprising a target sequence comprising contacting the target nucleic acid with any of the above-described engineered CRISPR-Cas systems. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, or an animal cell. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.
In some embodiments of any of the methods of modifying a target nucleic acid described above, the target nucleic acid is cleaved or a target sequence in the target nucleic acid is altered by the engineered CRISPR-Cas system. In some embodiments, expression of the target nucleic acid is altered by the engineered CRISPR-Cas system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition.
In some embodiments of any of the methods of modifying a target nucleic acid described above, the engineered CRISPR-Cas system comprises a precursor guide RNA array encoding a plurality of crrnas, wherein each crRNA comprises a different guide sequence.
In an embodiment of any one of the methods of modifying a target nucleic acid according to the above, the method is performed at a temperature of about 4 ℃ to about 67 ℃ (e.g., about 4 ℃ to about 15 ℃, about 15 ℃ to 40 ℃, about 4 ℃ to about 37 ℃, or about 40 ℃ to about 67 ℃).
Another aspect of the present application provides a method of treating a disease or disorder associated with a target nucleic acid in a cell of an individual, comprising modifying the target nucleic acid in the cell of the individual using any of the methods using the engineered CRISPR-Cas system described above, thereby treating the disease or disorder. In some embodiments, the disease or disorder is selected from the group consisting of: cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, ocular disease, bacterial infection, and viral infection.
Another aspect of the present application provides an engineered cell comprising a modified target nucleic acid, wherein the target nucleic acid has been modified using one of the methods described above.
In some embodiments, the present application provides an engineered non-human animal comprising one or more of the engineered cells described above.
Another aspect of the present application provides a method of modifying a target sequence in a target nucleic acid comprising contacting the target nucleic acid with an engineered CRISPR-Cas system at a temperature of about 40 ℃ to about 67 ℃, wherein the engineered CRISPR-Cas system comprises: (a) A Cas12i2 effector protein comprising a Cas12i2 nuclease or a functional derivative thereof; (b) A crRNA comprising a guide sequence complementary to the target sequence; wherein the Cas12i2 effector protein and the crRNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence.
Another aspect of the present application provides engineered crrnas that replace one or more uridine (U) residues with one non-U residue in a repeat sequence comprising at least four U residues. In some embodiments, the engineered crRNA comprises a spacer sequence of about 17-25 nucleotides in length. In some embodiments, the engineered crRNA comprises a spacer sequence of about 20 nucleotides in length. In some embodiments, the engineered crRNA comprises a repeat sequence comprising the nucleic acid sequence of SEQ ID NO. 173.
Compositions, kits and articles of manufacture for use in any of the above methods are also provided.
In particular, the present application also relates to the following:
1. a method of engineering an enzyme comprising:
(a) Obtaining a plurality of engineered enzymes, each engineered enzyme comprising one or more mutations that increase the flexibility of a flexible region of a plurality of flexible regions of a reference enzyme; and
(b) Selecting one or more engineered enzymes from the plurality of engineered enzymes, wherein the one or more engineered enzymes have increased activity compared to the reference enzyme.
2. The method of item 1, wherein the plurality of flexible regions are determined based on the amino acid sequence of the reference enzyme.
3. The method of item 2, wherein the plurality of flexible regions are determined without reference to the three-dimensional structure of the reference enzyme or equivalent thereof.
4. The method of item 3, wherein the plurality of flexible regions are determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine.
5. The method of item 4, wherein the plurality of flexible regions are determined using DynaMine.
6. The method of any one of claims 1-5, further comprising:
(i) Calculating a flexibility score for each amino acid residue of the reference enzyme, wherein a higher flexibility score indicates a lower conformational flexibility;
(ii) Select at X i Multiple peak amino acid residues at positions, whereThe flexibility score for each peak amino acid residue is below a predetermined threshold and below at X i -5 to X i -1、X i +1 and X i A flexibility score for the amino acid residue at position +5; and
(iii) Defining the plurality of flexible regions as amino acid residues X i -2 to X i +2。
7. The method of any of claims 1-6, wherein the plurality of flexible regions are located at random convolutions.
8. The method of any one of claims 1-7, wherein the one or more mutations comprises inserting one or more glycine (G) residues in the flexible region.
9. The method of claim 8, wherein the one or more mutations comprises inserting two G residues in the flexible region.
10. The method of claim 8 or 9, wherein the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P).
11. The method of any one of claims 1-10, wherein the one or more mutations comprises replacing one or more non-G residues with one or more G residues.
12. The method of claim 11, wherein the one or more mutations comprises replacing a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W).
13. The method of any one of claims 1-12, wherein the enzyme is a bacterial or archaeal enzyme.
14. The method of claim 13, wherein the enzyme is a Cas nuclease.
15. The method of claim 14, wherein the Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX.
16. The method of claim 14 or 15, wherein the plurality of flexible regions are in a domain of the reference Cas nuclease that interacts with DNA and/or RNA.
17. The method of any one of claims 14-16, wherein the activity is a site-specific nuclease activity.
18. The method of any one of claims 14-17, wherein the activity is a gene editing activity in a eukaryotic cell.
19. The method of claim 18, wherein the activity is a gene editing activity in a human cell.
20. The method of claim 18 or 19, wherein the gene editing efficiency of the engineered Cas nuclease selected in step (b) is at least about 20% greater than the reference Cas nuclease at one genomic site in the cell.
21. The method of any one of claims 18-20, wherein the average gene editing efficiency of the engineered Cas nuclease selected in step (b) is at least about 20% higher than the reference Cas nuclease at the plurality of genomic sites in the cell.
22. The method of item 20 or 21, wherein the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
23. An engineered Cas nuclease obtained using the method of any one of claims 14-22.
24. An engineered Cas nuclease comprising one or more mutations that increase flexibility of a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has increased activity compared to the reference Cas nuclease.
25. The engineered Cas nuclease of claim 24, wherein the flexible region is determined based on the amino acid sequence of the reference Cas nuclease.
26. The engineered Cas nuclease of claim 25, wherein the flexible region is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine.
27. The engineered Cas nuclease of claim 26, wherein the flexible region is determined using DynaMine, and wherein the flexibility score S of the amino acid residue with the highest flexibility in the flexible region 2 Prediction Not greater than about 0.8.
28. The engineered Cas nuclease of any one of claims 24-27, wherein the flexible region has 5 consecutive amino acid residues, wherein amino acid residue 3 has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility.
29. The engineered Cas nuclease of any one of claims 24-28, wherein the flexible region is located at a random coil.
30. The engineered Cas nuclease of any one of claims 24-29, wherein the flexible region is in a domain of the reference Cas nuclease that interacts with DNA and/or RNA.
31. The engineered Cas nuclease of any one of claims 24-30, wherein the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX.
32. An engineered Cas12b nuclease comprising one or more mutations that increase the flexibility of the flexible region corresponding to amino acid residues 835 to 839 in a reference Cas12b nuclease, wherein the amino acid residue numbering is based on SEQ ID NO:1, wherein the engineered Cas12b nuclease has increased activity compared to the reference Cas12b nuclease.
33. The engineered Cas12b nuclease of claim 32, comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID No. 2.
34. An engineered Cas12i nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas12i nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779, and amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8, wherein the engineered Cas12i nuclease has increased activity compared to the reference Cas12i nuclease.
35. The engineered Cas12i nuclease of claim 34, wherein the flexible region corresponds to amino acid residues 439-443 or amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8.
36. The engineered Cas12i nuclease of claim 34 or 35, comprising an amino acid sequence having at least about 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 18 and 20.
37. An engineered Cas9 nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas9 nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 39-43, amino acid residues 135-139, amino acid residues 176-180, amino acid residues 274-278, amino acid residues 351-355, amino acid residues 389-393, amino acid residues 521-525, amino acid residues 541-545, amino acid residues 755-759, amino acid residues 774-778, amino acid residues 786-790, amino acid residues 811-815, amino acid residues 848-852, amino acid residues 855-859, amino acid residues 874-878, amino acid residues 891-895, amino acid residues 1019-1023, and amino acid residues 1036-1040, wherein the amino acid residue numbering is based on SEQ ID NO:25, wherein the engineered Cas9 nuclease has enhanced activity compared to the reference Cas9 nuclease.
38. The engineered Cas9 nuclease of claim 37, wherein the flexible region is selected from the group of regions corresponding to: amino acid residues 135-139, amino acid residues 176-180, amino acid residues 541-545, amino acid residues 755-759, and amino acid residues 811-815, wherein the amino acid residue numbering is based on SEQ ID NO. 25.
39. The engineered Cas9 nuclease of claim 37 or 38, comprising an amino acid sequence having at least about 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 27, 28, 33, 34, and 41.
40. An engineered Cas9 nuclease comprising one or more mutations that increase flexibility of a flexible region in a reference Cas9 nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 45-49, amino acid residues 84-88, amino acid residues 116-120, amino acid residues 128-132, amino acid residues 216-220, amino acid residues 318-322, amino acid residues 387-391, amino acid residues 497-501, amino acid residues 583-587, amino acid residues 594-598, amino acid residues 614-618, amino acid residues 696-700, and amino acid residues 739-743, wherein the amino acid residue numbering is based on SEQ ID NO:53, wherein the engineered Cas9 nuclease has increased activity compared to the reference Cas9 nuclease.
41. The engineered Cas9 nuclease of claim 40, wherein the flexible region corresponds to amino acid residues 45-49 or amino acid residues 116-120, wherein the amino acid residue numbering is based on SEQ ID No. 53.
42. The engineered Cas9 nuclease of claim 40 or 41, comprising an amino acid sequence having at least about 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 54 and 58-59.
43. The engineered Cas nuclease of any one of claims 24-42, wherein the one or more mutations comprise insertion of one or more G residues in the flexible region.
44. The engineered Cas nuclease of claim 43, wherein the one or more mutations comprises insertion of two G residues in the flexible region.
45. The engineered Cas nuclease of claim 43 or 44, wherein the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P).
46. The engineered Cas nuclease of any one of claims 24-45, wherein the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W).
47. The engineered Cas nuclease of any one of claims 24-46, comprising one or more mutations that increase the flexibility of two or more flexible regions in the reference Cas nuclease.
48. The engineered Cas nuclease of any one of claims 24-47, wherein the activity is a site-specific nuclease activity.
49. The engineered Cas nuclease of any one of claims 24-48, wherein the activity is a gene editing activity in a eukaryotic cell.
50. The engineered Cas nuclease of claim 49, wherein the activity is gene editing activity in a human cell.
51. The engineered Cas nuclease of claim 49 or 50, wherein the gene editing efficiency of the engineered Cas nuclease is at least about 20% greater than the reference Cas nuclease at one genomic site in the cell.
52. The engineered Cas nuclease of any one of claims 49-51, wherein the average gene editing efficiency of the engineered Cas nuclease is at least about 20% higher than the reference Cas nuclease at multiple genomic sites in the cell.
53. The engineered Cas nuclease of any one of claims 49-52, wherein the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
54. An engineered Cas nuclease comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 14, 18, 20, 27, 28, 33, 34, 41, 54 and 58-59.
55. An engineered Cas effector protein comprising the engineered Cas nuclease of any one of claims 23-54 or a functional derivative thereof.
56. The engineered Cas effector protein of claim 55, wherein the engineered Cas nuclease or functional derivative thereof has enzymatic activity.
57. The engineered Cas effector protein of claim 56, wherein the effector protein is capable of inducing a double strand break in a DNA molecule.
58. The engineered Cas effector protein of claim 56, wherein the effector protein is capable of inducing a single strand break in a DNA molecule.
59. The engineered Cas effector protein of claim 55, wherein the effector protein comprises an enzyme-inactivating mutant of the engineered Cas nuclease.
60. The engineered Cas effector protein of any one of claims 55-59, further comprising a functional domain fused to the engineered Cas nuclease or functional derivative thereof.
61. The engineered Cas effector protein of item 60, wherein the functional domain is selected from the group consisting of: translation initiation domains, transcription repression domains, transactivation domains, epigenetic modification domains, nucleobase editing domains, reverse transcriptase domains, reporter domains and nuclease domains.
62. The engineered Cas effector protein of any one of claims 55-61, comprising: a first polypeptide comprising an N-terminal portion of the engineered Cas nuclease or a functional derivative thereof and a second polypeptide comprising a C-terminal portion of the engineered Cas nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex with a target nucleic acid comprising a target sequence complementary to the guide sequence.
63. The engineered Cas effector protein of claim 62, wherein the first polypeptide and the second polypeptide each comprise a dimerization domain.
64. The engineered Cas effector protein of claim 63, wherein the first dimerization domain and the second dimerization domain are associated with each other in the presence of an inducer.
65. The engineered Cas effector protein of claim 62, wherein the first polypeptide and the second polypeptide do not comprise a dimerization domain.
66. An engineered CRISPR-Cas system comprising:
(a) The engineered Cas effector protein of any one of claims 55-65; and
(b) A guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA,
wherein the engineered Cas effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence.
67. The engineered CRISPR-Cas system of claim 66, wherein the guide RNA is a crRNA comprising the guide sequence.
68. The engineered CRISPR-Cas system of claim 66 or 67, comprising a precursor guide RNA array encoding a plurality of crrnas.
69. The engineered CRISPR-Cas system of claim 66, wherein the guide RNA comprises crRNA and tracrRNA.
70. The engineered CRISPR-Cas system of claim 69, wherein the guide RNA is a single guide RNA (sgRNA).
71. The engineered CRISPR-Cas system of any of claims 66-70, comprising one or more vectors encoding the engineered Cas effect protein.
72. The engineered CRISPR-Cas system of item 71, wherein the one or more vectors are adeno-associated virus (AAV) vectors.
73. The engineered CRISPR-Cas system of claim 72, wherein the AAV vector further encodes the guide RNA.
74. A method of detecting a target nucleic acid in a sample, comprising:
(a) Contacting a sample with the engineered CRISPR-Cas system of any of items 66-68 and a tagged detection nucleic acid that is single stranded and that does not hybridize to a guide sequence of the guide RNA; and
(b) Measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas effector protein, thereby detecting the target nucleic acid.
75. A method of modifying a target nucleic acid comprising a target sequence, comprising contacting the target nucleic acid with the engineered CRISPR-Cas system of any one of items 66-73.
76. The method of claim 75, wherein the method is performed in vitro.
77. The method of claim 75, wherein the target nucleic acid is present in a cell.
78. The method of claim 77, wherein the cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, or an animal cell.
79. The method of claim 77 or 78, wherein the method is performed ex vivo.
80. The method of claim 77 or 78, wherein said method is performed in vivo.
81. The method of any one of claims 75-80, wherein the target nucleic acid is cleaved or a target sequence in the target nucleic acid is altered by the engineered CRISPR-Cas system.
82. The method of any one of claims 75-80, wherein the expression of the target nucleic acid is altered by the engineered CRISPR-Cas system.
83. The method of any one of claims 75-82, wherein the target nucleic acid is genomic DNA.
84. The method of any one of claims 75-83, wherein the target sequence is associated with a disease or disorder.
85. The method of any one of claims 75-84, wherein the engineered CRISPR-Cas system comprises a precursor guide RNA array encoding a plurality of crrnas, wherein each crRNA comprises a different guide sequence.
86. The method of any one of claims 75-85, wherein the method is performed at a temperature of about 4 ℃ to about 67 ℃.
87. The use of the engineered CRISPR-Cas system of any one of claims 66-73 in a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual.
88. The use of claim 87, wherein the disease or disorder is selected from the group consisting of: cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, ocular disease, bacterial infection, and viral infection.
89. An engineered cell comprising a modified target nucleic acid, wherein the target nucleic acid has been modified using the method of any one of claims 75-86.
90. An engineered non-human animal comprising one or more engineered cells of claim 89.
It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments that relate to particular method steps, reagents or conditions, or composition components, are expressly contemplated by this disclosure and are disclosed herein as if each and every combination were individually and specifically disclosed.
Drawings
Fig. 1A shows an exemplary design pipeline for engineering Cas proteins with increased activity. In this example, candidate flexible regions were identified using DynaMine software, and glycine substitutions (single underlined) or insertions (double underlined) were introduced to generate Cas protein variants. The variants were then cloned into expression vectors along with eGFP and cells were transfected with the variant cas+gfp plasmid and sgRNA or crRNA plasmid to test the editing efficiency of the variants.
Fig. 1B shows the determination of peak amino acids from the obtained Cas protein S2 sequence parameter score spectrum.
Fig. 2 shows the flexibility (S2) score spectrum of BhCas12b 4.
Figure 3 shows the indels (compared to enBhCas12b 4) generated by wild type BhCas12b4 using the indicated sgrnas in human cells. The last panel in fig. 3 shows the% indels generated for all sites tested.
Fig. 4 shows the flexibility (S2) score spectrum of Cas12i 2.
Fig. 5 shows the computationally determined secondary structural region of Cas12i 2.
Figure 6 shows the gene editing efficiency (% indels) of Cas12i2 variants compared to WT Cas12i using four different crrnas.
Figure 7 shows the gene editing efficiency (% indels) of Cas12i2 variants (named enas 12i 2) with combined mutations of variants 2.2+6.1 compared to WT Cas12i alone or variants 2.2 and 6.1. enCas12i2 (2.2+6.1) shows improved editing efficiency.
Fig. 8A shows the overall genome editing efficiency of enCas12i2 in human cells compared to SpCas9 and BhCas12b-v 4. Editing efficiency (% indels) was analyzed at 46 sites for enCas12i2, 18 sites for SpCas9 and 23 sites for BhCas12bv 4.
Fig. 8B shows the average editing efficiency of enCas12i2 at different Protospacer Adjacent Motif (PAM) sites including NTTA, NTTC, NTTG and NTTT and ATTN, CTTN, GTTN and TTTN in human 293T cells.
Figure 9 shows the processing of precursor crrnas by enCas12i2 in vivo. Using a precursor crRNA targeting 3 sites, enCas12i2 has comparable genome editing activity compared to using a single crRNA.
Figure 10 shows in vitro cleavage of DNA plasmids by Cas12i2 and enCas12i 2.
Figure 11A shows detection of dsDNA containing an XBP target sequence by wild-type Cas12i2 and engineered Cas12i2 variants.
Fig. 11B shows the results demonstrating that wild-type Cas12i2 can cleave fluorescent reporter molecules linked via an rU in a nucleic acid detection experiment.
Fig. 12 shows the flexibility (S2) score spectrum of GeoCas 9.
Fig. 13 shows the calculated secondary structural regions of GeoCas 9.
Fig. 14 shows the results of a targeted depth sequencing (Targeted deep sequencing) assay to determine the efficiency of indels generated by engineered GeoCas9 variants. Arrows indicate engineered GeoCas9 variants with significantly improved editing efficiency.
Figure 15 shows the location of selected flexible regions and corresponding domains of SaCas 9.
Fig. 16 shows the results of the T7 enzyme cleavage assay to test the editing efficiency of the SaCas9 variants. Engineered SaCas9 variants 1.1, 3.1, 3.2 (represented by box outlines) showed significantly improved gene editing efficiency.
Fig. 17A shows in vitro dsDNA cleavage of wild-type Cas12i1 and Cas12i 2.
Fig. 17B shows the crRNA sequences used to test cleavage of Cas12i1 or Cas12i 2.
Figure 18 shows the percent indels produced by wild-type Cas12i1 and Cas12i2 using crrnas at human genomic target sites. Gene editing efficiency was measured using a T7 endonuclease 1 (T7E 1) assay. We found that Cas12i2 is capable of producing indels in a broader target, suggesting that it is a promising candidate for engineering.
Fig. 19 shows the editing efficiency of the illustrated crRNA mutants compared to the wild-type crRNA sequence using Cas12i 1.
Figure 20 shows the results demonstrating that Cas12i2 has enzymatic activity (i.e., is capable of cleaving dsDNA) over a broad temperature range of 12 ℃ -67 ℃.
Figure 21 shows Cas12i2 editing using crRNA arrays (precursor crrnas) at multiple genomic target sites in human cells.
Figure 22 shows the results of defining the seed sequence of Cas12i2 by testing the ability of Cas12i2 to produce indels using crrnas having a single base mismatch at one of 1-19 bases of the crrnas.
Figure 23 shows the identification of the 20bp optimal spacer length for Cas12i 2. Cas12i2 was tested for its ability to generate indels using two different crrnas (CCR 5-1 and CCR 5-2) using a 17-25bp spacer length range.
Detailed Description
The present application provides methods for engineering enzymes by introducing amino acid mutations that enhance flexibility in the flexible region of the enzyme, which results in increased in vitro and in vivo enzyme activity. The methods described herein are applicable to a variety of Cas nucleases, including Cas12b, cas12i, and Cas9. Notably, the methods described herein do not rely on the three-dimensional structure of the Cas nuclease. For example, the methods described herein have been successfully applied to Cas12i to produce a variety of engineered Cas12i proteins with improved genome editing efficiency over a broad range of loci. Engineered Cas effector proteins (e.g., cas12b, cas12i, and Cas 9) and methods of use thereof are also provided.
I. Definition of the definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As used herein, "effector protein" refers to a protein having an activity such as site-specific binding activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, single-stranded RNA cleavage activity, or transcriptional regulation activity.
As used herein, "guide RNA" and "gRNA" are used interchangeably herein to refer to an RNA capable of forming a complex with a Cas effector protein and a target nucleic acid (e.g., double-stranded DNA). The guide RNA may comprise a single RNA molecule or be bound to each other by hybridization of two or more RNA molecules by complementary regions in the two or more RNA molecules. When used in combination with a double RNA-guided Cas nuclease, such as Cas9, the guide RNAs comprise a crRNA and a tracrRNA. When used in combination with a single RNA-guided Cas nuclease, such as Cas12i, the guide RNA does not comprise a tracrRNA or additional transactivating RNA. Also contemplated herein are precursor guide RNA arrays that can be processed into multiple crrnas, and for certain CRISPR/Cas systems, the processed crrnas are further bound to a tracrRNA or another transactivating RNA (e.g., a scoutna) to guide Cas effector proteins. "crRNA" or "CRISPR RNA" comprises a guide sequence having sufficient complementarity to a target sequence of a target nucleic acid (e.g., double-stranded DNA) that directs sequence-specific binding of a CRISPR complex to the target nucleic acid. "tracrRNA" or "transactivation CRISPR RNA" are partially complementary to and base pair with crRNA and may play a role in the maturation of crRNA. A "single guide RNA" or "sgRNA" is an engineered guide RNA that has crRNA and tracrRNA fused to each other in a single molecule.
The terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to polymeric forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof. "oligonucleotide" and "oligonucleotide" are used interchangeably and refer to short polynucleotides having no more than about 50 nucleotides.
As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick (Watson-Crick) base pairing. Percent complementarity means the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10 of 10 moieties, complementary by about 50%, 60%, 70%, 80%, 90% and 100%, respectively). "fully complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100 complementary, or to two nucleic acids that hybridize under stringent conditions.
As used herein, "stringent conditions" for hybridization refers to conditions under which nucleic acids having complementarity to a target sequence hybridize predominantly to the target sequence, but do not substantially hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and will vary depending on a number of factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes, section one, chapter two, "Overview of principles of hybridization and the strategy of nucleic acid probe assay," Elsevier, N, Y.
"hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding may occur through watson crick base pairing, hopogstein (Hoogstein) binding, or in any other sequence-specific manner. Sequences capable of hybridizing to a given sequence are referred to as the "complement" of the given sequence.
"percent (%) sequence identity" for a nucleic acid sequence is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a particular nucleic acid sequence after aligning the sequences by allowing gaps (gaps) to achieve the maximum percent sequence identity, if necessary. "percent (%) sequence identity" for a peptide, polypeptide, or protein sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or amino acid sequence after aligning the sequences by allowing for gaps, if necessary, to achieve the greatest percent sequence homology. For the purpose of determining the percentage of amino acid sequence identity, the alignment may be accomplished in various ways within the skill of the art, e.g., using techniques such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software, and the like. One skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.
The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. The protein may have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component).
As used herein, "variant" is interpreted as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains the requisite properties. Typical variants of a polynucleotide differ from the nucleic acid sequence of another reference polynucleotide. Variations in the variant nucleic acid sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. In general, the differences are limited such that the sequences of the reference polypeptides and variants are very similar overall and identical in many regions. The amino acid sequences of the variant and reference polypeptides may differ by any combination of one or more substitutions, additions, deletions. The amino acid residues that are replaced or inserted may or may not be those encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring (such as allelic variants), or may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of skill in the art.
As used herein, the term "wild-type" has a meaning commonly understood by those skilled in the art, meaning a typical form of an organism, strain, gene or trait that distinguishes it from mutants or variants when it is present in the nature. It can be isolated from resources in nature and is not deliberately modified.
As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably to refer to human participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or which naturally occurs.
As used herein, the term "ortholog" has the meaning commonly understood by one of ordinary skill in the art. As a further guidance, an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or similar function as the protein as its ortholog.
As used herein, the term "identity" is used to denote sequence matching between two polypeptides or between two nucleic acids. When one position in both comparison sequences is occupied by the same base or amino acid monomer subunit (e.g., one position in each of two DNA molecules is occupied by adenine, or one position in each of two polypeptides is occupied by lysine), then each molecule is identical at that position. The "percent identity" between these two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 matches out of 6 positions total). Typically, such a comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved, for example, by the method in Needleman et al (1970) J.mol.biol.48:443-453, which may conveniently be carried out by a computer program such as the alignment program (DNAstar, inc.). PAM 120 weight residue tables can also be used and integrated into the ALIGN program (version 2.0) using the algorithm of e.meyers and w.miller (comp. Appl biosci.,4:11-17 (1988)). Gap length penalty 12 and gap penalty 4 are used to determine the percent identity between two amino acid sequences. Furthermore, the Blossum 62 matrix or PAM250 matrix, with a GAP weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6, can be used to determine the percent identity between two amino acid sequences using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm integrated into the GAP program of the GCG software package (available from www.gcg.com).
As used herein, "cell" is understood to refer not only to a particular single cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term herein.
As used herein, the terms "transduction" and "transfection" include methods known in the art for introducing DNA into a cell to express a protein or molecule of interest using an infectious agent (e.g., a virus) or otherwise. In addition to viruses or virus-like agents, there are also chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes or cationic polymers (e.g. DEAE-dextran or polyethylenimine); non-chemical methods such as electroporation, cell extrusion (cell squeezing), sonoporation (sonoporation), optical transfection, puncture transfection (impalefection), protoplast fusion, plasmid delivery or transposons; particle-based methods, such as using gene gun, magnetic transfection or magnet-assisted transfection, particle bombardment; and hybridization methods (such as nuclear transfection).
As used herein, the term "transfected", "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected", "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid.
The term "in vivo" refers to the organism from which the cells were obtained. "ex vivo" or "in vitro" refers to the organism from which cells are obtained.
As used herein, "treatment" is a method for achieving a beneficial or desired result, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the progression of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) remission of the disease, reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving quality of life, and/or prolonging survival. "treating" also includes reducing the pathological consequences of cancer. The methods of the present invention contemplate any one or more of these processing aspects.
As used herein, the term "effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease (e.g., to ameliorate, alleviate, mitigate, and/or delay one or more symptoms thereof). As understood in the art, an "effective amount" may be administered in one or more administrations, i.e., a single administration or multiple such administrations may be required to reach the desired treatment endpoint.
"subject," "individual," or "patient" are used interchangeably herein for therapeutic purposes to refer to any animal classified as a mammal, including humans, domestic animals, and farm animals, as well as zoos, gyms, or pet animals such as dogs, horses, cats, cattle, etc. In some embodiments, the subject is a human subject.
It is to be understood that embodiments of the invention described herein include embodiments that "consist of and/or" consist essentially of.
References herein to "about" a value or parameter include (and describe) variations with respect to the value or parameter itself. For example, reference to a description of "about X" includes a description of "X".
As used herein, reference to "no" values or parameters generally means and describes "other than …" values or parameters. For example, the method is not used to treat type X cancers, meaning that the method is used to treat cancers other than type X.
As used herein, the term "about X-Y" has the same meaning as "about X to about Y".
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. This statement is therefore intended to serve as antecedent basis for use of exclusive terminology such as "only," "only," etc., or use of the limitation "no" in connection with recitation of claim elements.
As used herein, the term "and/or" in words such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, as used herein, the term "and/or" in words such as "A, B and/or C" is intended to include each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).
Methods for engineering enzymes
The present application provides methods of engineering an enzyme comprising: (a) Obtaining a plurality of engineered enzymes, each engineered enzyme comprising one or more mutations that increase the flexibility of a flexible region of a plurality of flexible regions of a reference enzyme; and (b) selecting one or more engineered enzymes from the plurality of engineered enzymes, wherein the one or more engineered enzymes have increased activity compared to the reference enzyme. In some embodiments, the method further comprises determining a plurality of flexible regions in the reference enzyme. In some embodiments, the three-dimensional structure of the reference enzyme or equivalent thereof is not available. In some embodiments, the enzyme is a bacterial or archaeal enzyme. In some embodiments, the activity is measured in eukaryotic cells, such as mammalian cells (e.g., human cells).
In some embodiments, methods of engineering a Cas nuclease are provided, comprising: (a) Determining a plurality of flexible regions in the reference Cas nuclease (e.g., based on a primary sequence of the reference Cas nuclease); (b) Obtaining a plurality of engineered Cas nucleases, each comprising one or more mutations that increase the flexibility of a flexible region of a plurality of flexible regions of the reference Cas nuclease; and (c) selecting one or more engineered Cas nucleases from the plurality of engineered Cas nucleases, wherein the one or more engineered Cas nucleases have increased activity compared to a reference Cas nuclease. In some embodiments, the plurality of flexible regions is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. In some embodiments, the Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the plurality of flexible regions are located at random convolutions. In some embodiments, the plurality of flexible regions are in domains that interact with DNA and/or RNA of a reference Cas nuclease. In some embodiments, the flexible region is at least about 5 (e.g., 5) amino acids in length. In some embodiments, the one or more mutations include insertion of one or more (e.g., 2) glycine (G) residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P). In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise the substitution of one or more G residues for one or more non-G residues. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W). In some embodiments, the activity is a site-specific nuclease activity. In some embodiments, the activity is a gene editing activity in a eukaryotic cell (e.g., a human cell). In some embodiments, the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
In some embodiments, methods of engineering a Cas nuclease are provided, comprising: (a) Calculating a flexibility score for each amino acid residue of a reference Cas nuclease (e.g., based on the primary sequence of the reference Cas nuclease), wherein a higher flexibility score indicates lower conformational flexibility; (b) Select at X i A plurality of peak amino acid residues at positions, wherein each peak amino acid residue has a flexibility score below a predetermined threshold and below that at X i -5 to X i -1、X i +1 and X i A flexibility score for the amino acid residue at position +5; (c) Multiple flexible regions (as amino acid residues X i -2 to X i +2); (d) Obtain a plurality ofAn engineered Cas nuclease, each engineered Cas nuclease comprising one or more mutations that increase flexibility of a flexible region in a plurality of flexible regions of the reference Cas nuclease, wherein the one or more mutations comprise inserting one or more (e.g., 2) G residues in the flexible region, and/or replacing one or more non-G residues with one or more G residues; and (e) selecting one or more engineered Cas nucleases from the plurality of engineered Cas nucleases, wherein the one or more engineered Cas nucleases have increased activity compared to the reference Cas nuclease. In some embodiments, the plurality of flexible regions is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. In some embodiments, the Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the plurality of flexible regions are located at random convolutions. In some embodiments, the plurality of flexible regions are in domains of the reference Cas nuclease that interact with DNA and/or RNA. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P). In some embodiments, the flexible amino acid residues are selected according to the following priorities: g >S>N>D>H>M>T>E>Q>K>R>A>P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: leucine (L), isoleucine (I), valine. (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W). In some embodiments, the activity is a site-specific nuclease activity. In some embodiments, the activity is a gene editing activity in a eukaryotic cell (e.g., a human cell). In one placeIn some embodiments, the gene editing efficiency is measured using the following method: t7 endonuclease 1 (T7E 1) assay, sequencing of target DNA, insertion deletion by split chase (tid) assay or insertion deletion detection by amplicon analysis (IDAA) assay.
Also provided are engineered enzymes (e.g., engineered Cas nucleases) obtained using any of the methods described herein, as well as libraries comprising the various engineered enzymes described herein.
