JP2001218584A - Human protein and complementary dna [4) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a refined human protein, DNA fragment coding for the protein and containing complete length of cDNA (complementary DNA), an expression vector for the DNA fragment, a transduced cell by the expression vector and an antibody for the protein. SOLUTION: This refined human protein has an amino acid sequence represented by sequence number either of 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 in the specification. The DNA fragment has a base sequence in an open reading frame represented by sequence number 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 in the specification. The expression vector is for expressing the DNA fragment. The transduced cell is obtained from the expression vector. The antibody is for the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a purified human protein, a DNA fragment encoding the protein, an expression vector for the DNA fragment, various cells transformed with the expression vector, and an antibody against the protein. It is about. The protein of the present invention can be used as a pharmaceutical or as an antigen for producing an antibody against the protein. Also, this protein
It can be used as a research reagent for elucidating an intracellular protein network or as a protein source for screening for a protein that binds to a low-molecular-weight drug.
The human cDNA of the present invention can be used as a probe for gene diagnosis or a gene source for gene therapy. Also,
It can be used as a gene source for mass-producing the protein encoded by this cDNA. These DNs
An expression vector capable of in vitro translation or expression of A in a host cell can be used for producing the protein of the present invention in vitro or in various host cells. Cells into which these genes have been introduced to overexpress proteins can be used for detection of corresponding receptors and ligands, screening of new low-molecular-weight drugs, and the like. The antibody against the protein of the present invention is used for purifying the protein or for examining the expression level and localization of the protein in cells.

[0002]

BACKGROUND OF THE INVENTION Human proteins are a fundamental element of the cells that make up our bodies. These include (1) cytoskeletal proteins that maintain cell morphology and are involved in intracellular substance transport and cell movement, (2) metabolic enzymes involved in intracellular substance metabolism, and (3) energy -Proteins involved in production,
(4) signaling proteins involved in cell growth and division,
(5) a translation-related protein involved in protein synthesis, (6) a protease-related protein involved in protein degradation, (7) a protein involved in genome replication, (8) a transcription factor involved in gene transcription, ( 9) Includes nuclear proteins involved in mRNA splicing. These proteins are not only important in elucidating the function of human cells, but also useful in drug development. Many of the low molecular compound drugs known so far exhibit their efficacy by binding to a specific protein in cells and enhancing or blocking the action of the protein. Therefore, having a complete set of human proteins is a powerful tool for screening these low-molecular-weight drugs.

Hitherto, to obtain a human protein, a method has been adopted in which human tissues and cultured cells are ground and then a single protein is purified by combining various separation methods. Proteins with a high content and known activity, such as those known so far, can be easily isolated and purified by conventional methods, but many proteins that have not yet been analyzed have a low content, and It is difficult to isolate depending on its properties.
Also, many human tissues are difficult to obtain. Therefore,
With conventional methods for isolating and purifying proteins, it is almost impossible to prepare all human proteins.

[0004] On the other hand, since the structural information of human proteins is written in human genomic DNA, the primary structure of all human proteins can be estimated by reading all this information. This is one of the goals of the Human Genome Project. However, as a result of genome decoding, only the DNA sequence information is obtained, and the protein itself cannot be obtained. In cells, genomic information is first transcribed into mRNA,
The protein is synthesized by translating the sequence information of A. Therefore, if a cDNA prepared using this mRNA as a template can be synthesized, a corresponding protein can also be synthesized using the cDNA. Therefore, a so-called EST project for synthesizing cDNA using mRNA isolated from various cells as a template and determining a partial base sequence of cDNA is in progress.

[0005]

In order to obtain a protein, an essential requirement for a cDNA is that it includes all of the protein's translation region, that is, a so-called full-length cDN.
A. However, the ratio of cDNA synthesized by the conventional method to full length is low, and it is difficult to determine whether the obtained cDNA is full length. That is, most of what is known as ESTs are cDNA fragments containing only a part of the protein translation region.

In contrast, the inventors of the present application have completed a unique full-length cDNA synthesis technique (Kato,
S. et al., Gene 150: 243-250, 1994). By analyzing human full-length cDNA clones synthesized by this technique, it became possible to obtain human proteins in the form of full-length cDNA. Using this technique, human full-length cD
It has been desired to clone all NA to prepare a human protein bank.

[0007] As a result of research on human diseases, it is becoming clear that most diseases are caused by some form of genetic abnormality.
In order to treat these diseases, gene therapy in which a normal gene is introduced instead of an abnormal gene is expected to be promising. In this case, the human full-length cDNA can be used as a gene source for gene therapy.

[0008] The invention of this application has been made in view of the circumstances described above, and comprises a novel purified human protein,
It is an object of the present invention to provide a DNA fragment encoding the protein, an expression vector for the DNA fragment, a cell transformed with the expression vector, and an antibody against the protein.

[0009]

This application provides the following inventions (1) to (7) to solve the above-mentioned problems. (1) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 1
A purified human protein having any one of 6, 18 and 20 amino acid sequences. (2) A DNA fragment encoding the protein of the invention (1). (3) A human cDNA encoding the protein of the invention (1), which comprises 1, 3, 5, 7, 9, 11, 13, 15, 17
Or a DNA fragment having the nucleotide sequence of 19 translation regions. (4) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 1
The DNA fragment according to the invention (3), comprising the nucleotide sequence of any one of 5, 17 and 19. (5) An expression vector capable of in vitro translation or expression in a host cell of the DNA fragment according to any one of the inventions (2) to (4). (6) A transformant using the expression vector of the invention (5), which is capable of producing the protein of the invention (1). (7) An antibody against the protein of the invention (1).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the invention (1) can be isolated from human organs, cell lines, etc., by preparing a peptide by chemical synthesis based on the amino acid sequence provided by this application, or Inventions (2) to (4) DN
Although it can be obtained by a method of producing the fragment A by recombinant DNA technology, a method of obtaining by recombinant DNA technology is preferably used. For example, the invention (3)
Alternatively, a protein can be expressed in vitro by preparing RNA by in vitro transcription from a vector having the DNA fragment (cDNA) of (4) and performing in vitro translation using this as a template. Also, by recombining the translation region into an appropriate expression vector by a known method, E. coli,
In prokaryotic cells such as Bacillus subtilis and eukaryotic cells such as yeast, insect cells, mammalian cells, and plant cells, a large amount of proteins encoded by DNA fragments can be expressed.

When the protein of the invention (1) is produced by expressing a DNA fragment by in vitro translation, for example, the translation region of the DNA fragment of the invention (3) or (4) may be replaced with RNA.
Recombined into a vector having a polymerase promoter,
The protein of the invention (1) can be produced in vitro by adding it to an in vitro translation system such as a rabbit reticulocyte lysate or a wheat germ extract containing an RNA polymerase corresponding to the promoter. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T7
18, pT7 / 319, pBluescript II and the like.

When the protein of the invention (1) is produced by expressing a DNA fragment in a microorganism such as Escherichia coli, it has an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator and the like which can be replicated in the microorganism. Expression vector, for example, the invention (3) or (4)
After preparing an expression vector in which the translation region of the DNA fragment has been recombined and transforming a host cell with this expression vector,
By culturing the obtained transformant, the protein encoded by this DNA fragment can be mass-produced in a microorganism. At this time, a protein fragment containing an arbitrary region can be obtained by adding a start codon and a stop codon before and after an arbitrary translation region for expression. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving this fusion protein with an appropriate protease, only the protein portion encoded by the cDNA can be obtained. Examples of expression vectors for Escherichia coli include the pUC system,
Examples include pBluescript II, pET expression system, pGEX expression system, and the like.

[0013] The protein of the invention (1) can be used in a eukaryotic cell by DN.
When the A fragment is expressed and produced, for example, the translation region of the DNA fragment of the invention (3) or (4) may be a promoter, a splicing region, an expression vector for eukaryotic cells having a poly (A) addition site and the like. When the recombinant is introduced into eukaryotic cells, the protein of the invention (1) can be produced in eukaryotic cells. Examples of expression vectors include pKA1, pCD
M8, pSVK3, pMSG, pSVL, pBK-CM
V, pBK-RSV, EBV vector, pRS, pYE
S2 and the like can be exemplified. Also, pIND / V5-Hi
s, pFLAG-CMV-2, pEGFP-N1, pE
If GFP-C1 or the like is used as an expression vector, Hi
It can also be expressed as a fusion protein to which various tags such as s tag, FLAG tag, and GFP are added. Examples of eukaryotic cells include mammalian cultured cells such as monkey kidney cells COS7, Chinese hamster ovary cells CHO, budding yeast,
Fission yeast, silkworm cells, Xenopus egg cells and the like are generally used, and any eukaryotic cell may be used as long as it can express the protein of the invention (1). In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used.

After the protein of the invention (1) is expressed in prokaryotic or eukaryotic cells, the target protein can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzymatic digestion, salting out or solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAG
E, isoelectric focusing, ion exchange chromatography,
Examples include hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

The protein of the invention (1) includes SEQ ID NO: 2,
4, 6, 8, 10, 12, 14, 16, 18, or 20
And a peptide fragment (5 or more amino acid residues) consisting of any partial amino acid sequence of the above amino acid sequence. These peptide fragments can be used as antigens for producing antibodies. Many of the proteins of the invention (1) undergo various modifications in cells after being translated. Therefore, these modified proteins are also included in the scope of the protein of the invention (1). Such post-translational modifications include:
Examples include N-terminal methionine elimination, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.

The DNA fragments of the inventions (2) to (4) include
All DNAs encoding the protein of (1) are included.
This DNA fragment can be obtained using a method based on chemical synthesis, a method based on cDNA cloning, a method for screening a human genomic library, and the like.

The DNA fragment of the invention (3) or (4) (cD
NA) can be cloned, for example, from a cDNA library derived from human cells. cDNA is synthesized using poly (A) ⁺ RNA extracted from human cells as a template. The human cells may be cells extracted from a human body by surgery or the like or cultured cells. The cDNA was prepared by the Okayama-Berg method (Okayama, H. and Berg, P., Mol. Cell. Biol. 2: 161
-170, 1982), Gubler-Hoffman method (Gubler, U. and Hof
fman, J., Gene 25: 263-269, 1983), but in order to obtain a full-length clone efficiently, the capping method (Kato , S. et al., Gene 150: 243-250, 1994). A commercially available human cDNA library can also be used. To clone a cDNA of interest from a cDNA library, the cDNA of the invention (3) or (4) provided by this application (SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 1
Oligonucleotides may be synthesized based on the base sequence of any part of 9), and screening may be performed by colony or plaque hybridization by a known method using this as a probe. In addition, an oligonucleotide that hybridizes to both ends of the cDNA fragment of interest is synthesized, and using this as a primer,
The cDNA fragment of the invention (3) or (4) can also be prepared from mRNA isolated from human cells by RT-PCR.

[0018] The DNA fragment of the invention (3) may be any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 1
9 is a cDNA having a nucleotide sequence of 9 translation regions (Open Reading Frame: ORF), and the DNA fragment of the invention (4) is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
CDNA consisting of either 17 or 19 nucleotide sequence
It is. Each clone number (HP number), cDN
Table 1 summarizes the cells from which the A clone was obtained, the total number of bases in the cDNA, and the number of amino acid residues in the encoded protein.

[0019]

[Table 1]

In addition, SEQ ID NOs: 1, 3, 5, 7, 9, 1
Using an oligonucleotide probe synthesized based on any of the base sequences of 1, 13, 15, 17 or 19, c prepared from a human cell line or a human tissue shown in Table 1
By screening a DNA library,
A clone identical to the cDNA of the inventions (3) and (4) can be easily obtained.

Generally, polymorphisms due to individual differences are frequently observed in human genes. Accordingly, cDNAs in which one or more nucleotides are added, deleted and / or substituted by other nucleotides in SEQ ID NOS: 11 to 30 are also included in the scope of the present invention.

