JP2000135095A - High-affinity ip3 bonded polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new polypeptide containing a specific amino acid sequence, having bond activity of high affinity for inositol 1,4,5-triphosphate (IP3), useful for an IP3-inducing Ca antagonist, a therapeutic agent for diseases following Ca production. SOLUTION: This new recombinant polypeptide is a polypeptide containing an amino acid sequence of the formula, or a polypeptide containing an amino acid sequence in which at least one amino acid is deleted from, added to or substituted for that of the amino acid sequence of the formula and having bond activity of high affinity for inositol 1,4,5-triphosphate (IP3), or a polypeptide containing an amino acid sequence containing >=70% homology to the amino acid sequence of the formula and having bond activity of high affinity for IP3, and is useful for an IP3-inducing Ca antagonist, a therapeutic agent for diseases following Ca production or a gene therapeutic agent containing an IP3, or the like. The polypeptide is obtained by expressing a gene selected from a mouse cerebellum cDNA library.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to inositol 1,4,
Polypeptide having high affinity binding activity to 5-triphosphate, gene encoding the polypeptide, recombinant vector containing the gene, transformant containing the vector, and inositol 1,4,5-trisulfate The present invention relates to a method for producing a polypeptide having a high affinity for phosphate.

[0002]

2. Description of the Related Art Inositol 1,4,5-triphosphate (hereinafter referred to as I)
P3) is one of the second messengers produced by activating inositol phospholipid metabolism in response to extracellular stimuli such as hormones, growth factors and neurotransmitters. IP3 is a substance that induces an increase in intracellular calcium concentration, and this IP3-induced increase in calcium is a highly versatile and important signaling mechanism that controls various cell functions in a wide variety of animal species. For example, IP
3 are nematodes [nematodes], Drosophila [arthropods],
Fertilization and embryo development in a wide range, from invertebrates such as squid [mollusks] to vertebrates such as mice and humans,
It controls many physiological functions such as development and differentiation, cell proliferation, secretion, immunity, muscle contraction, and cranial nerves (taste, vision, memory, learning, etc.).

[0003] The molecular mechanism begins with the binding of IP3 to its target, the IP3 receptor. That is, I
When P3 binds to IP3 receptors (IP3-sensitive calcium channels) located at intracellular calcium storage sites (such as the endoplasmic reticulum), the channels open and release calcium from the calcium storage sites into the cytoplasm, resulting in many calcium-dependent It controls the activity of proteins and enzymes.

Compounds involved in IP3-induced calcium signaling include heparin, adenophosphin (a metabolic compound of mold) and zestspondin (a metabolic compound of sponge). However, although heparin inhibits IP3 binding, it has many intracellular targets and low specificity. Adenophostin is also an antagonist of IP3 binding and a potent activator of IP3 receptor channels, but its use is limited due to low yield from mold and lack of membrane permeability. Zestspondin has recently been reported to be an inhibitor of the IP3 receptor channel that does not affect IP3 binding, but its yield is low and its specificity is still largely unknown. Thus, at present, few useful compounds act on IP3-induced calcium signaling. In particular, there is no substance or system that inhibits IP3-induced calcium signaling by specifically trapping the increased IP3 at the cellular level.

[0005]

SUMMARY OF THE INVENTION The present invention provides a polypeptide having a high affinity binding to inositol 1,4,5-triphosphate,
It is intended to provide a gene encoding the polypeptide, a recombinant vector containing the gene, a transformant containing the vector, and a method for producing a polypeptide having high affinity binding to inositol 1,4,5-triphosphate. Aim.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a protein having a partial amino acid sequence at the N-terminal side of the IP3 receptor has an extremely high affinity for IP3. The present inventors have succeeded in isolating a polypeptide having sexual binding activity and have completed the present invention.

That is, the present invention provides the following (a), (b) or
It is a recombinant polypeptide of (c). (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 wherein at least one amino acid has been deleted, substituted or added; A polypeptide having a high affinity binding activity to 4,5-triphosphate (c) having a homology of 70 to the amino acid sequence represented by SEQ ID NO: 2;
% Or more amino acid sequence, and inositol 1,4,
Polypeptide having high affinity binding activity to 5-triphosphate

Further, the present invention provides a gene encoding the polypeptide of the above (a), (b) or (c), or a nucleotide sequence having a homology of 70% or more with the gene, and inositol 1,4 , A gene encoding a polypeptide having high affinity binding activity to 5-triphosphate.