Determining a plurality of flexible regions
The plurality of flexible regions in the reference enzyme may be determined using any method known in the art. In some embodiments, the plurality of flexible regions is determined based solely on the amino acid sequence of the reference enzyme. In some embodiments, a plurality of flexible regions are determined based on structural information of the reference enzyme, including, for example, secondary structure, crystal structure, NMR structure, and the like. In some embodiments, the plurality of flexible regions is determined without reference to structural information (e.g., three-dimensional structure) of the reference enzyme. In some embodiments, the three-dimensional structure of the reference enzyme or equivalent thereof is not available.
In some embodiments of the methods provided herein, the plurality of flexible regions is determined based on the amino acid sequence of the reference enzyme. In some embodiments, the plurality of flexible regions is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. For example, yu et al have described methods for determining flexible regions based on amino acid sequences (Engineering proteins for thermostability through rigidifying flexible sites. Biotechnology Advances Volume 32, issue 2, march-April 2014, pages 308-315).
In some embodiments, the plurality of flexible regions is determined based on NMR chemical shift data of the protein in solution. In some embodiments, dynaMine is used to define multiple flex regions (Cilia et al DynaMine webserver: predicting protein dynamics from sequence. Nucleic Acids Res.2014Jul 1;42 (Web Server issue): W264-W270). DynaMine uses NMR chemical shift data of proteins in solution to gain a quantitative understanding of the relationship between amino acid sequence and backbone dynamics and predict flexible regions. DynaMine predictors developed from these data can predict the residue level potential of proteins for skeletal dynamics based on sequence information alone, in contrast to the approach of 3D structural information. This approach reveals a large number of available protein sequences that lack structural information for kinetic analysis. In some embodiments, the flexibility profile is an S2 score profile, wherein a lower S2 score indicates higher flexibility.
In some embodiments, molecular dynamics simulation (e.g., simulated root mean square fluctuations) is used to determine the plurality of flexible regions. In some embodiments, the plurality of flexible regions is determined based on factor B (e.g., crystallographic) data. In some embodiments, the plurality of flex regions are defined using PredyFlexy described by Tarun et al (In silico prediction of protein flexibility with local structure app. Biochimie (165), october 2019, pages 150-155).
In some embodiments, the expected average number of contacts per residue is used as an indicator of whether a given region is folded or unfolded, such as using FoldUnfold (Oxana et al FoldUnfold: web server for the prediction of disordered regions in protein chain, bioinformation, volume 22,Issue 23,1December 2006,Pages 2948-2949).
In some embodiments, the plurality of flexible regions is determined by calculating normalized B values from the amino acid sequence, such as using PROFbval (Schlesinger et al PROFbval: predict flexible and rigid residues in proteins.Bioinformatics, volume 22,Issue 7,1April 2006,Pages 891-893).
In some embodiments, multiple coarse-grained methods, a combination of structural databases and atomic models are used to determine the multiple flexible regions, such as using Flexserv (Camps et al FlexServ: an integrated tool for the analysis of protein Flexibility. Bioinformation 2009Jul1;25 (13): 1709-10).
In some embodiments, the plurality of flexible regions are determined using a trained supervised pattern recognition method, support Vector Machines (SVMs), such as using a FlexPred Web server (Kuznetsov 1 et al FlexPred: a Web-server for predicting residue positions involved in conformational switches in proteins.Bioinformation.2008;3 (3): 134-136).
In some embodiments, the plurality of flexible regions are determined based on protein turbulence calculated using a recurrent neural network, such as using DisoMine (Orlando et al prediction of disordered regions in proteins with recurrent Neural Networks and protein dynamics, bioRxiv2020.05.25.115253; doi: https:// doi.org/10.1101/2020.05.25.115253).
In some embodiments, determining the plurality of flexible regions comprises: (i) Calculating a flexibility score for each amino acid residue of the reference enzyme, wherein a higher flexibility score indicates a lower conformational flexibility; (ii) Select at X i A plurality of peak amino acid residues at positions, wherein each peak amino acid residue has a flexibility score below a predetermined threshold and below that at X i -5 to X i -1、X i +1 and X i A flexibility score for the amino acid residue at position +5; and (iii) defining the plurality of flexible regions as amino acid residues X i -2 to X i +2. In some embodiments, the flexible region is determined using DynaMine, and the flexibility score S of the amino acid residue with the highest flexibility in the flexible region 2 Prediction Not greater than about 0.8 (e.g., not greater than about 0.75 or 0.7).
In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility.
In some embodiments, the flexible region is located in a random coil or linker region of the protein. In some embodiments, random convolutions or linker regions are determined based on available structural data. In some embodiments, random convolutions or linker regions are determined based on homology to orthologs of known structure. In some embodiments, random convolutions or linker regions are determined based on the amino acid sequence. In some embodiments, no 3D structural data is available for the reference protein or ortholog of the reference protein. In some embodiments, the flexible region is not located at random convolutions or in the linker region. In some embodiments, the flexible region is located within about 10 amino acids from the random coil or linker region, such as any of about 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In some embodiments, the flexible region is longer than the random crimp or linker region. In some embodiments, the flexible region is shorter than the random coil or linker region. In some embodiments, at least a portion of the flexible region is located in an alpha helix or beta chain.
In some embodiments, the flexible region is located in a functional domain of the reference enzyme. In some embodiments, the flexible region is located near the catalytic site of the reference enzyme. In some embodiments, the flexible region is not located in a functional domain of the reference enzyme. In some embodiments, the reference enzyme is a reference nuclease. In some embodiments, the flexible region is in the domain of a reference nuclease (e.g., cas nuclease) that interacts with DNA and/or RNA. In some embodiments, the flexible region is not located in the domain of a reference nuclease that interacts with DNA and/or RNA. In some embodiments, the reference enzyme is a Cas nuclease selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In the "other engineered Cas effector proteins" section of the third section, other Cas nucleases that can be engineered using the methods described herein are discussed. In some embodiments, the reference enzyme is an Argonaut protein.
In some embodiments, the engineered enzyme (e.g., cas nuclease) comprises one or more mutations that increase two or more (e.g., 2, 3, 4, 5, 6) flexible regions flexibility. In some embodiments, one or more mutations in different flexible regions have a synergistic effect in increasing the activity of the engineered enzyme relative to the reference enzyme.
Producing variants with mutations that increase flexibility
In some embodiments, the method comprises: creating an insertion of one or more G residues and/or a G substitution of a hydrophobic amino acid residue in a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has enhanced activity compared to the reference Cas nuclease. In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments, the one or more mutations include insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P). In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P.
In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas nuclease comprises one or more mutations that increase the flexibility of two or more flexible regions. In some embodiments, the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the method comprises: resulting in the insertion of one or more G or S residues and/or the substitution of a hydrophobic amino acid residue with a G or S residue in the flexible region. In some embodiments, the method comprises: resulting in replacement of hydrophobic residues (e.g., L, I, V, C, Y, F or W) or less flexible amino acid residues with more flexible amino acid residues. In some embodiments, the more flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P.
Selection of engineered enzymes with enhanced activity
In some embodiments, the method comprises selecting one or more engineered enzymes from a plurality of engineered enzymes, wherein the one or more engineered enzymes have increased activity compared to the reference enzyme, wherein the activity is a site-specific nuclease activity. In some embodiments, the activity is a gene editing activity in a eukaryotic cell. In some embodiments, the activity is a gene editing activity in a human cell.
In some embodiments, the increased activity is a site-specific nuclease activity. In some embodiments, the site-specific nuclease activity is determined in vitro. In some embodiments, the site-specific nuclease activity is determined in a cell. The activity of the site-specific nuclease can be assessed using methods known in the art, including, for example, in vitro cleavage assays based on agarose gel electrophoresis as described in the examples provided herein. In some embodiments, the engineered enzyme (e.g., engineered Cas nuclease) variant has a site-specific nuclease activity that is increased by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more as compared to the selected reference enzyme.
In some embodiments, the activity is a gene editing activity in a cell, such as a prokaryotic cell or eukaryotic cell. Methods known in the art can be used to determine the gene editing efficiency of an engineered enzyme in a cell, including, for example, T7 endonuclease 1 (T7E 1) assays, target DNA sequencing (including, for example, sanger sequence and second generation sequencing), indel Detection (IDAA) assays by split chase indels (TIDEs) assays, or by amplicon analysis. See, e.g., sentmaat MF et al, "A survey of validation strategies for CRISPR-Cas9 coding," Scientific Reports,2018,8, article No. 888, which is incorporated herein by reference in its entirety. In some embodiments, for example, targeted second generation sequencing (NGS) is used to measure the gene editing efficiency of an engineered enzyme in a cell, as described in the examples herein.
In some embodiments, the activity is a gene editing activity in a eukaryotic cell, such as a plant cell or a mammalian cell. In some embodiments, an enzyme engineered in a eukaryotic cell (e.g., an engineered Cas nuclease) variant has a site-specific nuclease activity that is increased by at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more compared to a selected reference enzyme.
In some embodiments, the activity is a gene editing activity in a human cell, such as a 293T cell. In some embodiments, the enzyme (e.g., engineered Cas nuclease) variant engineered in the human cell has a site-specific nuclease activity that is increased by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more as compared to the selected reference enzyme.
In some embodiments, the method further comprises the step of combining mutations from one or more engineered enzymes of the plurality of engineered enzymes, wherein the one or more engineered enzymes have increased activity compared to a reference enzyme. In some embodiments, the combined mutations comprise mutations in one or more (e.g., 2, 3, 4, or more) different flexible regions.
Engineered Cas effector proteins
The present application provides engineered Cas effector proteins with improved activity, such as target binding, double strand cleavage activity, nickase activity, and/or gene editing activity. In some embodiments, the engineered Cas nuclease is obtained using any one of the engineering methods described in section II. In some embodiments, an engineered Cas effector protein (e.g., a Cas nuclease, a Cas nickase, a Cas fusion effector protein, or a split (split) Cas effector protein) is provided that comprises any of the engineered Cas nucleases described herein or functional derivatives thereof.
In some embodiments, an engineered Cas nuclease is provided comprisingOne or more mutations that increase flexibility of a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has increased activity compared to the reference Cas enzyme. In some embodiments, the flexible region is determined based on the amino acid sequence of the reference Cas nuclease, for example, using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine. In some embodiments, the flexible region is determined using DynaMine, and wherein the flexibility score S of the amino acid residue with the highest flexibility in the flexible region 2 Prediction Not greater than about 0.8 (e.g., not greater than about 0.75 or 0.7). In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments, the flexible region is located at a random curl. In some embodiments, the flexible region is in a domain that interacts with DNA and/or RNA of a reference Cas nuclease. In some embodiments, the engineered Cas nuclease comprises one or more mutations that increase the flexibility of two or more flexible regions. In some embodiments, the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX.
In some embodiments, an engineered Cas nuclease is provided that comprises insertion of one or more G residues and/or substitution of hydrophobic amino acid residues in a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has enhanced activity compared to the reference Cas nuclease. In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments, the one or more mutations comprise insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas nuclease comprises one or more mutations that increase the flexibility of two or more flexible regions. In some embodiments, the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX.
In some embodiments, an engineered Cas effector protein is provided, comprising an engineered Cas nuclease or a functional derivative thereof, wherein the engineered Cas nuclease comprises one or more mutations that increase flexibility of a flexible region comprising at least 5 consecutive amino acid residues of a reference Cas nuclease, wherein the engineered Cas nuclease has increased activity compared to the reference Cas nuclease. In some embodiments, the flexible region is determined using DynaMine, and wherein the flexibility score S of the amino acid residue with the highest flexibility in the flexible region 2 pred Not greater than about 0.8 (e.g., not greater than about 0.75 or 0.7). In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments, the engineered Cas nuclease comprises insertion of one or more G residues and/or substitution of hydrophobic amino acid residues in the flexible region. In some embodiments, the one or more mutations comprise insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted into the N of the flexible amino acid residues in the flexible region A terminal end, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g>S>N>D>H>M>T>E>Q>K>R>A>P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas nuclease comprises one or more mutations that increase the flexibility of two or more flexible regions. In some embodiments, the reference Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the functional derivative of the engineered Cas nuclease is an enzymatically active Cas nuclease, a Cas nickase, a Cas that loses enzymatic activity, a fusion effector protein (e.g., a transcriptional activator, a transcriptional repressor, a base editor, or a primer editor), or a split Cas effector protein (e.g., an inducible split Cas effector protein or an self-induced split Cas effector protein).
Also provided are engineered CRISPR-Cas systems comprising any of the engineered Cas effector proteins (e.g., engineered Cas nucleases) and guide RNAs (including precursor guide RNA arrays, crrnas, single guide RNAs, or crrnas and tracrrnas) described herein. In some embodiments, the engineered CRISPR-Cas system comprises one or more nucleic acid molecules encoding the engineered Cas effector protein and/or the guide RNA. In some embodiments, the engineered CRISPR-Cas system comprises one or more vectors encoding the engineered Cas effector protein and/or the guide RNA. In some embodiments, vectors encoding the engineered Cas effector proteins are provided. In some embodiments, the vector further comprises a guide RNA. In some embodiments, the carrier is selected from the group consisting of: retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors. In some embodiments, the vector is an adeno-associated virus (AAV) vector.
The engineered Cas nucleases described herein have mutations that increase conformational flexibility of one or more (e.g., 1, 2, 3, or more) flexible regions in a reference Cas nuclease (e.g., a naturally occurring wild-type Cas nuclease). Mutations in two or more flexible regions can be combined to provide a synergistic increase in activity in the engineered Cas nuclease as compared to the reference Cas nuclease. As described in section II, the flexible region in the reference Cas nuclease can be determined using methods known in the art, e.g., based on one or more of the methods described above in section II, "methods of engineering enzymes". In some embodiments, the flexible region of the reference Cas is determined based on the amino acid sequence of the reference Cas nuclease. In some embodiments, the flexible region of the reference Cas nuclease is determined using a procedure selected from the group consisting of: predyFlexy, foldUnfold, PROFbval, flexserv, flexPred, dynaMine and discomine.
The flexible region can comprise 5 or more (e.g., 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more) consecutive amino acid residues of the reference Cas nuclease. In some embodiments, the flexible region has 5 consecutive amino acid residues of the reference Cas nuclease. The flexible region can be defined by first selecting a peak amino acid residue at position X of the reference Cas nuclease, wherein the peak amino acid residue has a peak amino acid residue that is below a predetermined threshold (e.g., S as determined by DynaMine 2 pred 0.8 or less), and wherein the flexibility score of the peak amino acid residue is lower than the flexibility score of the amino acid residues X-5 to X-1 (i.e., the 5 amino acid residues flanking the N-terminus of the peak amino acid residue) and x+1 to x+5 (i.e., the 5 amino acid residues flanking the C-terminus of the peak amino acid residue), wherein a higher flexibility score indicates lower conformational flexibility. In some embodiments, the flexible region has 5 consecutive amino acid residues, wherein the 3 rd amino acid residue has the lowest flexibility score, and wherein a higher flexibility score indicates lower conformational flexibility.
In some embodiments, the flexible region of the reference Cas nuclease is determined using DynaMine. In some embodiments, the amino acid residue with the highest flexibility (i.e., peak amino acid residue) in the flexible region of the flexible region is located in a context-dependent flexible region as determined by DynaMine. In some embodiments, the amino acid residue with the highest flexibility (i.e., peak amino acid residue) in the flexible region of the flexible region is located in the flexible region as determined by DynaMine. In some embodiments, the flexibility score S of the amino acid residue with the highest flexibility in the flexible region (i.e., the peak amino acid residue) based on the calculation in DynaMine 2 pred No greater than about 0.8, such as no greater than any of about 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72,0.71,0.7, or less. In some embodiments, the amino acid residues of the flexible region are in a context-dependent flexible region and/or in a flexible region as determined by DynaMine. In some embodiments, the flexibility score S for each amino acid residue in the flexible region is based on calculations in DynaMine 2 pred No greater than about 0.8, e.g., no greater than any of about 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72,0.71,0.7, or less.
In some embodiments, the flexible region of the reference Cas nuclease is located at the random coil. In some embodiments, the flexible region of the reference Cas nuclease is not located in the alpha helix. In some embodiments, the flexible region of the reference Cas nuclease is not located in the β -strand. In some embodiments, the flexible region of the reference Cas nuclease is not located at random coils. In some embodiments, at least a portion of the flexible region of the reference Cas nuclease is located in the alpha helix or beta strand.
In some embodiments, the flexible region of the reference Cas nuclease is in the domain of the reference Cas nuclease that interacts with DNA. In some embodiments, the flexible region of the reference Cas nuclease is in the domain of the reference Cas nuclease that interacts with RNA (e.g., guide RNA, such as crRNA and/or tracrRNA). In some embodiments, the flexible region of the reference Cas nuclease is in a domain of the reference Cas nuclease that does not interact with DNA or RNA. In some embodiments, the flexible region of the reference Cas nuclease is located between the functional domains of the reference Cas nuclease.
Any abrupt change that increases the flexibility of the flexible region may be used. Flexibility of 20 naturally occurring amino acid residues has been characterized based on experimental data (e.g., crystal structure, NMR, and other protein kinetic studies). Flexible amino acid residues include G, T, R, S, N, Q, D, P, E, K, A, M; rigid amino acid residues include W, Y, F, C, I, V, H, L. In some embodiments, the flexibility of an amino acid depends on the identity of its neighboring amino acids. See, e.g., smith DK et al, "Improved amino acid flexibility parameters," Protein Sci.,2003,12 (5): 1060-1072.
In some embodiments, the one or more mutations of the flexible region include the insertion of one or more (e.g., 1, 2, 3, or more) amino acid residues associated with the flexible conformation. The insertion may occur anywhere in the flexible region. In some embodiments, the insertion is at the N-terminus or C-terminus of the peak amino acid in the flexible region. In some embodiments, the insertion is N-terminal or C-terminal to a flexible amino acid residue, e.g., with the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, wherein there is more than one preferred flexible amino acid (e.g., two G) in the flexible region, the insertion is made at the N-terminus or C-terminus of the preferred flexible amino acid residue that is closer to the peak amino acid residue. In some embodiments, wherein two preferred flexible amino acid residues (e.g., two G) are present in the flexible region and are the same distance from the peak amino acid residue, the insertion is made N-terminal or C-terminal to the preferred flexible amino acid residue with adjacent amino acid residues having a higher priority in terms of flexibility than adjacent amino acid residues with other preferred flexible amino acid residues (i.e., G > S > N > D > H > M > T > E > Q > K > R > a > P). In some embodiments, wherein there are two preferred flexible amino acid residues (e.g., two G) in the flexible region that are the same distance from the peak amino acid residue and their adjacent amino acid residues have the same priority in terms of flexibility, the insertion is made at the N-terminus or C-terminus of the preferred flexible amino acid residue that is closer to the N-terminus of the reference Cas nuclease. Glycine (G) is widely accepted as a flexible amino acid residue. In some embodiments, the one or more mutations of the flexible region include inserting one or more (e.g., 1, 2, 3, or more) G's in the flexible region. In some embodiments, the one or more mutations comprise insertion of two G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. Other flexible amino acid residues or groups of flexible amino acid residues are also contemplated, such as S, T, GS, SG, GGS and the like.
In some embodiments, the one or more mutations of the flexible region include replacing one or more amino acid residues (e.g., rigid amino acid residues) associated with a conformation having less flexibility with one or more amino acid residues (e.g., flexible amino acid residues) having a conformation having more flexibility. In some embodiments, the one or more mutations of the flexible region include substitution of hydrophobic amino acid residues in the flexible region with flexible amino acid residues. In some embodiments, the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the one or more mutations of the flexible region include substitution of two or more (e.g., 2, 3, or 4) hydrophobic amino acid residues in the flexible region with flexible amino acid residues. In some embodiments, the flexible amino acid residue is G, S, T, N, D, H, K or R. In some embodiments, the flexible amino acid residue is G, S or T. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. Combinations of insertions and substitutions in the flexible regions described herein are also contemplated if the mutation does not adversely affect folding and/or activity of the engineered Cas nuclease.
In some embodiments, the engineered Cas nuclease is a Cas effector protein of a class 1 or class 2 CRISPR-Cas system. In some embodiments, the engineered Cas nuclease is Sup>A Cas effector protein of type I-Sup>A, type I-B, type I-C, type I-D, type I-E, type I-F, type II-Sup>A, type II-B, type II-C, type III-Sup>A, type III-B, type IV-Sup>A, type IV-B, type V-Sup>A, type V-B, type V-F, type V-U3, type V-U4, type V-U2, type V-U1, type V-C, type V-D, type V-E, type V-U5, type V-G, type V-H, type V-I, type V-K, and type VI CRISPR-Cas system. See, e.g., makarova KS and Koonin EV, "Annotation and Classification of CRISPR-Cas Systems", methods mol. Biol.,2015,1311:47-75; markarova KS et al, "An updated evolutionary classification of CRISPR-Cas systems," Nat. Rev. Microbiol.2015,13:722-736; yan WX et al, "Functionally diverse type V CRISPR-Cas systems," Science,2019,363 (6422):88-91; and pinella-Redondo R.et al, "Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids," Nucleic Acids Research,2020,48 (4): 2000-2012, which are incorporated herein by reference in their entirety. In some embodiments, the engineered Cas nuclease is selected from the group consisting of: cas9, cas12a, cas12b, cas12c, cas12d, cas12f, cas12g, cas12h, cms1, cas12i, cas12j, cas12k and CasX. In some embodiments, the engineered Cas nuclease is Cas9. In some embodiments, the engineered Cas nuclease is Cas12i. In some embodiments, the engineered Cas nuclease is Cas12b.
The engineered Cas nuclease has increased activity as compared to the reference Cas nuclease. In some embodiments, the activity is a target DNA binding activity. In some embodiments, the activity is a site-specific nuclease activity. In some embodiments, the activity is double stranded DNA cleavage activity. In some embodiments, the activity is a single strand DNA cleavage activity, including, for example, a site-specific DNA cleavage activity or a non-specific DNA cleavage activity. In some embodiments, the activity is a single stranded RNA cleavage activity, e.g., a site-specific RNA cleavage activity or a non-specific RNA cleavage activity. In some embodiments, the activity is measured in vitro. In some embodiments, the activity is measured in a cell, such as a bacterial cell, a plant cell, or a eukaryotic cell. In some embodiments, the activity is measured in a mammalian cell, such as a rodent cell or a human cell. In some embodiments, the activity is measured in a human cell, such as a 293T cell. In some embodiments, the engineered Cas nuclease has a site-specific nuclease activity that is increased by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more as compared to a reference Cas nuclease. The site-specific nuclease activity of the engineered Cas nuclease can be measured using methods known in the art, including, for example, gel migration assays.
In some embodiments, the activity is a gene editing activity in a cell. In some embodiments, the cell is a bacterial cell, a plant cell, or a eukaryotic cell. In some embodiments, the cell is a mammalian cell, such as a rodent cell or a human cell. In some embodiments, the cell is a 293T cell. In some embodiments, the activity is an indel-forming activity of the target genomic site in the cell, e.g., site-specific cleavage of the target nucleic acid by the engineered Cas nuclease and DNA repair by a non-homologous end joining (NHEJ) mechanism. In some embodiments, the activity is insertion of an exogenous nucleic acid sequence at a target genomic site in the cell, e.g., site-specific cleavage of the target nucleic acid by the engineered Cas nuclease and DNA repair by Homologous Recombination (HR) mechanisms. In some embodiments, the engineered Cas nuclease increases gene editing (e.g., indel formation) activity at the genomic locus of a cell (e.g., a human cell such as a 293T cell) by at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more as compared to a reference Cas nuclease. In some embodiments, the engineered Cas nuclease increases gene editing (e.g., indel formation) activity of any one of at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more at a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) genomic sites of a cell (e.g., a human cell such as a 293T cell) as compared to a reference Cas nuclease. In some embodiments, the engineered Cas nuclease is capable of editing a greater number of genomic sites than the reference Cas nuclease. In some embodiments, the consensus PAM sequence of the engineered Cas nuclease is the same as the reference Cas nuclease.
The gene editing efficiency of an engineered Cas nuclease in a cell can be determined using methods known in the art, including, for example, T7 endonuclease 1 (T7E 1) assays, sequencing of target DNA (including, for example, sanger sequences, and second generation sequencing), insertion loss detection (IDAA) assays by split chase insertion loss (TIDE) assays, or by amplicon analysis. See, e.g., sentmaat MF et al, "A survey of validation strategies for CRISPR-Cas9 coding," Scientific Reports,2018,8, article No. 888, which is incorporated herein by reference in its entirety. In some embodiments, for example, as described in the examples herein, targeted second generation sequencing (NGS) is used to measure the gene editing efficiency of the engineered Cas nuclease in the cell. Exemplary genomic loci for determining the gene editing efficiency of the engineered Cas nuclease include, but are not limited to CCR5, AAVS, CD34, RNF2, and EMX1.
The present application also provides engineered Cas effector proteins based on any of the engineered Cas nucleases described herein. In some embodiments, the engineered Cas effector protein comprises a functional derivative of the engineered Cas nuclease, such as any one of the functional derivatives described in the "functional derivative" section below.
In some embodiments, the engineered Cas effector protein has site-specific nuclease activity. In some embodiments, the engineered Cas effector protein can induce a double strand break in a target DNA molecule. In some embodiments, the engineered Cas effector protein comprises an engineered Cas nuclease having enzymatic activity. In some embodiments, the engineered Cas effector protein is a Cas nickase that can induce single strand breaks in a target DNA molecule. In some embodiments, the engineered Cas effector protein comprises a nickase mutant of the engineered Cas nuclease.
In some embodiments, the engineered Cas effector protein comprises an enzyme-inactivating (i.e., losing enzyme activity) mutant of the engineered Cas nuclease. In some embodiments, the engineered Cas effector protein further comprises one or more functional domains fused to the engineered Cas nuclease or functional derivative thereof. In some embodiments, the engineered Cas effector protein comprises a functional domain fused to an enzymatically inactive mutant of the engineered Cas nuclease. In some embodiments, the functional domain is selected from the group consisting of: a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain.
In some embodiments, the engineered Cas effector protein comprises a split Cas polypeptide based on the engineered Cas nuclease or a functional derivative thereof. In some embodiments, the engineered Cas effector protein comprises: a first polypeptide comprising an N-terminal portion of the engineered Cas nuclease or a functional derivative thereof and a second polypeptide comprising a C-terminal portion of the engineered Cas nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a CRISPR complex that specifically binds to a target nucleic acid comprising a target sequence complementary to the guide sequence. In some embodiments, the split Cas effector protein is inducible, e.g., each split Cas polypeptide comprises a dimerization domain capable of associating with each other in the presence of an inducer (e.g., rapamycin). In some embodiments, the split Cas effector protein is self-induced, e.g., the split Cas polypeptide does not comprise a dimerization domain, and they are capable of associating with each other in the presence of a guide RNA.
Engineered Cas12b effector proteins
The present application provides engineered Cas12b effector proteins (e.g., cas12b nucleases, cas12b nickases, cas12b fusion effector proteins, split Cas12b effector proteins) with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity).
In some embodiments, an engineered Cas12b nuclease is provided that comprises one or more mutations that increase flexibility of the flexible region corresponding to amino acid residues 835-839, wherein the numbering of the amino acid residues is based on SEQ ID NO:1, wherein the engineered Cas12b nuclease has enhanced activity compared to a reference Cas12b nuclease. In some embodiments, the reference Cas12b nuclease is BhCas12b (e.g., bhCas12bv 4). In some embodiments, the one or more mutations include insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas12b nuclease comprises the amino acid sequence of SEQ ID No. 83. In some embodiments, the engineered Cas12b nuclease comprises an amino acid sequence that has at least about 85% (e.g., any of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the sequence of SEQ ID NO: 2.
In some embodiments, an engineered Cas12b nuclease is provided that comprises the amino acid sequence of SEQ ID No. 2.
SEQ ID NO. 1: bacillus exovillakii (Bacillus hisashii) Cas12b version 4 (BhCAs 12b-v 4) amino acid sequence
MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYGERSRFENSRLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCRVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM
SEQ ID NO. 2: enBhCAs12bv4 (BhCAs 12b-v4 1.1) amino acid sequence
MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPGGERSRFENSRLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCRVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM
The V-B type CRISPR-Cas12B (also known as C2C 1) system has been identified as a double RNA (i.e., crRNA and tracrRNA) -directed DNA endonuclease system with different features than Cas9 and Cas12a (Shmakov, s.et al mol. Cell 60,385-397 (2015)). First, cas12b reportedly produces cohesive ends in vitro away from the Protospacer Adjacent Motif (PAM) site upon double helical recombination with crRNA/tracrRNA. Second, although the RuvC domain of Cas12b is similar to Cas9 and Cas12a, its putative Nuc domain has no sequence or structural similarity to the HNH domain of Cas9 and the Nuc domain of Cas12 a. Furthermore, the Cas12b protein is smaller than the most widely used SpCas9 and Cas12a (e.g., aacCas12b:1,129 amino acids (aa); spCas9:1,369aa; asCas12a:1,353aa; lbCAS12a:1,228 aa), which makes Cas12b suitable for adeno-associated virus (AAV) -mediated in vivo delivery in gene therapy. Cas12b recognizes simpler PAM sequences (e.g., aacas 12b:5'-TTN-3' (SEQ ID NO: 3)) compared to small-sized Cas9 proteins such as SaCas9 and CjCas9, and significantly increases the targeting range of Cas12b in the genome compared to SaCas9:5'-NNGRRT-3' (SEQ ID NO: 4), cjCas9:5'-NNNNRYAC-3' (SEQ ID NO: 5). In addition, cas12b has minimal off-target effects and thus can be a safer choice for therapeutic and clinical applications.
Cas12b (C2C 1) nucleases from a variety of organisms can be used as reference Cas12b nucleases to provide the engineered Cas12b effector proteins of the present application. Exemplary Cas12b nucleases have been described, for example, in Shmakov, s.et al mol. Cell 60,385-397 (2015); shmakov, S.et al, nat. Rev. Microbiol.15,169-182 (2017); WO2016205764 and WO2020/087631 are incorporated herein by reference in their entirety.
In some embodiments, the engineered Cas12b effector protein is based on a reference Cas12b protein (e.g., cas12b nuclease) selected from the group consisting of: cas12b protein (AaCas 12 b) of alicyclobacillus acidophilus (Alicyclobacillus acidiphilus), cas12b (AkCas 12 b) of kakegawensis alicyclobacillus (Alicyclobacillus kakegawensis), cas12b (AmCas 12 b) of alicyclobacillus megaterium (Alicyclobacillus macrosporangiidus), cas12b (BhCas 12 b) of Bacillus exophthalmus (Bacillus hisashii), bsCas12b (Bacillus) of Bacillus, bs3Cas12b of Bacillus, cas12b (dicocas 12 b) of vibrio most desulphus (Desulfovibrio inopinatus), cas12b (LsCas 12 b) of ralstonia schwanani (Laceyella sediminis), cas12b (SbCas 12 b) of spirochete (Spirochaetes bacterium), cas12b (TcCas 12 b) of Bacillus megaterium (Tuberibacillus calidus) and functional derivatives thereof. The sequence of a naturally occurring Cas12b protein is known, for example, in UniProtKB IDs: T0D7A2, A0A6I3SPI6 and AOA6I7FUC4, the contents of which are incorporated herein by reference in their entirety.
In some embodiments, the reference Cas12b protein is Cas12b nuclease (BhCas 12 b) from bacillus cereus or a functional derivative thereof. In some embodiments, the engineered Cas12b effector protein is based on a reference Cas12b protein comprising an amino acid sequence having at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID No. 6. In some embodiments, the engineered Cas12b effector protein is based on a reference Cas12b nuclease comprising the amino acid sequence of SEQ ID No. 6. Exemplary flexible regions in BhCas12b and mutations that increase the flexibility of the flexible regions are shown in fig. 2-3.
In some embodiments, the reference Cas12b protein is a Cas12b nuclease (AaCas 12 b) from alicyclobacillus acidophilus or a functional derivative thereof. In some embodiments, the engineered Cas12b effector protein is based on a reference Cas12b protein comprising an amino acid sequence having at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID No. 7. In some embodiments, the engineered Cas12b effector protein is based on a reference Cas12b nuclease comprising the amino acid sequence of SEQ ID No. 7.
Note that orthologs or fragments thereof having some sequence identity (e.g., any of at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) to a reference Cas12b protein can be used as a basis for designing the engineered Cas12b effector proteins of the present application. The skilled artisan can determine the percent sequence identity of Cas12b orthologs or fragments thereof suitable for use in the present application based on the purpose and application. Methods for determining sequence identity values can be found in: computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988; biocomputing: informatics and Genome Projects, smith, d.w., ed., academic Press, new York,1993; computer Analysis of Sequence Data Part I, griffin, a.m., and Griffin, h.g., eds., humana Press, new Jersey,1994; sequence Analysis in Molecular Biology von Heinje, g., academic Press,1987; and Sequence Analysis Primer, gribskov, m.and deveeux, j., eds., M stock Press, new York,1991. Various orthologs of Cas12b are described in WO2020/087631, which application is incorporated herein by reference in its entirety.