Similarly, proteins in which one or more amino acids are added, deleted and / or substituted by other amino acids resulting from these changes are also proteins having the amino acid sequences of SEQ ID NOS: 1 to 10. As long as it has the activity of, it is included in the scope of the present invention.

The DNA fragments of the inventions (3) and (4) include DNA fragments (10 bp or more) consisting of any partial nucleotide sequence of the nucleotide sequences of SEQ ID NOS: 11 to 30. Further, a DNA fragment consisting of a sense strand and an antisense strand is also included in this range. These DNA fragments can be used as probes for gene diagnosis.

The antibody of the invention (7) can be obtained from serum after immunization of an animal using the protein of the invention (1) as an antigen. As the antigen, a peptide chemically synthesized based on the amino acid sequence of SEQ ID NOS: 1 to 10 or a protein expressed in eukaryotic cells or prokaryotic cells can be used.
Alternatively, it can be prepared by introducing the above eukaryotic cell expression vector into an animal muscle or skin by injection or gene gun, and then collecting serum (for example,
Method described in JP-A-7-313187). As animals, mice, rats, rabbits, goats, chickens and the like are used. By fusing B cells collected from the spleen of the immunized animal with myeloma to produce a hybridoma,
A monoclonal antibody against the protein of the invention (1) can be produced.

[0025]

Next, the present invention will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples. In the following examples, basic operations and enzymatic reactions relating to DNA recombination are described in the literature ("Molecular Cloning. A Laborato
ry Manual ", Cold Spring Harbor Laboratory, 1989)
Was followed. Restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo Co., Ltd. unless otherwise specified. The buffer composition and reaction conditions for each enzyme reaction were in accordance with the attached instructions. cDNA synthesis is described in the literature (Kato, S. et al., Gene 150:
243-250, 1994). Example 1: cDNA cloning As a cDNA library, a human full-length cDNA library (WO97 / 33993, WO98 / 1121)
7, WO98 / 21328). A full-length cDNA clone was selected from each library, and its entire nucleotide sequence was determined. The resulting clone (A)
Details of (J) are as follows. (A) HP02644 human fibrosarcoma cell line HT-1080 cDNA
C of clone HP02644 obtained from the library
When the total base sequence of the DNA insert was determined, 72
bp 5 'untranslated region, 2580 bp ORF, 268
bp 3 ′ untranslated region (SEQ ID NO: 1). ORF is 859 amino acid residues (SEQ ID NO: 2)
And a translation product of 150 kDa, which is larger than the molecular weight of 96,271 predicted from the ORF, was generated as a result of in vitro translation (Example 2). Expression of the fusion protein of this protein and GFP was observed in the nucleolus (Example 4).

When a protein database was searched using the amino acid sequence of this protein, the nematode RNA helicase-like protein CELF55F8 (accession number AA
B37806). In FIG.
2 shows a comparison of the amino acid sequences of the human protein encoded by the protein (A) and the nematode RNA helicase-like protein. -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,.
Represents a similar amino acid residue to the protein of the present invention. It had 31.6% homology over the entire region. RNA helicase-like proteins include ribosome formation, transcription, splicing, RNA maturation, RNA transport, RNA
It is involved in many processes involving RNA, such as degradation and translation.

When GenBank was searched using the nucleotide sequence of clone (A) cDNA, those having a homology of 90% or more (for example, accession number Z4
8570 and A74673), all of which are shorter than the clone (A) cDNA. Also, EST
Among them, those having 90% or more homology (for example, accession number AA788907) were registered,
Since it is a partial sequence, it cannot be determined whether clone (A) encodes the same protein as the encoded protein. (B) HP03233 human fibrosarcoma cell line HT-1080 cDNA
C of clone HP03233 obtained from library
When the total base sequence of the DNA insert was determined, 14
bp 5 'untranslated region, 984 bp ORF, 504b
It had a structure consisting of the 3 'untranslated region of p (SEQ ID NO: 3). The ORF encodes a protein consisting of 327 amino acid residues (SEQ ID NO: 4), and as a result of in vitro translation, a translation product of 37 kDa was generated, which is almost the same as the molecular weight of 37,116 predicted from the ORF (Example 2). A fusion protein of this protein and GFP was found in the Golgi apparatus and the endoplasmic reticulum (Example 4).

When a protein database was searched using the amino acid sequence of this protein, it was similar to the fission yeast putative ubiquinone biosynthetic methyltransferase (accession number CAB09781). FIG.
A comparison of the human protein encoded by clone (B) with the putative ubiquinone biosynthetic methyltransferase from fission yeast is shown below. -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. Represents a similar amino acid residue to the protein of the present invention. 43.7% over all areas
Had the same homology.

When GenBank was searched using the nucleotide sequence of clone (B) cDNA, ESTs having a homology of 90% or more (eg, accession number AA338101) were registered. However, since it is a partial sequence, it cannot be determined whether clone (B) encodes the same protein as the encoded protein. (C) HP10384 When the entire nucleotide sequence of the cDNA insert of clone HP10384 obtained from the human epidermoid carcinoma cell line KB cDNA library was determined, the 5 'untranslated region of 126 bp, the ORF of 261 bp, the 3' untranslated region of 350 bp (SEQ ID NO: 5). ORF is 8
It encoded a protein consisting of 6 amino acid residues (SEQ ID NO: 6). As a result of the in vitro translation, a translation product of 10 kDa, which was almost the same as the molecular weight predicted from the ORF of 10,128, was produced (Example 2). The fusion protein of this protein and GFP was expressed in whole cells or in the form of granules or aggregates (Example 4).

When GenBank was searched using the base sequence of clone (C) cDNA, ESTs having a homology of 90% or more (eg, accession number AF150406) were registered. However, since it is a partial sequence, it cannot be determined whether clone (C) encodes the same protein as the encoded protein. (D) HP10431 clone H obtained from human liver cDNA library
When the entire nucleotide sequence of the cDNA insert of P10431 was determined, it had a structure consisting of an 84 bp 5 ′ untranslated region, a 537 bp ORF, and a 282 bp 3 ′ untranslated region (SEQ ID NO: 7). The ORF encodes a protein consisting of 178 amino acid residues (SEQ ID NO: 8). As a result of in vitro translation, a molecular weight of 20,
A translation product of 23 kDa slightly larger than 277 was produced (Example 2). The fusion protein of this protein and GFP is
It was observed in the whole cells, and granular aggregates were also observed therein (Example 4).

When GenBank was searched using the nucleotide sequence of clone (D) cDNA, ESTs having a homology of 90% or more (eg, accession number AW160991) were registered. However, since it is a partial sequence, it cannot be determined whether clone (D) encodes the same protein as the encoded protein. (E) HP10437 Clone H obtained from human gastric cancer cDNA library
When the entire nucleotide sequence of the cDNA insert of P10437 was determined, a 186 bp 5 ′ untranslated region, 354 bp
Had a structure consisting of a 630 bp 3 ′ untranslated region (SEQ ID NO: 9). ORF encodes a protein consisting of 117 amino acid residues (SEQ ID NO: 10),
As a result of in vitro translation, the expected molecular weight 1
A translation product of 22 kDa greater than 3,616 was produced (Example 2). The fusion protein of this protein and GFP is
Localization was observed in the whole cell or in the nucleus (Example 4).

When a protein database was searched using the amino acid sequence of this protein, it was similar to the human pp21 homolog (accession number AAF17229). FIG. 3 shows a comparison between the human protein encoded by clone (E) and the human pp21 homolog. -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. Represents a similar amino acid residue to the protein of the present invention. It had 39.4% homology over the entire region. pp21 is an analog of transcription elongation factor SII.

When GenBank was searched using the base sequence of clone (E) cDNA, ESTs having a homology of 90% or more (eg, accession number AA3222053) were registered. However, since it is a partial sequence, it cannot be determined whether clone (E) encodes the same protein as the encoded protein. (F) HP10525 Clone HP obtained from human gastric cancer DNA library
When the entire nucleotide sequence of the 10525 cDNA insert was determined, it had a structure consisting of a 104 bp 5 ′ untranslated region, a 261 bp ORF, and a 39 bp 3 ′ untranslated region (SEQ ID NO: 11). The ORF encodes a protein consisting of 86 amino acid residues (SEQ ID NO: 12).
A 14 kDa translation product slightly larger than 0,110 was produced (Example 2). Expression of the fusion protein of this protein and GFP was observed in the whole cell (Example 4).

A search of the protein database using the amino acid sequence of this protein revealed that the fission yeast hypothetical protein SPAC8C9.11 (accession number AAC71).
096). FIG. 4 shows a comparison of the amino acid sequences of the human protein encoded by clone (F) and the fission yeast hypothetical protein SPAC8C9.11. −
Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. Represents a similar amino acid residue to the protein of the present invention. It had 44.0% homology over the entire region.

When GenBank was searched using the nucleotide sequence of clone (F) cDNA, ESTs having a homology of 90% or more (eg, accession number AA310786) were registered. However, since it is a partial sequence, it cannot be determined whether clone (F) encodes the same protein as the encoded protein. (G) HP10533 When the entire nucleotide sequence of the cDNA insert of clone HP10533 obtained from the human osteosarcoma cell line Saos-2 cDNA library was determined, a 9 bp 5 'untranslated region, a 537 bp ORF, and a 276 bp 3' untranslated It had a structure consisting of regions (SEQ ID NO: 13). ORF
Encodes a protein consisting of 178 amino acid residues (SEQ ID NO: 14). As a result of in vitro translation, a 26 kDa translation product having a molecular weight larger than the predicted molecular weight of 19,508 was produced (Example 2). This protein and GFP
Was recognized as an aggregate and a part of the whole cell (Example 4).

When GenBank was searched using the nucleotide sequence of clone (G) cDNA, ESTs having a homology of 90% or more (for example, accession number AI929383) were registered. However, since it is a partial sequence, it cannot be determined whether clone (G) encodes the same protein as the encoded protein. (H) HP10543 human fibrosarcoma cell line HT-1080 cDNA
C of clone HP10543 obtained from library
When the total nucleotide sequence of the DNA insert was determined, 94
bp 5 'untranslated region, 540 bp ORF, 118b
It had a structure consisting of the 3 'untranslated region of p (SEQ ID NO: 15). ORF has 179 amino acid residues (SEQ ID NO: 1).
As a result of in vitro translation, a 30 kDa translation product having a molecular weight larger than that predicted from the ORF of 19,070 was produced (Example 2).
The fusion protein of this protein and GFP was expressed in the whole cell or in the nucleus (Example 4).

When a protein database was searched using the amino acid sequence of this protein, mouse leucine-rich domain interacting protein 1 (accession number AA
D17989). FIG.
3 shows a comparison of the amino acid sequences of human protein encoded by protein (H) and mouse leucine-rich domain interacting protein 1. -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. The amino acid residues are similar to those of the protein of the present invention. 138 amino acid residues at the C-terminal side are 6
It had 9.6% homology.

When GenBank was searched using the nucleotide sequence of clone (H) cDNA, ESTs having a homology of 90% or more (eg, accession number AA434567) were registered. However, since it is a partial sequence, it cannot be determined whether clone (H) encodes the same protein as the encoded protein. (I) HP10565 Clone H obtained from human gastric cancer cDNA library
The total nucleotide sequence of the cDNA insert of P10565 was determined, and the 218 bp 5 ′ untranslated region, 570 bp
Had a structure consisting of a 434 bp 3 ′ untranslated region (SEQ ID NO: 17). The ORF encodes a protein consisting of 189 amino acid residues (SEQ ID NO: 18), and as a result of in vitro translation, a translation product of 23 kDa, which is slightly larger than the molecular weight predicted from the ORF of 20,663, was generated (Example 2). The fusion protein of this protein and GFP was localized in the Golgi apparatus and the endoplasmic reticulum (Example 4).