Further, the present invention provides the following (d) or (e):
It is a gene containing A. (d) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 (e) DNA comprising the nucleotide sequence having a homology of 70% or more with the DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, and inositol
DNA encoding a polypeptide having high affinity binding activity to 1,4,5-triphosphate

[0010] Further, the present invention is a recombinant vector containing the gene. Furthermore, the present invention is a transformant containing the recombinant vector. Further, the present invention provides
Culturing the transformant, and collecting a polypeptide having high affinity binding activity to inositol 1,4,5-triphosphate from the resulting culture. .

Further, the present invention is an IP3-induced calcium antagonist or a therapeutic agent for a disease associated with calcium production, comprising the protein, or a gene therapeutic agent for a disease associated with calcium production, comprising the gene. Diseases include nervous, vascular, respiratory, digestive, lymphatic,
At least one selected from the group consisting of those occurring in tissues of the urinary system and the reproductive system is included. Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the present invention is a protein having extremely high affinity for IP3 and specifically binding to IP3, and a partial amino acid sequence at the N-terminal side of a natural type IP3 receptor. (Also referred to as a cutting type). The polypeptide of the present invention is also referred to as a high affinity IP3-binding polypeptide.

1. Cloning of Gene Encoding IP3 Receptor In order to obtain the high-affinity IP3 binding polypeptide of the present invention, first, a gene encoding a native IP3 receptor protein is cloned. The nucleotide sequence of the IP3 receptor gene is already known (Nucleic Acid Res. 17: 5385-5386, 198).
9; Nature 342: 32-38, 1989), and can also be prepared by genetic engineering techniques as follows.

(1) Preparation and Screening of cDNA Library Encoding IP3 Receptor Preparation of mRNA for IP3 receptor can be performed by a commonly used technique. For example, tissues or cells such as mouse cerebellum are treated with a guanidine reagent,
After obtaining RNA, poly (A +) RNA (mRN) is prepared by affinity column method using oligo dT-cellulose or poly-U-sepharose using Sepharose 2B as a carrier, or batch method.
Get A). Using the resulting mRNA as a template and synthesizing single-stranded cDNA using oligo dT primer and reverse transcriptase,
A double-stranded cDNA is synthesized from the single-stranded cDNA. The double-stranded cDNA thus obtained is inserted into an appropriate cloning vector to prepare a recombinant vector. A cDNA library can be obtained by transforming Escherichia coli or the like using the obtained recombinant vector and selecting transformants using tetracycline resistance and ampicillin resistance as indices.

The transformation of Escherichia coli was carried out according to the method of Hanahan [Hanahan, D .: J. Mol. Biol. 166: 557-580 (198).
3)], that is, a method of adding a recombinant vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, it is necessary to contain a drug resistance gene such as tetracycline or ampicillin. In addition, a cloning vector other than a plasmid, for example, λ phage or the like can also be used.

As a screening method for selecting a strain having the target DNA from the transformant obtained as described above, for example, "expression cloning" by immunoscreening using an antibody, or a method of synthesizing a primer from a known sequence However, there is a method of performing a polymerase chain reaction (PCR) using the same.

The DNA fragment or the amplified DNA fragment encoding the antibody epitope thus obtained is labeled with ³² P, ³⁵ S, biotin or the like to obtain a probe, which is used to denature and fix the DNA of the transformant. Other DNA fragments can be screened by hybridizing with a nitrocellulose filter and searching the obtained positive strain.

(2) Determination of Nucleotide Sequence The nucleotide sequence of the obtained clone is determined. The nucleotide sequence can be determined by the Maxam-Gilbert chemical modification method or M
It can be carried out by a known method such as dideoxynucleotide chain termination using 13 phage, but usually the sequence is determined using an automatic base sequencer (for example, 373A DNA sequencer manufactured by PERKIN-ELMER). SEQ ID NO: 3
Shows the nucleotide sequence of the natural (full-length) gene encoding the IP3 receptor, and SEQ ID NO: 4 shows the full-length amino acid sequence of the IP3 receptor.