Naturally occurring Cas12b nucleases have a variety of domains. In some embodiments, the reference Cas12b nuclease comprises from N-terminus to C-terminus: a first WED domain (WED-1; also known as an OBD-1 domain), a first REC domain (REC 1), a second WED domain (WED-II; also known as an OBD-II domain), a first RuvC domain (RuvC-I), a bridge-helix (BH) domain, a second RuvC domain (RuvC-II), a first Nuc domain (Nuc-I; also known as a UK-I domain), a third RuvC domain (RuvC-III) and a second Nuc domain (Nuc-II; also known as a UK-II domain). Domain boundaries can be determined using methods known in the art, such as based on the crystal structure of the reference Cas12b nuclease (e.g., PDB ID Nos:5U30, 5U31, 5U33, 5U34, and 5WQE for AaCas12 b) and/or sequence homology to known functional domains in the reference Cas12b nuclease. In some embodiments, aaCas12b has the following domains: a WEB-I domain (amino acid residues 1-14), a REC1 domain (amino acid residues 15-386), a WED-II domain (amino acid residues 387-518), a RuvC-I domain (amino acid residues 519-628), a BH domain (amino acid residues 629-658), a REC2 domain (amino acid residues 659-784), a RuvC-II domain (amino acid residues 785-900), a Nuc-I domain (amino acid residues 901-974), a RuvC-III domain (amino acid residues 975-993), and a Nuc-II domain (amino acid residues 994-1129), wherein the amino acid numbering is based on SEQ ID NO 7.
The crystal structure of alicyclobacillus acidoterrestris (Alicyclobacillus acidoterrestris) Cas12b bound to sgrnas as a binary complex and to target DNA as a ternary complex has been described in Yang h., et al cell 167:1814-1828 (2016) and Liu l.et al mol. Cell 65:310-322 (2017). Briefly, the crystal structure shows 2 discrete REC (recognition region, residues 15-386, 658-784) and NUC (nuclease, residues 1-14, 387-658 and 785-1129) leaves (lobe, consisting of multiple domains). crRNA (or single guide RNA, sgRNA) binds in the central channel between two leaves. PAM recognition is sequence specific and occurs primarily through interactions with REC1 (helix-1) and WED-II (OBD-II) domains. The sgRNA-target DNA heteroduplex binds to REC leaves mainly in a sequence independent manner.
In some embodiments, the engineered Cas12b nuclease is based on a functional variant of a naturally occurring Cas12b nuclease. In some embodiments, the functional variants have one or more mutations, such as amino acid substitutions, insertions, and deletions. For example, the functional variant can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions as compared to the wild-type naturally occurring Cas12b nuclease. In some embodiments, the one or more substitutions is a conservative substitution. In some embodiments, the functional variant has all domains of a naturally occurring Cas12b nuclease. In some embodiments, the functional variant does not have one or more domains of a naturally occurring Cas12b nuclease.
Also provided are engineered Cas12b effector proteins based on any of the engineered Cas12b nucleases described herein. In some embodiments, the engineered Cas12b effector protein has enzymatic activity. In some embodiments, the engineered Cas12b effector protein is a nuclease that cleaves both strands of a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the engineered Cas12b effector protein is a nickase, i.e., a single strand that cleaves a target double-helical nucleic acid (e.g., double-helical DNA). In some embodiments, the engineered Cas12b effector protein comprises an enzyme-inactivating mutant of the engineered Cas12b nuclease. Mutations in one or more amino acid residues in the Cas12b nuclease active site result in Cas12b that loses enzymatic activity. For example, the R785A, R911A or D977A mutant of AaCas12b has no nuclease activity in human cells. See, e.g., teng f.et al, cell Discovery,4, article No.: 63 (2018), which is incorporated herein by reference in its entirety. In some embodiments, the engineered Cas12b effector protein comprises an engineered Cas12b having a mutation corresponding to the R785A mutation of AaCas12 b. In some embodiments, the engineered Cas12b effector protein comprises an engineered Cas12b having one or more mutations corresponding to R785A, R911A or D977A of AaCas12 b. In some embodiments, the engineered Cas12b effector protein comprises an engineered Cas12b having a mutation corresponding to the R911A mutation of AaCas12 b. In some embodiments, the engineered Cas12b effector protein comprises an engineered Cas12b having a mutation corresponding to the D977A mutation of AaCas12 b.
In some embodiments, an engineered Cas12b nickase is provided. In some embodiments, engineered Cas12b fusion effector proteins are provided that comprise an engineered Cas12b nuclease or a functional derivative thereof (e.g., an enzyme-inactivating mutant of an engineered Cas12b nuclease) fused to a functional domain, such as a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain. In some embodiments, an engineered split Cas12b effector protein is provided. Split Cas12b effector proteins have been described, for example, in PCT/CN2020/111057, which is incorporated herein by reference in its entirety.
Also provided are engineered CRISPR-Cas12b systems comprising any of the engineered Cas12b effector proteins described herein, and a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas12b effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising a target sequence. In some embodiments, the guide RNA comprises crRNA and tracrRNA. In some embodiments, the guide RNA is sgRNA. In some embodiments, the guide RNA is a precursor molecule that can be processed into crRNA and tracrRNA. In some embodiments, the guide RNA is a precursor RNA array encoding a plurality of crrnas, and wherein each processed crRNA is associated with a tracrRNA. In some embodiments, the engineered Cas12b effector protein and/or the guide RNA is encoded by one or more vectors, such as AAV vectors. In some embodiments, the engineered CRISPR-Cas12b system is a Ribonucleoprotein (RNP) complex comprising the engineered Cas12b effector protein bound to the guide RNA.
Engineered Cas12i effector proteins
The present application provides engineered Cas12i effector proteins with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity).
In some embodiments, an engineered Cas12i nuclease is provided that comprises one or more mutations that increase flexibility of a flexible region in a reference Cas12i nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 228-232, amino acid residues 439-443, amino acid residues 478-482, amino acid residues 500-504, amino acid residues 775-779, and amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID No. 8, wherein the engineered Cas12i nuclease has increased activity compared to a reference Cas12i nuclease. In some embodiments, the flexible region corresponds to amino acid residues 439-443 or amino acid residues 925-929, wherein the amino acid residue numbering is based on SEQ ID NO. 8. In some embodiments, the reference Cas12i nuclease is Cas12i2. In some embodiments, the one or more mutations comprise insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas12i nuclease comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs 96-105. In some embodiments, the engineered Cas12i nuclease comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs 99 and 104-105. In some embodiments, the engineered Cas12i nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 8.
In some embodiments, an engineered Cas12i2 nuclease comprising the amino acid sequence of SEQ ID No. 99 is provided. In some embodiments, the engineered Cas12i2 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 14. In some embodiments, an engineered Cas12i nuclease comprising the amino acid sequence of SEQ ID No. 14 is provided.
In some embodiments, an engineered Cas12i2 nuclease comprising the amino acid sequence of SEQ ID No. 104 is provided. In some embodiments, the engineered Cas12i2 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 19. In some embodiments, an engineered Cas12i nuclease comprising the amino acid sequence of SEQ ID No. 18 is provided.
In some embodiments, an engineered Cas12i2 nuclease comprising the amino acid sequence of SEQ ID No. 105 is provided. In some embodiments, the engineered Cas12i2 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 18. In some embodiments, an engineered Cas12i nuclease comprising the amino acid sequence of SEQ ID No. 19 is provided.
In some embodiments, an engineered Cas12i2 nuclease is provided that comprises the amino acid sequence of SEQ ID No. 99 and the amino acid sequence of SEQ ID No. 104. In some embodiments, the engineered Cas12i2 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 20. In some embodiments, an engineered Cas12i nuclease comprising the amino acid sequence of SEQ ID No. 20 is provided.
SEQ ID NO. 8: wild Cas12i2 amino acid sequence
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO. 14: cas12i2-2.2 amino acid sequence MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGGGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO. 18: cas12i2-6.1 amino acid sequence
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO. 19: cas12i2-6.2 amino acid sequence
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
SEQ ID NO. 20: enCas12i2 (Cas 12i 2-2.2+6.1) amino acid sequence MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGGGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNGGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS
Type V-I CRISPR-Cas12I has been identified as an RNA-guided DNA endonuclease system. Unlike CRISPR-Cas systems such as Cas12b or Cas9, cas12 i-based CRISPR systems do not require a tracrRNA sequence. In some embodiments, the RNA guide sequence comprises crRNA. Typically, crrnas described herein include direct repeat sequences and spacer sequences. In certain embodiments, the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence. In some embodiments, the crrnas include direct repeat sequences, spacer sequences, and direct repeat sequences (DR-spacer-DR), which are typical features of precursor crRNA (pre-crRNA) configurations in other CRISPR systems. In some embodiments, the crRNA includes truncated direct repeat sequences and spacer sequences, which are typical features of processed or mature crrnas. In some embodiments, the CRISPR-Cas effect protein forms a complex with an RNA guide sequence, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid that is complementary to the spacer sequence.
In some embodiments, the engineered Cas12i of the present application is an endonuclease that binds to a specific site of a target sequence and cleaves under the direction of a guide RNA, and has DNA and RNA endonuclease activity. In some embodiments, the Cas12i is capable of autonomous crRNA biogenesis by processing a precursor crRNA array. Autonomous precursor crRNA processing can facilitate delivery of Cas12i, enabling a double nick application, as two separate genomic sites can be targeted by a single crRNA transcript. The Cas12i protein then processes the CRISPR array into two homologous crrnas, forming a paired nick complex. Multiplexing (Multiplexing) of type V-I (Cas 12I) effector proteins is done using the precursor crRNA processing capabilities of the effector proteins, where multiple targets with different sequences can be programmed on a single RNA guide sequence. In this way, multiple genes or DNA targets can be manipulated simultaneously for therapeutic applications. In some embodiments, the guide RNA comprises a precursor crRNA expressed by a CRISPR array consisting of target sequences interleaved with unprocessed DR sequences, repeated by the intrinsic precursor crRNA processing of the effector protein to enable simultaneous targeting of one, two or more sites.
In some embodiments, a type VI CRISPR-Cas effect protein is capable of recognizing or consists of a Protospacer Adjacent Motif (PAM), and the target nucleic acid comprises or consists of the nucleic acid sequence 5'-TTN-3' (SEQ ID NO: 21), 5'-TTH-3' (SEQ ID NO: 22), 5'-TTY-3' (SEQ ID NO: 23), or 5'-TTC-3' (SEQ ID NO: 24).
Cas12i nucleases from a variety of organisms can be used as the reference Cas12i nuclease to provide the engineered Cas12i effector proteins of the present application. Exemplary Cas12i nucleases have been described in, for example, WO2019/201331A1 and US2020/0063126A1, which are incorporated herein by reference in their entirety. In some embodiments, the reference Cas12i protein has enzymatic activity. In some embodiments, the reference Cas12i is a nuclease, i.e., cleaves both strands of a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the reference Cas12i is a nickase, i.e., a single strand that cleaves a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the reference Cas12i protein is enzyme inactivated. In some embodiments, the reference Cas12i nuclease is Cas12i1 (e.g., SEQ ID NO: 9), cas12i2 (e.g., SEQ ID NO: 8), or Cas12i-Phi (e.g., SEQ ID NO: 10). In some embodiments, the reference Cas12i nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 8-10. Orthologs having a certain sequence identity (e.g., at least about any of 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) to Cas12i or a functional derivative thereof may be used as a basis for designing the engineered Cas12i effector proteins of the present application.
In some embodiments, the engineered Cas12i protein is based on a functional variant of a naturally occurring Cas12i protein. In some embodiments, the functional variants have one or more mutations, such as amino acid substitutions, insertions, and deletions. For example, the functional variant can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions as compared to the wild-type naturally occurring Cas12i protein. In some embodiments, the one or more substitutions is a conservative substitution. In some embodiments, the functional variant has all domains of a naturally occurring Cas12i protein. In some embodiments, the functional variant does not have one or more domains of a naturally occurring Cas12i protein.
Also provided are engineered Cas12i effector proteins based on any of the engineered Cas12i nucleases described herein. In some embodiments, the engineered Cas12i effector protein has enzymatic activity. In some embodiments, the engineered Cas12i effector protein is a nuclease that cleaves both strands of a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the engineered Cas12i effector protein is a nickase, i.e., a single strand that cleaves a target double-helical nucleic acid (e.g., double-helical DNA). In some embodiments, the engineered Cas12i effector protein comprises an enzyme-inactivating mutant of the engineered Cas12i nuclease. Mutations in one or more amino acid residues in the Cas12i nuclease active site result in Cas12i losing enzymatic activity. In some embodiments, the engineered Cas12i enzymes provided herein can be modified to have reduced nuclease activity, e.g., the nuclease is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% inactivated as compared to the wild-type Cas12i enzyme. The nuclease activity can be reduced by several methods, for example, introducing mutations into the PAM interaction domain of the nuclease or Cas12i enzyme. In some embodiments, catalytic residues of nuclease activity are identified, and these amino acid residues can be replaced with different amino acid residues (e.g., glycine or alanine) to reduce the nuclease activity. Examples of such mutations of Cas12i1 include D647A, E894A or D948A. Examples of such mutations of Cas12i2 include D599A, E833A or D886A.
In some embodiments, an engineered Cas12i nickase is provided. In some embodiments, an engineered Cas12i fusion effector protein is provided that comprises an engineered Cas12i nuclease or a functional derivative thereof (e.g., an enzyme-inactivating mutant of the engineered Cas12i nuclease) fused to a functional domain, such as a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain. In some embodiments, an engineered Cas12i base editor is provided that comprises a catalytically inactive variant of any of the engineered Cas12i nucleases described herein (e.g., enCas12i2 or SEQ ID NO: 20) fused to a cytosine deaminase domain or an adenosine deaminase domain. In some embodiments, an engineered Cas12i master editor is provided that comprises a catalytically inactive variant of any of the engineered Cas12i nucleases described herein (e.g., enCas12i2 or SEQ ID NO: 20) fused to a reverse transcriptase domain. In some embodiments, an engineered split Cas12i effector protein is provided.
Also provided are engineered CRISPR-Cas12i systems comprising any of the engineered Cas12i effector proteins described herein, and a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas12i effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising a target sequence. In some embodiments, the guide RNA comprises crRNA. In some embodiments, tracrRNA is not required. In some embodiments, the guide RNA comprises a precursor crRNA expressed from a CRISPR array consisting of target sequences interleaved with unprocessed DR sequences, repeated by intrinsic precursor crRNA processing of the effector protein, to enable simultaneous targeting of one, two or more sites. In some embodiments, the guide RNA is a precursor RNA array encoding a plurality of crrnas. In some embodiments, the engineered Cas12i effector protein and/or the guide RNA is encoded by one or more vectors, such as AAV vectors. In some embodiments, the engineered CRISPR-Cas12i system is a Ribonucleoprotein (RNP) complex comprising an engineered Cas12i effector protein that binds to the guide RNA.
Engineered Cas9 effector proteins
The present application provides engineered Cas9 effector proteins with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity).
In some embodiments, an engineered Cas9 nuclease is provided that comprises one or more mutations that increase flexibility of a flexible region in a reference Cas9 nuclease, the flexible region selected from the group of regions corresponding to: amino acid residues 39-43, amino acid residues 135-139, amino acid residues 176-180, amino acid residues 274-278, amino acid residues 351-355, amino acid residues 389-393, amino acid residues 521-525, amino acid residues 541-545, amino acid residues 755-759, amino acid residues 774-778, amino acid residues 786-790, amino acid residues 811-815, amino acid residues 848-852, amino acid residues 855-859, amino acid residues 874-878, amino acid residues 891-895, amino acid residues 1019-1023, and amino acid residues 1036-1040, wherein the amino acid residue numbering is based on SEQ ID NO:25, and wherein the engineered Cas9 nuclease has enhanced activity compared to the reference Cas9 nuclease. In some embodiments, the reference Cas9 nuclease is GeoCas9. In some embodiments, the flexible region in the reference Cas9 nuclease is selected from the group of regions corresponding to: amino acid residues 135-139, amino acid residues 176-180, amino acid residues 541-545, amino acid residues 755-759, and amino acid residues 811-815, wherein the amino acid residue numbering is based on SEQ ID NO. 25. In one embodiment, the one or more mutations comprise the insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas9 nuclease comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs 138-175. In some embodiments, the engineered Cas9 nucleases comprise one or more sequences selected from the group consisting of SEQ ID NOs: 139. 140, 145-146, and 153. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 25.
In some embodiments, provided is an engineered GeoCas9 nuclease comprising the amino acid sequence of SEQ ID NO 139. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 27. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO:27 is provided.
In some embodiments, an engineered GeoCas9 nuclease comprising the amino acid sequence of SEQ ID NO. 140 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 29. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID No. 28 is provided.
In some embodiments, an engineered GeoCas9 nuclease comprising the amino acid sequence of SEQ ID NO:145 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 33. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO 33 is provided.
In some embodiments, an engineered GeoCas9 nuclease comprising the amino acid sequence of SEQ ID NO. 146 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 34. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO 34 is provided.
In some embodiments, an engineered GeoCas9 nuclease comprising the amino acid sequence of SEQ ID NO. 153 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 40. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID No. 41 is provided.
SEQ ID NO. 25: wild GeoCas9 amino acid sequence
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO. 27: geoCas9-2.1 amino acid sequence
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERGGSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO. 28: geoCas9-3.1 amino acid sequence
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGGGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO. 33: geoCas9-8.1 amino acid sequence
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGGGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO. 34: geoCas9-9.1 amino acid sequence MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHGPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD
SEQ ID NO. 41: geoCas9-12.1 amino acid sequence
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAAIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQPVFVSRMPKRSGTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETVRPLQSTRD in some embodiments, an engineered Cas9 nuclease is provided that comprises one or more mutations that increase the flexibility of a flexible region in a reference Cas9 nuclease selected from the group of regions corresponding to: amino acid residues 45-49, amino acid residues 84-88, amino acid residues 116-120, amino acid residues 128-132, amino acid residues 216-220, amino acid residues 318-322, amino acid residues 387-391, amino acid residues 497-501, amino acid residues 583-587, amino acid residues 594-598, amino acid residues 614-618, amino acid residues 696-700, and amino acid residues 739-743, wherein the amino acid residue numbering is based on SEQ ID NO:53, and wherein the engineered Cas9 nuclease has increased activity compared to a reference Cas9 nuclease. In some embodiments, the flexible region in the reference Cas9 nuclease corresponds to amino acid residues 45-49 or amino acid residues 116-120, wherein the amino acid residue numbering is based on SEQ ID No. 53. In some embodiments, the reference Cas9 nuclease is SaCas9. In some embodiments, the one or more mutations comprise insertion of one or more (e.g., 2) G residues in the flexible region. In some embodiments, the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. s, N, D, H, M, T, E, Q, K, R, A and P. In some embodiments, the flexible amino acid residues are selected according to the following priorities: g > S > N > D > H > M > T > E > Q > K > R > A > P. In some embodiments, the one or more mutations comprise a substitution of a hydrophobic amino acid residue in the flexible region with a G residue, wherein the hydrophobic amino acid residue is selected from the group consisting of: l, I, V, C, Y, F and W. In some embodiments, the engineered Cas9 nuclease comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs 199-217. In some embodiments, the engineered Cas9 nucleases comprise one or more sequences selected from the group consisting of SEQ ID NOs: 199. 203 and 204. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:53.
In some embodiments, an engineered SaCas9 nuclease comprising the amino acid sequence of SEQ ID NO 199 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 54. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO:54 is provided.
In some embodiments, an engineered SaCas9 nuclease comprising the amino acid sequence of SEQ ID NO. 203 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID No. 58. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO:58 is provided.
In some embodiments, an engineered SaCas9 nuclease comprising the amino acid sequence of SEQ ID NO:204 is provided. In some embodiments, the engineered Cas9 nuclease comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 59. In some embodiments, an engineered Cas9 nuclease comprising the amino acid sequence of SEQ ID NO:59 is provided.
SEQ ID NO. 53: wild SaCas9 amino acid sequence
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO. 54: saCas9-1.1 amino acid sequence
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGGGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO. 58: saCas9-3.1 amino acid sequence
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGGHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
SEQ ID NO 59: saCas9-3.2 amino acid sequence
MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGGGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGS
The type II CRISPR-Cas9 system is a double RNA-guided (i.e., crRNA and tracrRNA) DNA endonuclease system. Mature crrnas base-paired with transactivation crRNA (tracrRNA) form a double RNA structure that directs CRISPR-associated protein Cas9 to introduce double-strand (ds) breaks in target DNA. At the site complementary to the crRNA guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, while the Cas9 RuvC-like domain cleaves the non-complementary strand. When the double-tracrRNA crRNA is engineered into a single RNA chimera, it also directs sequence-specific Cas9 dsDNA cleavage. DNA-targeting RNAs (also referred to herein as "crRNAs"; or "guide RNAs"; or "gRNAs") include: i) A first fragment comprising a nucleotide sequence complementary to a target sequence in a target DNA; ii) a second fragment which interacts with a site-directed polypeptide; iii) Transcription terminators.
Cas9 nucleases from a variety of organisms can be used as the reference Cas9 nuclease to provide the engineered Cas9 effector proteins of the present application. Exemplary Cas9 proteins have been described, for example, in US8697359, US10266850, and US20170145425, which are incorporated herein by reference in their entirety.
In some embodiments, the engineered Cas9 effector protein is based on a reference Cas9 protein (e.g., cas9 nuclease) selected from Cas9 proteins from the following bacteria: streptococcus pneumoniae (Streptococcus pneumoniae) (Csn 1), streptococcus pyogenes (Streptococcus pyogenes) (SpCas 9) or streptococcus thermophilus (Streptococcus thermophiles) (StCas 9), staphylococcus aureus (Staphylococcus aureus) (SaCas 9), neisseria meningitidis (Neisseria meningitides) (Nm 2Cas 9), campylobacter jejuni (Campylobacter jejuni) (CjCas 9), geobacillus stearothermophilus (Geobacillus stearothermophilus) (GeoCas 9) and treponema (Treponema denticola) (TdCas 9), and may include mutations Cas9 derived from these organisms. In some embodiments, the reference Cas9 protein may have desirable properties for certain applications, such as targeting thermophilic bacteria. For example, geoCas9 is active at temperatures up to 70 ℃ compared to 45 ℃ for streptococcus pyogenes Cas9 (SpCas 9). In some embodiments, the reference Cas9 nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 25, 53, or 72-73. Orthologs having a certain sequence identity (e.g., at least about any of 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) to Cas9 or a functional derivative thereof may be used as a basis for designing the engineered Cas9 effector proteins of the present application.
Naturally occurring Cas9 nucleases have a variety of domains. In some embodiments, the reference Cas9 nuclease comprises a domain architecture having a central HNH endonuclease domain and a split RuvC/RNaseH domain. In some embodiments, the reference Cas9 nuclease comprises 4 key motifs with conserved architecture. Motifs 1, 2 and 4 are RuvC-like motifs, while motif 3 is HNH motif. Domain boundaries can be determined using methods known in the art, such as based on the crystal structure of the reference Cas9 nuclease (e.g., PDB ID Nos:5CZZ, 4OGC, 5X2G, 6 JOO).
Also provided are engineered Cas9 effector proteins based on any of the engineered Cas9 nucleases described herein. In some embodiments, the engineered Cas9 effector protein has enzymatic activity. In some embodiments, the engineered Cas9 effector protein is a nuclease that cleaves both strands of a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the engineered Cas9 effector protein is a nickase, i.e., a single strand that cleaves a target duplex nucleic acid (e.g., duplex DNA). For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves one single strand). Other examples of mutations that make Cas9 a nickase include, but are not limited to: H840A, N854A and N863A. In some embodiments, the engineered Cas9 effector protein comprises an enzyme-inactivating mutant of the engineered Cas9 nuclease. Mutations in one or more amino acid residues in the Cas9 nuclease active site can result in Cas9 enzyme inactivation. For example, two or more catalytic domains of Cas9 (RuvC I, ruvC II, and RuvC III) can be mutated to produce a mutated Cas9 that lacks substantially all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A or N863A mutations to produce a Cas9 enzyme that lacks substantially all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to lack substantially all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less relative to its non-mutant form. Other mutations may be useful; if the Cas9 or other CRISPR enzyme is from a species other than streptococcus pyogenes, the corresponding amino acid can be mutated to achieve a similar effect.
In some embodiments, an engineered Cas9 nickase is provided. In some embodiments, an engineered Cas9 fusion effector protein is provided that comprises an engineered Cas9 nuclease or a functional derivative thereof (e.g., an enzyme-inactivating mutant of an engineered Cas9 nuclease) fused to a functional domain, such as a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), or a nuclease domain. In some embodiments, an engineered split Cas9 effector protein is provided. Split Cas9 effector proteins have been described, for example, in WO2016/112242, which is incorporated herein by reference in its entirety.
Also provided are engineered CRISPR-Cas9 systems comprising any of the engineered Cas9 effector proteins described herein, and a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas9 effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence. In some embodiments, the guide RNA comprises crRNA and tracrRNA. In some embodiments, the guide RNA is sgRNA. In some embodiments, the guide RNA is a precursor molecule that can be processed into crRNA and tracrRNA. In some embodiments, the guide RNA is a precursor RNA array encoding a plurality of crrnas, and wherein each processed crRNA is associated with a tracrRNA. In some embodiments, the engineered Cas9 effector protein and/or the guide RNA is encoded by one or more vectors, such as AAV vectors. In some embodiments, the engineered CRISPR-Cas9 system is a Ribonucleoprotein (RNP) complex comprising the engineered Cas9 effector protein bound to the guide RNA.
Other engineered Cas effector proteins
The present application provides engineered Cas effector proteins (e.g., cas nucleases, cas nickases, cas fusion effector proteins, split Cas effector proteins) with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity). Suitable reference Cas proteins for engineering may include, for example: casl, caslB, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9, csnl, csxl2, caslO, csyl, csy2, csy3, csel, cse2, cscl, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmrl, cmr3, cmr4, cmr5, cmr6, csbl, csb2, csb3, csxl7, csxl4, csxlO, csxl6, csaX, csx3, csxl5, csfl, csf2, csf3, csf4, cpfl (also known as Casl2 a), casX (Cas 12 e), 12c, cas12d, cas12g, cas12k, 12i, cas12f, 12j, cas12w, cas12v, casX, casY, casZ, or Cas equivalent thereof. For a description of exemplary Cas reference proteins, see review articles: zhang F (2019), development of CRISPR-Cas systems for genome editing and beyond. Quantily Reviews of Biophysics, e6,1-31; li Y & Peng N.endogenous CRISPR-Cas system-based genome editing and antimicrobials: review and procopys. Front in microbiology 10 (2019): 2471.
In some embodiments, the reference Cas nuclease is Cas12a, cas12b (e.g., any one of the reference proteins described in the section "engineered Cas12b effector protein"), cas9 (e.g., any one of the reference proteins described in the section "engineered Cas9 effector protein"), cas12i (e.g., any one of the reference proteins described in the section "engineered Cas12i effector protein"), cas12f, cas12j, or CasX (Cas 12 e).
In some embodiments, the reference protein is Sup>A Cas12 Sup>A protein of the V-Sup>A type CRISPR-Cas system (previously referred to as Cpf 1). The V-A system does not require tracrrnSup>A and can simplify the design of guide RNA. Cas12a (Cpf 1) nucleases from a variety of organisms can be used as reference Cas12a nucleases to provide the engineered Cas12a effector proteins of the present application. Exemplary Cas12a nucleases have been described in, for example, US10648020; US10669540; US9790490; US20180282713 and WO2018188571, which are incorporated herein by reference in their entirety.
In some embodiments, the engineered Cas12a effector protein is based on a reference Cas12a protein (e.g., cas12a nuclease) selected from Cas12a proteins from the following species: prevolvulella (Prevoltella) and Francisella (Francisella) such as Francisella neo (Francisella novicida) (FnCas 12a or FnCpf 1), or amino acid coccus (Acidomicrococcus) (AsCas 12a or AsCpf 1) or Lachnospiraceae (LbCAs 12a or LbCPf1 p). In some embodiments, the reference Cas12a nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 74-75. Orthologs having a certain sequence identity (e.g., at least about any of 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) to Cas12a or a functional derivative thereof may be used as a basis for designing Cas12a effector proteins engineered in the present application.
The crystal structure of the amino acid coccus Cas12 a-crRNA-target DNA complex has been described in Yamano T, nishimasu H, zetsche B, et al crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell.2016;165:949-962, available as PDB code 5B 43. The crystal structure of LbCas12a (LbCpf 1) may also be obtained (e.g., PDB codes 5XUU, 5XH6, and 5 XUT).
In some embodiments, the engineered Cas12a effector protein comprises one or more mutations that reduce or eliminate nuclease activity. For example, suitable mutated amino acid positions in the FnCpf1 RuvC domain include, but are not limited to: D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257A. In some embodiments, the point mutation that substantially reduces nuclease activity comprises a mutation in a putative second nuclease domain, such as N580A, N584A, T587A, W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A and Y629A. In another example, mutations in the AsCpf1p RuvC domain are D908A, E993A and D1263A, wherein the D908A, E993A and D1263A mutations completely inactivate the DNA cleavage activity of the AsCpf1 effector protein. In some embodiments, mutations in the LbCpf1pRuvC domain include, but are not limited to 832, 947, or 1180. In some embodiments, the mutation in the LbCpf1 RuvC domain is LbD832A, E925A, D947A or D1180A, wherein the LbD832A, E925A, D947A or D1180A mutation completely inactivates the DNA cleavage activity of the Cas12a protein. Mutations at positions in other engineered Cas12a corresponding to those described for FnCpf1 and LbCpf1 are contemplated herein.
In some embodiments, the reference protein is a CasX (also known as Cas12 e) protein. CasX nucleases from a variety of organisms can be used as the reference CasX nuclease to provide the engineered CasX effector proteins of the present application. In some embodiments, the engineered CasX protein of the methods and/or compositions of the invention is (or is derived from) a naturally occurring (wild-type) protein. Exemplary CasX nucleases have been described, for example, in US10570415, WO2018/202800 and WO2019/084148, which are incorporated herein by reference in their entirety.
In some embodiments, the engineered CasX effector protein is based on a reference CasX protein (e.g., casX nuclease) selected from the group consisting of CasX proteins from delta-proteobacteria (deltaproteobacteria) (DpbCasX or CasX 1) and superficial fungi (plantamycin) (plmcassx or CasX 2). In some embodiments, the reference CasX nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 76-77. Orthologues having a certain sequence identity (e.g., at least about any of 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) to CasX or a functional derivative thereof may be used as a basis for designing the CasX effector proteins engineered herein.
In some embodiments, the CasX protein is short compared to the CRISPR-Cas endonuclease previously identified, and thus the use of the protein as a surrogate provides the advantage that the nucleotide sequence encoding the protein is relatively short. For example, this is useful where a nucleic acid encoding the engineered CasX protein is desired, such as where a viral vector (e.g., AAV vector) is employed for delivery to a cell such as a eukaryotic cell (e.g., mammalian cell, human cell, mouse cell, in vitro, ex vivo, in vivo) for research and/or clinical application. It should also be noted that bacteria with naturally occurring CasX CRISPR sites are present in environmental samples collected at low temperature (e.g. 10-17 ℃). Thus, it is expected that CasX will perform well at low temperatures (e.g., 10-14 ℃, 10-17 ℃, 10-20 ℃).
Upon analysis of the frozen EM structure of the DpbCAsX-sgRNA-DNA complex (PDB codes 6NY2, 6NY1 and 6NY 3), non-target binding (NTSB) and Target Strand Loading (TSL) domains were found (Liu-J-J, orlovaN, oakesBL, et al, casX enzymes comprise a distinct family of RNA-guide genome editors. Nature.2019; 566:218-223). The position of the TSL domain is similar to that of the so-called "Nuc" domain in Cas12a (PDB 5B 43). These domains perform similar functions in CasX and Cas12a enzymes: after the non-target DNA strands are cleaved by RuvC domains, they bend the sgRNA-DNA duplex. This conformational change allows the target DNA strand to be cleaved by the RuvC domain. Thus, both Cas12e and Cas12a rely on a single nuclease domain for double-stranded DNA cleavage, in contrast to Cas9 which uses different domains HNH and RuvC to cleave each DNA strand. In Cas12e and Cas12a, the larger structural changes change the accessibility of the DNA strand to RuvC nuclease and in this way complements the lack of a second nuclease domain.