When GenBank was searched using the base sequence of clone (I) cDNA, ESTs having a homology of 90% or more (eg, accession number AA258633) were registered. However, since it is a partial sequence, it cannot be determined whether clone (I) encodes the same protein as the encoded protein. (J) HP10570 human fibrosarcoma cell line HT-1080 cDNA
C of clone HP10570 obtained from library
When the total nucleotide sequence of the DNA insert was determined, 94
bp 5 'untranslated region, 354 bp ORF, 761b
It had a structure consisting of the 3 'untranslated region of p (SEQ ID NO: 19). ORF is composed of 117 amino acid residues (SEQ ID NO: 2)
0), and a translation product of 14 kDa was produced as a result of in vitro translation, which is almost the same as the molecular weight of 12,767 predicted from the ORF (Example 2).
The fusion protein of this protein and GFP was localized in the Golgi apparatus and the endoplasmic reticulum (Example 4).

When GenBank was searched using the nucleotide sequence of clone (J) cDNA, ESTs having a homology of 90% or more (eg, accession number W07113) were registered. However, since it is a partial sequence, it cannot be determined whether clone (J) encodes the same protein as the encoded protein. Example 2: Protein Synthesis Example 1 by in vitro translation using a plasmid vector having the isolated cDNA, was subjected to in vitro transcription / translation by T _N T rabbit reticulocyte lysate kit (Promega).
At this time, [ ³⁵ S] methionine was added, and the expression product was labeled with a radioisotope. All reactions were performed according to the protocol attached to the kit.

The specific method is as follows. Plasmid 2μg, T _N T rabbit reticulocyte lysate 12.5
μl, buffer (included in the kit) 0.5 μl, amino acid mixture (without methionine) 2 μl, [ ³⁵ S] methionine (Amersham) 2 μl (0.37 MBq / μm
l), 0.5 μl of T7 RNA polymerase, RNasi
n ° C at 90 ° C in a total volume of 25 μl
Allowed to react for minutes. To 3 μl of the reaction solution, add SDS sampling buffer (125 mM Tris-HCl buffer, pH 6.8,
2 μl of 120 mM 2-mercaptoethanol, 2% SDS solution, 0.025% bromophenol blue, 20% glycerol) was added, and the mixture was heated at 95 ° C. for 3 minutes, and then subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography was performed to determine the molecular weight of the translation product. Example 3 Expression in COS7 Cells Escherichia coli transformed with the expression vector carrying the cDNA isolated in Example 1 was cultured in 2 ml of 2 × YT medium containing 100 μg / ml ampicillin at 37 ° C. for 2 hours, followed by helper phage M13KO7 (50 μl). ) Was added and the cells were cultured at 37 ° C. overnight. Single-stranded phage particles were obtained from the supernatant separated by centrifugation by polyethylene glycol precipitation. This was added to 100 μl of 1 mM Tris-0.
The cells were suspended in 1 mM EDTA, pH 8 (TE).

Monkey kidney-derived cultured cells COS7 are 10%
Dulbecco's modified Eagle containing fetal bovine serum (DME
M) The cells were cultured at 37 ° C. in the presence of 5% CO 2 in a medium. 1
x10 ⁵ pieces of COS7 cells 6 well plates planted (Nunc, 3cm diameter hole), 5% CO ₂ presence 2 at 37 ° C.
Incubated for 2 hours. After removing the medium, the cell surface was washed with a phosphate buffer, and further washed again with DMEM (TDMEM) containing 50 mM Tris-HCl (pH 7.5). 1 μl of single-stranded phage suspension, 0.6 m of DMEM medium
1, 3 μl of TRANSFECTAM ^™ (IBF) was added, and the cells were cultured at 37 ° C. for 3 hours in the presence of 5% CO ₂ . After removing the sample solution, the cell surface was washed with TDMEM, 2 ml of DMEM containing 10% fetal bovine serum was added per well, and the cells were cultured at 37 ° C. for 2 days in the presence of 5% CO ₂ . The medium is [ ³⁵ S] cysteine or [ ³⁵ S]
After changing to a medium containing methionine, the cells were cultured for 1 hour.
After separating the medium and the cells by centrifugation, the protein of the cell fraction was subjected to SDS-PAGE. Example 4 Expression of Green Fluorescent Protein (GFP) Fusion Protein Using a 26-mer sense primer starting from a translation initiation codon having an EcoRI recognition site and a 26-mer antisense primer having a stop codon having a BamHI recognition site, CDNA encoding the target protein
Was used as a template to amplify the translation region by PCR. PCR
The product is digested with EcoRI and BamHI, and the GFP fusion protein expression vector-pEGFP-N1 (Clontec)
At the EcoRI-BamHI site. After confirming the nucleotide sequence, the obtained fusion gene expression vector was transfected into COS7 cells by the method described in Example 3. The distribution of green fluorescence was observed with a fluorescence microscope, and the localization site of the target protein was examined. Example 5: Preparation of antibody Using a 26-mer sense primer starting from a translation initiation codon having an EcoRI recognition site added thereto and a 26-mer antisense primer having a stop codon having a SalI recognition sequence added thereto, translation was performed by PCR using each cDNA as a template as a template. The region was amplified. PCR products were EcoRI and Sal
The resulting fragment was digested with I and inserted into EcoRI and SalI sites of pGEX-5X-1 (Pharmacia). After confirming the nucleotide sequence, the host E. coli JM109 was transformed. L
The cells were cultured in a B medium at 37 ° C. for 5 hours, IPTG was added to a final concentration of 0.4 mM, and the cells were further cultured at 37 ° C. for 4 hours. The cells were separated by centrifugation and the lysis solution (50 m
M Tris-HCl pH 7.5, 1 mM EDTA,
(0.2 mM PMF), frozen once at −80 ° C. and thawed, and then sonicated. 10,000xg
Centrifugation for 30 minutes, and glutathione Sepharose 4B was added to the supernatant.
Was added and incubated at 4 ° C. for 1 hour. After thoroughly washing the beads, the elution solution (50 mM Tris-HCl
The fusion protein was eluted with 50 mM glutathione (pH 7.5). Using the obtained fusion protein as an antigen, a rabbit was immunized by a conventional method to obtain an antiserum. The antiserum was prepared by first subjecting the 40% saturated ammonium sulfate precipitated fraction to GST affinity column chromatography.
The ST antibody was removed. The flow-through fraction was further purified by a GST fusion protein antigen column.

[0043]

As described above in detail, according to the present application, a novel purified human protein, a DNA fragment encoding these proteins, an expression vector of this DNA fragment, a cell transformed with this expression vector, and this protein Are provided. Since all of the proteins provided by this application are considered to be proteins functioning in cells, as intracellular target proteins,
It can be used for detection of corresponding receptors and ligands, screening of new small molecule drugs, and the like. In addition, it can be used as an antigen for preparing an antibody against this protein. The DNA fragment provided by this application can be used as a probe for gene diagnosis or a gene source for gene therapy. By using this DNA fragment, the protein can be expressed in a large amount. Cells into which these proteins have been expressed by introducing these genes can be used to obtain modified forms of the proteins. The antibody provided by this application can be used for detection, quantification, purification, etc. of the protein of the present invention.

[0044]