Cas12a and Cas12X both produce a product with an adhesive end. In contrast, cas9 proteins produce predominantly blunt ends. Notably, liu Junjie (Jun-Jie Liu) et al found that the cohesive ends produced by DpbCasX were approximately 10 nucleotides long, which were longer than the 3-5nt overhangs typically produced by Cas12a proteins. The 5' -overhangs produced by Cas12a and CasX can be used to insert DNA fragments into the genome by direct DNA ligation in vivo or in vitro.
In contrast to Cas12a, casX enzymes require tracrRNA in addition to crRNA for DNA target recognition.
In some embodiments, the reference Cas nuclease is Cas12f. Exemplary Cas12f nucleases have been disclosed in, for example, WO 2020088450. In some embodiments, the reference Cas nuclease is Cas12i. Exemplary Cas12i nucleases have been disclosed in, for example, WO 2020098772.
In some embodiments, the reference Cas nuclease is Cas12j (e.g., SEQ ID NO: 78).
Variants
The present application provides engineered Cas effector proteins comprising functional variants of the engineered Cas nucleases described herein. In some embodiments, the amino acid sequence of the functional variant has a difference (e.g., has a deletion, insertion, substitution, and/or fusion) of at least one amino acid residue when compared to the amino acid sequence of the corresponding engineered Cas nuclease. In some embodiments, the functional variants have one or more mutations, such as amino acid substitutions, insertions, and/or deletions. For example, a functional variant can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions as compared to an engineered Cas nuclease. In some embodiments, the one or more substitutions is a conservative substitution. In some embodiments, the functional variant has all domains of an engineered Cas nuclease. In some embodiments, the functional variant does not have one or more domains of an engineered Cas nuclease.
For any of the Cas variant proteins described herein (e.g., nickase Cas protein, inactivated or catalytically inactivated Cas (dCas), fused Cas), the Cas variants can include Cas protein sequences having the same parameters (e.g., domains present, percent identity, etc.) described above.
Catalytic Activity
In some embodiments, the functional variant has a different catalytic activity than the non-mutated version of its engineered Cas nuclease. In some embodiments, the mutation (e.g., amino acid substitution, insertion, and/or deletion) is in a catalytic domain (e.g., ruvC domain) of the Cas effect protein. In some embodiments, the variants comprise mutations in multiple catalytic domains. Cas effector proteins that cleave one strand of a double-stranded target nucleic acid without cleaving the other strand are referred to herein as "nickases" (e.g., "nickases Cas"). Herein, a Cas protein having substantially no nuclease activity is referred to as an inactivated Cas protein ("dCas") (stating that in the case of fusion Cas effect proteins, a heterologous polypeptide (fusion partner) can provide nuclease activity, as will be described in detail below). In some embodiments, a Cas effector protein is considered to lack substantially all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less relative to its non-mutant form.
Exemplary mutations of Cas functional variants are described in Cas12b, cas12i and Cas9 subsections described above, as well as WO2016205764, WO2020/087631, WO2019/201331A1, US2020/0063126A1, US8697359, US10266850, US20170145425, US10648020, US10669540, US9790490, US20180282713, WO2018188571, US10570415, WO2018/202800 and WO2019/084148, which are incorporated herein by reference in their entirety.
Split Cas effector proteins
The present application also provides split Cas effector proteins based on any of the engineered Cas effector proteins described herein. Split Cas effector proteins may be advantageous for delivery. In some embodiments, the engineered Cas effector protein is split into two portions of the enzyme, which can be reconstituted together to provide a substantially functional Cas effector protein. Split versions of Cas effector proteins (e.g., cas12 and Cas9 proteins) have been described, for example, in WO2016/112242, WO2016/205749, and PCT/CN2020/111057, which are incorporated herein by reference in their entirety.
In some embodiments, a split Cas effect protein is provided comprising a first polypeptide comprising an N-terminal portion of any one of the engineered Cas nucleases described herein or a functional derivative thereof and a second polypeptide comprising a C-terminal portion of the engineered Cas nuclease or a functional derivative thereof, wherein the first and second polypeptides are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a CRISPR complex that specifically binds to a target nucleic acid comprising a target sequence that is complementary to the guide sequence. In some embodiments, the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first and second dimerization domains are associated with each other in the presence of an inducer (e.g., rapamycin). In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the split Cas effector protein is self-induced.
The segmentation may be performed in a manner that does not affect the catalytic domain. Cas effector proteins may be used as nucleases (including nicking enzymes) or may be inactivated enzymes, which are essentially RNA-guided DNA-binding proteins with little or no catalytic activity (e.g., due to mutations in their catalytic domains).
In some embodiments, the nuclease leaf and the α -helical leaf of the Cas protein are expressed as separate polypeptides. Although the leaves do not interact by themselves, the RNA guide sequence recruits them into a complex that summarizes the activity of the full-length Cas enzyme and catalyzes site-specific DNA cleavage. In some embodiments, the modified RNA guide sequences may be used to eliminate the activity of the split enzyme by preventing dimerization, thereby allowing the development of an inducible dimerization system. Such split enzymes are described, for example, in Wright, addison V., et al, "Rational design of a split-Cas9 enzyme complex," Proc.Nat' l.Acad.Sci.,112.10 (2015): 2984-2989, which is incorporated herein by reference in its entirety.
The split Cas effector protein portions described herein can be designed to be split (i.e., split) into two halves at a split position, which is the point at which the N-and C-terminal portions of the reference Cas effector protein are separated, by splitting the reference engineered Cas effector protein (e.g., full-length engineered Cas12b, cas12i, cas9, cas12a, or CasX effector protein, or functional variants thereof). In some embodiments, the N-terminal portion comprises amino acid residues 1 to X, and the C-terminal portion comprises amino acid residues x+1 to the C-terminus of the reference Cas effect protein. In this example, numbering is continuous, but this is not required, as it is also contemplated that amino acids (or nucleotides encoding them) may be trimmed from any of the ends of the split and/or mutations (e.g., insertions, deletions, and substitutions) in the interior region of the polypeptide chain, provided that the reconstituted Cas effector protein retains sufficient DNA binding activity (if desired), DNA nickase or cleavage activity, e.g., at least 40%, 50%, 60%, 70%, 80%, 90% or 95% activity compared to the reference Cas effector protein.
The spots can be designed by computer (in silico) and cloned into the construct. In this process, mutations can be introduced into the split Cas effector protein, and nonfunctional domains can be removed. In some embodiments, the two portions or fragments of the split Cas effector protein (i.e., the N-terminal and C-terminal fragments) can form a complete Cas effector protein comprising, for example, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the complete Cas effector protein sequence.
The split Cas effector proteins may each comprise one or more dimerization domains. In some embodiments, the first polypeptide comprises a first dimerization domain fused to a first split Cas effect protein moiety and the second polypeptide comprises a second dimerization domain fused to a second split Cas effect protein moiety. The dimerization domain may be fused to the split Cas effector protein moiety by a peptide linker (e.g., a flexible peptide linker such as a GS linker) or a chemical bond. In some embodiments, the dimerization domain is fused to the N-terminus of the split Cas effector protein portion. In some embodiments, the dimerization domain is fused to the C-terminus of the split Cas effector protein portion.
In some embodiments, the split Cas effector protein does not comprise any dimerization domain.
In some embodiments, the dimerization domain facilitates association of two split Cas effector protein portions. In some embodiments, the split Cas effector protein moiety is induced by an inducer to associate or dimerize to a functional Cas effector protein. In some embodiments, the split Cas effector protein comprises an inducible dimerization domain. In some embodiments, the dimerization domain is not an inducible dimerization domain, i.e., the dimerization domain dimerizes in the absence of an inducer.
The inducer may be an induction energy source or induction molecule other than a guide RNA (e.g., sgRNA). The inducer reconstitutes the two split Cas effector protein portions into a functional Cas effector protein by dimerization of the induced dimerization domains. In some embodiments, the inducer brings together the two split Cas effector protein portions by the action of induced association of the inducible dimerization domain. In some embodiments, the two split Cas effector protein moieties are not associated with each other to reconstruct into a functional Cas effector protein in the absence of an inducer. In some embodiments, in the absence of an inducer, two separate Cas effector protein portions can associate with each other in the presence of a guide RNA (e.g., sgRNA) to reconstruct into a functional Cas effector protein.
The inducers of the present application may be thermal, ultrasonic, electromagnetic energy or chemical compounds. In some embodiments, the inducer is an antibiotic, a small molecule, a hormone derivative, a steroid, or a steroid derivative. In some embodiments, the inducer is abscisic acid (ABA), doxycycline (DOX), cumate (cumate), rapamycin, 4-hydroxy tamoxifen (4 OHT), an estrogen, or an ecdysone. In some embodiments, the split Cas effect system is an inducer controlled system selected from the group consisting of: antibiotic-based induction systems, electromagnetic energy-based induction systems, small molecule-based induction systems, nuclear receptor-based induction systems, and hormone-based induction systems. In some embodiments, the split Cas effect system is an inducer controlled system selected from the group consisting of: a tetracycline (Tet)/DOX induction system, a photoinduction system, an ABA induction system, a cymene repressor/operator system, a 4 OHT/estrogen induction system, an ecdysone-based induction system, and an FKBP12/FRAP (FKBP 12-rapamycin complex) induction system. Such inducers are also discussed herein and in PCT/US2013/051418, which is incorporated herein by reference in its entirety. The FRB/FKBP/rapamycin system has been described in Paulmurugan and Gambhir, cancer Res, august 15,2005 65;7413, and Crabtree et al Chemistry & Biology 13,99-107, jan 2006, which is incorporated herein by reference in its entirety.
In some embodiments, the paired split Cas effector proteins are separate and inactive until dimerization of the dimerization domains (e.g., FRB and FKBP) is induced, which leads to reassembly of the functional Cas effector protein nuclease. In some embodiments, the first split Cas effector protein comprising the first half of the inducible dimer (e.g., FRB) is delivered separately and/or in a separate location from the second split Cas effector protein comprising the second half of the inducible dimer (e.g., FKBP).
Other exemplary FKBP-based induction systems that can be used in the inducer-controlled split Cas effect systems described herein include, but are not limited to: FKBP dimerized with Calcineurin (CNA) in the presence of FK 506; FKBP dimerizing with CyP-Fas in the presence of FKCsA; FKBP dimerized with FRB in the presence of rapamycin; gyrB dimerized with GryB in the presence of coumaromycin; GAI dimerized with GID1 in the presence of gibberellin; or Snap-tag dimerized with HaloTag in the presence of HaXS.
Alternatives within the FKBP family itself are also contemplated. For example, FKBPs undergo homodimerization in the presence of FK1012 (i.e., one FKBP dimerizes with another FKBP).
In some embodiments, the dimerization domain is FKBP and the inducer is FK1012. In some embodiments, the dimerization domain is GryB and the inducer is coumarone. In some embodiments, the dimerization domain is ABA and the inducer is gibberellin.
In some embodiments, the split Cas effector protein moiety may be automatically induced (i.e., auto-activated or auto-induced) to associate/dimerize to a functional Cas effector protein in the absence of an inducer. Without being bound by any theory or hypothesis, the automatic induction of the split Cas effector protein moiety may be mediated by binding to a guide RNA, such as sgRNA. In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the first polypeptide and the second polypeptide comprise a dimerization domain.
In some embodiments, the reconstituted Cas effector proteins of the split Cas effector systems described herein (including inducer controlled and auto-induced systems) have an editing efficiency of at least 70% (e.g., at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more efficiency, or 100% efficiency) relative to the reference Cas effector protein editing efficiency.
In some embodiments, the reconstituted Cas effector protein of the split Cas effector systems controlled by the inducers described herein has an editing efficiency of no more than 50% (e.g., no more than any of about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less efficient, or 0% efficient) relative to the reference Cas effector protein editing efficiency in the absence of the inducer (i.e., due to auto-induction).
Fusion Cas effect proteins
The present application also provides engineered Cas effector proteins comprising additional protein domains and/or components, such as linkers, nuclear localization/export sequences, functional domains, and/or reporter proteins.
In some embodiments, the engineered Cas effector protein is a protein complex comprising one or more heterologous protein domains (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) and a nucleic acid targeting domain of the engineered Cas nuclease or a functional derivative thereof. In some embodiments, the engineered Cas effector protein is a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains) fused to the engineered Cas nuclease.
In some embodiments, an engineered Cas effector protein of the present application may comprise (e.g., by fusion proteins, such as by one or more peptide linkers, e.g., GS peptide linkers, etc.) one or more functional domains or associations thereto (e.g., by co-expression of multiple proteins). In some embodiments, the one or more functional domains are enzyme domains. These functional domains may have a variety of activities, such as DNA and/or RNA methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switching activity (e.g., photoinduced). In some embodiments, the one or more functional domains are transcriptional activation domains (i.e., transactivation domains) or repressor domains. In some embodiments, the one or more functional domains are histone modification domains. In some embodiments, the one or more functional domains are a transposase domain, an HR (homologous recombination) mechanism domain, a recombinase domain, and/or an integrase domain. In some embodiments, the functional domain is a Kruppel related cassette (KRAB), VP64, VP16, fok1, P65, HSF1, myoD1, biotin-APEX, APOBEC1, AID, pmCDA1, tad1, and M-MLV reverse transcriptase. In some embodiments, the functional domain is selected from the group consisting of: a translation initiation domain, a transcription repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (e.g., a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (e.g., a fluorescent domain), and a nuclease domain.
In some embodiments, the localization of one or more functional domains in the engineered Cas effector protein allows for the correct spatial orientation of functional domains to affect targets with the assigned functional role. For example, if the functional domain is a transcriptional activator (e.g., VP16, VP64, or p 65), the transcriptional activator is placed in a spatial orientation that enables it to affect transcription of the target. Likewise, a transcriptional repressor is positioned to affect transcription of a target, and a nuclease (e.g., fok 1) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is located at the N-terminus of the engineered Cas effector protein. In some embodiments, the functional domain is located at the C-terminus of the engineered Cas effector protein. In some embodiments, the engineered Cas effector protein comprises a first functional domain at the N-terminus and a second functional domain at the C-terminus. In some embodiments, the engineered Cas effector protein comprises a catalytically inactive mutant of any of the engineered Cas nucleases described herein fused to one or more functional domains.
In some embodiments, the engineered Cas effector protein is a transcriptional activator. In some embodiments, the engineered Cas effector protein comprises an enzyme-inactivating variant of any of the engineered Cas nucleases described herein fused to a transactivation domain. In some embodiments, the transactivation domain is selected from the group consisting of: VP64, p65, HSF1, VP16, myoD1, HSF1, RTA, SET7/9, and combinations thereof. In some embodiments, the transactivation domain comprises VP64, p65 and HSF1. In some embodiments, the engineered Cas effector protein comprises two split Cas effector polypeptides, each fused to a transactivation domain.
In some embodiments, the engineered Cas effector protein is a transcriptional repressor. In some embodiments, the engineered Cas effector protein comprises an enzyme-inactivating variant of any of the engineered Cas nucleases described herein fused to a transcription repression domain. In some embodiments, the transcriptional repressor domain is selected from the group consisting of: kruppel-related cassettes (KRABs), enR, nuE, ncoR, SID, SID X and combinations thereof. In some embodiments, the engineered Cas effect protein comprises two split Cas effect polypeptides, each fused to a transcription repression domain.
In some embodiments, the engineered Cas effector protein is a base editor, such as a cytosine editor or an adenosine editor. In some embodiments, the engineered Cas effector protein comprises an enzyme-inactivating variant of any of the engineered Cas nucleases described herein fused to a nucleobase editing domain, such as a Cytosine Base Editing (CBE) domain or an Adenosine Base Editing (ABE) domain. In some embodiments, the nucleobase editing domain is a DNA editing domain. In some embodiments, the nucleobase editing domain has deaminase activity. In some embodiments, the nucleobase editing domain is a cytosine deaminase domain. In some embodiments, the nucleobase editing domain is an adenosine deaminase domain. Exemplary base editors based on Cas nucleases are described, for example, in WO2018/165629A1 and WO2019/226953A1, which are incorporated herein by reference in their entirety. Exemplary CBE domains include, but are not limited to: activation-induced cytidine deaminase or AID (e.g., hAID), apolipoprotein B mRNA editing complex or apodec (e.g., rat apodec 1, hapobeec 3A/B/C/D/E/F/G), and PmCDA1. Exemplary ABE domains include, but are not limited to: tadA, ABE8 and variants thereof (see, e.g., gaudelli et al, 2017, nature551:464-471;and Richter et al.,2020,Nature Biotechnology 38:883-891). In some embodiments, the functional domain is an apodec 1 domain, e.g., a rat apodec 1 domain comprising the amino acid sequence of SEQ ID No. 218. In some embodiments, the functional domain is a TadA domain, e.g., an e.coli (e.coli) TadA domain comprising the amino acid sequence of SEQ ID NO: 219. In some embodiments, the engineered Cas effector protein further comprises one or more nuclear localization sequences.
In some embodiments, the engineered Cas effector protein is a master editor. A Cas 9-based master editor is described, for example, in a.anzalone et al, nature,2019,576 (7785):149-157, which is incorporated herein by reference in its entirety. In some embodiments, the engineered Cas effector protein comprises a nickase variant of any of the engineered Cas nucleases described herein fused to a reverse transcriptase domain. In some embodiments, the functional domain is a reverse transcriptase domain. In some embodiments, the reverse transcriptase domain is an M-MLV reverse transcriptase or variant thereof, e.g., an M-MLV reverse transcriptase having one or more mutations of D200N, T306K, W313F, T P and L603W. In some embodiments, the reverse transcriptase domain comprises the amino acid sequence of SEQ ID NO. 220 or 221. In some embodiments, an engineered CRISPR/Cas system is provided that includes the master editor. In some embodiments, the engineered CRISPR/Cas system further comprises a second Cas nickase, e.g., based on the same engineered Cas nuclease as the main editor. In some embodiments, the engineered CRISPR/Cas system comprises a master editor guide RNA (pegRNA) comprising a primer binding site and a Reverse Transcriptase (RT) template sequence.
In some embodiments, the present application provides a split Cas effector system having one or more (e.g., 1, 2, 3, 4, 5, 6, or more) functional domains associated (i.e., bound or fused) with one or both of the split Cas effector protein moieties. The functional domain may be provided as part of the first and/or second split Cas effector protein as a fusion within the construct. The functional domain is typically fused to other portions of the split Cas effector protein (e.g., split Cas effector protein portions) by a peptide linker, such as a GS linker. These functional domains can be used to re-alter the function of the split Cas effect system based on the catalytic inactivation of Cas effect proteins.
In some embodiments, the engineered Cas effector protein comprises one or more Nuclear Localization Sequences (NLS) and/or one or more Nuclear Export Sequences (NES). Exemplary NLS sequences include, for example, PKKKRKVPG (SEQ ID NO: 79) and ASPKKKRKV (SEQ ID NO: 80). The NLS and/or NES can be operably linked to the N-terminus and/or C-terminus of the engineered Cas effector protein or a polypeptide chain in the engineered Cas effector protein.
In some embodiments, the engineered Cas effector protein may encode additional components, such as a reporter protein. In some embodiments, the engineered Cas effector protein comprises a fluorescent protein, such as GFP. Such a System may allow for imaging of genomic loci (see, e.g., "Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System" Chen B et al cell 2013). In some embodiments, the engineered Cas effect protein is an inducible split Cas effect system that can be used to image genomic sites.
Engineered CRISPR-Cas systems
Also provided is an engineered CRISPR-Cas system comprising: (a) Any of the engineered Cas effector proteins described herein (e.g., engineered Cas nucleases, nickases, split Cas, transcription repressors, transcription activators, base editors, or main editors); and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered Cas effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence. In some embodiments, the engineered CRISPR-Cas system comprises one or more nucleic acids encoding the engineered Cas effect protein and/or the guide RNA. In some embodiments, the engineered CRISPR-Cas system comprises a precursor guide RNA array that can be processed into a plurality of crrnas, e.g., by the engineered Cas effector protein. In some embodiments, the engineered CRISPR-Cas system comprises one or more vectors encoding the engineered Cas effector protein and/or the guide RNA. In some embodiments, the engineered CRISPR-Cas system comprises a Ribonucleoprotein (RNP) complex comprising the engineered Cas effect protein bound to the guide RNA.
The engineered CRISPR-Cas system of the present application may comprise any suitable guide RNA. The guide RNA (gRNA) can comprise a guide sequence that is capable of hybridizing to a target sequence in a target nucleic acid of interest, such as a target genomic site in a cell. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) sequence that comprises the guide sequence. In some embodiments, the gRNA comprises a transactivation CRISPR RNA (tracrRNA) sequence. In some embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the sgrnas comprise tracrRNA and crRNA.
In some embodiments, the CRISPR-Cas systems provided herein do not require a tracrRNA sequence (e.g., a CRISPR-Cas12i or CRISPR-Cas12a system). In some embodiments, the guide RNA comprises crRNA. Typically, crrnas described herein include direct repeat sequences and spacer sequences. In certain embodiments, the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence. In some embodiments, the crrnas include direct repeat sequences, spacer sequences, and direct repeat sequences (DR-spacer sequence-DR), which are typical features of precursor crRNA (pre-crRNA) configurations. In some embodiments, the crRNA includes truncated direct repeat sequences and spacer sequences, which are typical features of processed or mature crrnas. In some embodiments, the CRISPR-Cas effect protein forms a complex with an RNA guide sequence, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid that is complementary to the spacer sequence.
Some V-type Cas enzymes, such as Cas12c/d enzymes, require a scoutna. See Harrington et al 2020, mol. Cell, 79:1-9. In some embodiments, the guide RNA comprises crRNA and scoutna.
In some embodiments, the guide RNA is crRNA comprising a guide sequence. In some embodiments, the engineered CRISPR-Cas system comprises a precursor guide RNA array encoding a plurality of crrnas. In some embodiments, the Cas effector protein cleaves the precursor guide RNA array to generate a plurality of crrnas. In some embodiments, the engineered CRISPR-Cas system comprises a precursor guide RNA array encoding a plurality of crrnas, wherein each crRNA comprises a different guide sequence. In some embodiments, the crRNA encoded by the precursor guide RNA array is associated with a tracrRNA or a scoutna.
The guide sequence may be of a suitable length. In some embodiments, the guide sequence is between about 18 to about 35 nucleotides, including, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. The guide sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementarity to a target sequence of a target nucleic acid.
Constructs and vectors
Also provided herein are constructs, vectors, and expression systems encoding any of the engineered Cas-effect proteins described herein. In some embodiments, the construct, vector, or expression system further comprises one or more grnas (e.g., sgrnas) or crRNA arrays.
A "vector" is a composition of matter that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to: linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In general, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers that function in at least one organism. The term "vector" should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to: adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York), among other virology and molecular biology manuals.
Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used.
In certain embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV2, AAV8 or AAV9, which may be constructed to comprise at least 1 x 10 5 A single administration of individual particles (also referred to as particle units, pu) administers adenovirus or adeno-associated virus. In some embodiments, the amount administered is at least about 1 x 10 6 The particles of the composition are mixed with each other,at least about 1X 10 7 Individual particles of at least about 1X 10 8 Individual particles, or at least about 1X 10 9 Adeno-associated virus of individual particles. Methods of delivery and amounts of administration are described, for example, in WO 2016205764 and U.S. patent No.8,454,972, which are incorporated herein by reference in their entirety.
In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, modified AAV vector delivery may be used. The modified AAV vector may be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV 6) and pseudotyped AAV (e.g., AAV2/8, AAV2/5, and AAV 2/6). Exemplary AAV vectors and techniques that can be used to produce rAAV particles are known in the art (see, e.g., apnte-Ubillus et al (2018) appl. Microbiol. Biotechnol.102 (3): 1045-54; zhong et al (2012) J. Genet. Syndr. Gene Ther. S1:008; west et al (1987) Virology 160:38-47 (1987); tratsche et al (1985) mol. Cell. Biol.5:3251-60; U.S. Pat. Nos.4,797,368 and 5,173,414; international publication Nos. WO2015/054653 and WO93/24641, each of which is incorporated herein by reference).
Any known AAV vector for delivering Cas9 and other Cas proteins may be used to deliver the engineered Cas systems of the present application.
Methods for introducing vectors into mammalian cells are known in the art. The vector may be transferred into the host cell by physical, chemical or biological means.
The physical methods for introducing the vector into the host cell include: calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2001) Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, new York. In some embodiments, the vector is introduced into the cell by electroporation.
Biological methods for introducing heterologous nucleic acids into host cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.
Chemical methods for introducing the vector into the host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In some embodiments, the engineered CRISPR-Cas system is delivered in the form of RNP in a nanoparticle.
In some embodiments, the vector or expression system encoding the CRISPR-Cas system or components thereof comprises one or more selectable or detectable markers that provide a means to isolate or effectively select cells that contain and/or have been modified by the CRISPR-Cas system (e.g., at an early stage and large scale).
Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue, and the expression of the encoded polypeptide is evidenced by certain readily detectable properties (e.g., enzymatic activity). The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al FEBS Letters 479:79-82 (2000)).
Other methods of confirming the presence of heterologous nucleic acid in a host cell include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blots, RT-PCR, and PCR; biochemical assays detect the presence or absence of a particular peptide, for example, by immunological methods (e.g., ELISA and Western blot).
In some embodiments, the nucleic acid sequence encoding the engineered Cas effector protein and/or the guide RNA is operably linked to a promoter. In some embodiments, the promoter is an endogenous promoter relative to a cell engineered using the engineered CRISPR-Cas system. For example, the nucleic acid encoding the engineered Cas effector protein may be knocked into the genome of an engineered mammalian cell downstream of the endogenous promoter using any method known in the art. In some embodiments, the endogenous promoter is a protein-rich (e.g., β -actin) promoter. In some embodiments, the endogenous promoter is an inducible promoter, e.g., inducible by an endogenous activation signal of an engineered mammalian cell. In some embodiments, wherein the engineered mammalian cell is a T cell, the promoter is a T cell activation-dependent promoter (such as an IL-2 promoter, NFAT promoter, or nfkb promoter).
In some embodiments, the promoter is a heterologous promoter relative to a cell engineered using the engineered CRISPR-Cas system. A variety of promoters have been explored to express genes in mammalian cells, and any promoter known in the art may be used in the present application. Promoters can be broadly classified as constitutive or regulated, e.g., inducible.
In some embodiments, the nucleic acid sequence encoding the engineered Cas effector protein and/or the guide RNA is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to: the Cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF 1 alpha), ubiquitin C promoter (Ubic), phosphoglycerate kinase Promoter (PGK), simian virus 40 early promoter (SV 40), and chicken beta-actin promoter are coupled to the CMV early enhancer (CAG). In some embodiments, the promoter is a CAG promoter comprising a Cytomegalovirus (CMV) early enhancer element, a promoter, a first exon and a first intron of a chicken β -actin gene, and a splice acceptor of a rabbit β -globin gene.
In some embodiments, the nucleic acid sequence encoding the engineered CRISPR-Cas protein and/or the guide RNA is operably linked to an inducible promoter. Inducible promoters belong to the regulated promoter subtype. The inducible promoter may be induced by one or more conditions such as physical conditions, microenvironment or physiological state of the host cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the induction conditions are selected from the group consisting of: an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of cells to be engineered by the engineered CRISPR-Cas system. In some embodiments, the promoter may be induced by a small molecule inducer such as a compound. In some embodiments, the small molecule is selected from the group consisting of: doxycycline, tetracycline, alcohol, metal, or steroid. Chemically inducible promoters have been most widely studied. Such promoters include those whose transcriptional activity is regulated by the presence or absence of small molecule chemicals such as doxycycline, tetracyclines, alcohols, steroids, metals, and other compounds. Doxycycline induction systems with a reverse tetracycline-controlled transactivator (rtTA) and a tetracycline responsive element promoter (TRE) are the most mature systems at present. WO9429442 describes the strict control of gene expression in eukaryotic cells by tetracycline responsive promoters. WO9601313 discloses tetracycline-regulated transcriptional modulators. In addition, tet technology such as the Tet-on system has been described, for example, in TetSystems. In this application, any known chemically regulated promoter can be used to drive expression of the engineered CRISPR-Cas protein and/or the guide RNA.
In some embodiments, the nucleic acid sequence encoding the engineered Cas effect protein (e.g., enCas12i 2) is codon optimized.
In some embodiments, expression constructs are provided comprising a codon optimized sequence encoding the engineered Cas effector protein linked to a BPK2104-ccdB vector. In some embodiments, the expression construct encodes a tag (e.g., a 10xHis tag) operably linked to the C-terminus of the engineered Cas effector protein.
In some embodiments, each engineered split Cas construct encodes a fluorescent protein such as GFP or RFP. The reporter protein can be used to assess co-localization and/or dimerization of the engineered Cas protein, for example using a microscope. The nucleic acid sequence encoding the engineered Cas effect protein may be fused to a nucleic acid sequence encoding an additional component using a sequence encoding a self-cleaving peptide such as a T2A, P2A, E2A or F2A peptide.
In some embodiments, expression constructs for mammalian cells (e.g., human cells) are provided that comprise a nucleic acid sequence encoding the engineered Cas-effector protein. In some embodiments, the expression construct comprises a codon optimized sequence encoding the engineered Cas effect protein inserted into the pCAG-2A-eGFP vector, thereby operably linking the Cas protein to the eGFP. In some embodiments, a second vector is provided for expressing a guide RNA (e.g., sgRNA, crRNA, or precursor crRNA array) in a mammalian cell (e.g., a human cell). In some embodiments, the sequence encoding the guide RNA is expressed in the pUC19-U6-i2-cr RNA vector backbone. An exemplary dual vector expression system is shown in FIG. 1.
IV method of use
One aspect of the present application provides methods of detecting a target nucleic acid or modified nucleic acid in vitro, ex vivo, or in vivo using any of the engineered Cas effector proteins or CRISPR-Cas systems described herein, and methods of treating or diagnosing using the engineered Cas effector proteins or CRISPR-Cas systems. Also provided are uses of the engineered Cas effector proteins or CRISPR-Cas systems described herein for detecting or modifying nucleic acids in cells, and for treating or diagnosing a disease or condition in a subject; and the use of a composition comprising one or more components of the engineered CRISPR-Cas system or any of the engineered Cas effector proteins in the manufacture of a medicament for detecting or modifying a nucleic acid in a cell and for treating or diagnosing a disease or condition in a subject.
Methods of using Cas12i
One aspect of the present application provides methods of cleaving a target nucleic acid and performing genome editing in a mammalian cell (e.g., a human cell) using Cas12i (including wild-type or engineered Cas12i effector proteins).
In some embodiments, the present application provides a method of modifying a target sequence in a target nucleic acid comprising contacting the target nucleic acid with an engineered CRISPR-Cas system at a temperature of about 40 ℃ to about 67 ℃, wherein the engineered CRISPR-Cas system comprises: (a) A Cas12i effector protein comprising a Cas12i nuclease or a functional derivative thereof; (b) A crRNA comprising a guide sequence complementary to the target sequence, wherein the Cas12i effector protein and the crRNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence. In some embodiments, the Cas12i effector protein is a Cas12i2 nuclease or a functional derivative thereof. In some embodiments, the method is performed at an elevated temperature, for example at any of about 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 67 ℃. In some embodiments, the method is performed at a temperature of about 40 ℃ to 50 ℃,50 ℃ to 60 ℃,45 ℃ to 55 ℃,55 ℃ to 65 ℃,40 ℃ to 60 ℃, or 50 ℃ to 67 ℃. In some embodiments, the Cas12i effector protein has non-specific single-stranded RNA cleavage activity.
In some embodiments, the methods of using Cas12i effector proteins (e.g., engineered Cas12i2 proteins) described herein are performed at a temperature of from 4 ℃ to about 40 ℃, such as at a temperature of about 4-10, 10-20, 20-30, 30-40, 15-37, 4-20, or 20-40 ℃.
The present application also provides engineered crrnas that improve the gene editing efficacy of Cas12i nucleases (e.g., cas12i1 nucleases). In some embodiments, the engineered crRNA increases the gene editing activity of the Cas12i2 nuclease in a human cell by at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80% or more) compared to a crRNA comprising an endogenous repeat sequence (e.g., SEQ ID NO: 171) corresponding to the Cas12i2 nuclease.
In some embodiments, an engineered crRNA is provided that replaces one or more uridine (U) residues with non-U residues in a repeat sequence comprising at least four U residues. In some embodiments, an engineered precursor guide RNA array encoding a plurality of engineered crrnas described herein is provided.