[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Human Proteins and cDNAs then (4) <130> NP00041-YS <140> <141> <160> 20 <170> PatentIn Ver. 2.1 <210> 1 <211> 2920 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (73) .. (2652) <400> 1 aggtacagcg gcggtttctg aggttcttca ctcgcgactg acggagctgc ggtggcgtct 60 ccacacgcaa cc atg aag ttg aag gac aca aaa tca agg cca aag cag tca 111 Met Lys Leu Lys Asp Thr Lyc Serg Serp Pro aag gga atc aaa gtt gtg gga aaa tgg 159 Ser Cys Gly Lys Phe Gln Thr Lys Gly Ile Lys Val Val Gly Lys Trp 15 20 25 aag gaa gtg aag att gac cca aat atg ttt gca gat gga cag atg gat 207 Lys Glu Val Lys Ile Asp Pro Asn Met Phe Ala Asp Gly Gln Met Asp 30 35 40 45 gac ttg gtg tgc ttt gag gaa ttg aca gat tac cag ttg gtc tcc cct 255 Asp Leu Val Cys Phe Glu Glu Leu Thr Asp Tyr Gln Leu Val Ser Pro 50 55 60 gcc aag aat ccc tcc agt ctc ttc tca aag gaa gca ccc aag aga aag 303 Ala Lys Asn Pro Ser Ser Leu Phe Ser Lys Glu Ala Pro Lys Arg Lys 65 70 75 gca caa gct gtt tca gaa gaa gag gag gag gag gag gga aag tct agc 351 Ala Gln Ala Val Ser Glu Glu Glu Glu Glu Glu Glu Gly Lys Ser Ser 80 85 90 tca cca aag aaa aag atc aag ttg aag aaa agt aaa aat gta gca act 399 Ser Pro Lys Lys Ly s Ile Lys Leu Lys Lys Ser Lys Asn Val Ala Thr 95 100 105 gaa gga acc agt acc cag aaa gaa ttt gaa gtg aaa gat cct gag ctg 447 Glu Gly Thr Ser Thr Gln Lys Glu Phe Glu Val Lys Asp Pro Glu Leu 110 115 120 125 gag gcc cag gga gat gac atg gtt tgt gat gat ccg gag gct ggg gag 495 Glu Ala Gln Gly Asp Asp Met Val Cys Asp Asp Pro Glu Ala Gly Glu 130 135 140 atg aca tca gaa aac ctg gtc caa act gct cca aaa aag aag aaa aat 543 Met Thr Ser Glu Asn Leu Val Gln Thr Ala Pro Lys Lys Lys Lys Asn 145 150 155 aaa ggg aaa aaa ggg ttg gag cct tct cag agc act gct gcc aag gtg 591 Lys Gly Lys Lys Gly Leu Glu Pro Ser Gln Ser Thr Ala Ala Lys Val 160 165 170 ccc aaa aaa gcg aag aca tgg att cct gaa gtt cat gat cag aaa gca 639 Pro Lys Lys Ala Lys Thr Trp Ile Pro Glu Val His Asp Gln Lys Ala 175 180 185 gat gtg tca gct tgg aag gac ctg ttt gtt ccc agg ccg gtt ctc cga 687 Asp Val Ser Ala Trp Lys Asp Leu Phe Val Pro Arg Pro Val Leu Arg 190 195 200 205 gca ctc agc ttt cta ggc ttc tct gca ccc aca cca atc caa gcc ctg Al a Leu Ser Phe Leu Gly Phe Ser Ala Pro Thr Pro Ile Gln Ala Leu 210 215 220 acc ttg gca cct gcc atc cgt gac aaa ctg gac atc ctt ggg gct gct 783 Thr Leu Ala Pro Ala Ile Arg Asp Lys Leu Asp Ile Leu Gly Ala Ala 225 230 235 gag aca gga agt ggg aaa act ctt gcc ttt gcc atc cca atg att cat 831 Glu Thr Gly Ser Gly Lys Thr Leu Ala Phe Ala Ile Pro Met Ile His 240 245 250 gcg gtg ttg cag tgg cag aag agg aat gct gcc cct cct cca agt aac 879 Ala Val Leu Gln Trp Gln Lys Arg Asn Ala Ala Pro Pro Pro Ser Asn 255 260 265 acc gaa gca cca cct gga gag acc aga act gag gcc gga gct gag act 927 Thr Glu Ala Pro Pro Gly Glu Thr Arg Thr Glu Ala Gly Ala Glu Thr 270 275 280 285 aga tca cca ggc aag gct gaa gct gag tct gat gca ttg cct gac gat 975 Arg Ser Pro Gly Lys Ala Glu Ala Glu Ser Asp Ala Leu Pro Asp Asp 290 295 300 act gta att gag agt gaa gca ctg ccc agt gat att gca gcc gag gcc 1023 Thr Val Ile Glu Ser Glu Ala Leu Pro Ser Asp Ile Ala Ala Glu Ala 305 310 315 aga gcc aag act gga ggc act gtc tca gac cag gcg ttg ctg t tt ggt 1071 Arg Ala Lys Thr Gly Gly Thr Val Ser Asp Gln Ala Leu Leu Phe Gly 320 325 330 gac gat gat gct ggt gaa ggg cct tct tcc ctg atc agg gag aaa cct 1119 Asp Asp Asp Ala Gly Glu Gly Pro Ser Ser Leu Ile Arg Glu Lys Pro 335 340 345 gtt ccc aaa cag aat gag aat gag gag gaa aat ctt gat aaa gag cag 1167 Val Pro Lys Gln Asn Glu Asn Glu Glu Glu Asn Leu Asp Lys Glu Gln 350 355 360 365 act gga aat cta aaa cag gag ttg gat gac aaa agc gcc acc tgt aag 1215 Thr Gly Asn Leu Lys Gln Glu Leu Asp Asp Lys Ser Ala Thr Cys Lys 370 375 380 gca tat cca aag cgt cct ctg ctt gga ctg gtt ctg act ccc act cga 1263 Ala Pro Lys Arg Pro Leu Leu Gly Leu Val Leu Thr Pro Thr Arg 385 390 395 gag ctg gcc gtc cag gtc aaa cag cac att gat gct gtg gcc agg ttt 1311 Glu Leu Ala Val Gln Val Lys Gln His Ile Asp Ala Val Ala Arg Phe 400 405 410 aca gga att aaa act gct att ttg gtt ggt gga atg tcc acg cag aaa 1359 Thr Gly Ile Lys Thr Ala Ile Leu Val Gly Gly Met Ser Thr Gln Lys 415 420 425 cag cag agg atg ctg aac cgt cgt cct g ag att gtg gtt gct act cca 1407 Gln Gln Arg Met Leu Asn Arg Arg Pro Glu Ile Val Val Ala Thr Pro 430 435 440 445 ggc cgg ctg tgg gaa tta att aaa gaa aag cat tat cat ttg agg aac 1455 Gly Arg Leu Tlu Glu Leu Ile Lys Glu Lys His Tyr His Leu Arg Asn 450 455 460 ctt cgg cag ctc agg tgc ctg gta gtg gat gag gct gac cgg atg gtt 1503 Leu Arg Gln Leu Arg Cys Leu Val Val Asp Glu Ala Asp Arg Met Val 465 470 470 gag aaa ggc cat ttt gct gag ctc tca cag ctg cta gag atg ctc aat 1551 Glu Lys Gly His Phe Ala Glu Leu Ser Gln Leu Leu Glu Met Leu Asn 480 485 490 gac tcc caa tac aac cca aag aga caatt cct gtt gcc aca 1599 Asp Ser Gln Tyr Asn Pro Lys Arg Gln Thr Leu Val Phe Ser Ala Thr 495 500 505 ctc acc ctg gtg cat cag gct cct gct cga atc ctt cat aag aag cac 1647 Leu Thr Leu Val His Gln Ala Pro Ala Arg Ile Leu His Lys Lys His 510 515 520 525 acc aag aaa atg gat aaa aca gcc aaa ctt gac ctc ctt atg cag aaa 1695 Thr Lys Lys Met Asp Lys Thr Ala Lys Leu Asp Leu Leu Met Gln Lys 530 535 540 att ggc atg ag ag g ggc aag ccc aag gtc att gac ctc aca agg aat gag 1743 Ile Gly Met Arg Gly Lys Pro Lys Val Ile Asp Leu Thr Arg Asn Glu 545 550 555 gcc acg gtg gag acg cta aca gag acc aag atc cat tgt gag act gat 1791 Ala Thr Val Glu Thr Leu Thr Glu Thr Lys Ile His Cys Glu Thr Asp 560 565 570 gag aaa gac ttc tac ttg tac tac ttc ctg atg cag tat cca ggc cgc 1839 Glu Lys Asp Phe Tyr Leu Tyr Tyr Phe Leu Met Gln Pro Gly Arg 575 580 585 agc tta gtg ttt gcc aac agt atc tcc tgc atc aaa cgc ctc tct ggg 1887 Ser Leu Val Phe Ala Asn Ser Ile Ser Cys Ile Lys Arg Leu Ser Gly 590 595 600 605 ctc ctc at g at c gat g ccc ttg acc ctg cat gcc tgt atg 1935 Leu Leu Lys Val Leu Asp Ile Met Pro Leu Thr Leu His Ala Cys Met 610 615 620 cac cag aag cag agg ctc aga aac ctg gag cag ttt gcc cgt ctg gaa 1983 His Gln Lys Gln Leu Arg Asn Leu Glu Gln Phe Ala Arg Leu Glu 625 630 635 gac tgt gtt ctc ttg gca aca gat gtg gca gct cgg ggt ctg gat att 2031 Asp Cys Val Leu Leu Ala Thr Asp Val Ala Ala Arg Gly Leu Asp Ile 640 650 cct aaa gtc cag cat gtc atc cat tac cag gtc cca cgt acc tcg gag 2079 Pro Lys Val Gln His Val Ile His Tyr Gln Val Pro Arg Thr Ser Glu 655 660 665 att tat gtc cac cga agt ggt cga act gct cga gct acc aat gaa ggc 2127 Ile Tyr Val His Arg Ser Gly Arg Thr Ala Arg Ala Thr Asn Glu Gly 670 675 680 685 ctc agt ctg atg ctc att ggg cct gag gat gtg atc aac ttt aag aag 2175 Leu Ser Leu Met Leu Ile G Pro Asp Val Ile Asn Phe Lys Lys 690 695 700 att tac aaa acg ctc aag aaa gat gag gat atc cca ctg ttc ccc gtg 2223 Ile Tyr Lys Thr Leu Lys Lys Asp Glu Asp Ile Pro Leu Phe Pro Val 705 710 710 715 cag aca aaat atg gat gtg gtc aag gag cga atc cgt tta gct cga 2271 Gln Thr Lys Tyr Met Asp Val Val Lys Glu Arg Ile Arg Leu Ala Arg 720 725 730 cag att gag aaa tct gag tat cgg aac ttc cag gct tgc ctg cac aac Ile Glu Lys Ser Glu Tyr Arg Asn Phe Gln Ala Cys Leu His Asn 735 740 745 tct tgg att gag cag gca gca gct gcc ctg gag att gag ctg gaa gaa 2367 Ser Trp Ile Glu Gln Ala Ala Ala Ala Leu Glu Glu Leu Glu Glu 750 755 760 765 gac atg tat aag gga gga aaa gct gac cag caa gaa gaa cgt cgg aga 2415 Asp Met Tyr Lys Gly Gly Lys Ala Asp Gln Gln Glu Glu Arg Arg Arg 770 775 780 caa aag cag atgag aag aag gag ctg cgc cac ctg ctg tcc 2463 Gln Lys Gln Met Lys Val Leu Lys Lys Glu Leu Arg His Leu Leu Ser 785 790 795 cag cca ctg ttt acg gag agc cag aaa acc aag tat ccc act cag tct 2511 Gln Pro Le Thr Glu Ser Gln Lys Thr Lys Tyr Pro Thr Gln Ser 800 805 810 ggc aag ccg ccc ctg ctt gtg tct gcc cca agt aag agc gag tct gct 2559 Gly Lys Pro Pro Leu Leu Val Ser Ala Pro Ser Lys Ser Glu Ser Ala 815 820 825 ttg agc tgt ctc tcc aag cag aag aag aag aag aca aag aag ccg aag 2607 Leu Ser Cys Leu Ser Lys Gln Lys Lys Lys Lys Thr Lys Lys Pro Lys 830 835 840 845 gag cca cag ccg gaa cag cca cag cagt gca aat taa 2652 Glu Pro Gln Pro Glu Gln Pro Gln Pro Ser Thr Ser Ala Asn 850 855 860 ctggtcaagt gtgtcagtga ctgcacattg gtttctgttc tctggctatt tgcaaaacct 2712 ctcccaccct tgtgtttcac tccaccacca acccca ggta aaaaagtctc cctctcttcc 2772 actcacaccc atagcgggag agacctcatg cagatttgca ttgttttgga gtaagaattc 2832 aatgcagcag cttaattttt ctgtattgca gtgtttatag gcttcttgtg tgttaaactt 2892 gatttcatag <210> 2 <211> 859 <212> PRT <213> Homo sapiens <400> 2 Met Lys Leu Lys Asp Thr Lys Ser Arg Pro Lys Gln Ser Ser Cys Gly 1 5 10 15 Lys Phe Gln Thr Lys Gly Ile Lys Val Val Gly Lys Trp Lys Glu Val 20 25 30 Lys Ile Asp Pro Asn Met Phe Ala Asp Gly Gln Met Asp Asp Leu Val 35 40 45 Cys Phe Glu Glu Leu Thr Asp Tyr Gln Leu Val Ser Pro Ala Lys Asn 50 55 60 Pro Ser Ser Leu Phe Ser Lys Glu Ala Pro Lys Arg Lys Ala Gln Ala 65 70 75 80 Val Ser Glu Glu Glu Glu Glu Glu Glu Gly Lys Ser Ser Ser Pro Lys 85 90 95 Lys Lys Ile Lys Leu Lys Lys Ser Lys Asn Val Ala Thr Glu Gly Thr 100 105 110 Ser Thr Gln Lys Glu Phe Glu Val Lys Asp Pro Glu Leu Glu Ala Gln 115 120 125 Gly Asp Asp Met Val Cys Asp Asp Pro Glu Ala Gly Glu Met Thr Ser 130 135 140 Glu Asn Leu Val Gln Thr Ala Pro Lys Lys Lys Lys Asn Lys Gly Lys 145 150 155 160 Lys Gly Leu Glu Pro Ser Gln Ser Thr Ala Ala Lys Val Pro Lys Lys 165 170 175 Ala Lys Thr Trp Ile Pro Glu Val His Asp Gln Lys Ala Asp Val Ser 180 185 190 Ala Trp Lys Asp Leu Phe Val Pro Arg Pro Val Leu Arg Ala Leu Ser 195 200 205 Phe Leu Gly P he Ser Ala Pro Thr Pro Ile Gln Ala Leu Thr Leu Ala 210 215 220 Pro Ala Ile Arg Asp Lys Leu Asp Ile Leu Gly Ala Ala Glu Thr Gly 225 230 235 240 Ser Gly Lys Thr Leu Ala Phe Ala Ile Pro Met Ile His Ala Val Leu 245 250 255 Gln Trp Gln Lys Arg Asn Ala Ala Pro Pro Pro Ser Asn Thr Glu Ala 