In some embodiments, the engineered crRNA comprises a spacer sequence of about 17-25 (e.g., any of 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides long. In some embodiments, the engineered crRNA comprises a spacer sequence about 20 nucleotides long.
In some embodiments, the engineered crRNA comprises a repeat sequence comprising the nucleic acid sequence of SEQ ID NO. 173.
In some embodiments, an engineered CRISPR-Cas system is provided, comprising: (a) A Cas12i effector protein comprising a Cas12i1 nuclease or a functional derivative thereof; and (b) crRNA that replaces one or more uridine (U) residues with non-U residues in a repeat sequence comprising at least four U residues and a guide sequence complementary to the target sequence; wherein the Cas12i effector protein and the crRNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence. In some embodiments, methods of modifying a target sequence in a target nucleic acid using the engineered CRISPR-Cas system are provided.
Also contemplated herein are methods, methods of treatment, methods of detection, and the like, of modifying a target nucleic acid in a mammalian cell using Cas12i (including wild-type or engineered Cas12i effector proteins) and/or the engineered crrnas described herein according to any of the methods described in this section IV, "methods of use".
Method of modification
In some embodiments, the present application provides methods of modifying a target nucleic acid comprising a target sequence, the method comprising contacting the target nucleic acid with any of the engineered CRISPR-Cas systems described herein. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, or an animal cell. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.
In some embodiments, the target nucleic acid is cleaved or a target sequence in the target nucleic acid is altered by the engineered CRISPR-Cas system. In some embodiments, the expression of the target nucleic acid is altered by the engineered CRISPR-Cas system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition. In some embodiments, the engineered CRISPR-Cas system comprises a precursor guide RNA array encoding a plurality of crrnas, wherein each crRNA comprises a different guide sequence. In some embodiments, the process is conducted at a temperature of about 4 ℃ to about 67 ℃, such as about 12 ℃ to 67 ℃,4 ℃ to 25 ℃,25 ℃ to about 37 ℃, about 37 ℃ to about 45 ℃, about 45 ℃ to about 50 ℃, about 50 ℃ to about 60 ℃, or any of about 40 ℃ to about 67 ℃. In some embodiments, the process is carried out at a low temperature (e.g., about 4 ℃ to about 12 ℃). In some embodiments, the process is conducted at an elevated temperature (e.g., about 40 ℃ to about 67 ℃).
In some embodiments, the present application provides methods of treating a disease or condition associated with a target nucleic acid in a cell of an individual, comprising modifying the target nucleic acid in the cell of the individual using any of the methods described herein, thereby treating the disease or condition. In some embodiments, the disease or condition is selected from the group consisting of: cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, ocular disease, bacterial infection, and viral infection.
The engineered CRISPR-Cas systems described herein can modify a target nucleic acid in a cell in a variety of ways, depending on the type of Cas effector protein engineered in the CRISPR-Cas system. In some embodiments, the methods induce site-specific cleavage in a target nucleic acid. In some embodiments, the method cleaves genomic DNA in a cell, such as a bacterial cell, a plant cell, or an animal cell (e.g., a mammalian cell). In some embodiments, the method kills the cell by cleaving genomic DNA in the cell. In some embodiments, the method cleaves viral nucleic acid in a cell.
In some embodiments, the method alters (e.g., increases or decreases) the expression level of the target nucleic acid in the cell. In some embodiments, the methods use engineered Cas effector proteins to increase the expression level of the target nucleic acid in a cell, e.g., based on an enzymatically inactive Cas protein fused to a transactivation domain. In some embodiments, the methods use engineered Cas effector proteins to reduce the expression level of the target nucleic acid in a cell, e.g., based on an enzymatically inactive Cas protein fused to a transcription repression domain. In some embodiments, the methods are based on, for example, an enzymatically inactive Cas protein fused to an epigenetic modification domain, using an engineered Cas effector protein to introduce an epigenetic modification to the target nucleic acid in a cell. The engineered Cas systems described herein can be used to introduce additional modifications to the target nucleic acid, depending on the functional domain comprised by the engineered Cas effector protein.
In some embodiments, the method alters a target sequence in the target nucleic acid in a cell. In some embodiments, the method introduces a mutation into the target nucleic acid in a cell. In some embodiments, the methods use one or more endogenous DNA repair pathways, such as non-homologous end joining (NHEJ) or Homologous Directed Recombination (HDR), to repair double strand breaks induced in target DNA in cells as a result of sequence specific cleavage by a CRISPR complex. Exemplary mutations include, but are not limited to: insertions, deletions, substitutions and frameshifts. In some embodiments, the method inserts donor DNA at the target site. In some embodiments, insertion of the donor DNA results in the introduction of a selectable marker or reporter protein into the cell. In some embodiments, insertion of the donor DNA results in knock-in of the gene. In some embodiments, the insertion of the donor DNA results in a knockout mutation. In some embodiments, the insertion of the donor DNA results in a substitution mutation such as a single nucleotide substitution. In some embodiments, the method induces a phenotypic change in the cell.
In some embodiments, the engineered CRISPR-Cas system is used for a portion of a genetic circuit (genetic circuit), or for inserting a genetic circuit into genomic DNA of a cell. The inducer-controlled engineered split Cas effector proteins described herein are particularly useful as components of genetic circuits. The gene circuit can be used for gene therapy. Methods and techniques for designing and using genetic circuits are known in the art. Further reference may be made, for example, to Brophy, jennifer AN, and Christopher A.Voigt. "Principles of genetic circuit design." Nature methods 11.5 (2014): 508.
The engineered CRISPR-Cas systems described herein can be used to modify a variety of target nucleic acids. In some embodiments, the target nucleic acid is in a cell. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is extrachromosomal DNA. In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is a viral nucleic acid such as viral DNA. In some embodiments, the target nucleic acid is a plasmid in a cell. In some embodiments, the target nucleic acid is a horizontally transferred (horizontally transferred) plasmid. In some embodiments, the target nucleic acid is RNA.
In some embodiments, the target nucleic acid is an isolated nucleic acid such as an isolated DNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector such as a plasmid. In some embodiments, the target nucleic acid is an isolated linear DNA fragment.
The methods described herein are applicable to any suitable cell type. In some embodiments, the cell is a bacterium, yeast cell, fungal cell, algal cell, plant cell, or animal cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cells are cells of natural origin, such as isolated from a tissue biopsy. In some embodiments, the cell is a cell isolated from an in vitro cultured cell line. In some embodiments, the cells are from a primary cell line. In some embodiments, the cells are from an immortalized cell line. In some embodiments, the cell is a genetically engineered cell.
In some embodiments, the cell is an animal cell of an organism selected from the group consisting of: cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits and fish.
In some embodiments, the cell is a plant cell of an organism selected from the group consisting of: corn, wheat, barley, oat, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet, maize, sorghum, canola, industrial hemp, vegetable crops, feed crops, industrial crops, woody crops, and biomass crops.
In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a human embryonic kidney 293T (HEK 293T or 293T) cell or a HeLa cell. In some embodiments, the cell is a human embryonic kidney (HEK 293T) cell. In some embodiments, the mammalian cell is selected from the group consisting of: immune cells, liver cells, tumor cells, stem cells, zygotes, muscle cells and skin cells.
In some embodiments, the cell is an immune cell selected from the group consisting of: cytotoxic T cells, helper T cells, natural Killer (NK) T cells, iNK-T cells, NK-T-like cells, γδ T cells, tumor infiltrating T cells, and Dendritic Cell (DC) activated T cells. In some embodiments, the method produces a modified immune cell, such as a CAR-T cell or a TCR-T cell.
In some embodiments, the cell is an Embryonic Stem (ES) cell, an Induced Pluripotent Stem (iPS) cell, a gamete progenitor cell, a gamete, a zygote, or a cell in an embryo.
The methods described herein can be used to modify a target cell in vivo, ex vivo, or in vitro, and can be performed in a manner that alters the cell such that, once modified, the progeny or cell line of the modified cell retains an altered phenotype. The modified cells and offspring may be part of a multicellular organism, such as a plant or animal with ex vivo or in vivo applications (e.g., genome editing and gene therapy).
In some embodiments, the method is performed ex vivo. In some embodiments, the modified cells (e.g., mammalian cells) are propagated ex vivo after the engineered CRISPR-Cas system is introduced into the cells. In some embodiments, the modified cells are cultured to propagate for at least any one of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the modified cells are cultured for no more than about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the modified cells are further evaluated or screened to select cells having one or more desired phenotypes or characteristics.
In some embodiments, the target sequence is a sequence associated with a disease or condition. Exemplary diseases or conditions include, but are not limited to: cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, ocular disease, bacterial infection, and viral infection. In some embodiments, the disease or condition is a genetic disease. In some embodiments, the disease or condition is a monogenic disease or condition. In some embodiments, the disease or condition is a polygenic disease or condition.
In some embodiments, the target sequence has a mutation compared to the wild-type sequence. In some embodiments, the target sequence has a Single Nucleotide Polymorphism (SNP) associated with a disease or condition.
In some embodiments, the donor DNA inserted into the target nucleic acid encodes a biological product selected from the group consisting of: reporter proteins, antigen-specific receptors, therapeutic proteins, antibiotic-resistant proteins, RNAi molecules, cytokines, kinases, antigens, antigen-specific receptors, cytokine receptors, and suicide polypeptides. In some embodiments, the donor DNA encodes a therapeutic protein. In some embodiments, the donor DNA encodes a therapeutic protein useful in gene therapy. In some embodiments, the donor DNA encodes a therapeutic antibody. In some embodiments, the donor DNA encodes an engineered receptor, such as a Chimeric Antigen Receptor (CAR) or an engineered TCR. In some embodiments, the donor DNA encodes a therapeutic RNA, such as a small RNA (e.g., siRNA, shRNA, or miRNA) or a long non-coding RNA (lincRNA).
The methods described herein can be used to perform multiplex gene editing or modulation at two or more (e.g., 2, 3, 4, 5, 6, 8, 10, or more) different target sites. In some embodiments, the method detects or modifies a plurality of target nucleic acids or target nucleic acid sequences. In some embodiments, the method comprises contacting a target nucleic acid with a guide RNA comprising a plurality (e.g., 2, 3, 4, 5, 6, 8, 10, or more) crRNA sequences, wherein each crRNA comprises a different target sequence.
Also provided are engineered cells comprising modified target nucleic acids, the cells produced using any of the methods described herein. The engineered cells may be used in cell therapies. Autologous or allogeneic cells can be used to prepare engineered cells using the methods described herein for cell therapy.
The methods described herein can also be used to generate isogenic lines of cells (e.g., mammalian cells) to study genetic variants.
Also provided are engineered non-human animals comprising the engineered cells described herein. In some embodiments, the engineered non-human animal is a genome-edited non-human animal. The engineered non-human animals can be used as disease models.
Techniques for generating non-human genome editing or transgenic animals are well known in the art and include, but are not limited to: prokaryotic microinjection, viral infection, transformation of embryonic stem cells and Induced Pluripotent Stem (iPS) cells. Detailed methods that may be used include, but are not limited to, the method described by Sundberg and Ichiki (2006,Genetically Engineered Mice Handbook,CRC Press) and the method described by Gibson (2004,A Primer Of Genome Science 2nd ed.Sunderland,Mass.: sinauer).
The engineered animal may be of any suitable species including, but not limited to: cattle, horses, sheep, dogs, deer, felines, goats, pigs, primates, and less well known mammals such as elephants, deer, zebras or camels.
Treatment method
Further provided are methods of treatment using any of the methods of modifying a target nucleic acid in a cell according to the disclosure, and diagnostic methods using any of the methods of detecting a target nucleic acid according to the disclosure.
In some embodiments, the present application provides a method of treating a disease or condition associated with a target nucleic acid in a cell of an individual comprising contacting the target nucleic acid with any of the engineered CRISPR-Cas systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas effector protein and the guide RNA associate with each other to bind to the target nucleic acid to modify the target nucleic acid, thereby allowing the disease or condition to be treated. In some embodiments, a mutation (e.g., a knockout or knock-in mutation) is introduced into the target nucleic acid. In some embodiments, expression of the target nucleic acid is enhanced. In some embodiments, expression of the target nucleic acid is inhibited.
In some embodiments, the present application provides a method of treating a disease or condition in an individual comprising administering to the individual an effective amount of any of the engineered CRISPR-Cas systems described herein and a donor DNA encoding a therapeutic agent, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid of the individual, wherein the engineered Cas effector protein and the guide RNA bind to each other to bind to the target nucleic acid and insert donor DNA into the target sequence, thereby allowing the disease or condition to be treated.
In some embodiments, the present application provides a method of treating a disease or condition in an individual comprising administering to the individual an effective amount of an engineered cell comprising a modified target nucleic acid, wherein the engineered cell is prepared by contacting the cell with any of the engineered CRISPR-Cas systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered Cas effector protein and the guide RNA associate with each other to bind to the target nucleic acid to modify the target nucleic acid. In some embodiments, the engineered cell is an immune cell.
In some embodiments, the individual is a human. In some embodiments, the individual is an animal, for example a model animal such as a rodent, pet or farm animal. In some embodiments, the subject is a mammal.
In some embodiments, the disease or condition is selected from the group consisting of: cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, ocular disease, bacterial infection, and viral infection. In some embodiments, the target nucleic acid is PCSK9. In some embodiments, the disease or condition is a cardiovascular disease. In some embodiments, the disease or condition is coronary artery disease. In some embodiments, the method reduces cholesterol levels in the subject. In some embodiments, the method treats diabetes in an individual.
Detection method
The present application also provides methods of detecting a target nucleic acid using any of the engineered Cas effector proteins or CRISPR-Cas systems with improved activity. The use of Cas effector proteins as detection reagents can utilize the following findings: once activated by detection of target DNA, V-type CRISPR/Cas proteins (e.g., cas12 proteins, such as Cas12a, cas12b, cas12c, cas12d, cas12e (CasX), and Cas12 i) can promiscuously cleave non-targeted single-stranded DNA (ssDNA). Methods of using Cas proteins as detection reagents are described, for example, in US10253365 and WO2020/056924, which are incorporated herein by reference in their entirety.
In some embodiments, once a V-type CRISPR/Cas effect protein (e.g., a Cas12 protein, such as Cas12a, cas12b, cas12c, cas12d, cas12e (CasX), or Cas12 i) is activated by a guide RNA that occurs when the sample contains target DNA hybridized to the guide RNA (i.e., the sample contains target DNA), the Cas effect protein becomes a nuclease that can promiscuously cleave single-stranded nucleic acids (e.g., non-target ssDNA or RNA, i.e., single-stranded nucleic acids to which the guide sequence of the guide RNA does not hybridize). Thus, when target DNA (double-stranded or single-stranded) is present in the sample (e.g., in some cases exceeding a threshold amount), the result is cleavage of single-stranded nucleic acid in the sample, which can be detected using any convenient detection method (e.g., using a tagged single-stranded detection nucleic acid such as DNA or RNA). Cas12i can cleave ssDNA and ssRNA.
In some embodiments, methods of detecting target DNA (e.g., double-stranded or single-stranded) in a sample are provided, comprising: (a) contacting the sample with: (i) Any of the engineered V-type CRISPR/Cas effect proteins described herein (e.g., cas12 protein, such as Cas12a, cas12b, cas12c, cas12d, cas12e (CasX), or Cas12 i); (ii) A guide RNA comprising a guide sequence that hybridizes to the target DNA; and (iii) a detection nucleic acid that is single-stranded (i.e., a "single-stranded detection nucleic acid") and that does not hybridize to the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage of the single stranded detection nucleic acid by the engineered V-type CRISPR/Cas effect protein. In some cases, the single-stranded detection nucleic acid comprises a dye pair that emits fluorescence (e.g., the dye pair that emits fluorescence is a Fluorescence Resonance Energy Transfer (FRET) pair, a quencher/fluorescence pair). In some cases, the target DNA is viral DNA (e.g., papilloma virus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, etc.). In some embodiments, the single stranded detection nucleic acid is DNA. In some embodiments, the single stranded detection nucleic acid is RNA. In some embodiments, the engineered Cas effector protein is an engineered Cas12i nuclease, such as enas 12i2.
The method for detecting target DNA (single-stranded or double-stranded) in a sample of the present disclosure can detect target DNA with high sensitivity. In some cases, the methods of the present disclosure can be used to detect target DNA present in a sample comprising a plurality of DNA (including the target DNA and a plurality of non-target DNA), wherein the target DNA is present at every 10 7 One or more copies of the non-target DNA are present (e.g., every 10 6 One or more copies of each 10 of non-target DNA 5 One or more copies of each 10 of non-target DNA 4 One or more copies of each 10 of non-target DNA 3 One or more copies of each 10 of non-target DNA 2 One or more copies of each of the non-target DNA, one or more copies of each of the 50 non-target DNA, one or more copies of each of the 20 non-target DNA, one or more copies of each of the 10 non-target DNA, or one or more copies of each of the 5 non-target DNA).
In some embodiments, the engineered Cas effector proteins described herein can detect target DNA with greater sensitivity than the reference Cas nuclease. In some embodiments, the engineered Cas effector protein can detect target DNA with a sensitivity of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the reference Cas nuclease.
Delivery method
In some embodiments, the engineered CRISPR-Cas systems described herein or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof, can be delivered to host cells (e.g., any of the vectors described in the "constructs and vectors" section above) by a variety of delivery systems, such as plasmids or viruses. In some embodiments or methods, the engineered CRISPR-Cas system can be delivered by other methods, such as nuclear transfection or electroporation of ribonucleoprotein complexes consisting of the engineered Cas effector protein and one or more homologous RNA guide sequences thereof.
In some embodiments, delivery is by nanoparticles or exosomes.
In some embodiments, the paired Cas nickase complexes can be delivered directly using nanoparticles or other direct protein delivery methods, such that complexes comprising two paired crRNA elements are co-delivered. In addition, the proteins can be delivered to the cells either through viral vectors or directly, followed by direct delivery of CRISPR arrays comprising two paired spacers for double nicks. In some cases, for direct RNA delivery, RNA can be conjugated to at least one sugar moiety such as N-acetylgalactosamine (GalNAc) (particularly triple antenna GalNAc).
V. kit and article of manufacture
Also provided are compositions, kits, unit agents, and articles of manufacture comprising one or more components of any of the engineered Cas effect proteins or engineered CRISPR-Cas systems described herein.
In some embodiments, a kit is provided comprising: one or more AAV vectors encoding any of the engineered Cas effector proteins or engineered CRISPR-Cas systems described herein. In some embodiments, the kit further comprises one or more guide RNAs. In some embodiments, the kit further comprises donor DNA. In some embodiments, the kit further comprises cells such as human cells.
The kit may comprise one or more additional components, such as containers, reagents, media, cytokines, buffers, antibodies, etc., to allow for the propagation of the engineered cells. The kit may also comprise a device for administering the composition.
The kit can further comprise instructions for using the engineered CRISPR-Cas systems described herein, such as methods of detecting or modifying a target nucleic acid. In some embodiments, the kit comprises instructions for treating or diagnosing a disease or condition. Instructions relating to the use of the kit components typically include information regarding the amount of administration, the schedule of administration, and the route of administration for the deliberate treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. For example, a kit (which comprises a sufficient dose of a composition disclosed herein) may be provided to provide effective treatment of an individual over an extended period of time. The kit may also include a plurality of unit doses of the composition and instructions for use in a packaged quantity sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compound pharmacy).
The kits of the invention are in suitable packaging. Suitable packages include, but are not limited to: vials, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), and the like. The kit may optionally provide additional components such as buffers and explanatory information. Accordingly, the present application also provides an article of manufacture comprising a vial (e.g., a sealed vial), bottle, jar, flexible package, or the like.
The article may comprise a container, a label or package insert on or adhered to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. Typically, the container contains a composition effective to treat the diseases or conditions described herein, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert will further include instructions for administering the composition to an individual.
Package inserts refer to instructions that are typically contained in commercial packages of therapeutic products that contain information about the indication, usage, dosage, administration, contraindications, and/or warnings of using such therapeutic products.
In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. From a commercial and user perspective, it may also include other materials, including other buffers, diluents, filters, needles and syringes.
Examples
The following examples are intended to be merely illustrative of the invention and should not be construed as limiting the invention in any way. The following examples and detailed description are provided by way of example and not by way of limitation.
Example 1: engineering a pipeline of enzymes with improved efficiency
This example provides a strategy to design Cas enzymes with enhanced conformational transition kinetics, which results in better catalytic efficiency of Cas endonucleases. The exemplary methods provided herein allow engineering Cas proteins with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity).
We believe that the conditions for Cas conformational transition in human cells may not be optimal, and enhanced conformational transition kinetics may result in better catalytic efficiency of the Cas enzyme. To design Cas proteins with enhanced conformational transition kinetics, we designed a strategy to increase the flexibility of the flexible region in Cas enzymes. Fig. 1 shows a pipeline for designing a SaCas9 variant as an example of this design workflow.
Enhanced Cas enzyme design pipeline (pipeline)
First, we determined the flexible region of the Cas enzyme using DynaMine molecular dynamics predictor (Cilia et al, the DynaMine webserver: predicting protein dynamics from sequence. Nucleic Acids Res.2014Jul 1;42 (Web Server issue): W264-W270). From the predictions of molecular dynamics, we obtained an S2-sequence parameter score profile for Cas protein. The range of S2 order parameters may be 0-1, where 1 corresponds to a rigid key vector and 0 corresponds to complete flexibility.
Based on the S2-order parameter score spectrum obtained from DynaMine, we define the peak amino acid (peak aa, amino acid peak) as the amino acid with the lowest score compared to the score of 5 adjacent amino acids on either side (either before or after the amino acid peak). Based on this definition we have determined the peak amino acids. We selected a region comprising one peak amino acid on either side (before or after peak aa) and its adjacent 2 amino acids as candidate flexible regions for engineering.
Next, we engineered Cas variants with enhanced flexibility in the candidate flexible region. In this embodiment, we engineered candidate flexible regions with enhanced flexibility using the following strategies:
(a) Glycine substitution
If a highly hydrophobic amino acid (aa; L, I, V, C, Y, F, W) is present in the candidate flexible region, the hydrophobic residue is replaced by glycine (G).
(b) Glycine insertion
Within the candidate flexible region, we have inserted two G's in front of the most flexible aa of the flexible region. The most flexible aa is selected according to the following priority order: g > S > N > D > H > M > T > E > Q > K > R > A > P.
If there are a plurality of equal most flexibilities aa in the flexible region, we define the most flexibilities aa as: amino acid (aa) closer to said peak aa.
If there are a plurality of equal most flexibilities aa in the region and the distance between two most flexibilities aa and the peak aa is the same, we define the most flexibilities aa as: adjacent aa have a priority aa.
Cloning and purification as described in the "methods" section below gave Cas variants and cleavage efficiencies of the variants were measured in human cells.
Method
Plasmid construction
The coding sequences of BhCas12bv4, cas12i2 and GeoCas9 are codon optimized (human) and synthesized. Variants of Cas protein were generated by PCR-based site-directed mutagenesis. For prokaryotic expression, the coding sequence for Cas12i2 and variants thereof was ligated into BPK2104-ccdB expression vector using T4 ligase, which was digested with XmaI and SpeI. The fusion construct comprises a fused 10xHis tag at the C-terminus of the protein. Cas effector proteins are expressed in human 293T cells by the pCAG-2A-eGFP vector. DNA encoding Cas protein is inserted between XmaI and NheI. Vectors expressing sgrnas or crrnas of BhCas12bv4, geoCas9, and Cas12i2 in 293T were constructed by ligating annealed oligonucleotides containing target sequences into BasI digested pUC19-U6-i2-crRNA backbones.
Protein purification
The prokaryotic expression plasmid was transformed into E.coli (E.coli) BL21 strain (λDE 3) and the transformant was inoculated onto solid LB containing chloramphenicol (CmR). 3-5 clones were picked and cultured overnight at 37℃in 15ml of liquid LB containing CmR. 2ml of the culture was then transferred to 300ml of liquid LB for cultivation until OD600 reached 1.2, and then induced with IPTG at 16℃for 16h. The cell pellet was resuspended in lysis buffer and then sonicated. The supernatant was retained after centrifugation for future purification. The target protein is obtained by a one-step purification method using a Ni column. The target protein was sterilized with a 0.22 μm filter and then stored as an aliquot. The concentration was determined by Bradford protein assay with BSA as standard.
In vitro RNA transcription
PCR amplified dsDNA containing the T7 promoter was used as an in vitro transcription template using the HISRIBETM T7 rapid high yield RNA synthesis kit (NEB) to generate crRNA. Using Oligo Clean&The transcribed crRNA was purified using a Concentrator kit (ZYMOTM Research) and was isolated on a NANODROP TM 2000 (Thermo Fisher Scientific) quantitative analysis was performed.
Cell culture, transfection and Fluorescence Activated Cell Sorting (FACS)
HEK293T cells were cultured in DMEM (Gibco) with 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the degree of fusion reached 70%. 600ng of the plasmid encoding the Cas protein and 3000ng of the plasmid encoding the crRNA were transfected into each 24-cell culture dish using Lipofectamine 3000 (Invitrogen). After 68h of transfection, HEK293T cells were digested with trypsin-EDTA (0.05%) (Gibco) Fluorescence Activated Cell Sorting (FACS). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.
T7 endonuclease I (T7 EI) assay and targeted deep sequencing analysis for genomic modification
FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55 ℃ for 3 hours, followed by 10 minutes at 95 ℃. PCR amplification was performed on dsDNA fragments containing the target sites in different genomic sites using corresponding primer pairs. For the T7E1 assay, 200-400 ng of PCR product was used and ddH was added 2 O to a final volume of 10. Mu.L. The mixture is then subjected to a re-annealing procedure to form heteroduplex dsDNA. Then, the mixture was treated with 1/10 volume of NEBUFFER at 37 DEG C TM 2.1 and 0.2. Mu. LT7EI (NEB) treatment mixtures 50 minAnd (3) a clock. The digested products were analyzed by-3% agarose gel electrophoresis. Indels are calculated based on previous methods. The PCR product identified by the T7E1 assay containing the mutation was cloned into a TA cloning vector and then transformed into competent E.coli. After overnight incubation, colonies were randomly picked and sent for Sanger sequencing. For targeted deep sequencing, the target sites were amplified directly by barcode (barcoded) PCR using cell lysate directly as template. The PCR products were purified and pooled into several libraries for high throughput sequencing. Indels (%) were analyzed using CRISPResso2 by calculating the ratio of reads (reads) containing insertions or deletions. Reads less than 0.05% of the total reads were discarded.
Example 2: design and characterization of engineered BhCAs12b
This example describes the use of the design pipeline described in example 1 to design and characterize engineered variants of bacillus ectovillage (Bacillus hisashii) Cas12bv4 (BhCas 12bv 4) with improved gene editing activity in human cells.
The flexible region of Cas12bv4 was determined computer-wise using DynaMine as described in example 1 above. As described in example 1 above, peaks aa and candidate flexible regions for engineering were identified. Fig. 2 shows the flexibility (S2 score) spectrum of BhCas12bv4, with selected peaks aa indicated by circles. Table 1 below shows the flexible region sequence (SEQ ID NO: 81) and Y > G substitution of the engineered variant (SEQ ID NO: 82). The amino acid position is based on SEQ ID NO. 1.BhCAs12bv4 has no resolved crystal structure, but highly homologous BhCAs12b (> 98% homology) has a useful crystal structure, so the linker of BhCAs12bv4 as shown in Table 1 was determined from the structure and homology of BhCAs 122 b.
Table 1: candidate flexible regions and modified sequences of enBhCas12b 4.
The resulting BhCAs12bv4 variant (enbhCAs 12bv4 1.1, as shown in SEQ ID NO: 2) was cloned and purified as described in example 1. Then, we tested the efficiency of editing of the enBhCas12bv4 1.1 for 11 genomic loci in human 293T cells, and found a significant improvement in the efficiency of editing of the engineered variants compared to the efficiency of wild-type BhCas12bv4 (fig. 3). The most significant improvement in editing efficiency was observed at the less efficient editing of the wild-type BhCAs12bv4 genomic sites such as RNF2-5, CCR5-8 and CCR5-1 (FIG. 3).
Example 3: design and characterization of engineered Cas12i2
This example describes the use of the design pipeline described in example 1 to design and characterize Cas12i2 engineered variants with improved gene editing activity in human cells.
Cas12i2 has no known 3D structure nor its structural equivalent has been resolved. Thus, we sought to test whether our approach that does not require resolution of the structure can be used to engineer Cas12i2 variants with improved activity (e.g., target binding, double strand cleavage activity, nickase activity, and/or gene editing activity).
The flexible region of Cas12i2 was determined computer-wise using DynaMine as described in example 1 above. The flexibility (S2 score) spectrum of Cas12i2 is shown in fig. 4. Based on the S2 score spectrum, we selected the peak with S2 score less than 0.71 as the flexible region. Although there is no structural information of resolved Cas12i2 or closely related equivalents, we found that the flexible region we selected was related to the Cas12i2 linker region as determined by calculation of secondary structure predictions, as shown in fig. 5 (Buchan DWA, jones DT (2019). The PSIPRED Protein Analysis Workbench:20years on.Nucleic Acids Research).
The selected flexible region amino acid sequences and modified amino acid sequences of the engineered flexible variant regions are shown in table 2 below. The amino acid position is based on SEQ ID NO. 8.
Table 2: selected candidate flexible regions and modified sequences of engineered Cas12i 2.
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Determination of genome editing
To analyze the activity of the engineered Cas12i2 variants in human cells, four target sites (CCR 5-3, CCR5-2, RNF2-7 and CCR 5-8) were selected in the human genome. The editing efficiency (% indels) of Cas12i2 variants compared to wild-type Cas12i2 was determined at each site. Among the variants tested, cas12i2-2.2 (SEQ ID NO: 14) and Cas12i2-6.1 (SEQ ID NO: 18) significantly improved gene editing efficiency. Notably, variant 6.1 significantly improved editing efficiency at all four target sites (fig. 6).
Based on the increased editing efficiency of Cas12i2 variants 2.2 and 6.1, we then tested whether editing efficiency at 5 target sites (CCR 5-3, CCR5-2, AAVS1-1, CCR5-15, RNF 2-7) could be further improved by binding to variants 2.2 and 6.1 mutations. Combining variants 2.2+6.1 further improves the editing efficiency of Cas12i2 (fig. 7). Herein, we refer to the combined variants Cas12i2-2.2+6.1 as "enCas12i2" (SEQ ID NO: 20).
Next, we tested the overall genome editing efficiency of enCas12i2 in human cells compared to SpCas9 and BhCas12b-v 4. Editing efficiency (indel%) was analyzed for enCas12i2 at 46 sites, sp Cas9 at 18 sites, and BhCas12bv4 at 23 sites (fig. 8A). We demonstrate that enas 12i2 can efficiently recognize Protospacer Adjacent Motif (PAM) sites NTTA, NTTC, NTTG and NTTT, as well as ATTN, CTTN, GTTN and TTTN (fig. 8B). Finally, we demonstrate that enCas12i2 retains the ability to process precursor crrnas (fig. 9). The efficiency of gene editing for the precursor crRNA form is equivalent to that of the single crRNA form.
Determination of in vitro cleavage
We also compared the in vitro plasmid cleavage activity of enCas12i2 with wild-type Cas12i2. A detailed description of in vitro cleavage assays can be found in example 6. Briefly, the target plasmid was incubated with different concentrations of RNP formed by Cas12i2 or enCas12i2 plus crRNA. The reaction was carried out at 37℃for 10 minutes. As shown in fig. 10, the purified enCas12i2 protein showed increased dsDNA cleavage activity at 37 ℃ compared to the wild-type Cas12i2 protein.
Determination of nucleic acid detection
Finally, we used wild-type and engineered Cas12i2 in the assays of nucleic acid detection. Briefly, 60nM of Cas12i2 or engineered variants thereof were mixed with 108nM crRNA with 1 XNEBuffer 2, 40nM activator, RNase inhibitor, 200nM synthetic FQ-ssDNA-5T detector (5 '-6-FAM-TTTTT-BHQ 1-3') in a single 20. Mu.l reaction system. The reaction was run in a Applied Biosystems 7500 real-time PCR system (Thermo Fisher) at 37℃for 15 cycles and the FAM channel was measured every minute. The ΔRn value is derived and analyzed by SigmaPlot software. The sequence of the trans XBP activator is shown in SEQ ID NO. 230, and the XBP1 target site is shown in SEQ ID NO. 231.
As shown in fig. 11A, both wild-type Cas12i2 and engineered Cas12i2 variants (2.2, 6.1, and 2.2+6.1) are capable of detecting double-stranded DNA containing an XBP target site (i.e., trans XBP activator). The Δrn value corresponds to the level of detection agent nucleic acid cleavage. The engineered Cas12i2 variants were shown to have higher detection activity than wild-type Cas12i2.
Furthermore, in the nucleic acid detection experiments, we used the detection nucleic acid containing RNA nucleotides, found that wild-type Cas12i2 can cleave RNA-based fluorescent reporter molecules containing rU (5 '-6-FAM-uuuuu-BHQ 1-3') (fig. 11B).
Taken together, these results demonstrate that Cas12i2 can be effectively modified by our method of engineering Cas proteins with improved activity. The method is independent of three-dimensional structural information of Cas12i 2.
Example 4: design and characterization of engineered GeoCas9
This example describes the use of the design pipeline described in example 1 to design and characterize GeoCas9 engineered variants with improved gene editing activity in human cells.
To demonstrate that our methods are widely applicable to Cas proteins other than Cas12 proteins, we next generated and tested engineered variants of GeoCas 9.
The flexible region of GeoCas9 was determined computer-wise using DynaMine as described in example 1 above. The flexibility (S2 score) spectrum of GeoCas9 is shown in fig. 12. Based on the S2 score spectrum, we selected a flexible region from 18 amino acid peaks, where the S2 score was below 0.71. The flexible region we selected was associated with a GeoCas9 linker region determined by calculation of secondary structure predictions as shown in FIG. 13 (Buchan DWA, jones DT (2019). The PSIPRED Protein Analysis Workbench:20years on.Nucleic Acids Research).
The selected flexible region amino acid sequences and modified amino acid sequences of the engineered flexible variant regions are shown in table 3. The amino acid position is based on SEQ ID NO. 25.
Table 3: selected candidate flexible regions and modified sequences of engineered GeoCas9.
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Determination of targeted depth sequencing
To screen engineered GeoCas9 variants to improve gene editing activity, we performed targeted depth sequencing to determine the efficiency of indels generated by GeoCas9. Cells expressing the engineered GeoCas9 variants were first isolated by FACS sorting GFP-positive 293T cells. The cells were lysed in buffer L and incubated at 55 ℃ for 3 hours, followed by 10 minutes at 95 ℃. The target site comprising CD34-2 was amplified directly by barcode (barcoded) PCR using the cell lysate as a template. The PCR products were purified and pooled into several libraries for high throughput sequencing. Indels (%) were analyzed using CRISPResso2 by calculating the ratio of reads (reads) containing insertions or deletions. Reads less than 0.05% of the total reads were discarded. The results of targeted sequencing are shown in FIG. 14. The arrow indicates the engineered GeoCas9 whose editing efficiency is significantly improved. Mutants that improve efficiency are: 2.1 3.1 8.1 9.1 12.1.
Method
HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until the degree of fusion reached 70%. 600ng of the plasmid encoding the Cas protein and 3000ng of the plasmid encoding the crRNA were transfected into each 24-well plate by using Lipofectamine 3000 (Invitrogen). After 68 hours of transfection, HEK293T cells were digested with trypsin-EDTA (0.05%) (Gibco) and Fluorescent Activated Cell Sorting (FACS). Cell sorting was performed using MoFlo XDP (Beckman Coulter) with GFP signal.
FACS sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55 ℃ for 3 hours, followed by 10 minutes at 95 ℃. PCR amplification was performed using corresponding primer pairs for dsDNA fragments containing target sites in different genomic sites (table 4 below). For the T7E1 assay, 200-400 ng of PCR product was used and ddH was added 2 O to a final volume of 10. Mu.L. The mixture is then subjected to a re-annealing procedure to form heteroduplex dsDNA. Then, the mixture was treated with 1/10 volume of NEBUFFER at 37 DEG C TM 2.1 and 0.2. Mu. L T7EI (NEB) the mixture was treated for 50 minutes. The digested product was analyzed by-3% agarose gel electrophoresis. Indels were calculated according to previous methods (Cong et al, 2013). The PCR product containing the mutation identified by the T7E1 assay was cloned into a TA cloning vector, which was subsequently transformed into competent E.coli. After overnight incubation, colonies were randomly picked and sent for Sanger sequencing.
Table 4: primers used.
Example 5: design and characterization of engineered SaCas9
This example describes the use of the design pipeline described in example 1 to design and characterize SaCas9 engineered variants with improved gene editing activity in human cells.
The flexible region of SaCas9 was determined computer-wise using DynaMine as described in example 1 above. The flexible (S2 score) spectrum of SaCas9 is shown in figure 1. Based on the S2 score spectrum, we selected a flexible region from 13 amino acid peaks, where the S2 score was below 0.71 (circled peak in fig. 1).
The positions of the selected flexible region and corresponding domain of SaCas9 are shown in figure 15. The amino acid sequences and modified amino acid sequences of selected flexible regions of the engineered flexible variant regions are shown in table 5. The amino acid position is based on SEQ ID NO. 53.
Table 5: an engineered SaCas9 selected flexible region amino acid sequence and a modified amino acid sequence.
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To screen engineered SaCas9 variants to improve gene editing activity, we performed a T7 enzyme cleavage assay according to the same method described above for GeoCas9 (fig. 16). The engineered SaSaSaSa 9 variants 1.1 (SEQ ID NO: 54), 3.1 (SEQ ID NO: 58) and 3.2 (SEQ ID NO: 59) (indicated by the boxes in FIG. 16) showed significantly improved gene editing efficiency.
Example 6: genome editing of Cas12i in mammalian cells
This example describes characterization of wild-type Cas12i1 and Cas12i2.
Here we show that Cas12i1 and Cas12i2 can cleave dsDNA in vitro. Cas12i1 and Cas12i2 produced cleaved dsDNA products in vitro, which were resolved by gel electrophoresis (fig. 17A). The cRNA used in the in vitro cleavage assay is shown in figure 17B.
We tested whether Cas12i1 and Cas12i2 are capable of genome editing in human cells. We designed multiple crrnas to target AAVs1 (2 crrnas), CCR5 (7 crrnas), CD34 (2 crrnas), and RNF2 (10 crrnas), respectively, and tested Cas12i1 and Cas12i2 for the ability to generate indels using each crRNA. We found that Cas12i2 was able to create indels in a broader target, suggesting that it is a promising candidate for engineering (fig. 18).
Based on the wild-type crRNA sequence of Cas12i1, we designed three mutated crRNA sequences:
we found that using crRNA with t3c point mutation, cas12i1 was able to generate indels with significantly higher efficiency than using wild-type crRNA sequence (FIG. 19).
Based on our discovery that Cas12i2 is capable of generating indels in a broad range of targets, we next sought to further characterize Cas12i2. Notably, we found that Cas12i2 has enzymatic activity (i.e., is capable of cleaving dsDNA) over a broad temperature range from 12 ℃ -67 ℃ (fig. 20). These data indicate that Cas12i2 has potential for use in a variety of organisms living under different temperature conditions or having different body temperatures.
In addition, we found that wild-type Cas12i2 was able to edit multiple genomic target sites in human cells using crRNA arrays (fig. 21).
We next determined the seed sequence of wild-type Cas12i 2. Target binding of the CRISPR-Cas ribonucleoprotein effector begins by the Cas protein moiety recognizing the double-stranded PAM motif, followed by destabilizing, local melting and interrogation (interaction) of the target by the guide of the CRISPR RNA moiety. The latter procedure depends on the seed sequence, i.e. the target portion which must be strictly complementary to the CRISPR RNA guide sequence. Mismatches between the target outside the seed and the CRISPR RNA guide sequence have little effect on target binding, thus leading to off-target activity of the CRISPR-Cas effect protein. Here we define the seed sequence.
To define the seed sequence of Cas12i2, we tested the ability of Cas12i2 to generate AAVs1-1 and RNF2-1 crRNA indels with single base mismatches at one of bases 1-19 of the crRNA. We found that the seed sequence of Cas12i2 is within 1-10bp (fig. 22).
Finally, we determine the optimal spacer length for Cas12i 2. We tested the ability of Cas12i2 to generate indels with two different crRNAs (CCR 5-1 and CCR 5-2) using fragments with spacer lengths from 17bp to 25 bp. We found that the optimal spacer length for editing of the Cas12i2 gene was 20bp (FIG. 23).
Method
Cas12i expression
For prokaryotic expression, the coding sequence for Cas12i2 and variants thereof was ligated into BPK2104-ccdB expression vector digested with XmaI and SpeI using T4 ligase.
In vitro cleavage assay
Templates for in vitro cleavage assays were created by PCR and passed through DNA Clean&Concentator kit (ZYMO) TM Research) was purified. In a single in vitro cleavage assay, 100nM template was used, along with 1mM Cas12i protein and 2mM crRNA. The reaction is carried out in a 1xNEBUFFER TM 3.1 (for Cas12i 1) and 1x NEBUFFER TM 2 (for Cas12i 2) at 37 ℃ for 60 minutes. To determine the thermal stability of Cas12i2, the cleavage mixtures were subjected to a large range in their cleavage buffersIncubation was performed for 1 hour at ambient temperature (4 ℃ C. -67 ℃ C.). To perform the plasmid cleavage assay, plasmids were incubated with different concentrations of RNP formed by Cas12i2 or enCas12i2 plus crRNA. In 1xNEBUFFER TM 2 at 37℃for 10 minutes. After incubation with protease (Takara) for 20 min at 37℃the reaction was terminated by adding RNase mixture (Thermo Fisher Scientific) to digest crRNA at 37℃for 20 min. The reaction was resolved by agarose gel electrophoresis and ethidium bromide staining.
Sequence listing
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Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile
785 790 795 800
Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln
805 810 815
Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn
820 825 830
Tyr Asn Pro Gly Gly Glu Arg Ser Arg Phe Glu Asn Ser Arg Leu Met
835 840 845
Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu
850 855 860
Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg
865 870 875 880
Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Arg Val Val Thr
885 890 895
Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu
900 905 910
Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu
915 920 925
Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg
930 935 940
Lys Cys Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln
945 950 955 960
Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys
965 970 975
Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp
980 985 990
Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu
995 1000 1005
Lys Asp Gly Val Tyr Glu Trp Val Asn Ala Gly Lys Leu Lys Ile Lys
1010 1015 1020
Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser Asp Ile
1025 1030 1035 1040
Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly Glu Lys Leu
1045 1050 1055
Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro Ser Asp Lys Trp
1060 1065 1070
Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu Arg Ile Leu Ile Ser
1075 1080 1085
Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr Ile Glu Asp Asp Ser Ser
1090 1095 1100
Lys Gln Ser Met
1105
<210> 3
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 3
Thr Thr Asn
1
<210> 4
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 4
Asn Asn Gly Arg Arg Thr
1 5
<210> 5
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 5
Asn Asn Asn Asn Arg Tyr Ala Cys
1 5
<210> 6
<211> 1108
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 6
Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val
1 5 10 15
Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala
20 25 30
Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu
35 40 45
His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu
50 55 60
Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn
65 70 75 80
Ser Phe Thr His Glu Val Asp Lys Asp Glu Val Phe Asn Ile Leu Arg
85 90 95
Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu
100 105 110
Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn
115 120 125
Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp
130 135 140
Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys
145 150 155 160
Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly
165 170 175
Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Tyr Thr Asp
180 185 190
Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg
195 200 205
Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu
210 215 220
Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu
225 230 235 240
Tyr Glu Lys Val Glu Lys Glu Tyr Lys Thr Leu Glu Glu Arg Ile Lys
245 250 255
Glu Asp Ile Gln Ala Leu Lys Ala Leu Glu Gln Tyr Glu Lys Glu Arg
260 265 270
Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu
275 280 285
Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu
290 295 300
Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys
305 310 315 320
Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr
325 330 335
Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro
340 345 350
Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys
355 360 365
Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn
370 375 380
His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn
385 390 395 400
Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys
405 410 415
Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly
420 425 430
Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg
435 440 445
Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His
450 455 460
Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr
465 470 475 480
Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr
485 490 495
Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met
500 505 510
Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys
515 520 525
Ile His Arg Asp Asp Phe Pro Lys Val Val Asn Phe Lys Pro Lys Glu
530 535 540
Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly
545 550 555 560
Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly
565 570 575
Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys
580 585 590
Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu
595 600 605
Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr
610 615 620
Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu
625 630 635 640
Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe
645 650 655
Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp
660 665 670
Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu
675 680 685
Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val
690 695 700
Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys
705 710 715 720
Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu
725 730 735
Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys
740 745 750
Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg
755 760 765
Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu
770 775 780
Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile
785 790 795 800
Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln
805 810 815
Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn
820 825 830
Tyr Asn Pro Tyr Gly Glu Arg Ser Arg Phe Glu Asn Ser Arg Leu Met
835 840 845
Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu
850 855 860
Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg
865 870 875 880
Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Arg Val Val Thr
885 890 895
Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu
900 905 910
Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu
915 920 925
Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg
930 935 940
Lys Cys Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln
945 950 955 960
Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys
965 970 975
Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp
980 985 990
Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu
995 1000 1005
Lys Asp Gly Val Tyr Glu Trp Val Asn Ala Gly Lys Leu Lys Ile Lys
1010 1015 1020
Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser Asp Ile
1025 1030 1035 1040
Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly Glu Lys Leu
1045 1050 1055
Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro Ser Asp Lys Trp
1060 1065 1070
Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu Arg Ile Leu Ile Ser
1075 1080 1085
Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr Ile Glu Asp Asp Ser Ser
1090 1095 1100
Lys Gln Ser Met
1105
<210> 7
<211> 1129
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 7
Met Ala Val Lys Ser Met Lys Val Lys Leu Arg Leu Asp Asn Met Pro
1 5 10 15
Glu Ile Arg Ala Gly Leu Trp Lys Leu His Thr Glu Val Asn Ala Gly
20 25 30
Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu
35 40 45
Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Tyr Lys Thr
50 55 60
Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln
65 70 75 80
Val Glu Asn Gly His Cys Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu
85 90 95
Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly
100 105 110
Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu
115 120 125
Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn
130 135 140
Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu
145 150 155 160
Glu Glu Lys Ala Lys Ala Glu Ala Arg Lys Ser Thr Asp Arg Thr Ala
165 170 175
Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg
180 185 190
Val Tyr Thr Asp Ser Asp Met Ser Ser Val Gln Trp Lys Pro Leu Arg
195 200 205
Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala
210 215 220
Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Glu
225 230 235 240
Ala Tyr Ala Lys Leu Val Glu Gln Lys Ser Arg Phe Glu Gln Lys Asn
245 250 255
Phe Val Gly Gln Glu His Leu Val Gln Leu Val Asn Gln Leu Gln Gln
260 265 270
Asp Met Lys Glu Ala Ser His Gly Leu Glu Ser Lys Glu Gln Thr Ala
275 280 285
His Tyr Leu Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu
290 295 300
Lys Trp Glu Lys Leu Asp Pro Asp Ala Pro Phe Asp Leu Tyr Asp Thr
305 310 315 320
Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His
325 330 335
Asp Leu Phe Ala Lys Leu Ala Glu Pro Lys Tyr Gln Ala Leu Trp Arg
340 345 350
Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Val
355 360 365
Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp
370 375 380
Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn
385 390 395 400
Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Gly Arg His
405 410 415
Ala Ile Arg Phe Gln Lys Leu Leu Thr Val Glu Asp Gly Val Ala Lys
420 425 430
Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Ala Gln Leu Asp
435 440 445
Asp Leu Leu Pro Arg Asp Pro His Glu Leu Val Ala Leu Tyr Phe Gln
450 455 460
Asp Tyr Gly Ala Glu Gln His Leu Ala Gly Glu Phe Gly Gly Ala Lys
465 470 475 480
Ile Gln Tyr Arg Arg Asp Gln Leu Asn His Leu His Ala Arg Arg Gly
485 490 495
Ala Arg Asp Val Tyr Leu Asn Leu Ser Val Arg Val Gln Ser Gln Ser
500 505 510
Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu
515 520 525
Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp
530 535 540
Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu
545 550 555 560
Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser
565 570 575
Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro
580 585 590
Asn Ser Glu Gly Arg Val Pro Phe Cys Phe Pro Ile Glu Gly Asn Glu
595 600 605
Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly
610 615 620
Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg
625 630 635 640
Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val
645 650 655
Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys
660 665 670
Leu Ile Glu Gln Pro Met Asp Ala Asn Gln Met Thr Pro Asp Trp Arg
675 680 685
Glu Ala Phe Glu Asp Glu Leu Gln Lys Leu Lys Ser Leu Tyr Gly Ile
690 695 700
Cys Gly Asp Arg Glu Trp Thr Glu Ala Val Tyr Glu Ser Val Arg Arg
705 710 715 720
Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp Val
725 730 735
Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Gln Lys Asp Val Val
740 745 750
Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr Lys
755 760 765
Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val Ile
770 775 780
Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His Ile
785 790 795 800
Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile Ile
805 810 815
Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Asp Glu Arg Gly Lys
820 825 830
Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu
835 840 845
Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn
850 855 860
Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Leu
865 870 875 880
Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala
885 890 895
Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys
900 905 910
Arg Arg Val Pro Ala Arg Cys Ala Arg Glu Gln Asn Pro Glu Pro Phe
915 920 925
Pro Trp Trp Leu Asn Lys Phe Val Ala Glu His Lys Leu Asp Gly Cys
930 935 940
Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Phe Phe
945 950 955 960
Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala
965 970 975
Asp Leu Asn Ala Ala Gln Asn Leu Gln Arg Arg Leu Trp Ser Asp Phe
980 985 990
Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly
995 1000 1005
Glu Pro Val Leu Ile Pro Arg Thr Thr Gly Lys Arg Thr Ala Asp Ser
1010 1015 1020
Tyr Gly Asn Lys Val Phe Tyr Thr Lys Thr Gly Val Thr Tyr Tyr Glu
1025 1030 1035 1040
Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln Glu Glu Leu
1045 1050 1055
Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp Glu Ala Arg Glu
1060 1065 1070
Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly Ile Ile Asn Arg Gly
1075 1080 1085
Asp Trp Thr Arg Gln Lys Glu Phe Trp Ser Met Val Asn Gln Arg Ile
1090 1095 1100
Glu Gly Tyr Leu Val Lys Gln Ile Arg Ser Arg Val Arg Leu Gln Glu
1105 1110 1115 1120
Ser Ala Cys Glu Asn Thr Gly Asp Ile
1125
<210> 8
<211> 1054
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 8
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 9
<211> 1093
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 9
Met Ser Asn Lys Glu Lys Asn Ala Ser Glu Thr Arg Lys Ala Tyr Thr
1 5 10 15
Thr Lys Met Ile Pro Arg Ser His Asp Arg Met Lys Leu Leu Gly Asn
20 25 30
Phe Met Asp Tyr Leu Met Asp Gly Thr Pro Ile Phe Phe Glu Leu Trp
35 40 45
Asn Gln Phe Gly Gly Gly Ile Asp Arg Asp Ile Ile Ser Gly Thr Ala
50 55 60
Asn Lys Asp Lys Ile Ser Asp Asp Leu Leu Leu Ala Val Asn Trp Phe
65 70 75 80
Lys Val Met Pro Ile Asn Ser Lys Pro Gln Gly Val Ser Pro Ser Asn
85 90 95
Leu Ala Asn Leu Phe Gln Gln Tyr Ser Gly Ser Glu Pro Asp Ile Gln
100 105 110
Ala Gln Glu Tyr Phe Ala Ser Asn Phe Asp Thr Glu Lys His Gln Trp
115 120 125
Lys Asp Met Arg Val Glu Tyr Glu Arg Leu Leu Ala Glu Leu Gln Leu
130 135 140
Ser Arg Ser Asp Met His His Asp Leu Lys Leu Met Tyr Lys Glu Lys
145 150 155 160
Cys Ile Gly Leu Ser Leu Ser Thr Ala His Tyr Ile Thr Ser Val Met
165 170 175
Phe Gly Thr Gly Ala Lys Asn Asn Arg Gln Thr Lys His Gln Phe Tyr
180 185 190
Ser Lys Val Ile Gln Leu Leu Glu Glu Ser Thr Gln Ile Asn Ser Val
195 200 205
Glu Gln Leu Ala Ser Ile Ile Leu Lys Ala Gly Asp Cys Asp Ser Tyr
210 215 220
Arg Lys Leu Arg Ile Arg Cys Ser Arg Lys Gly Ala Thr Pro Ser Ile
225 230 235 240
Leu Lys Ile Val Gln Asp Tyr Glu Leu Gly Thr Asn His Asp Asp Glu
245 250 255
Val Asn Val Pro Ser Leu Ile Ala Asn Leu Lys Glu Lys Leu Gly Arg
260 265 270
Phe Glu Tyr Glu Cys Glu Trp Lys Cys Met Glu Lys Ile Lys Ala Phe
275 280 285
Leu Ala Ser Lys Val Gly Pro Tyr Tyr Leu Gly Ser Tyr Ser Ala Met
290 295 300
Leu Glu Asn Ala Leu Ser Pro Ile Lys Gly Met Thr Thr Lys Asn Cys
305 310 315 320
Lys Phe Val Leu Lys Gln Ile Asp Ala Lys Asn Asp Ile Lys Tyr Glu
325 330 335
Asn Glu Pro Phe Gly Lys Ile Val Glu Gly Phe Phe Asp Ser Pro Tyr
340 345 350
Phe Glu Ser Asp Thr Asn Val Lys Trp Val Leu His Pro His His Ile
355 360 365
Gly Glu Ser Asn Ile Lys Thr Leu Trp Glu Asp Leu Asn Ala Ile His
370 375 380
Ser Lys Tyr Glu Glu Asp Ile Ala Ser Leu Ser Glu Asp Lys Lys Glu
385 390 395 400
Lys Arg Ile Lys Val Tyr Gln Gly Asp Val Cys Gln Thr Ile Asn Thr
405 410 415
Tyr Cys Glu Glu Val Gly Lys Glu Ala Lys Thr Pro Leu Val Gln Leu
420 425 430
Leu Arg Tyr Leu Tyr Ser Arg Lys Asp Asp Ile Ala Val Asp Lys Ile
435 440 445
Ile Asp Gly Ile Thr Phe Leu Ser Lys Lys His Lys Val Glu Lys Gln
450 455 460
Lys Ile Asn Pro Val Ile Gln Lys Tyr Pro Ser Phe Asn Phe Gly Asn
465 470 475 480
Asn Ser Lys Leu Leu Gly Lys Ile Ile Ser Pro Lys Asp Lys Leu Lys
485 490 495
His Asn Leu Lys Cys Asn Arg Asn Gln Val Asp Asn Tyr Ile Trp Ile
500 505 510
Glu Ile Lys Val Leu Asn Thr Lys Thr Met Arg Trp Glu Lys His His
515 520 525
Tyr Ala Leu Ser Ser Thr Arg Phe Leu Glu Glu Val Tyr Tyr Pro Ala
530 535 540
Thr Ser Glu Asn Pro Pro Asp Ala Leu Ala Ala Arg Phe Arg Thr Lys
545 550 555 560
Thr Asn Gly Tyr Glu Gly Lys Pro Ala Leu Ser Ala Glu Gln Ile Glu
565 570 575
Gln Ile Arg Ser Ala Pro Val Gly Leu Arg Lys Val Lys Lys Arg Gln
580 585 590
Met Arg Leu Glu Ala Ala Arg Gln Gln Asn Leu Leu Pro Arg Tyr Thr
595 600 605
Trp Gly Lys Asp Phe Asn Ile Asn Ile Cys Lys Arg Gly Asn Asn Phe
610 615 620
Glu Val Thr Leu Ala Thr Lys Val Lys Lys Lys Lys Glu Lys Asn Tyr
625 630 635 640
Lys Val Val Leu Gly Tyr Asp Ala Asn Ile Val Arg Lys Asn Thr Tyr
645 650 655
Ala Ala Ile Glu Ala His Ala Asn Gly Asp Gly Val Ile Asp Tyr Asn
660 665 670
Asp Leu Pro Val Lys Pro Ile Glu Ser Gly Phe Val Thr Val Glu Ser
675 680 685
Gln Val Arg Asp Lys Ser Tyr Asp Gln Leu Ser Tyr Asn Gly Val Lys
690 695 700
Leu Leu Tyr Cys Lys Pro His Val Glu Ser Arg Arg Ser Phe Leu Glu
705 710 715 720
Lys Tyr Arg Asn Gly Thr Met Lys Asp Asn Arg Gly Asn Asn Ile Gln
725 730 735
Ile Asp Phe Met Lys Asp Phe Glu Ala Ile Ala Asp Asp Glu Thr Ser
740 745 750
Leu Tyr Tyr Phe Asn Met Lys Tyr Cys Lys Leu Leu Gln Ser Ser Ile
755 760 765
Arg Asn His Ser Ser Gln Ala Lys Glu Tyr Arg Glu Glu Ile Phe Glu
770 775 780
Leu Leu Arg Asp Gly Lys Leu Ser Val Leu Lys Leu Ser Ser Leu Ser
785 790 795 800
Asn Leu Ser Phe Val Met Phe Lys Val Ala Lys Ser Leu Ile Gly Thr
805 810 815
Tyr Phe Gly His Leu Leu Lys Lys Pro Lys Asn Ser Lys Ser Asp Val
820 825 830
Lys Ala Pro Pro Ile Thr Asp Glu Asp Lys Gln Lys Ala Asp Pro Glu
835 840 845
Met Phe Ala Leu Arg Leu Ala Leu Glu Glu Lys Arg Leu Asn Lys Val
850 855 860
Lys Ser Lys Lys Glu Val Ile Ala Asn Lys Ile Val Ala Lys Ala Leu
865 870 875 880
Glu Leu Arg Asp Lys Tyr Gly Pro Val Leu Ile Lys Gly Glu Asn Ile
885 890 895
Ser Asp Thr Thr Lys Lys Gly Lys Lys Ser Ser Thr Asn Ser Phe Leu
900 905 910
Met Asp Trp Leu Ala Arg Gly Val Ala Asn Lys Val Lys Glu Met Val
915 920 925
Met Met His Gln Gly Leu Glu Phe Val Glu Val Asn Pro Asn Phe Thr
930 935 940
Ser His Gln Asp Pro Phe Val His Lys Asn Pro Glu Asn Thr Phe Arg
945 950 955 960
Ala Arg Tyr Ser Arg Cys Thr Pro Ser Glu Leu Thr Glu Lys Asn Arg
965 970 975
Lys Glu Ile Leu Ser Phe Leu Ser Asp Lys Pro Ser Lys Arg Pro Thr
980 985 990
Asn Ala Tyr Tyr Asn Glu Gly Ala Met Ala Phe Leu Ala Thr Tyr Gly
995 1000 1005
Leu Lys Lys Asn Asp Val Leu Gly Val Ser Leu Glu Lys Phe Lys Gln
1010 1015 1020
Ile Met Ala Asn Ile Leu His Gln Arg Ser Glu Asp Gln Leu Leu Phe
1025 1030 1035 1040
Pro Ser Arg Gly Gly Met Phe Tyr Leu Ala Thr Tyr Lys Leu Asp Ala
1045 1050 1055
Asp Ala Thr Ser Val Asn Trp Asn Gly Lys Gln Phe Trp Val Cys Asn
1060 1065 1070
Ala Asp Leu Val Ala Ala Tyr Asn Val Gly Leu Val Asp Ile Gln Lys
1075 1080 1085
Asp Phe Lys Lys Lys
1090
<210> 10
<211> 1046
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 10
Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro
1 5 10 15
Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp
20 25 30
His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala
35 40 45
Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe
50 55 60
Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys
65 70 75 80
Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu
85 90 95
Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr
100 105 110
Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp Cys Arg
115 120 125
Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser
130 135 140
Leu Lys Ile Asp Ala Cys Thr Leu Phe Met Asn Lys Leu Ile Pro Val
145 150 155 160
Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe
165 170 175
Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met Leu Glu
180 185 190
Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp Glu Glu
195 200 205
Tyr Gln Asp Leu Ile Lys Arg Thr Phe Asp Val Ser Asn Tyr Lys Glu
210 215 220
Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu
225 230 235 240
Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Leu
245 250 255
Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Lys
260 265 270
Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala
275 280 285
Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn
290 295 300
Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr
305 310 315 320
Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys
325 330 335
Lys Asp Ile Leu Leu Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly
340 345 350
Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu
355 360 365
Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp Gly Ile
370 375 380
Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val
385 390 395 400
Lys Ala Leu Leu Gln Tyr Ile Tyr Pro His Phe Asp Lys Ile Thr Ala
405 410 415
Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn
420 425 430
Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn
435 440 445
Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln
450 455 460
Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr
465 470 475 480
Met Lys Val Ile Asp Asn Gly Arg Trp Val Ser His His Leu Pro Phe
485 490 495
His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu
500 505 510
Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly
515 520 525
Ser Ile Ile Ser Ala Thr Lys Leu Ala Ala Leu Lys Ser Gln Glu Glu
530 535 540
Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser
545 550 555 560
Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys
565 570 575
Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr
580 585 590
Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile
595 600 605
Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr
610 615 620
Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe
625 630 635 640
His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser
645 650 655
Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile
660 665 670
Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu
675 680 685
Arg Ser Ile Asn Glu Asp Tyr Val Ile Lys Ser Asp Asn Trp Arg Asn
690 695 700
Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp
705 710 715 720
Val Met Lys Val Asn Lys Asp Lys Asp Val Gln Asn Thr Phe Arg Ala
725 730 735
Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser
740 745 750
Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile
755 760 765
Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Asp Tyr Asp Gln Glu
770 775 780
Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys
785 790 795 800
Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val
805 810 815
Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val Glu Gly
820 825 830
Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys
835 840 845
Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly
850 855 860
Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr
865 870 875 880
Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val
885 890 895
Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu
900 905 910
Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys
915 920 925
Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Pro Leu Tyr Gln
930 935 940
Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe Asp Ser
945 950 955 960
Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly Asp His
965 970 975
Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr
980 985 990
Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu
995 1000 1005
Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val Leu
1010 1015 1020
Arg Gly Ile Lys Asp Glu Asp Glu Gln Pro Asp Asp Val Lys Lys Gln
1025 1030 1035 1040
Ala Leu Ala Arg Thr Lys
1045
<210> 11
<211> 1054
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 11
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Gly Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 12
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 12
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Gly Gly Ala Pro Ser Thr Leu Glu Lys
225 230 235 240
Phe Ile Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln
245 250 255
Thr Asp Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp
260 265 270
Cys Cys Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln
275 280 285
Tyr Asp Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala
290 295 300
Leu Lys Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu
305 310 315 320
Glu Gln Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys
325 330 335
Lys Leu Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr
340 345 350
Tyr Thr Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu
355 360 365
Tyr Lys Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val
370 375 380
Val Leu Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg
385 390 395 400
Asn Ile Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln
405 410 415
Asp Ile Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys
420 425 430
Ser Gln Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 13
<211> 1054
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 13
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Gly Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 14
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 14
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Gly Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 15
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 15
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Gly Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 16
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 16
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Gly Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 17
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 17
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Gly Gly Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 18
<211> 1054
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 18
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Gly Gly Thr
915 920 925
Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu
930 935 940
Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys
945 950 955 960
Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu
965 970 975
Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile
980 985 990
Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe
995 1000 1005
Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala Ala
1010 1015 1020
Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser Asp Glu
1025 1030 1035 1040
Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050
<210> 19
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 19
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn
435 440 445
Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser
450 455 460
Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly
465 470 475 480
Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln
485 490 495
Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg
500 505 510
Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
515 520 525
Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg
530 535 540
Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys
545 550 555 560
Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile
565 570 575
Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly
580 585 590
Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr
595 600 605
Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly
610 615 620
Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu
625 630 635 640
Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr
645 650 655
Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu
660 665 670
Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu
675 680 685
Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr
690 695 700
Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly
705 710 715 720
Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe
725 730 735
Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser
740 745 750
Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe
755 760 765
Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln
770 775 780
Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu
785 790 795 800
Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn
805 810 815
Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly
820 825 830
Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn
835 840 845
Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg
850 855 860
Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu
865 870 875 880
Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala
885 890 895
Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp
900 905 910
Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Gly
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 20
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 20
Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu
1 5 10 15
Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly
20 25 30
Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr
35 40 45
Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp
50 55 60
Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp
65 70 75 80
Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu
85 90 95
Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser
100 105 110
Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr
115 120 125
Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His
130 135 140
Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu
145 150 155 160
Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu
165 170 175
Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu
180 185 190
Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln
195 200 205
Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln
210 215 220
Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile
225 230 235 240
Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp
245 250 255
Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys
260 265 270
Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp
275 280 285
Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys
290 295 300
Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln
305 310 315 320
Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu
325 330 335
Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr
340 345 350
Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys
355 360 365
Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu
370 375 380
Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile
385 390 395 400
Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile
405 410 415
Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln
420 425 430
Lys Ala Asn Pro Ser Val Leu Gly Gly Gly Asn Gln Gly Phe Thr Trp
435 440 445
Thr Asn Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro
450 455 460
Asn Ser Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro
465 470 475 480
Asp Gly Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe
485 490 495
Phe Gln Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln
500 505 510
Phe Arg Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln
515 520 525
Thr Ala Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu
530 535 540
Ala Arg Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn
545 550 555 560
Leu Lys Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val
565 570 575
Arg Ile Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln
580 585 590
Ile Gly Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His
595 600 605
Ala Tyr Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu
610 615 620
Leu Gly Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile
625 630 635 640
Thr Glu Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu
645 650 655
Ala Tyr Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val
660 665 670
Ser Leu Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr
675 680 685
Val Glu Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg
690 695 700
Leu Tyr Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val
705 710 715 720
Arg Gly Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe
725 730 735
Arg Phe Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser
740 745 750
Leu Ser Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser
755 760 765
Tyr Phe Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp
770 775 780
Glu Gln Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys
785 790 795 800
Leu Glu Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile
805 810 815
Ala Asn Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile
820 825 830
Arg Gly Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys
835 840 845
Ala Asn Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys
850 855 860
Ile Arg Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly
865 870 875 880
Ser Leu Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp
885 890 895
Lys Ala Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly
900 905 910
Asp Trp Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Gly
915 920 925
Gly Thr Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His
930 935 940
Tyr Glu Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp
945 950 955 960
Arg Lys Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu
965 970 975
Lys Leu Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly
980 985 990
Arg Ile Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile
995 1000 1005
Val Phe Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala
1010 1015 1020
Ala Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser
1025 1030 1035 1040
Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr Ser
1045 1050 1055
<210> 21
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 21
Thr Thr Asn
1
<210> 22
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 22
Thr Thr His
1
<210> 23
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 23
Thr Thr Tyr
1
<210> 24
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 24
Thr Thr Cys
1
<210> 25
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 25
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 26
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 26
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Gly Gly Glu Ser Leu
35 40 45
Ala Leu Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg
50 55 60
Arg Lys His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly
65 70 75 80
Ile Leu Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu
85 90 95
Ile Asp Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn
100 105 110
Asn Asp Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly
115 120 125
Phe Lys Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr
130 135 140
Met Leu Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg
145 150 155 160
Thr Val Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys
165 170 175
Arg Asn Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 27
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 27
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Gly Gly Ser Asn Lys Glu Asn Ser Thr
130 135 140
Met Leu Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg
145 150 155 160
Thr Val Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys
165 170 175
Arg Asn Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 28
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 28
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Gly Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu
180 185 190
Glu Arg Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn
195 200 205
Met Ser Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala
210 215 220
Ser Gln Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly
225 230 235 240
Phe Cys Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr
245 250 255
Thr Phe Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu
260 265 270
Ile Ser Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 29
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 29
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Gly Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu
275 280 285
Leu Tyr Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile
290 295 300
Arg Thr Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val
305 310 315 320
Tyr Asp Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu
325 330 335
Glu Leu Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr
340 345 350
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 30
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 30
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Gly Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe
355 360 365
Gly Tyr Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr
370 375 380
Leu Arg Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 31
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 31
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Gly Gly Lys Arg Met Pro Asn Leu Ala
385 390 395 400
Asn Lys Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser
405 410 415
Phe Thr Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu
420 425 430
Pro Tyr Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala
435 440 445
Gly Tyr Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu
450 455 460
Pro Asn Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr
465 470 475 480
Gln Ala Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro
485 490 495
Val Ser Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp
500 505 510
Glu Arg Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 32
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 32
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Gly Gly Asn Arg Lys Lys Asn
515 520 525
Glu Thr Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro
530 535 540
Thr Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 33
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 33
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
Gly Gly His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly
545 550 555 560
Arg Cys Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu
565 570 575
Pro Gly Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu
580 585 590
Asp Asp Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg
595 600 605
Glu Lys Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu
610 615 620
Arg Trp Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser
625 630 635 640
Lys Lys Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu
645 650 655
Glu Thr Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser
660 665 670
Arg Phe Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser
675 680 685
Asp Asp Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His
690 695 700
Leu Arg Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu
705 710 715 720
His His Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp
725 730 735
Ile Ala Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu
740 745 750
Leu Ala Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 34
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 34
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Gly Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 35
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 35
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro Gly Gly His Phe Pro Gln Pro Trp Pro His Phe
755 760 765
Ala Asp Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 36
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 36
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Gly Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 37
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 37
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Gly Gly Ser Lys His Pro Lys Glu Ser Ile
770 775 780
Lys Ala Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 38
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 38
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Gly Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 39
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 39
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Gly Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 40
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 40
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Gly Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu
785 790 795 800
Gln Pro Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 41
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 41
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Gly Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 42
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 42
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Gly Gly Ala
805 810 815
Ala His Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser
820 825 830
Gly Lys Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu
835 840 845
Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 43
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 43
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Gly Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 44
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 44
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly Gly Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 45
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 45
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Gly Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 46
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 46
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Gly Gly Lys Glu Ser Asp Pro Arg
850 855 860
Thr Tyr Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 47
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 47
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Gly Gly Asn Asp Pro
865 870 875 880
Lys Lys Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 48
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 48
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Gly Gly Glu
885 890 895
Pro Gly Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln
900 905 910
Val Ile Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile
915 920 925
Val Arg Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro
930 935 940
Val Tyr Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile
945 950 955 960
Glu Pro Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr
965 970 975
Thr Phe Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu
980 985 990
Pro Arg Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val
995 1000 1005
Lys Asp Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 49
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 49
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Gly Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 50
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 50
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Gly Gly
1010 1015 1020
Leu Glu Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 51
<211> 1087
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 51
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Gly Gly Ser Arg
1025 1030 1035 1040
Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly Asn Ile
1045 1050 1055
Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser Ala His
1060 1065 1070
Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg Asp
1075 1080 1085
<210> 52
<211> 1089
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 52
Met Arg Tyr Lys Ile Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp
1 5 10 15
Ala Val Met Asn Leu Asp Ile Pro Arg Ile Glu Asp Leu Gly Val Arg
20 25 30
Ile Phe Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu Ala Leu
35 40 45
Pro Arg Arg Leu Ala Arg Ser Ala Arg Arg Arg Leu Arg Arg Arg Lys
50 55 60
His Arg Leu Glu Arg Ile Arg Arg Leu Val Ile Arg Glu Gly Ile Leu
65 70 75 80
Thr Lys Glu Glu Leu Asp Lys Leu Phe Glu Glu Lys His Glu Ile Asp
85 90 95
Val Trp Gln Leu Arg Val Glu Ala Leu Asp Arg Lys Leu Asn Asn Asp
100 105 110
Glu Leu Ala Arg Val Leu Leu His Leu Ala Lys Arg Arg Gly Phe Lys
115 120 125
Ser Asn Arg Lys Ser Glu Arg Ser Asn Lys Glu Asn Ser Thr Met Leu
130 135 140
Lys His Ile Glu Glu Asn Arg Ala Ile Leu Ser Ser Tyr Arg Thr Val
145 150 155 160
Gly Glu Met Ile Val Lys Asp Pro Lys Phe Ala Leu His Lys Arg Asn
165 170 175
Lys Gly Glu Asn Tyr Thr Asn Thr Ile Ala Arg Asp Asp Leu Glu Arg
180 185 190
Glu Ile Arg Leu Ile Phe Ser Lys Gln Arg Glu Phe Gly Asn Met Ser
195 200 205
Cys Thr Glu Glu Phe Glu Asn Glu Tyr Ile Thr Ile Trp Ala Ser Gln
210 215 220
Arg Pro Val Ala Ser Lys Asp Asp Ile Glu Lys Lys Val Gly Phe Cys
225 230 235 240
Thr Phe Glu Pro Lys Glu Lys Arg Ala Pro Lys Ala Thr Tyr Thr Phe
245 250 255
Gln Ser Phe Ile Ala Trp Glu His Ile Asn Lys Leu Arg Leu Ile Ser
260 265 270
Pro Ser Gly Ala Arg Gly Leu Thr Asp Glu Glu Arg Arg Leu Leu Tyr
275 280 285
Glu Gln Ala Phe Gln Lys Asn Lys Ile Thr Tyr His Asp Ile Arg Thr
290 295 300
Leu Leu His Leu Pro Asp Asp Thr Tyr Phe Lys Gly Ile Val Tyr Asp
305 310 315 320
Arg Gly Glu Ser Arg Lys Gln Asn Glu Asn Ile Arg Phe Leu Glu Leu
325 330 335
Asp Ala Tyr His Gln Ile Arg Lys Ala Val Asp Lys Val Tyr Gly Lys
340 345 350
Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp Phe Asp Thr Phe Gly Tyr
355 360 365
Ala Leu Thr Leu Phe Lys Asp Asp Ala Asp Ile His Ser Tyr Leu Arg
370 375 380
Asn Glu Tyr Glu Gln Asn Gly Lys Arg Met Pro Asn Leu Ala Asn Lys
385 390 395 400
Val Tyr Asp Asn Glu Leu Ile Glu Glu Leu Leu Asn Leu Ser Phe Thr
405 410 415
Lys Phe Gly His Leu Ser Leu Lys Ala Leu Arg Ser Ile Leu Pro Tyr
420 425 430
Met Glu Gln Gly Glu Val Tyr Ser Ser Ala Cys Glu Arg Ala Gly Tyr
435 440 445
Thr Phe Thr Gly Pro Lys Lys Lys Gln Lys Thr Met Leu Leu Pro Asn
450 455 460
Ile Pro Pro Ile Ala Asn Pro Val Val Met Arg Ala Leu Thr Gln Ala
465 470 475 480
Arg Lys Val Val Asn Ala Ile Ile Lys Lys Tyr Gly Ser Pro Val Ser
485 490 495
Ile His Ile Glu Leu Ala Arg Asp Leu Ser Gln Thr Phe Asp Glu Arg
500 505 510
Arg Lys Thr Lys Lys Glu Gln Asp Glu Asn Arg Lys Lys Asn Glu Thr
515 520 525
Ala Ile Arg Gln Leu Met Glu Tyr Gly Leu Thr Leu Asn Pro Thr Gly
530 535 540
His Asp Ile Val Lys Phe Lys Leu Trp Ser Glu Gln Asn Gly Arg Cys
545 550 555 560
Ala Tyr Ser Leu Gln Pro Ile Glu Ile Glu Arg Leu Leu Glu Pro Gly
565 570 575
Tyr Val Glu Val Asp His Val Ile Pro Tyr Ser Arg Ser Leu Asp Asp
580 585 590
Ser Tyr Thr Asn Lys Val Leu Val Leu Thr Arg Glu Asn Arg Glu Lys
595 600 605
Gly Asn Arg Ile Pro Ala Glu Tyr Leu Gly Val Gly Thr Glu Arg Trp
610 615 620
Gln Gln Phe Glu Thr Phe Val Leu Thr Asn Lys Gln Phe Ser Lys Lys
625 630 635 640
Lys Arg Asp Arg Leu Leu Arg Leu His Tyr Asp Glu Asn Glu Glu Thr
645 650 655
Glu Phe Lys Asn Arg Asn Leu Asn Asp Thr Arg Tyr Ile Ser Arg Phe
660 665 670
Phe Ala Asn Phe Ile Arg Glu His Leu Lys Phe Ala Glu Ser Asp Asp
675 680 685
Lys Gln Lys Val Tyr Thr Val Asn Gly Arg Val Thr Ala His Leu Arg
690 695 700
Ser Arg Trp Glu Phe Asn Lys Asn Arg Glu Glu Ser Asp Leu His His
705 710 715 720
Ala Val Asp Ala Ala Ile Val Ala Cys Thr Thr Pro Ser Asp Ile Ala
725 730 735
Lys Val Thr Ala Phe Tyr Gln Arg Arg Glu Gln Asn Lys Glu Leu Ala
740 745 750
Lys Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His Phe Ala Asp
755 760 765
Glu Leu Arg Ala Arg Leu Ser Lys His Pro Lys Glu Ser Ile Lys Ala
770 775 780
Leu Asn Leu Gly Asn Tyr Asp Asp Gln Lys Leu Glu Ser Leu Gln Pro
785 790 795 800
Val Phe Val Ser Arg Met Pro Lys Arg Ser Val Thr Gly Ala Ala His
805 810 815
Gln Glu Thr Leu Arg Arg Tyr Val Gly Ile Asp Glu Arg Ser Gly Lys
820 825 830
Ile Gln Thr Val Val Lys Thr Lys Leu Ser Glu Ile Lys Leu Asp Ala
835 840 845
Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser Asp Pro Arg Thr Tyr
850 855 860
Glu Ala Ile Arg Gln Arg Leu Leu Glu His Asn Asn Asp Pro Lys Lys
865 870 875 880
Ala Phe Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly
885 890 895
Pro Val Ile Arg Thr Val Lys Ile Ile Asp Thr Lys Asn Gln Val Ile
900 905 910
Pro Leu Asn Asp Gly Lys Thr Val Ala Tyr Asn Ser Asn Ile Val Arg
915 920 925
Val Asp Val Phe Glu Lys Asp Gly Lys Tyr Tyr Cys Val Pro Val Tyr
930 935 940
Thr Met Asp Ile Met Lys Gly Ile Leu Pro Asn Lys Ala Ile Glu Pro
945 950 955 960
Asn Lys Pro Tyr Ser Glu Trp Lys Glu Met Thr Glu Asp Tyr Thr Phe
965 970 975
Arg Phe Ser Leu Tyr Pro Asn Asp Leu Ile Arg Ile Glu Leu Pro Arg
980 985 990
Glu Lys Thr Val Lys Thr Ala Ala Gly Glu Glu Ile Asn Val Lys Asp
995 1000 1005
Val Phe Val Tyr Tyr Lys Thr Ile Asp Ser Ala Asn Gly Gly Leu Glu
1010 1015 1020
Leu Ile Ser His Asp His Arg Phe Ser Leu Arg Gly Val Gly Gly Gly
1025 1030 1035 1040
Ser Arg Thr Leu Lys Arg Phe Glu Lys Tyr Gln Val Asp Val Leu Gly
1045 1050 1055
Asn Ile Tyr Lys Val Arg Gly Glu Lys Arg Val Gly Leu Ala Ser Ser
1060 1065 1070
Ala His Ser Lys Thr Gly Glu Thr Val Arg Pro Leu Gln Ser Thr Arg
1075 1080 1085
Asp
<210> 53
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 53
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 54
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 54
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Gly
35 40 45
Gly Arg Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg
50 55 60
His Arg Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu
65 70 75 80
Thr Asp His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val
85 90 95
Lys Gly Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu
100 105 110
Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 55
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 55
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Gly Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 56
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 56
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Gly Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 57
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 57
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Gly Gly Ile Asn Pro Tyr Glu Ala Arg Val
85 90 95
Lys Gly Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu
100 105 110
Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 58
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 58
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Gly His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 59
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 59
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Gly Gly Val His Asn Val Asn Glu Val Glu
115 120 125
Glu Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 60
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 60
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Gly Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn
130 135 140
Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg
145 150 155 160
Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr
165 170 175
Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala
180 185 190
Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu
195 200 205
Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 61
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 61
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Gly Gly Ser Pro Phe
210 215 220
Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys
225 230 235 240
Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala
245 250 255
Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg
260 265 270
Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu
275 280 285
Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys
290 295 300
Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser
305 310 315 320
Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 62
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 62
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Gly Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys
325 330 335
Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp
340 345 350
Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln
355 360 365
Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu
370 375 380
Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 63
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 63
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Gly Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 64
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 64
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Gly Gly Tyr Thr Gly Thr His Asn Leu Ser Leu
385 390 395 400
Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn
405 410 415
Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp
420 425 430
Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile
435 440 445
Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile
450 455 460
Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu
465 470 475 480
Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu
485 490 495
Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 65
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 65
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Gly Gly Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile
500 505 510
Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys
515 520 525
Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile
530 535 540
Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His
545 550 555 560
Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val
565 570 575
Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 66
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 66
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Gly Gly Asn Arg Thr Pro Phe
580 585 590
Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 67
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 67
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Gly Gly Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys
595 600 605
Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 68
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 68
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Gly Gly Arg Ile Ser Lys Thr
610 615 620
Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val
625 630 635 640
Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr
645 650 655
Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu
660 665 670
Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg
675 680 685
Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 69
<211> 1056
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 69
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Gly Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys
740 745 750
Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys
755 760 765
Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu
770 775 780
Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr
785 790 795 800
Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys
805 810 815
Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His
820 825 830
His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr
835 840 845
Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn
850 855 860
Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys
865 870 875 880
Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp
885 890 895
Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro
900 905 910
Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr
915 920 925
Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn
930 935 940
Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln
945 950 955 960
Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn
965 970 975
Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg
980 985 990
Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn
995 1000 1005
Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys
1010 1015 1020
Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr
1025 1030 1035 1040
Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser
1045 1050 1055
<210> 70
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 70
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Gly Gly Tyr Lys His His
690 695 700
Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu
705 710 715 720
Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe
725 730 735
Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 71
<211> 1058
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 71
Met Gly Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser
1 5 10 15
Val Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala
20 25 30
Gly Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg
35 40 45
Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg
50 55 60
Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp
65 70 75 80
His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly
85 90 95
Leu Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His
100 105 110
Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp
115 120 125
Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys
130 135 140
Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys
145 150 155 160
Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp
165 170 175
Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His
180 185 190
Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr
195 200 205
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
210 215 220
Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr
225 230 235 240
Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu
245 250 255
Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu
260 265 270
Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val
275 280 285
Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile
290 295 300
Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly
305 310 315 320
Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile
325 330 335
Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile
340 345 350
Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu
355 360 365
Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile
370 375 380
Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala
385 390 395 400
Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile
405 410 415
Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser
420 425 430
Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser
435 440 445
Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala
450 455 460
Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala
465 470 475 480
Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln
485 490 495
Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr
500 505 510
Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His
515 520 525
Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu
530 535 540
Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile
545 550 555 560
Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val
565 570 575
Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr
580 585 590
Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His
595 600 605
Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys
610 615 620
Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys
625 630 635 640
Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly
645 650 655
Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val
660 665 670
Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys
675 680 685
Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu
690 695 700
Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys
705 710 715 720
Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu
725 730 735
Lys Gln Ala Glu Gly Gly Ser Met Pro Glu Ile Glu Thr Glu Gln Glu
740 745 750
Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp
755 760 765
Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg
770 775 780
Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly
785 790 795 800
Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn
805 810 815
Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met
820 825 830
Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu
835 840 845
Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr
850 855 860
Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile
865 870 875 880
Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile
885 890 895
Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu
900 905 910
Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe
915 920 925
Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu
930 935 940
Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser
945 950 955 960
Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys
965 970 975
Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu
980 985 990
Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu
995 1000 1005
Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala
1010 1015 1020
Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn
1025 1030 1035 1040
Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050 1055
Gly Ser
<210> 72
<211> 1369
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 72
Met Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser
1 5 10 15
Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
20 25 30
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu
35 40 45
Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg
50 55 60
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
65 70 75 80
Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp
85 90 95
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
100 105 110
Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala
115 120 125
Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val
130 135 140
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala
145 150 155 160
His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn
165 170 175
Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr
180 185 190
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
195 200 205
Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu
210 215 220
Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly
225 230 235 240
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn
245 250 255
Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr
260 265 270
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala
275 280 285
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser
290 295 300
Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala
305 310 315 320
Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
325 330 335
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe
340 345 350
Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala
355 360 365
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met
370 375 380
Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
385 390 395 400
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His
405 410 415
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
420 425 430
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg
435 440 445
Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala
450 455 460
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu
465 470 475 480
Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met
485 490 495
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
500 505 510
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val
515 520 525
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu
530 535 540
Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
545 550 555 560
Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
565 570 575
Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu
580 585 590
Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
595 600 605
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu
610 615 620
Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr
625 630 635 640
Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg
645 650 655
Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg
660 665 670
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
675 680 685
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
690 695 700
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
705 710 715 720
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
725 730 735
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
740 745 750
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn
755 760 765
Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg
770 775 780
Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His
785 790 795 800
Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr
805 810 815
Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn
820 825 830
Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
835 840 845
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn
850 855 860
Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met
865 870 875 880
Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
885 890 895
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu
900 905 910
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
915 920 925
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr
930 935 940
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
945 950 955 960
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val
965 970 975
Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
980 985 990
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu
995 1000 1005
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035 1040
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly
1045 1050 1055
Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu
1060 1065 1070
Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu
1075 1080 1085
Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly
1090 1095 1100
Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu
1105 1110 1115 1120
Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1125 1130 1135
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys
1140 1145 1150
Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1155 1160 1165
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu
1170 1175 1180
Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1185 1190 1195 1200
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala
1205 1210 1215
Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1220 1225 1230
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly
1235 1240 1245
Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275 1280
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn
1285 1290 1295
Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His
1300 1305 1310
Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
1315 1320 1325
Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu
1330 1335 1340
Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg
1345 1350 1355 1360
Ile Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 73
<211> 1082
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 73
Met Ala Ala Phe Lys Pro Asn Pro Ile Asn Tyr Ile Leu Gly Leu Asp
1 5 10 15
Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Glu
20 25 30
Glu Asn Pro Ile Arg Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg
35 40 45
Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu
50 55 60
Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu
65 70 75 80
Arg Ala Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asp
85 90 95
Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln
100 105 110
Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser
115 120 125
Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg
130 135 140
Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys
145 150 155 160
Gly Val Ala Asn Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr
165 170 175
Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile
180 185 190
Arg Asn Gln Arg Gly Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu
195 200 205
Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn
210 215 220
Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met
225 230 235 240
Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly
245 250 255
His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr
260 265 270
Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile
275 280 285
Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr
290 295 300
Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala
305 310 315 320
Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg
325 330 335
Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala
340 345 350
Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys
355 360 365
Lys Ser Pro Leu Asn Leu Ser Ser Glu Leu Gln Asp Glu Ile Gly Thr
370 375 380
Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys
385 390 395 400
Asp Arg Val Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser
405 410 415
Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val
420 425 430
Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile
435 440 445
Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu
450 455 460
Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala
465 470 475 480
Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly
485 490 495
Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser
500 505 510
Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys
515 520 525
Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe
530 535 540
Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu
545 550 555 560
Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Val
565 570 575
Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe
580 585 590
Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly
595 600 605
Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn
610 615 620
Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu
625 630 635 640
Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys
645 650 655
Phe Asp Glu Asp Gly Phe Lys Glu Cys Asn Leu Asn Asp Thr Arg Tyr