260 265 270 Pro Pro Gly Glu Thr Arg Thr Glu Ala Gly Ala Glu Thr Arg Ser Pro 275 280 285 Gly Lys Ala Glu Ala Glu Ser Asp Ala Leu Pro Asp Asp Thr Val Ile 290 295 300 Glu Ser Glu Ala Leu Pro Ser Asp Ile Ala Ala Glu Ala Arg Ala Lys 305 310 315 320 Thr Gly Gly Thr Val Ser Asp Gln Ala Leu Leu Phe Gly Asp Asp Asp 325 330 335 Ala Gly Glu Gly Pro Ser Ser Leu Ile Arg Glu Lys Pro Val Pro Lys 340 345 350 Gln Asn Glu Asn Glu Glu Glu Asn Leu Asp Lys Glu Gln Thr Gly Asn 355 360 365 Leu Lys Gln Glu Leu Asp Asp Lys Ser Ala Thr Cys Lys Ala Tyr Pro 370 375 380 Lys Arg Pro Leu Leu Gly Leu Val Leu Thr Pro Thr Arg Glu Leu Ala 385 390 395 400 Val Gln Val Lys Gln His Ile Asp Ala Val Ala Arg Phe Thr Gly Ile 405 410 415 Lys Thr Ala I le Leu Val Gly Gly Met Ser Thr Gln Lys Gln Gln Arg 420 425 430 Met Leu Asn Arg Arg Pro Glu Ile Val Val Ala Thr Pro Gly Arg Leu 435 440 445 Trp Glu Leu Ile Lys Glu Lys His Tyr His Leu Arg Asn Leu Arg Gln 450 455 460 Leu Arg Cys Leu Val Val Asp Glu Ala Asp Arg Met Val Glu Lys Gly 465 470 475 480 His Phe Ala Glu Leu Ser Gln Leu Leu Glu Met Leu Asn Asp Ser Gln 485 490 495 Tyr Asn Pro Lys Arg Gln Thr Leu Val Phe Ser Ala Thr Leu Thr Leu 500 505 510 Val His Gln Ala Pro Ala Arg Ile Leu His Lys Lys His Thr Lys Lys 515 520 525 Met Asp Lys Thr Ala Lys Leu Asp Leu Leu Met Gln Lys Ile Gly Met 530 535 540 Arg Gly Lys Pro Lys Val Ile Asp Leu Thr Arg Asn Glu Ala Thr Val 545 550 555 560 Glu Thr Leu Thr Glu Thr Lys Ile His Cys Glu Thr Asp Glu Lys Asp 565 570 575 Phe Tyr Leu Tyr Tyr Phe Leu Met Gln Tyr Pro Gly Arg Ser Leu Val 580 585 590 Phe Ala Asn Ser Ile Ser Cys Ile Lys Arg Leu Ser Gly Leu Leu Lys 595 600 605 Val Leu Asp Ile Met Pro Leu Thr Leu His Ala Cys Met His Gln Lys 610 615 620 Gln Arg Leu ArgAsn Leu Glu Gln Phe Ala Arg Leu Glu Asp Cys Val 625 630 635 640 640 Leu Leu Ala Thr Asp Val Ala Ala Arg Gly Leu Asp Ile Pro Lys Val 645 650 655 Gln His Val Ile His Tyr Gln Val Pro Arg Thr Ser Glu Ile Tyr Val 660 665 670 His Arg Ser Gly Arg Thr Ala Arg Ala Thr Asn Glu Gly Leu Ser Leu 675 680 685 Met Leu Ile Gly Pro Glu Asp Val Ile Asn Phe Lys Lys Ile Tyr Lys 690 695 700 Thr Leu Lys Lys Asp Glu Asp Ile Pro Leu Phe Pro Val Gln Thr Lys 705 710 715 715 720 Tyr Met Asp Val Val Lys Glu Arg Ile Arg Leu Ala Arg Gln Ile Glu 725 730 735 Lys Ser Glu Tyr Arg Asn Phe Gln Ala Cys Leu His Asn Ser Trp Ile 740 745 750 Glu Gln Ala Ala Ala Ala Leu Glu Ile Glu Leu Glu Glu Asp Met Tyr 755 760 765 Lys Gly Gly Lys Ala Asp Gln Gln Glu Glu Arg Arg Arg Gln Lys Gln 770 775 780 780 Met Lys Val Leu Lys Lys Glu Leu Arg His Leu Leu Ser Gln Pro Leu 785 790 795 800 Phe Thr Glu Ser Gln Lys Thr Lys Tyr Pro Thr Gln Ser Gly Lys Pro 805 810 815 Pro Leu Leu Val Ser Ala Pro Ser Lys Ser Glu Ser Ala Leu Ser Cys 820 825 830 Leu Ser Lys GlnLys Lys Lys Lys Thr Lys Lys Pro Lys Glu Pro Gln 835 840 845 Pro Glu Gln Pro Gln Pro Ser Thr Ser Ala Asn 850 855 <210> 3 <211> 1502 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (15) .. (998) <400> 3 actcgactac caag atg gcg gcc ccc ggg agc tgt gct cta tgg agc tat 50 Met Ala Ala Pro Gly Ser Cys Ala Leu Trp Ser Tyr 1 5 10 tgc ggc cgt ggg tgg tcg cgg gcg atg cgg ggc tgc cagct 98 Cys Gly Arg Gly Trp Ser Arg Ala Met Arg Gly Cys Gln Leu Leu Gly 15 20 25 ctt cgt agc tct tgg ccc ggg gac cta cta agt gct cgg ctc ttg tcc 146 Leu Arg Ser Ser Trp Pro Gly Asp Leu Leu Ser Ala Arg Leu Leu Ser 30 35 40 caa gag aag cgg gca gcg gaa acg cac ttt ggg ttt gag act gtg tcg 194 Gln Glu Lys Arg Ala Ala Glu Thr His Phe Gly Phe Glu Thr Val Ser 45 50 55 60 gaa gag gag aag ggg ggc aaa gtc tat cag gtg ttt gaa agt gtg gct 242 Glu Glu Glu Lys Gly Gly Lys Val Tyr Gln Val Phe Glu Ser Val Ala 65 70 75 aag aag tat gat gtg atg aat gat atg atg agt ctt ggt atc cat cgt 290 Lys Lys Tyr Asp Val Met Asn Asp Met Met Ser Leu Gly Ile His Arg 80 85 90 gtt tgg aag gat ttg ctg ctc tgg aag atg cac ccg ctt cct ggg acc 338 Val Trp Lys Asp Leu Leu Leu Trp Lys Met His Pro Leu Pro Gly Thr 95 100 105 cag ctg ctt gat gtt gct gga ggc aca ggt gac att gca ttc cgg ttc 386 Gln Leu Leu Asp Val Ala Gly Gly Thr Gly Asp Ile Ala Phe Arg Phe 110 115 120 ctt aat tat gtt cag tcc cag cat cag aga aaa cag aag agg cag tta 434 Leu Asn Tyr Val Gln Ser Gln His Gln Arg Lys Gln Lys Arg Gln Leu 125 130 135 140 agg gcc caa caa aat tta tcc tgg gaa gaa att gcc aaa gag tac cag 482 Arg Ala Gln Gln Asn Leu Ser Trp Glu Glu Ile Ala Lys Glu Tyr Gln 145 150 155 aat gaa gaa gat tcc ttg ggc ggg tct cgt gtc gtg gtg tgt gac atc 530 Asn Glu Glu Asp Ser Leu Gly Gly Ser Arg Val Val Vals Cys Asp Ile 160 165 170 aac aag gag atg cta aag gtt gga acag aaa gcc ttg gct caa gga 578 Asn Lys Glu Met Leu Lys Val Gly Lys Gln Lys Ala Leu Ala Gln Gly 175 180 185 tac aga gct gga ctt gca tgg gta tta gga gat gct gaa gaa ctg ccc 626 Tyr Arg Ala Gly Val Leu Gly Asp Ala Glu Glu Leu Pro 190 195 200 ttt gat gat gac aag ttt gat att tac acc att gcc ttt ggg atc cgg 674 Phe Asp Asp Asp Lys Phe Asp Ile Tyr Thr Ile Ala Phe Gly Ile Arg 205 210 215 220 aat gtc aca cac att gat cag gca ctc cag gaa gct cat cgg gtg ctg 722 Asn Val Thr His Ile Asp Gln Ala Leu Gln Glu Ala His Arg Val Leu 225 230 235 aaa cca gga gga cgg ttt ctc tgt ctg gaa tttgg ca ca aat 770 Lys Pro Gly Gly Arg Phe Leu Cys Leu Glu Phe Ser Gln Val Asn Asn 240 245 250 ccc ctc ata tcc agg ctt tat gat cta tat agc ttc cag gtc atc cct 818 Pro Leu Ile Ser Arg Leu Tyr Asp Leu Tyr Ser Phe Gln Val Ile Pro 255 260 265 gtc ctg gga gag gtc atc gct gga gac tgg aag tcc tat cag tac ctt 866 Val Leu Gly Glu Val Ile Ala Gly Asp Trp Lys Ser Tyr Gln Tyr Leu 270 275 280 gta gag agt atc cga agg ttt ccg tct cag gaa gag ttc aag gac atg 914 Val Glu Ser Ile Arg Arg Phe Pro Ser Gln Glu Glu Phe Lys Asp Met 285 290 295 300 ata gaa gat gca ggc ttt cac aag gtg act tac gaa agt cta aca tca 962 Ile Ala Gly Phe His Lys Val Thr Tyr Glu Ser Leu Thr Ser 305 310 315 ggc att gtg gcc att cat tct ggc ttc aaa ctt taa ttcctttcct 1008 Gly Ile Val Ala Ile His Ser Gly Phe Lys Leu 320 325 atcatggagc atgaacca gt catatcctgt tgaaagcctg gaactgaagg ataatctggc 1068 aaatgagaca gcagcagagc atctcctctt aaggatacgt gccttggact catgtttgaa 1128 tcgaacagtc tcaaagtgga agaacaaatt cttgtcactt ttttacagct ttctttggag 1188 ctgcttcagt ccatctccca gaggcatttg gtctgtatct ttgctcaact gctaatttct 1248 cttggctgta gggtgtgtgg ttaaggtaca accaccccta aagctcagtt ttgaagtgag 1308 tgtatttata gcttctctgc tggtgctgcc ttctagaggg atgatagatc atttgaaccc 1368 aatgacaatt tttaaccaga aaatttaatt gtacctgaat caacctttca gcctaggacg 1428 aagtctaggc ccaagtcaga gtattaatga tcatgagaat tgtgtgctga accagtaaac 1488 gagtttacct tttg 1502 <210> 4 <211> 327 <212> PRT <213> Homo sapiens <400> 4 Met Ala Ala Pro Gly Ser Cys Ala Leu Trp Ser Tyr Cys Gly Arg Gly 1 5 10 15 Trp Ser Arg Ala Met Arg Gly Cys Gln Leu Leu Gly Leu Arg Ser Ser 20 25 30 Trp Pro Gly Asp Leu Leu Ser Ala Arg Leu Leu Ser Gln Glu Lys Arg 35 40 45 Ala Ala Glu Thr His Phe Gly Phe Glu Thr Val Ser Glu Glu Glu Lys 50 55 60 Gly Gly Lys Val Tyr Gln Val Phe Glu Ser Val Ala Lys Lys Tyr Asp 65 70 75 80 Val Met Asn Asp Met Met Ser Leu Gly Ile His Arg Val Trp Lys Asp 85 90 95 Leu Leu Leu Trp Lys Met His Pro Leu Pro Gly Thr Gln Leu Leu Asp 100 105 110 Val Ala Gly Gly Thr Gly Asp Ile Ala Phe Arg Phe Leu Asn Tyr Val 115 120 125 Gln Ser Gln His Gln Arg Lys Gln Lys Arg Gln Leu Arg Ala Gln Gln 130 135 140 Asn Leu Ser Trp Glu Glu Ile Ala Lys Glu Tyr Gln Asn Glu Glu Asp 145 150 155 160 Ser Leu Gly Gly Ser Arg Val Val Val Cys Asp Ile Asn Lys Glu Met 165 170 175 Leu Lys Val Gly Lys Gln Lys Ala Leu Ala Gln Gly Tyr Arg Ala Gly 180 185 190 Leu Ala Trp Val Leu Gly Asp Ala Glu Glu Leu Pro Phe Asp Asp Asp 195 200 205 Lys Phe Asp I le Tyr Thr Ile Ala Phe Gly Ile Arg Asn Val Thr His 210 215 220 Ile Asp Gln Ala Leu Gln Glu Ala His Arg Val Leu Lys Pro Gly Gly 225 230 235 240 Arg Phe Leu Cys Leu Glu Phe Ser Gln Val Asn Asn Pro Leu Ile Ser 245 250 255 Arg Leu Tyr Asp Leu Tyr Ser Phe Gln Val Ile Pro Val Leu Gly Glu 260 265 270 Val Ile Ala Gly Asp Trp Lys Ser Tyr Gln Tyr Leu Val Glu Ser Ile 275 280 285 Arg Arg Phe Pro Ser Gln Glu Glu Phe Lys Asp Met Ile Glu Asp Ala 290 295 300 Gly Phe His Lys Val Thr Tyr Glu Ser Leu Thr Ser Gly Ile Val Ala 305 310 315 320 Ile His Ser Gly Phe Lys Leu 325 <210> 5 <211> 737 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (127) .. (387) <400> 5 aaaaattctt cttcgacggc gcggacctgg agcttccgcg cggtggcttc actctcctgt 60 aaaacgctag agcggcgagt tgttacctgc gtcctctgac ctgagagcga aggggaaagc 120 ggcgag atg act gac cgc tac acc atc cat agc cag ctg gag cac ctg 168 Met Thr Asp Arg Tyr Thr Ile His Ser Gln Leu Glu His Leu 1 5 10 cag tcc aag tac atc ggc acg ggc cac gcc gac acc acc aag tgg gag 216 Gln Ser Lys Tyr Ile Gly Thr Gly His Ala Asp Thr Thr Lys Trp Glu 15 20 25 30 tgg ctg gtg aac caa cac cgc gac tcg tac tgc tcc tac atg ggc cac 264 Trp Leu Val Asn Gln His Arg Asp Ser Tyr Cys Ser Tyr Met Gly His 35 40 45 ttc gac ctt ctc aac tac ttc gcc att gcg gag aat gag agc aaa gcg 312 Phe Asp Leu Leu Asn Tyr Phe Ala Ala Glu Asn Glu Ser Lys Ala 50 55 60 cga gtc cgc ttc aac ttg atg gaa aag atg ctt cag cct tgt gga ccg 360 Arg Val Arg Phe Asn Leu Met Glu Lys Met Leu Gln Pro Cys Gly Pro 65 70 75 cca gcc gac aag ccc gag gag aac tga gactctgcct taccacctca 407 Pro Ala Asp Lys Pro Glu Glu Asn 80 85 gtgcggggca cctctcccag cgtttctccg gtttgccaat cctcttaagt attcctgtct 467 ccaaaggacc ggctctccat ggctcctgcg cctcgtgctt tccgcgtaca gaagtgcttg 527 cccggggagt cccgcctgac ctgccttcat gtggaccctt agaacagcac tgggagacca 587 gcaggactcc tgagaactgt gctggtggag aggtcctaga gccggcgagc gtttgagaag 647 agggcatggc gctggagtga gatgggattt ggcgtctcgt ttttggctaa ttgattgtca 707 ttggcttttt ccataaagtt tagaaatcgt 737 <210> 6 <211> 86 <212> PRT <213> Homo sapiens <400> 6 Met Thr Asp Arg Tyr Thr Ile His Ser Gln Leu Glu His Leu Gln Ser 1 5 10 15 Lys Tyr Ile Gly Thr Gly His Ala Asp Thr Thr Lys Trp Glu Trp Leu 20 25 30 Val Asn Gln His Arg Asp Ser Tyr Cys Ser Tyr Met Gly His Phe Asp 35 40 45 Leu Leu Asn Tyr Phe Ala Ile Ala Glu Asn Glu Ser Lys Ala Arg Val 50 55 60 Arg Phe Asn Leu Met Glu Lys Met Leu Gln Pro Cys Gly Pro Pro Ala 65 70 75 80 Asp Lys Pro Glu Glu Asn 85 <210> 7 <211> 903 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (85) .. (621) <400> 7 ctgaggcccc acagcctccc aattccgggc agacccctga cacctgctgt ctggcccctt 60 ccggcctgaa gctgcagccg cgcc atg tcc acc cct ccg ttg gcc gcg tcg 111 Met Ser Thr Pro Pro Leu Ala Ag cg gcc cag gcc cag gcc cag gcc cag gcc cag gcc cag gcc cag gcc cag gcc cag ccc gcc 159 Gly Met Ala Pro Gly Pro Phe Ala Gly Pro Gln Ala Gln Gln Ala Ala 10 15 20 25 cgg gaa gtc aac acg gcg tcg ctg tgc cgc atc ggg cag gag aca gtg 207 Arg Glu Val Asn Thr Ala Ser Leu Cys Arg Ig Gly Gln Glu Thr Val 30 35 40 cag gac atc gtg tac cgc acc atg gag atc ttc cag ctc ctg agg aac 255 Gln Asp Ile Val Tyr Arg Thr Met Glu Ile Phe Gln Leu Leu Arg Asn 45 50 55 atg cag ctg cca aat ggt gtc act tac cac act gga aca tat caa gac 303 Met Gln Leu Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp 60 65 70 cgg tta aca aag cta cag gat aat ctt cgc caa ctt tca gtt ctc ttc 351 Arg Leu Thr Lys Leu Gln Asp Asn Leu Arg Gln Leu Ser Val Leu Phe 75 80 85 agg aag ctg aga ttg gta tat gac aaa tgc aat gaa aac tgt ggt ggg 399 Arg Lys Leu Arg Leu Val Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly 90 95 100 105 atg gat ccc att cca gtc gag caa ctt att cca tat gtg gaa gaa gat 447 Met Asp Pro Ile Pro Val Glu Gln Leu Ile Pro Tyr Val Glu Glu Asp 110 115 120 ggc tca aag aat gat gat cgg gct ggc cca cct cgt ttt gct agt gaa 495 Gly Ser Lys Asn Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu 125 130 135 gag agg cga gaa att gct gaa gta aat aaa aaa ctc aaa cag aag aat 543 Glu Arg Arg Glu Ile Ala Glu Val Asn Lys Lys Leu Lys Gln Lys Asn 140 145 150 caa cag ctg aaa caa att atg gat caa tta cga aat ctc atc tgg gat 591 Gln Gln Leu Lys Gln Ile Met Asp Gln Leu Arg Asn Leu Ile Asp 155 160 165 ata aat gcc atg ttg gca atg agg aac taa gctgatattt aaatttcctg 641 Ile Asn Ala Met Leu Ala Met Arg Asn 170 175 ctttacacat gttataccat tgttttttcc ctcaagtatt ttttccctgt gaagaagatt 701 atttatctgc ttttatttta gtcactaaaa ctaaagtttt tatttttaca ttgtgatttt 761 tacattaaaa tattaacttt ttttaatgct attttatgaa agattattgt aataaacttt 821 gatggggttt gtattttggt taatcttcat gaattgaata attgtttttt taaagcaaaa 881 taaagttttt taaataaatg tt 903 <210> 8 <211> 178 <212> PRT <213> Homo sapiens <400> 8 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala Pro Gly Pro Phe 1 5 10 15 Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Ser 20 25 30 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr 35 40 45 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val 50 55 60 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp 65 70 75 80 Asn Leu Arg Gln Leu Ser Val Leu Phe Arg Lys Leu Arg Leu Val Tyr 85 90 95 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Val Glu 100 105 110 Gln Leu Ile Pro Tyr Val Glu Glu Asp Gly Ser Lys Asn Asp Asp Arg 115 120 125 Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Ala Glu 130 135 140 Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu Lys Gln Ile Met 145 150 155 160 Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala Met Leu Ala Met 165 170 175 Arg Asn <210> 9 <211> 1170 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (187) .. (540) <400> 9 tatagtccag ggcctgtttc cctgtagcag ctccttattg ctggagaagg agaaaagtgc 60 ccaagatcct ttcaggatat ttggtttttt gggcgcgaca caaatcgagg tgagggaaga 120 gagaggaaaa tcccctgaat ccctgcagga ttaatttatt caaaaaggaa ataaaaaata 180 ctcaat atg caa aag tct tgt gaa gaa aat gag gga aaa cca cag aac 228 Met Gln Lys Ser Cys Glu Glu Asn Glu Gly Lys Pro Gln Asn 1 5 10 atg cca aag gcc gag gaa gat cgc cct ttg gag gat gta cca cag gag 276 Met Pro Lys Ala Glu Glu Asp Arg Pro Leu Glu Asp Val Pro Gln Glu 15 20 25 30 gca gaa gga aat cct caa cct tcc gaa gaa ggc gta agc cag gaa gca 324 Ala Glu Gly Asn Pro Gln Pro Ser Glu Glu Gly Val Ser Gln Glu Ala 35 40 45 gaa gga aac ccc aga gga ggg ccg aat cag cct ggc cag gga ttt aaa G372 Asn Pro Arg Gly Gly Pro Asn Gln Pro Gly Gln Gly Phe Lys 50 55 60 gag gac aca ccc gtt agg cat ttg gac cct gaa gaa atg ata aga gga 420 Glu Asp Thr Pro Val Arg His Leu Asp Pro Glu Glu Met Ile Arg Gly 65 70 75 gta gat gag ctt gaa agg ctt agg gaa gag ata aga aga gta aga aac 468 Val Asp Glu Leu Gl u Arg Leu Arg Glu Glu Ile Arg Arg Val Arg Asn 80 85 90 aag ttt gtg atg atg cat tgg aag caa aga cat tca cgc agc cgt cct 516 Lys Phe Val Met Met His Trp Lys Gln Arg His Ser Arg Ser Arg Pro 95 100 105 110 tat cct gtg tgc ttt agg cct tga attcattttt gcctaatatt aaaatctggc 570 Tyr Pro Val Cys Phe Arg Pro 115 cccagctttc tttctgttag cattttctga tgtatctttg acctccattt tacttttaat 630 catctgatga aattttgttt taggtaattt ccttggtacc agcatctcat tggattttgg 690 attttgaccc attttccagg tctatttttc aattggaaac tttcacacat ttgcatggga 750 atatgttcat tccatgttgt aaagtaaaac ataacaggtt atggcaaagc agcatattta 810 atatcagctc acatatgtag gataaaattc caaactttgt gtgtgtgcgt gtgtgtatac 870 atacatccat ataacatata tcacaaactt aaccaagctt atttctgtgt ggtgtgaaat 930 tttatttgtt ttcttctttt tgttcttttt gcttatatgt actttttaat gaacacgtgt 990 ctcacacaca aaaagaatta aggatttttt ttacaagtaa gagtcaaata atttgcaacc 1050 agcttatgag ggcaatgggg gcacctaaac tcttgatgaa agaactttaa aaagaaatgt 1110 aaacctcaaa ttacctctgg atctcttagc cagaggaata aactggcaat tattacaga t 1170 <210> 10 <211> 117 <212> PRT <213> Homo sapiens <400> 10 Met Gln Lys Ser Cys Glu Glu Asn Glu Gly Lys Pro Gln Asn Met Pro 1 5 10 15 Lys Ala Glu Glu Asp Arg Pro Leu Glu Asp Val Pro Gln Glu Ala Glu 20 25 30 Gly Asn Pro Gln Pro Ser Glu Glu Gly Val Ser Gln Glu Ala Glu Gly 35 40 45 Asn Pro Arg Gly Gly Pro Asn Gln Pro Gly Gln Gly Phe Lys Glu Asp 50 55 60 Thr Pro Val Arg His Leu Asp Pro Glu Glu Met Ile Arg Gly Val Asp 65 70 75 80 Glu Leu Glu Arg Leu Arg Glu Glu Ile Arg Arg Val Arg Asn Lys Phe 85 90 95 Val Met Met His Trp Lys Gln Arg His Ser Arg Ser Arg Pro Tyr Pro 100 105 110 Val Cys Phe Arg Pro 115 <210> 11 <211> 404 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (105) .. (365) <400> 11 tttttccagc ggaagtggct cctgtaaggc agcaaggtag cgtggccggc gcccgagctg 60 gggttgtgtc cctgctgggc tgccgttcca gctggactgc cgcc atg ga ctc ag g g Gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gc gag gc gag gc gag gc gag gg gag gc gag gc gag gc gg gc gag gc gag gc gg gag gg Leu Gln Arg Asp Leu Glu Ala Glu His 5 10 15 20 gtg gag gtg gag gac acg acc ctc aac cgt tgc tcc tgt agc ttc cga 212 Val Glu Val Glu Asp Thr Thr Leu Asn Arg Cys Ser Cys Ser Phe Arg 25 30 35 gtc ctg gtg gtg tcg gcc aag ttc gag ggg aaa ccg ctg ctt cag aga 260 Val Leu Val Val Ser Ala Lys Phe Glu Gly Lys Pro Leu Leu Gln Arg 40 45 50 cac agg ctg gtg aac gcg tgc cta gca gaagag atc cat 308 His Arg Leu Val Asn Ala Cys Leu Ala Glu Glu Leu Pro His Ile His 55 60 65 gcc ttt gaa cag aaa acc ctg acc cca gac cag tgg gca cgt gag cga 356 Ala Phe Glu Gln Lys Thr Leu Thr Pro Asp Gln Trp Ala Arg Glu Arg 70 75 80 cag aaa tga gggactggga tctgcacagc cattaaatta taaatctgg 404 Gln Lys 85 <210> 12 <211> 86 <212> PRT <213> Homo sapiens <400> 12 Met Glu Leu Ser Ala Glu Tyr Leu Arg Glu Lys Leu Gln Arg Asp Leu 1 5 10 15 Glu Ala Glu His Val Glu Val Glu Asp Thr Thr Leu Asn Arg Cys Ser 20 25 30 Cys Ser Phe Arg Val Leu Val Val Ser Ala Lys Phe Glu Gly Lys Pro 35 40 45 Leu Leu Gln Arg His Arg Leu Val Asn Ala Cys Leu Ala Glu Glu Leu 50 55 60 Pro His Ile His Ala Phe Glu Gln Lys Thr Leu Thr Pro Asp Gln Trp 65 70 75 80 Ala Arg Glu Arg Gln Lys 85 <210> 13 <211> 822 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (10) .. (546) <400> 13 attccaaac atg gcg gct cca cta ggg ggt atg ttt tct ggg cag cca ccc 51 Met Ala Ala Pro Leu Gly Gly Met Phe Ser Gly Gln Pro Pro 1 5 10 ggt ccc cct cag gcc ccg ccg ggc ctt ccg ggc caact tcg ctt ctt 99 Gly Pro Pro Gln Ala Pro Pro Gly Leu Pro Gly Gln Ala Ser Leu Leu 15 20 25 30 cag gca gct cca ggc gct cct aga cct tcc agc agt act ttg gtg gac 147 Gln Ala Ala Pro Gly Ala Pro Arg Pro Ser Ser Ser Thr Leu Val Asp 35 40 45 gag ttg gag tca tct ttc gag gct tgc ttt gca tct ctg gtg agt cag 195 Glu Leu Glu Ser Ser Phe Glu Ala Cys Phe Ala Ser Leu Val Ser Gln 50 55 60 gac tat gtc aat ggc acc gat cag gaa gaa att cga acc ggt gtt gat 243 Asp Tyr Val Asn Gly Thr Asp Gln Glu Glu Ile Arg Thr Gly Val Asp 65 70 75 cag tgt atc cag aag ttt ctg gat att gca aga cag aca gaa tgt ttt 291 Gln Cys Ile Gln Lys Phe Leu Asp Ile Ala Arg Gln Thr Glu Cys Phe 80 85 90 ttc tta caa aaa aga ttg cag tta tct gtc cag aaa cca gag caa gtt 339 Phe Leu Gln Lys Arg Leu Gln Leu Ser Val Gln Lys Pro Glu Gln Val 95 100 105 110 atc aaa gag gat gtg tca gaa cta agg aat gaa tta cag cgg aaa gat 387 Ile Lys Glu Asp Val Ser Glu Leu Arg Asn Glu Leu Gln Arg Lys Asp 115 120 125 gca cta gtc cag aag cac ttg aca aag ctg agg cat tgg cag gtg 435 Ala Leu Val Gln Lys His Leu Thr Lys Leu Arg His Trp Gln Gln Val 130 135 140 ctg gag gac atc aac gtg cag cac aaa aag ccc gcc gac atc cct cag 483 Leu Glu Asp Ile Asn Val Gln His Lys Lys Pro Ala Asp Ile Pro Gln 145 150 155 ggc tcc ttg gcc tac ctg gag cag gca tct gcc aac atc cct gca cct 531 Gly Ser Leu Ala Tyr Leu Glu Gln Ala Ser Ala Asn Ile Pro Ala Pro 160 165 170 ctg aag cca acg tga ggacagag gcctatgagt gggctgatgc 586 Leu Lys Pro Thr 175 gtgaggttgg ccacacattc cttcctgtgg acttgacatt ttggaagaac tctttgccag 646 ataatgagtt cattttagtt ttatgctccc attgaaaaat tttccactat ttttataagc 706 tgttaatttc ttgagtactt tataacatgt ctgtagcttg gataaaccaa gtaagtattt 766 tttttttgtc tttagcgaag tttagactgt gaatatgatg acacagattc tttttt 822 <210> 14 <211> 178 <212> PRT <213> Homo sapiens <400> 14 Met Ala Ala Pro Leu Gly Gly Met Phe Ser Gly Gln Pro Pro Gly Pro 1 5 10 15 Pro Gln Ala