660 665 670
Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp His Ile Leu Leu Thr
675 680 685
Gly Lys Gly Lys Arg Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn
690 695 700
Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp
705 710 715 720
Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala
725 730 735
Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala
740 745 750
Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Lys Val Leu His Gln
755 760 765
Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met
770 775 780
Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala
785 790 795 800
Asp Thr Pro Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser
805 810 815
Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg
820 825 830
Ala Pro Asn Arg Lys Met Ser Gly Ala His Lys Asp Thr Leu Arg Ser
835 840 845
Ala Lys Arg Phe Val Lys His Asn Glu Lys Ile Ser Val Lys Arg Val
850 855 860
Trp Leu Thr Glu Ile Lys Leu Ala Asp Leu Glu Asn Met Val Asn Tyr
865 870 875 880
Lys Asn Gly Arg Glu Ile Glu Leu Tyr Glu Ala Leu Lys Ala Arg Leu
885 890 895
Glu Ala Tyr Gly Gly Asn Ala Lys Gln Ala Phe Asp Pro Lys Asp Asn
900 905 910
Pro Phe Tyr Lys Lys Gly Gly Gln Leu Val Lys Ala Val Arg Val Glu
915 920 925
Lys Thr Gln Glu Ser Gly Val Leu Leu Asn Lys Lys Asn Ala Tyr Thr
930 935 940
Ile Ala Asp Asn Gly Asp Met Val Arg Val Asp Val Phe Cys Lys Val
945 950 955 960
Asp Lys Lys Gly Lys Asn Gln Tyr Phe Ile Val Pro Ile Tyr Ala Trp
965 970 975
Gln Val Ala Glu Asn Ile Leu Pro Asp Ile Asp Cys Lys Gly Tyr Arg
980 985 990
Ile Asp Asp Ser Tyr Thr Phe Cys Phe Ser Leu His Lys Tyr Asp Leu
995 1000 1005
Ile Ala Phe Gln Lys Asp Glu Lys Ser Lys Val Glu Phe Ala Tyr Tyr
1010 1015 1020
Ile Asn Cys Asp Ser Ser Asn Gly Arg Phe Tyr Leu Ala Trp His Asp
1025 1030 1035 1040
Lys Gly Ser Lys Glu Gln Gln Phe Arg Ile Ser Thr Gln Asn Leu Val
1045 1050 1055
Leu Ile Gln Lys Tyr Gln Val Asn Glu Leu Gly Lys Glu Ile Arg Pro
1060 1065 1070
Cys Arg Leu Lys Lys Arg Pro Pro Val Arg
1075 1080
<210> 74
<211> 1307
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 74
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn
1010 1015 1020
Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu
1025 1030 1035 1040
Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly
1045 1050 1055
Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys
1060 1065 1070
Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile
1075 1080 1085
Lys Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu
1090 1095 1100
His Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn
1105 1110 1115 1120
Arg Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp
1125 1130 1135
Asp Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr
1140 1145 1150
Pro Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg
1155 1160 1165
Phe Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala
1170 1175 1180
Leu Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu
1185 1190 1195 1200
Pro Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val
1205 1210 1215
Ala Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr
1220 1225 1230
Gly Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala
1250 1255 1260
Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His
1265 1270 1275 1280
Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln
1285 1290 1295
Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1300 1305
<210> 75
<211> 1228
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 75
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser
1010 1015 1020
Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu
1025 1030 1035 1040
Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp
1045 1050 1055
Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile
1060 1065 1070
Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu
1075 1080 1085
Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile
1090 1095 1100
Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp
1105 1110 1115 1120
Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln
1125 1130 1135
Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser
1140 1145 1150
Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu
1155 1160 1165
Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala
1170 1175 1180
Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys
1185 1190 1195 1200
Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys
1205 1210 1215
Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 76
<211> 986
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 76
Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn
1 5 10 15
Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Lys Thr Leu Leu Val
20 25 30
Arg Val Met Thr Asp Asp Leu Lys Lys Arg Leu Glu Lys Arg Arg Lys
35 40 45
Lys Pro Glu Val Met Pro Gln Val Ile Ser Asn Asn Ala Ala Asn Asn
50 55 60
Leu Arg Met Leu Leu Asp Asp Tyr Thr Lys Met Lys Glu Ala Ile Leu
65 70 75 80
Gln Val Tyr Trp Gln Glu Phe Lys Asp Asp His Val Gly Leu Met Cys
85 90 95
Lys Phe Ala Gln Pro Ala Ser Lys Lys Ile Asp Gln Asn Lys Leu Lys
100 105 110
Pro Glu Met Asp Glu Lys Gly Asn Leu Thr Thr Ala Gly Phe Ala Cys
115 120 125
Ser Gln Cys Gly Gln Pro Leu Phe Val Tyr Lys Leu Glu Gln Val Ser
130 135 140
Glu Lys Gly Lys Ala Tyr Thr Asn Tyr Phe Gly Arg Cys Asn Val Ala
145 150 155 160
Glu His Glu Lys Leu Ile Leu Leu Ala Gln Leu Lys Pro Glu Lys Asp
165 170 175
Ser Asp Glu Ala Val Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg Ala
180 185 190
Leu Asp Phe Tyr Ser Ile His Val Thr Lys Glu Ser Thr His Pro Val
195 200 205
Lys Pro Leu Ala Gln Ile Ala Gly Asn Arg Tyr Ala Ser Gly Pro Val
210 215 220
Gly Lys Ala Leu Ser Asp Ala Cys Met Gly Thr Ile Ala Ser Phe Leu
225 230 235 240
Ser Lys Tyr Gln Asp Ile Ile Ile Glu His Gln Lys Val Val Lys Gly
245 250 255
Asn Gln Lys Arg Leu Glu Ser Leu Arg Glu Leu Ala Gly Lys Glu Asn
260 265 270
Leu Glu Tyr Pro Ser Val Thr Leu Pro Pro Gln Pro His Thr Lys Glu
275 280 285
Gly Val Asp Ala Tyr Asn Glu Val Ile Ala Arg Val Arg Met Trp Val
290 295 300
Asn Leu Asn Leu Trp Gln Lys Leu Lys Leu Ser Arg Asp Asp Ala Lys
305 310 315 320
Pro Leu Leu Arg Leu Lys Gly Phe Pro Ser Phe Pro Val Val Glu Arg
325 330 335
Arg Glu Asn Glu Val Asp Trp Trp Asn Thr Ile Asn Glu Val Lys Lys
340 345 350
Leu Ile Asp Ala Lys Arg Asp Met Gly Arg Val Phe Trp Ser Gly Val
355 360 365
Thr Ala Glu Lys Arg Asn Thr Ile Leu Glu Gly Tyr Asn Tyr Leu Pro
370 375 380
Asn Glu Asn Asp His Lys Lys Arg Glu Gly Ser Leu Glu Asn Pro Lys
385 390 395 400
Lys Pro Ala Lys Arg Gln Phe Gly Asp Leu Leu Leu Tyr Leu Glu Lys
405 410 415
Lys Tyr Ala Gly Asp Trp Gly Lys Val Phe Asp Glu Ala Trp Glu Arg
420 425 430
Ile Asp Lys Lys Ile Ala Gly Leu Thr Ser His Ile Glu Arg Glu Glu
435 440 445
Ala Arg Asn Ala Glu Asp Ala Gln Ser Lys Ala Val Leu Thr Asp Trp
450 455 460
Leu Arg Ala Lys Ala Ser Phe Val Leu Glu Arg Leu Lys Glu Met Asp
465 470 475 480
Glu Lys Glu Phe Tyr Ala Cys Glu Ile Gln Leu Gln Lys Trp Tyr Gly
485 490 495
Asp Leu Arg Gly Asn Pro Phe Ala Val Glu Ala Glu Asn Arg Val Val
500 505 510
Asp Ile Ser Gly Phe Ser Ile Gly Ser Asp Gly His Ser Ile Gln Tyr
515 520 525
Arg Asn Leu Leu Ala Trp Lys Tyr Leu Glu Asn Gly Lys Arg Glu Phe
530 535 540
Tyr Leu Leu Met Asn Tyr Gly Lys Lys Gly Arg Ile Arg Phe Thr Asp
545 550 555 560
Gly Thr Asp Ile Lys Lys Ser Gly Lys Trp Gln Gly Leu Leu Tyr Gly
565 570 575
Gly Gly Lys Ala Lys Val Ile Asp Leu Thr Phe Asp Pro Asp Asp Glu
580 585 590
Gln Leu Ile Ile Leu Pro Leu Ala Phe Gly Thr Arg Gln Gly Arg Glu
595 600 605
Phe Ile Trp Asn Asp Leu Leu Ser Leu Glu Thr Gly Leu Ile Lys Leu
610 615 620
Ala Asn Gly Arg Val Ile Glu Lys Thr Ile Tyr Asn Lys Lys Ile Gly
625 630 635 640
Arg Asp Glu Pro Ala Leu Phe Val Ala Leu Thr Phe Glu Arg Arg Glu
645 650 655
Val Val Asp Pro Ser Asn Ile Lys Pro Val Asn Leu Ile Gly Val Asp
660 665 670
Arg Gly Glu Asn Ile Pro Ala Val Ile Ala Leu Thr Asp Pro Glu Gly
675 680 685
Cys Pro Leu Pro Glu Phe Lys Asp Ser Ser Gly Gly Pro Thr Asp Ile
690 695 700
Leu Arg Ile Gly Glu Gly Tyr Lys Glu Lys Gln Arg Ala Ile Gln Ala
705 710 715 720
Ala Lys Glu Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser Arg Lys Phe
725 730 735
Ala Ser Lys Ser Arg Asn Leu Ala Asp Asp Met Val Arg Asn Ser Ala
740 745 750
Arg Asp Leu Phe Tyr His Ala Val Thr His Asp Ala Val Leu Val Phe
755 760 765
Glu Asn Leu Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg Thr Phe Met
770 775 780
Thr Glu Arg Gln Tyr Thr Lys Met Glu Asp Trp Leu Thr Ala Lys Leu
785 790 795 800
Ala Tyr Glu Gly Leu Thr Ser Lys Thr Tyr Leu Ser Lys Thr Leu Ala
805 810 815
Gln Tyr Thr Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr Ile Thr Thr
820 825 830
Ala Asp Tyr Asp Gly Met Leu Val Arg Leu Lys Lys Thr Ser Asp Gly
835 840 845
Trp Ala Thr Thr Leu Asn Asn Lys Glu Leu Lys Ala Glu Gly Gln Ile
850 855 860
Thr Tyr Tyr Asn Arg Tyr Lys Arg Gln Thr Val Glu Lys Glu Leu Ser
865 870 875 880
Ala Glu Leu Asp Arg Leu Ser Glu Glu Ser Gly Asn Asn Asp Ile Ser
885 890 895
Lys Trp Thr Lys Gly Arg Arg Asp Glu Ala Leu Phe Leu Leu Lys Lys
900 905 910
Arg Phe Ser His Arg Pro Val Gln Glu Gln Phe Val Cys Leu Asp Cys
915 920 925
Gly His Glu Val His Ala Asp Glu Gln Ala Ala Leu Asn Ile Ala Arg
930 935 940
Ser Trp Leu Phe Leu Asn Ser Asn Ser Thr Glu Phe Lys Ser Tyr Lys
945 950 955 960
Ser Gly Lys Gln Pro Phe Val Gly Ala Trp Gln Ala Phe Tyr Lys Arg
965 970 975
Arg Leu Lys Glu Val Trp Lys Pro Asn Ala
980 985
<210> 77
<211> 978
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 77
Met Gln Glu Ile Lys Arg Ile Asn Lys Ile Arg Arg Arg Leu Val Lys
1 5 10 15
Asp Ser Asn Thr Lys Lys Ala Gly Lys Thr Gly Pro Met Lys Thr Leu
20 25 30
Leu Val Arg Val Met Thr Pro Asp Leu Arg Glu Arg Leu Glu Asn Leu
35 40 45
Arg Lys Lys Pro Glu Asn Ile Pro Gln Pro Ile Ser Asn Thr Ser Arg
50 55 60
Ala Asn Leu Asn Lys Leu Leu Thr Asp Tyr Thr Glu Met Lys Lys Ala
65 70 75 80
Ile Leu His Val Tyr Trp Glu Glu Phe Gln Lys Asp Pro Val Gly Leu
85 90 95
Met Ser Arg Val Ala Gln Pro Ala Pro Lys Asn Ile Asp Gln Arg Lys
100 105 110
Leu Ile Pro Val Lys Asp Gly Asn Glu Arg Leu Thr Ser Ser Gly Phe
115 120 125
Ala Cys Ser Gln Cys Cys Gln Pro Leu Tyr Val Tyr Lys Leu Glu Gln
130 135 140
Val Asn Asp Lys Gly Lys Pro His Thr Asn Tyr Phe Gly Arg Cys Asn
145 150 155 160
Val Ser Glu His Glu Arg Leu Ile Leu Leu Ser Pro His Lys Pro Glu
165 170 175
Ala Asn Asp Glu Leu Val Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg
180 185 190
Ala Leu Asp Phe Tyr Ser Ile His Val Thr Arg Glu Ser Asn His Pro
195 200 205
Val Lys Pro Leu Glu Gln Ile Gly Gly Asn Ser Cys Ala Ser Gly Pro
210 215 220
Val Gly Lys Ala Leu Ser Asp Ala Cys Met Gly Ala Val Ala Ser Phe
225 230 235 240
Leu Thr Lys Tyr Gln Asp Ile Ile Leu Glu His Gln Lys Val Ile Lys
245 250 255
Lys Asn Glu Lys Arg Leu Ala Asn Leu Lys Asp Ile Ala Ser Ala Asn
260 265 270
Gly Leu Ala Phe Pro Lys Ile Thr Leu Pro Pro Gln Pro His Thr Lys
275 280 285
Glu Gly Ile Glu Ala Tyr Asn Asn Val Val Ala Gln Ile Val Ile Trp
290 295 300
Val Asn Leu Asn Leu Trp Gln Lys Leu Lys Ile Gly Arg Asp Glu Ala
305 310 315 320
Lys Pro Leu Gln Arg Leu Lys Gly Phe Pro Ser Phe Pro Leu Val Glu
325 330 335
Arg Gln Ala Asn Glu Val Asp Trp Trp Asp Met Val Cys Asn Val Lys
340 345 350
Lys Leu Ile Asn Glu Lys Lys Glu Asp Gly Lys Val Phe Trp Gln Asn
355 360 365
Leu Ala Gly Tyr Lys Arg Gln Glu Ala Leu Leu Pro Tyr Leu Ser Ser
370 375 380
Glu Glu Asp Arg Lys Lys Gly Lys Lys Phe Ala Arg Tyr Gln Phe Gly
385 390 395 400
Asp Leu Leu Leu His Leu Glu Lys Lys His Gly Glu Asp Trp Gly Lys
405 410 415
Val Tyr Asp Glu Ala Trp Glu Arg Ile Asp Lys Lys Val Glu Gly Leu
420 425 430
Ser Lys His Ile Lys Leu Glu Glu Glu Arg Arg Ser Glu Asp Ala Gln
435 440 445
Ser Lys Ala Ala Leu Thr Asp Trp Leu Arg Ala Lys Ala Ser Phe Val
450 455 460
Ile Glu Gly Leu Lys Glu Ala Asp Lys Asp Glu Phe Cys Arg Cys Glu
465 470 475 480
Leu Lys Leu Gln Lys Trp Tyr Gly Asp Leu Arg Gly Lys Pro Phe Ala
485 490 495
Ile Glu Ala Glu Asn Ser Ile Leu Asp Ile Ser Gly Phe Ser Lys Gln
500 505 510
Tyr Asn Cys Ala Phe Ile Trp Gln Lys Asp Gly Val Lys Lys Leu Asn
515 520 525
Leu Tyr Leu Ile Ile Asn Tyr Phe Lys Gly Gly Lys Leu Arg Phe Lys
530 535 540
Lys Ile Lys Pro Glu Ala Phe Glu Ala Asn Arg Phe Tyr Thr Val Ile
545 550 555 560
Asn Lys Lys Ser Gly Glu Ile Val Pro Met Glu Val Asn Phe Asn Phe
565 570 575
Asp Asp Pro Asn Leu Ile Ile Leu Pro Leu Ala Phe Gly Lys Arg Gln
580 585 590
Gly Arg Glu Phe Ile Trp Asn Asp Leu Leu Ser Leu Glu Thr Gly Ser
595 600 605
Leu Lys Leu Ala Asn Gly Arg Val Ile Glu Lys Thr Leu Tyr Asn Arg
610 615 620
Arg Thr Arg Gln Asp Glu Pro Ala Leu Phe Val Ala Leu Thr Phe Glu
625 630 635 640
Arg Arg Glu Val Leu Asp Ser Ser Asn Ile Lys Pro Met Asn Leu Ile
645 650 655
Gly Ile Asp Arg Gly Glu Asn Ile Pro Ala Val Ile Ala Leu Thr Asp
660 665 670
Pro Glu Gly Cys Pro Leu Ser Arg Phe Lys Asp Ser Leu Gly Asn Pro
675 680 685
Thr His Ile Leu Arg Ile Gly Glu Ser Tyr Lys Glu Lys Gln Arg Thr
690 695 700
Ile Gln Ala Ala Lys Glu Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser
705 710 715 720
Arg Lys Tyr Ala Ser Lys Ala Lys Asn Leu Ala Asp Asp Met Val Arg
725 730 735
Asn Thr Ala Arg Asp Leu Leu Tyr Tyr Ala Val Thr Gln Asp Ala Met
740 745 750
Leu Ile Phe Glu Asn Leu Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg
755 760 765
Thr Phe Met Ala Glu Arg Gln Tyr Thr Arg Met Glu Asp Trp Leu Thr
770 775 780
Ala Lys Leu Ala Tyr Glu Gly Leu Pro Ser Lys Thr Tyr Leu Ser Lys
785 790 795 800
Thr Leu Ala Gln Tyr Thr Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr
805 810 815
Ile Thr Ser Ala Asp Tyr Asp Arg Val Leu Glu Lys Leu Lys Lys Thr
820 825 830
Ala Thr Gly Trp Met Thr Thr Ile Asn Gly Lys Glu Leu Lys Val Glu
835 840 845
Gly Gln Ile Thr Tyr Tyr Asn Arg Tyr Lys Arg Gln Asn Val Val Lys
850 855 860
Asp Leu Ser Val Glu Leu Asp Arg Leu Ser Glu Glu Ser Val Asn Asn
865 870 875 880
Asp Ile Ser Ser Trp Thr Lys Gly Arg Ser Gly Glu Ala Leu Ser Leu
885 890 895
Leu Lys Lys Arg Phe Ser His Arg Pro Val Gln Glu Lys Phe Val Cys
900 905 910
Leu Asn Cys Gly Phe Glu Thr His Ala Asp Glu Gln Ala Ala Leu Asn
915 920 925
Ile Ala Arg Ser Trp Leu Phe Leu Arg Ser Gln Glu Tyr Lys Lys Tyr
930 935 940
Gln Thr Asn Lys Thr Thr Gly Asn Thr Asp Lys Arg Ala Phe Val Glu
945 950 955 960
Thr Trp Gln Ser Phe Tyr Arg Lys Lys Leu Lys Glu Val Trp Lys Pro
965 970 975
Ala Val
<210> 78
<211> 757
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 78
Met Pro Lys Pro Ala Val Glu Ser Glu Phe Ser Lys Val Leu Lys Lys
1 5 10 15
His Phe Pro Gly Glu Arg Phe Arg Ser Ser Tyr Met Lys Arg Gly Gly
20 25 30
Lys Ile Leu Ala Ala Gln Gly Glu Glu Ala Val Val Ala Tyr Leu Gln
35 40 45
Gly Lys Ser Glu Glu Glu Pro Pro Asn Phe Gln Pro Pro Ala Lys Cys
50 55 60
His Val Val Thr Lys Ser Arg Asp Phe Ala Glu Trp Pro Ile Met Lys
65 70 75 80
Ala Ser Glu Ala Ile Gln Arg Tyr Ile Tyr Ala Leu Ser Thr Thr Glu
85 90 95
Arg Ala Ala Cys Lys Pro Gly Lys Ser Ser Glu Ser His Ala Ala Trp
100 105 110
Phe Ala Ala Thr Gly Val Ser Asn His Gly Tyr Ser His Val Gln Gly
115 120 125
Leu Asn Leu Ile Phe Asp His Thr Leu Gly Arg Tyr Asp Gly Val Leu
130 135 140
Lys Lys Val Gln Leu Arg Asn Glu Lys Ala Arg Ala Arg Leu Glu Ser
145 150 155 160
Ile Asn Ala Ser Arg Ala Asp Glu Gly Leu Pro Glu Ile Lys Ala Glu
165 170 175
Glu Glu Glu Val Ala Thr Asn Glu Thr Gly His Leu Leu Gln Pro Pro
180 185 190
Gly Ile Asn Pro Ser Phe Tyr Val Tyr Gln Thr Ile Ser Pro Gln Ala
195 200 205
Tyr Arg Pro Arg Asp Glu Ile Val Leu Pro Pro Glu Tyr Ala Gly Tyr
210 215 220
Val Arg Asp Pro Asn Ala Pro Ile Pro Leu Gly Val Val Arg Asn Arg
225 230 235 240
Cys Asp Ile Gln Lys Gly Cys Pro Gly Tyr Ile Pro Glu Trp Gln Arg
245 250 255
Glu Ala Gly Thr Ala Ile Ser Pro Lys Thr Gly Lys Ala Val Thr Val
260 265 270
Pro Gly Leu Ser Pro Lys Lys Asn Lys Arg Met Arg Arg Tyr Trp Arg
275 280 285
Ser Glu Lys Glu Lys Ala Gln Asp Ala Leu Leu Val Thr Val Arg Ile
290 295 300
Gly Thr Asp Trp Val Val Ile Asp Val Arg Gly Leu Leu Arg Asn Ala
305 310 315 320
Arg Trp Arg Thr Ile Ala Pro Lys Asp Ile Ser Leu Asn Ala Leu Leu
325 330 335
Asp Leu Phe Thr Gly Asp Pro Val Ile Asp Val Arg Arg Asn Ile Val
340 345 350
Thr Phe Thr Tyr Thr Leu Asp Ala Cys Gly Thr Tyr Ala Arg Lys Trp
355 360 365
Thr Leu Lys Gly Lys Gln Thr Lys Ala Thr Leu Asp Lys Leu Thr Ala
370 375 380
Thr Gln Thr Val Ala Leu Val Ala Ile Asp Leu Gly Gln Thr Asn Pro
385 390 395 400
Ile Ser Ala Gly Ile Ser Arg Val Thr Gln Glu Asn Gly Ala Leu Gln
405 410 415
Cys Glu Pro Leu Asp Arg Phe Thr Leu Pro Asp Asp Leu Leu Lys Asp
420 425 430
Ile Ser Ala Tyr Arg Ile Ala Trp Asp Arg Asn Glu Glu Glu Leu Arg
435 440 445
Ala Arg Ser Val Glu Ala Leu Pro Glu Ala Gln Gln Ala Glu Val Arg
450 455 460
Ala Leu Asp Gly Val Ser Lys Glu Thr Ala Arg Thr Gln Leu Cys Ala
465 470 475 480
Asp Phe Gly Leu Asp Pro Lys Arg Leu Pro Trp Asp Lys Met Ser Ser
485 490 495
Asn Thr Thr Phe Ile Ser Glu Ala Leu Leu Ser Asn Ser Val Ser Arg
500 505 510
Asp Gln Val Phe Phe Thr Pro Ala Pro Lys Lys Gly Ala Lys Lys Lys
515 520 525
Ala Pro Val Glu Val Met Arg Lys Asp Arg Thr Trp Ala Arg Ala Tyr
530 535 540
Lys Pro Arg Leu Ser Val Glu Ala Gln Lys Leu Lys Asn Glu Ala Leu
545 550 555 560
Trp Ala Leu Lys Arg Thr Ser Pro Glu Tyr Leu Lys Leu Ser Arg Arg
565 570 575
Lys Glu Glu Leu Cys Arg Arg Ser Ile Asn Tyr Val Ile Glu Lys Thr
580 585 590
Arg Arg Arg Thr Gln Cys Gln Ile Val Ile Pro Val Ile Glu Asp Leu
595 600 605
Asn Val Arg Phe Phe His Gly Ser Gly Lys Arg Leu Pro Gly Trp Asp
610 615 620
Asn Phe Phe Thr Ala Lys Lys Glu Asn Arg Trp Phe Ile Gln Gly Leu
625 630 635 640
His Lys Ala Phe Ser Asp Leu Arg Thr His Arg Ser Phe Tyr Val Phe
645 650 655
Glu Val Arg Pro Glu Arg Thr Ser Ile Thr Cys Pro Lys Cys Gly His
660 665 670
Cys Glu Val Gly Asn Arg Asp Gly Glu Ala Phe Gln Cys Leu Ser Cys
675 680 685
Gly Lys Thr Cys Asn Ala Asp Leu Asp Val Ala Thr His Asn Leu Thr
690 695 700
Gln Val Ala Leu Thr Gly Lys Thr Met Pro Lys Arg Glu Glu Pro Arg
705 710 715 720
Asp Ala Gln Gly Thr Ala Pro Ala Arg Lys Thr Lys Lys Ala Ser Lys
725 730 735
Ser Lys Ala Pro Pro Ala Glu Arg Glu Asp Gln Thr Pro Ala Gln Glu
740 745 750
Pro Ser Gln Thr Ser
755
<210> 79
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 79
Pro Lys Lys Lys Arg Lys Val Pro Gly
1 5
<210> 80
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 80
Ala Ser Pro Lys Lys Lys Arg Lys Val
1 5
<210> 81
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 81
Pro Tyr Gly Glu Arg
1 5
<210> 82
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 82
Asp Leu Ser Asn Tyr Asn Pro Tyr Gly Glu Arg
1 5 10
<210> 83
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 83
Pro Gly Gly Glu Arg
1 5
<210> 84
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 84
Gly Asn Leu Gly Ala
1 5
<210> 85
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 85
Leu Gly Asn Gln Gly
1 5
<210> 86
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 86
Pro Asp Gly Arg Trp
1 5
<210> 87
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 87
Ala Ala Gly Asn Ser
1 5
<210> 88
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 88
Ser Lys Asn Asn Pro
1 5
<210> 89
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 89
Asn Ile Gly Thr Gly
1 5
<210> 90
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 90
Ala Gly Asn Leu Gly Ala Pro
1 5
<210> 91
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 91
Leu Gly Asn Gln Gly Phe Thr
1 5
<210> 92
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 92
His Pro Asp Gly Arg Trp
1 5
<210> 93
<211> 28
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 93
Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg Thr Pro Arg Phe
1 5 10 15
Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala
20 25
<210> 94
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 94
Ser Lys Asn Asn Pro Ile Ser
1 5
<210> 95
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 95
Lys Asn Ile Gly Thr
1 5
<210> 96
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 96
Gly Asn Gly Gly Ala
1 5
<210> 97
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 97
Gly Asn Leu Gly Gly Gly Ala
1 5
<210> 98
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 98
Gly Gly Asn Gln Gly
1 5
<210> 99
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 99
Leu Gly Gly Gly Asn Gln Gly
1 5
<210> 100
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 100
Pro Asp Gly Arg Gly
1 5
<210> 101
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 101
Pro Asp Gly Gly Gly Arg Trp
1 5
<210> 102
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 102
Ala Ala Gly Gly Gly Asn Ser
1 5
<210> 103
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 103
Gly Gly Ser Lys Asn Asn Pro
1 5
<210> 104
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 104
Asn Gly Gly Thr Gly
1 5
<210> 105
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 105
Asn Ile Gly Gly Gly Thr Gly
1 5
<210> 106
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 106
Asn Pro Gln Thr Gly
1 5
<210> 107
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 107
Arg Ser Asn Lys Glu
1 5
<210> 108
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 108
Asn Lys Gly Glu Asn
1 5
<210> 109
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 109
Ser Gly Ala Arg Gly
1 5
<210> 110
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 110
Gly Lys Gly Lys Ser
1 5
<210> 111
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 111
Gln Asn Gly Lys Arg
1 5
<210> 112
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 112
Glu Asn Arg Lys Lys
1 5
<210> 113
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 113
Asn Pro Thr Gly His
1 5
<210> 114
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 114
Thr Glu Pro His Phe
1 5
<210> 115
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 115
Leu Ser Lys His Pro
1 5
<210> 116
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 116
Asn Leu Gly Asn Tyr
1 5
<210> 117
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 117
Val Thr Gly Ala Ala
1 5
<210> 118
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 118
Ala Ser Gly His Phe
1 5
<210> 119
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 119
Tyr Gly Lys Glu Ser
1 5
<210> 120
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 120
His Asn Asn Asp Pro
1 5
<210> 121
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 121
Lys Asn Gly Glu Pro
1 5
<210> 122
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 122
Ala Asn Gly Gly Leu
1 5
<210> 123
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 123
Gly Val Gly Ser Arg
1 5
<210> 124
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 124
Asp Arg Ala Glu Asn Pro Gln Thr Gly Glu Ser Leu
1 5 10
<210> 125
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 125
Arg Gly Phe Lys Ser Asn Arg Lys Ser Glu Arg Ser Asn
1 5 10
<210> 126
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 126
Asp Pro Lys Phe Ala Leu His Lys Arg Asn Lys Gly Glu Asn Tyr Thr
1 5 10 15
Asn Thr Ile Ala
20
<210> 127
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 127
Ser Pro Ser Gly Ala Arg Gly Leu Thr
1 5
<210> 128
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 128
Gly Lys Gly Lys Ser Ser Ser Phe Leu Pro Ile Asp
1 5 10
<210> 129
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 129
Glu Gln Asn Gly Lys Arg Met
1 5
<210> 130
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 130
Tyr Gly Leu Thr Leu Asn Pro Thr
1 5
<210> 131
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 131
Lys Thr Glu Pro His Phe Pro Gln Pro Trp Pro His
1 5 10
<210> 132
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 132
Lys His Pro
1
<210> 133
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 133
Leu Asn Leu Gly Asn Tyr Asp
1 5
<210> 134
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 134
Met Pro Lys Arg Ser Val Thr Gly
1 5
<210> 135
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 135
Ile Lys Leu Asp Ala Ser Gly His Phe Pro Met Tyr Gly Lys Glu Ser
1 5 10 15
Asp
<210> 136
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 136
His Asn Asn Asp
1
<210> 137
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 137
Gln Glu Pro Leu Tyr Lys Pro Lys Lys Asn Gly Glu Pro Gly Pro Val
1 5 10 15
<210> 138
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 138
Asn Pro Gln Thr Gly Gly Gly
1 5
<210> 139
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 139
Arg Gly Gly Ser Asn Lys Glu
1 5
<210> 140
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 140
Asn Lys Gly Gly Gly Glu Asn
1 5
<210> 141
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 141
Ser Gly Gly Gly Ala Arg Gly
1 5
<210> 142
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 142
Gly Lys Gly Gly Gly Lys Ser
1 5
<210> 143
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 143
Gln Asn Gly Gly Gly Lys Arg
1 5
<210> 144
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 144
Glu Gly Gly Asn Arg Lys Lys
1 5
<210> 145
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 145
Asn Pro Thr Gly Gly Gly His
1 5
<210> 146
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 146
Thr Glu Pro His Gly
1 5
<210> 147
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 147
Thr Glu Pro Gly Gly His Phe
1 5
<210> 148
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 148
Gly Ser Lys His Pro
1 5
<210> 149
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 149
Leu Gly Gly Ser Lys His Pro
1 5
<210> 150
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 150
Asn Gly Gly Asn Tyr
1 5
<210> 151
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 151
Asn Leu Gly Asn Gly
1 5
<210> 152
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 152
Asn Leu Gly Gly Gly Asn Tyr
1 5
<210> 153
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 153
Gly Thr Gly Ala Ala
1 5
<210> 154
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 154
Val Thr Gly Gly Gly Ala Ala
1 5
<210> 155
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 155
Ala Ser Gly His Gly
1 5
<210> 156
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 156
Ala Ser Gly Gly Gly His Phe
1 5
<210> 157
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 157
Gly Gly Lys Glu Ser
1 5
<210> 158
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 158
Tyr Gly Gly Gly Lys Glu Ser
1 5
<210> 159
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 159
His Asn Gly Gly Asn Asp Pro
1 5
<210> 160
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 160
Lys Asn Gly Gly Gly Glu Pro
1 5
<210> 161
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 161
Ala Asn Gly Gly Gly
1 5
<210> 162
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 162
Ala Asn Gly Gly Gly Gly Leu
1 5
<210> 163
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 163
Gly Gly Gly Ser Arg
1 5
<210> 164
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 164
Gly Val Gly Gly Gly Ser Arg
1 5
<210> 165
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 165
atttttgtgc ccatcgttgg cac 23
<210> 166
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 166
agaaatccgt ctttcattga cgg 23
<210> 167
<211> 35
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 167
atttaccctg acgatagagt gcaacttcac aagct 35
<210> 168
<211> 35
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 168
agcttgtgaa gttgcactct atcgtcaggg taaat 35
<210> 169
<211> 35
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 169
ggttctgatt ggagttgtcc aggtttttgg aagct 35
<210> 170
<211> 35
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 170
agcttccaaa aacctggaca actccaatca gaacc 35
<210> 171
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 171
atttttgtgc ccatcgttgg cac 23
<210> 172
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 172
atctttgtgc ccatcgttgg cac 23
<210> 173
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 173
attcttgtgc ccatcgttgg cac 23
<210> 174
<211> 23
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 174
ataattgtgc ccatcgttgg cac 23
<210> 175
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 175
Asn Glu Gly Arg Arg
1 5
<210> 176
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 176
Leu Ser Gly Ile Asn
1 5
<210> 177
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 177
Arg Arg Gly Val His
1 5
<210> 178
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 178
Asp Thr Gly Asn Glu
1 5
<210> 179
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 179
Pro Gly Glu Gly Ser
1 5
<210> 180
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 180
Ser Thr Gly Lys Pro
1 5
<210> 181
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 181
Leu Lys Gly Tyr Thr
1 5
<210> 182
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 182
Lys Arg Asn Arg Gln
1 5
<210> 183
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 183
Lys Lys Gly Asn Arg
1 5
<210> 184
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 184
Ser Ser Ser Asp Ser
1 5
<210> 185
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 185
Lys Gly Lys Gly Arg
1 5
<210> 186
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 186
Asn Lys Gly Tyr Lys
1 5
<210> 187
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 187
Ala Glu Ser Met Pro
1 5
<210> 188
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 188
Asn Leu Leu Thr Asp His Ser Glu Leu Ser Gly Ile
1 5 10
<210> 189
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 189
Arg Gly Val His
1
<210> 190
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 190
Glu Glu Asp Thr Gly Asn Glu Leu
1 5
<210> 191
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 191
Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp
1 5 10 15
Lys
<210> 192
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 192
Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro Glu Phe Thr Asn
1 5 10
<210> 193
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 193
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu
1 5 10
<210> 194
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 194
Asn Arg
1
<210> 195
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 195
Ser Ser Asp Ser Lys Ile
1 5
<210> 196
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 196
Lys Gly Lys Gly Arg Ile
1 5
<210> 197
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 197
Lys Phe Lys Lys Glu Arg Asn Lys Gly
1 5
<210> 198
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 198
Asn Gln Met Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu
1 5 10 15
<210> 199
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 199
Asn Glu Gly Gly Gly Arg Arg
1 5
<210> 200
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 200
Gly Ser Gly Ile Asn
1 5
<210> 201
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 201
Leu Ser Gly Gly Asn
1 5
<210> 202
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 202
Leu Ser Gly Gly Gly Ile Asn
1 5
<210> 203
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 203
Arg Arg Gly Gly His
1 5
<210> 204
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 204
Arg Arg Gly Gly Gly Val His
1 5
<210> 205
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 205
Asp Thr Gly Gly Gly Asn Glu
1 5
<210> 206
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 206
Pro Gly Glu Gly Gly Gly Ser
1 5
<210> 207
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 207
Ser Thr Gly Gly Gly Lys Pro
1 5
<210> 208
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 208
Gly Lys Gly Tyr Thr
1 5
<210> 209
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 209
Leu Lys Gly Gly Thr
1 5
<210> 210
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 210
Leu Lys Gly Gly Gly Tyr Thr
1 5
<210> 211
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 211
Lys Arg Gly Gly Asn Arg Gln
1 5
<210> 212
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 212
Lys Lys Gly Gly Gly Asn Arg
1 5
<210> 213
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 213
Ser Ser Gly Gly Ser Asp Ser
1 5
<210> 214
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 214
Lys Gly Lys Gly Gly Gly Arg
1 5
<210> 215
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 215
Asn Lys Gly Gly Lys
1 5
<210> 216
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 216
Asn Lys Gly Gly Gly Tyr Lys
1 5
<210> 217
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 217
Ala Glu Gly Gly Ser Met Pro
1 5
<210> 218
<211> 229
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 218
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys
225
<210> 219
<211> 167
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 219
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala
35 40 45
Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 220
<211> 677
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 220
Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro
675
<210> 221
<211> 677
<212> PRT
<213> artificial sequence
<220>
<223> Artificial
<400> 221
Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asn Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Trp Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro
675
<210> 222
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 222
tcagtctgaa gagcagagcc aggaa 25
<210> 223
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 223
ttatattgtt cctccgtgcg tcagt 25
<210> 224
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 224
cttgtcatgg tcatctgcta ctcgg 25
<210> 225
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 225
atatttcctg ctccccagtg gatcg 25
<210> 226
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 226
ttgaaatgag tttggtcagg gatgg 25
<210> 227
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 227
aactgtgtat ttccgtgctg attcc 25
<210> 228
<211> 25
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 228
ggagctgtag gcgattatag ttgaa 25
<210> 229
<211> 24
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 229
ttctcaaacc ctggaaagca cttt 24
<210> 230
<211> 100
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 230
ttaaagtagt tctttgatga atatgaatga gtagggaaag gattctttgt gatagtgata 60
cctctgtggt aagagaaggg tggtatgtga gttttagtct 100
<210> 231
<211> 20
<212> DNA/RNA
<213> artificial sequence
<220>
<223> Artificial
<400> 231
gtgatagtga tacctctgtg 20

Claims (16)

1. An engineered Cas12b nuclease comprising one or more mutations that increase the flexibility of the flexible region corresponding to amino acid residues 835 to 839 in a reference Cas12b nuclease, wherein the amino acid residue numbering is based on SEQ ID NO:1, wherein the engineered Cas12b nuclease has increased activity compared to the reference Cas12b nuclease;
Wherein the one or more mutations are insertion of one or more G residues in the flexible region, wherein the one or more G residues are inserted N-terminal to a flexible amino acid residue in the flexible region, wherein the flexible amino acid residue is selected from the group consisting of: G. serine (S), asparagine (N), aspartic acid (D), histidine (H), methionine (M), threonine (T), glutamic acid (E), glutamine (Q), lysine (K), arginine (R), alanine (a) and proline (P); or alternatively
Wherein the one or more mutations are substitutions of hydrophobic amino acid residues in the flexible region with G residues, wherein the hydrophobic amino acid residues are selected from the group consisting of: leucine (L), isoleucine (I), valine (V), cysteine (C), tyrosine (Y), phenylalanine (F) and tryptophan (W);
wherein the amino acid sequence of the engineered Cas12b nuclease at the flexible region is mutated to SEQ ID No. 83.
2. The engineered Cas12b nuclease of claim 1, wherein the engineered Cas12b effector protein is based on a reference Cas12b protein selected from the group consisting of: cas12b protein of alicyclobacillus acidophilus (Alicyclobacillus acidiphilus) (AaCas 12 b), cas12b of kakegawensis alicyclobacillus (Alicyclobacillus kakegawensis) (AkCas 12 b), cas12b of alicyclobacillus megaterium (Alicyclobacillus macrosporangiidus) (AmCas 12 b), cas12b of Bacillus ectovillans (Bacillus hisashii) (BhCas 12 b), bsCas12b of Bacillus (Bacillus), bs3Cas12b of Bacillus, cas12b of vibrio most desulphus (Desulfovibrio inopinatus) (diecas 12 b), cas12b of borrelia (Laceyella sediminis) (LsCas 12 b), cas12b of spirochete (Spirochaetes bacterium) (SbCas 12 b), and Cas12b of Bacillus thermogenesis (Tuberibacillus calidus) (TcCas 12 b).
3. The engineered Cas12b nuclease of claim 1, wherein the engineered Cas12b effector protein is based on BhCas12b.
4. The engineered Cas12b nuclease of claim 1, comprising an amino acid sequence having at least about 85% sequence identity to the sequence of SEQ ID No. 2.
5. An engineered Cas12b effector protein comprising the engineered Cas12b nuclease of any one of claims 1-4.
6. The engineered Cas12b effector protein of claim 5, wherein the effector protein is capable of inducing a double-strand break or single-strand break in a DNA molecule.
7. The engineered Cas12b effector protein of claim 5, wherein the engineered Cas12b nuclease further comprises an enzyme-inactivating mutant of R785A, R911A or D977A, wherein the amino acid residue numbering is based on SEQ ID No. 7.
8. The engineered Cas12b effector protein of claim 5, further comprising a functional domain fused to the engineered Cas12b nuclease.
9. The engineered Cas12b effector protein of claim 8, wherein the functional domain is selected from the group consisting of: translation initiation domains, transcription repression domains, transactivation domains, epigenetic modification domains, nucleobase editing domains, reverse transcriptase domains, reporter domains and nuclease domains.
10. The engineered Cas12b effector protein of claim 5, comprising a first polypeptide comprising the N-terminal portion amino acid residues 1-X of the engineered Cas12b nuclease of any one of claims 1-5 and a second polypeptide comprising the amino acid residues x+1 of the engineered Cas12b nuclease of any one of claims 1-5 to the C-terminus of the Cas12b nuclease, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex that specifically binds to a target nucleic acid comprising a target sequence complementary to the guide sequence.
11. An engineered CRISPR-Cas12b system, comprising:
(a) The engineered Cas12b effector protein of any one of claims 5-10; and
(b) A guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA,
wherein the engineered Cas12b effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to and induces modification of a target nucleic acid comprising the target sequence.
12. A method for detecting a target nucleic acid in a sample based on non-diagnostic and therapeutic purposes, comprising:
(a) Contacting the sample with the engineered CRISPR-Cas12b system of claim 11 and a tagged detection nucleic acid that is single stranded and does not hybridize to a guide sequence of the guide RNA; and
(b) Measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered Cas12b effector protein, thereby detecting the target nucleic acid.
13. A method of modifying a target nucleic acid comprising a target sequence based on non-diagnostic and therapeutic purposes, comprising contacting the target nucleic acid with the engineered CRISPR-Cas12b system of claim 11.
14. The method of claim 13, wherein the target nucleic acid is cleaved, a target sequence in the target nucleic acid is altered, or expression of the target nucleic acid is altered by the engineered CRISPR-Cas system.
15. An engineered cell comprising the target nucleic acid modified using the method of claim 13 or 14.
16. The use of the engineered CRISPR-Cas system of claim 11 in the manufacture of a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual.
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