Pro Pro Gly Leu Pro Gly Gln Ala Ser Leu Leu Gln Ala 20 25 30 Ala Pro Gly Ala Pro Arg Pro Ser Ser Ser Thr Leu Val Asp Glu Leu 35 40 45 Glu Ser Ser Phe Glu Ala Cys Phe Ala Ser Leu Val Ser Gln Asp Tyr 50 55 60 Val Asn Gly Thr Asp Gln Glu Glu Ile Arg Thr Gly Val Asp Gln Cys 65 70 75 80 Ile Gln Lys Phe Leu Asp Ile Ala Arg Gln Thr Glu Cys Phe Phe Leu 85 90 95 Gln Lys Arg Leu Gln Leu Ser Val Gln Lys Pro Glu Gln Val Ile Lys 100 105 110 Glu Asp Val Ser Glu Leu Arg Asn Glu Leu Gln Arg Lys Asp Ala Leu 115 120 125 Val Gln Lys His Leu Thr Lys Leu Arg His Trp Gln Gln Val Leu Glu 130 135 140 Asp Ile Asn Val Gln His Lys Lys Pro Ala Asp Ile Pro Gln Gly Ser 145 150 155 160 Leu Ala Tyr Leu Glu Gln Ala Ser Ala Asn Ile Pro Ala Pro Leu Lys 165 170 175 Pro Thr <210> 15 <211> 752 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (95) .. (634) <400> 15 ttttcgcttc cggctgccgc aggcgcttcg ctggtgcaga cgcagtgctg agcacacagc 60 taccggacaa agagtgacgc ccggagctgg agtt atg gc g gct acg gag cc g atg gc gg gc gg gc gg gc gg gc gg gc g at g Ala Ala Thr Gly Ser Pro Ala Ala Val Pro Pro Glu Lys Leu Glu 10 15 20 gga gcc ggt tcg agc tca gcc cct gag cgt aac tgt gtg ggc tcc tcg 211 Gly Ala Gly Ser Ser Ser Ala Pro Glu Arg Asn Cys Val Gly Ser Ser 25 30 35 ctg cca gag gcc tca ccg cct gcc cct gag cct tcc agt ccc aac gcc 259 Leu Pro Glu Ala Ser Pro Pro Ala Pro Glu Pro Ser Ser Pro Asn Ala 40 45 50 55 gcg gtc cct gaa gcc atc cct acg ccc cga gct gcg gcc tcc gcg gcc 307 Ala Val Pro Glu Ala Ile Pro Thr Pro Arg Ala Ala Ala Ser Ala Ala 60 65 70 ctg gag ctg cct ctc ggg ccc gca ccc gtg agc gta gcg cct cag gcc 355 Leu Glu Leu Gly Pro Ala Pro Val Ser Val Ala Pro Gln Ala 75 80 85 gaa gct gaa gcg cgc tcc aca cca ggc ccc gcc ggc tct aga ctc ggt 403 Glu Ala Glu Ala Arg Ser Thr Pro Gly Pro Ala Gly Ser Arg Leu Gly 90 95 100 ccc gag acg ttc cgc cag cgt ttc cgg cag ttc cgc tac cag gat gcg 451 Pro Glu Thr Phe Arg Gln Arg Phe Arg Gln Phe Arg Tyr Gln Asp Ala 105 110 115 gcg ggt ccc cgg gag ttc cgg cag ctg cgg gag ctg tcc cgc cag 499 Ala Gly Pro Arg Glu Ala Phe Arg Gln Leu Arg Glu Leu Ser Arg Gln 120 125 130 135 tgg ctg cgg cct gac atc cgc acc aag gag cag atc gtg gag atg ctg5 Arg Pro Asp Ile Arg Thr Lys Glu Gln Ile Val Glu Met Leu 140 145 150 gtg caa gag cag ctg ctc gcc atc ctg ccc gag gcg gct cgg gcc cgg 595 Val Gln Glu Gln Leu Leu Ala Ile Leu Pro Glu Ala Ala Arg Ala Arg 155 160 165 cgg atc cgc cgc cgc acg gat gtg cgc atc act ggc tga gcggtggagc 644 Arg Ile Arg Arg Arg Thr Asp Val Arg Ile Thr Gly 170 175 180 tgcggg ggcggc cagggccggg cgctggcc ggactggcc cat ggactgcc ggactggcc <210> 16 <211> 179 <212> PRT <213> Homo sapiens <400> 16 Met Ala Ala Thr Glu Pro Ile Leu Ala Ala Thr Gly Ser Pro Ala Ala 1 5 10 15 Val Pro Pro Glu Lys Leu Glu Gly Ala Gly Ser Ser Ser Ala Pro Glu 20 25 30 Arg Asn Cys Val Gly Ser Ser Leu Pro Glu Ala Ser Pro Pro Ala Pro 35 40 45 Glu Pro Ser Ser Pro Asn Ala Ala Val Pro Glu Ala Ile Pro Thr Pro 50 55 60 Arg Ala Ala Ala Ser Ala Ala Leu Glu Leu Pro Leu Gly Pro Ala Pro 65 70 75 80 Val Ser Val Ala Pro Gln Ala Glu Ala Glu Ala Arg Ser Thr Pro Gly 85 90 95 Pro Ala Gly Ser Arg Leu Gly Pro Glu Thr Phe Arg Gln Arg Phe Arg 100 105 110 Gln Phe Arg Tyr Gln Asp Ala Ala Gly Pro Arg Glu Ala Phe Arg Gln 115 120 125 Leu Arg Glu Leu Ser Arg Gln Trp Leu Arg Pro Asp Ile Arg Thr Lys 130 135 140 Glu Gln Ile Val Glu Met Leu Val Gln Glu Gln Leu Leu Ala Ile Leu 145 150 155 160 Pro Glu Ala Ala Arg Ala Arg Arg Ile Arg Arg Arg Thr Asp Val Arg 165 170 175 Ile Thr Gly <210> 17 <211> 1222 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (219) .. (788) <400> 17 ctcctgcctc agcctcccga gtagctggga ctacaggcgg ccgccaccat gcccggctaa 60 ttttttgtat ttttagtaga gacggggttt caccatgtta gccaggatgg cctcgatctc 120 ctgaccgcgt gatccgcccg cctcggcctc cgaaactgct gaaattacag gcgtgagcca 180 ccgcgcccgg ccctccctct tccgctgccg ccgtggga atg gaa aca tct gcc cca 236 Met Glu Thr Ser Ala Pro 1 5 cgt gcc gga agc caa gtg gtg gcg aca act gcg cgc cac tcc gcg gcc 284 Arg Ala Gly Ser Gln Val Val Ala Thr Thr Ala Arg His Ser Ala Ala 10 15 20 tac cgc gca gat cct cta cgt gtg tcc tcg cga gac aag ctc acc gaa 332 Tyr Arg Ala Asp Pro Leu Arg Val Ser Ser Arg Asp Lys Leu Thr Glu 25 30 35 atg gcc gcg tcc agt caa gga aac ttt gag gga aat ttt gag tca ctg 380 Met Ala Ala Ser Ser Gln Gly Asn Phe Glu Gly Asn Phe Glu Ser Leu 40 45 50 gac ctt gcg gaa ttt gct aag aag cag cca tgg tgg cgt aag ctg ttc 428 Asp Leu Ala Glu Phe Ala Lys Lys Gln Pro Trp Trp Arg Lys Leu Phe 55 60 65 70 ggg cag gaa tct gga cct tca gca gaaag agc gtg gca acc cag 476 Gly Gln Glu Ser Gly Pro Ser Ala Glu Lys Tyr Ser V al Ala Thr Gln 75 80 85 ctg ttc att gga ggt gtc act gga tgg tgc aca ggt ttc ata ttc cag 524 Leu Phe Ile Gly Gly Val Thr Gly Trp Cys Thr Gly Phe Ile Phe Gln 90 95 100 aag gtt gga aag ttg gct gca aca gct gtg gga ggt gga ttt ttt ctc 572 Lys Val Gly Lys Leu Ala Ala Thr Ala Val Gly Gly Gly Phe Phe Leu 105 110 115 ctt cag ctt gca aac cat act ggg tac atc aaa gtt gac tgg caa cga 620 Leu Gln Leu Ala Asn His Thr Gly Tyr Ile Lys Val Asp Trp Gln Arg 120 125 130 gtg gag aag gac atg aag aaa gcc aaa gag cag ctg aag atc cgt aag 668 Val Glu Lys Asp Met Lys Lys Ala Lys Glu Gln Leu Lys Ile Arg Lys 135 140 145 150 agc aat cag ata cct act gag gtc agg agc aaa gct gag gag gtg gtg 716 Ser Asn Gln Ile Pro Thr Glu Val Arg Ser Lys Ala Glu Glu Glu Val Val 155 160 165 tca ttt gtg aag aag aat gtt cta gta act ggg gga ttt ttc gga ggc 764 Ser Phe Val Lys Lys Asn Val Leu Val Thr Gly Gly Phe Phe Gly Gly 170 175 180 ttt ctg ctt ggc atg gca tcc taa ggaagatgac ctcatgttca ttgttcctgg 818 Phe Leu Leu Gly Met Ala Ser 185 tccag ccagcagcct ctacactcca tcataggaca tcgagtccct cctcctcttc 878 tcccatgcct tcttccctgc catggcaaat ctgagtggct tctctaagca tctgctggta 938 caagtcaatg tggcaccatg agcttcatgg tggcagaaga gacaatagtc cttagctctc 998 ctcccagtac accccctact tggccagtct gtaggccaac aagaaggttc ctttaccccc 1058 atgcaagaca cttatgagaa cacattacaa gatggctgac cgtggaggat gagtggatcc 1118 tgaaaggttg tcccaaactg ttgatttgga aaagaaataa gcacatagat aaccttattg 1178 tgtgctgcat ggaaaggaac tgaatacatt tgcctttaag catg 1222 <210> 18 <211> 189 <212> PRT <213> Homo sapiens <400> 18 Met Glu Thr Ser Ala Pro Arg Ala Gly Ser Gln Val Val Ala Thr Thr 1 5 10 15 Ala Arg His Ser Ala Ala Tyr Arg Ala Asp Pro Leu Arg Val Ser Ser 20 25 30 Arg Asp Lys Leu Thr Glu Met Ala Ala Ser Ser Gln Gly Asn Phe Glu 35 40 45 Gly Asn Phe Glu Ser Leu Asp Leu Ala Glu Phe Ala Lys Lys Gln Pro 50 55 60 Trp Trp Trp Arg Lys Leu Phe Gly Gln Glu Ser Gly Pro Ser Ala Glu Lys 65 70 75 80 Tyr Ser Val Ala Thr Gln Leu Phe Ile Gly Gly Val Thr Gly Trp Cys 85 90 95 Thr Gly Phe Ile Phe Gln Lys Val Gly Lys Leu Ala Ala Thr Ala Val 100 105 110 Gly Gly Gly Phe Phe Leu Leu Gln Leu Ala Asn His Thr Gly Tyr Ile 115 120 125 Lys Val Asp Trp Gln Arg Val Glu Lys Asp Met Lys Lys Ala Lys Glu 130 135 140 Gln Leu Lys Ile Arg Lys Ser Asn Gln Ile Pro Thr Glu Val Arg Ser 145 150 155 160 Lys Ala Glu Glu Val Val Ser Phe Val Lys Lys Asn Val Leu Val Thr 165 170 175 Gly Gly Phe Phe Gly Gly Phe Leu Leu Gly Met Ala Ser 180 185 <210> 19 <211> 1209 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (95) .. (448) <400> 19 tcgcgcttgc ctgtgtcccg ggcttgtctg tgaagtgggc gtgaagatcg ttgccacctt 60 ccaacctacc tcacaggggt gttgtgggga cacc atg atc tct gga ttg ttc atg 115 Met Ile Ser Gc tcg gc Lec Gc Lec Gc Lec Gc Lec Gc Lec Pg Leu Cys Cys Ala Gly Ser His Arg Pro Pro Glu Thr Gly Gln Leu 10 15 20 ccc tac gac cct agc gcc tcc gcc ctc cgc ggc ccc tct cct ctc ttc 211 Pro Tyr Asp Pro Ser Ala Ser Ala Leu Arg Gly Pro Ser Pro Leu Phe 25 30 35 ctg ctc tgt ccc tcc ttc tcc atc agg gag cag cgt gac ttc agc gag 259 Leu Leu Cys Pro Ser Phe Ser Ile Arg Glu Gln Arg Asp Phe Ser Glu 40 45 50 55 tcc cgc gag cac ctg gct aga cag tta aca agc acg tcc ttc cag cct 307 Ser Arg Glu His Leu Ala Arg Gln Leu Thr Ser Thr Ser Phe Gln Pro 60 65 70 gag cca gcg cag gtt tgg gag ggg gct tcc tgg ccc ccc cca cgg tgt 355 Glu Pro Ala Gln Val Trp Glu Gly Ala Ser Trp Pro Pro Pro Arg Cys 75 80 85 tcc agc ccc tcc tct ctt ccg ccc cct agt ctc cca ccc ttc cct ccc 403 Ser Ser Pro Ser Ser Leu Pro Pro Pro Ser Leu Pro Pro Phe Pro Pro 90 95 100 cgt agt gac caa ttc cta tct ctt ccc tct ccg cag gct caa tga 448 Arg Ser Asp Gln Phe Leu Ser Leu Pro Ser Pro Gln Ala Gln 105 110 115 atcgaatgaa tgtgaacttc ttcatctgtg aaaaatcttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttctt ttggcgagag agcgatggct gccgtgggga gtactgggga 568 gccctcgcgg caagcagggt gggggggact tgggggcatg ccgggccctc actctctcgc 628 ctgttctgtg tctcacatgc tttttctttc aaaattggga tccttccatg ttgagccagc 688 cagagaagat agcgagatct aaatctctgc caaaaaaaaa aaaaacttaa aaattaaaaa 748 cacaaagagc aaagcagaac ttataaaatt atatatatat atattaaaaa gtctctattc 808 ttcacccccc agccttcctg aacctgcctc tctgaggata aagcaattca ttttctccca 868 ccctcggccc tcttgttttt aaaataaact tttaaaaagg aaaaaaaaaa gtcactcttg 928 ctatttcttt tttttagtta gaggtggaac attccttgga ccaggtgttg tattgcagga 988 ccccttcccc cagcagccaa gccccctctt ctctccctcc cgccctggct cagctcccgc 1048 ggccccgccc gtcccccctc ccaggactgg tctcatgag tacagtagagagattag tagatca tca tca tca tga tga tca tca tca tga tga tga tga tga tg 1168 atcgctcgtt tttaacttca caaataaatg ataacaaaac c 1209 <210> 20 <211> 117 <212> PRT <213> Homo sapiens <400> 20 Met Ile Ser Gly Leu Phe Met Ser Leu Cys Cys Ala Gly Ser His Arg 1 5 10 15 Pro Pro Glu Thr Gly Gln Leu Pro Tyr Asp Pro Ser Ala Ser Ala Leu 20 25 30 Arg Gly Pro Ser Pro Leu Phe Leu Leu Cys Pro Ser Phe Ser Ile Arg 35 40 45 Glu Gln Arg Asp Phe Ser Glu Ser Arg Glu His Leu Ala Arg Gln Leu 50 55 60 Thr Ser Thr Ser Phe Gln Pro Glu Pro Ala Gln Val Trp Glu Gly Ala 65 70 75 80 Ser Trp Pro Pro Pro Arg Cys Ser Ser Pro Ser Ser Leu Pro Pro Pro 85 90 95 Ser Leu Pro Pro Phe Pro Pro Arg Ser Asp Gln Phe Leu Ser Leu Pro 100 105 110 Ser Pro Gln Ala Gln 115

[Brief description of the drawings]

FIG. 1 is a diagram comparing the amino acid sequences of human protein encoded by clone HP02644 and a nematode RNA helicase-like protein.

FIG. 2 is a diagram comparing human protein encoded by clone HP03233 with putative ubiquinone biosynthetic methyltransferase from fission yeast.

FIG. 3 shows a comparison between the human protein encoded by clone HP10437 and a human pp21 homolog.

FIG. 4 is a diagram comparing the amino acid sequences of human protein encoded by clone HP10525 and the fission yeast hypothetical protein SPAC8C9.11.

FIG. 5. Human protein encoded by clone HP10543 and mouse leucine-rich domain interacting protein 1
FIG. 7 is a diagram comparing the amino acid sequences of

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/08 5/10 C12N 15/00 ZNAA // C12P 21/08 5/00 A F Terms (reference) 4B024 AA11 BA41 BA61 BA80 CA04 CA09 DA03 DA06 EA04 GA11 GA18 GA19 HA12 HA17 4B064 AG26 AG27 CA10 CA20 CC24 CE12 DA13 4B065 AA26X AA90X AA93Y AB01 BA02 CA24 CA46 4H045 AA10 AA11 BA10 BA21 BA41

Claims (7)

[Claims] 1. SEQ ID NOs: 2, 4, 6, 8, 10, 12,
A purified human protein having any one of 14, 16, 18 and 20 amino acid sequences. 2. A DNA fragment encoding the protein of claim 1. 3. A human cD encoding the protein of claim 1.
NA, 1, 3, 5, 7, 9, 11, 13, 1
DN having a base sequence of 5, 17, or 19 translation regions
A fragment. 4. SEQ ID NO: 1, 3, 5, 7, 9, 11, 1
4. The DNA fragment according to claim 3, comprising the base sequence of any one of 3, 15, 17 and 19. 5. An expression vector capable of in vitro translation or expression in a host cell of the DNA fragment according to any one of claims 2 to 4. 6. A transformant using the expression vector according to claim 5, which is capable of producing the protein according to claim 1. 7. An antibody against the protein according to claim 1.