2. Design and synthesis of the gene encoding the high affinity IP3 binding polypeptide of the present invention (1) Design and synthesis of the gene encoding the high affinity IP3 binding polypeptide The high affinity IP3 binding polypeptide of the present invention is a natural type It contains the sequence of a partial region on the N-terminal side of the IP3 receptor protein, and includes amino acids 579 to 800, preferably 579 to 734, of the amino acid sequence of the full-length IP3 receptor protein (SEQ ID NO: 4). The amino acids after the 1st are truncated. In the present invention, this truncated polypeptide is referred to as IP3
It is called a sponge (Fig. 1). By making the polypeptide of the present invention in such a truncated form, it has extremely high affinity for IP3 and can specifically bind to IP3 (high affinity IP3 binding activity).

Here, “high affinity” refers to the IP3 of the present invention.
The affinity of sponge for IP3 is similar to that of natural IP3 receptor
100 to 1000 times (preferably 50
(0 to 1000 times) means higher.

In the present invention, the IP3 sponge contains at least the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence corresponds to amino acids 226 to 578 of the amino acid sequence represented by SEQ ID NO: 4.
In the present invention, the amino acid region in this range is defined as a core region. Then, the length of the fragment and the length of the DNA encoding the fragment can be arbitrarily set while maintaining the high affinity binding activity to IP3. For example, the amino acid sequence represented by SEQ ID NO: 4 may include amino acids 224 to 604 (encoded by the 670th to 1812th sequence of the nucleotide sequence represented by SEQ ID NO: 3). , First
The amino acid sequence may include amino acids at positions 1 to 604 (encoded by the sequences 1 to 1812 of the nucleotide sequence represented by SEQ ID NO: 3), and may include amino acids at positions 1 to 734 (represented by SEQ ID NO: 3). (Encoded by the 1st to 2202nd sequence of the nucleotide sequence).

In these fragments, primers are designed based on the sequence of an arbitrary region of the base sequence represented by SEQ ID NO: 3 which is a region outside the fragment, and DNA encoding the natural type IP3 receptor is prepared. (SEQ ID NO: 3; Nucleic Acid Res. 17: 5
385-5386, 1989; Nature 342: 32-38, 1989) as a template.

(2) Preparation of Gene Encoding Mutant of IP3 Sponge of the Present Invention (Variant IP3 Gene) In the present invention, a mutation can be introduced into at least a part of the amino acid sequence of the IP3 sponge. Therefore, the mutant is also included in the IP3 sponge of the present invention. In order to introduce a mutation into an amino acid, a technique for introducing a mutation into the nucleotide sequence of a gene encoding the amino acid is employed.

A mutation can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method or a method analogous thereto. For example, a site-directed mutagenesis method using a mutant oligonucleotide as a primer (Yoshikawa, F. et al., J. Biol. Chem. 271: 1
8277-18284, 1996), and a mutation introduction kit (for example, Mutant-K (TAKARA), Mutant-K
G (TAKARA), TAKARA LA PCR in vitro Mutagene
sis series kit) can be used to introduce the mutation.

First, of the gene encoding the IP3 sponge of the present invention (also referred to as the IP3 sponge gene), a primer containing a mutated base and a region of about 10 bases before and after the mutation site is synthesized. Then, a PCR reaction is performed using the IP3 sponge gene as a template and the above primers.
By purifying this product and treating it with an appropriate restriction enzyme, the desired mutant IP3 sponge gene can be obtained.

(3) Determination of Nucleotide Sequence The nucleotide sequence of the gene obtained as described in (1) or (2) above is determined. The nucleotide sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using M13 phage. Usually, an automatic nucleotide sequencer (for example, PERKIN-ELMER) is used.
The sequence is determined using a 373A DNA sequencer (trade name).

SEQ ID NO: 1 shows the nucleotide sequence of the IP3 sponge gene of the present invention, and SEQ ID NO: 2 shows the amino acid sequence of the IP3 sponge of the present invention. The polypeptide comprising this amino acid sequence has a high affinity for IP3. Yes, as long as it has an activity of specifically binding to IP3 (high-affinity IP3 binding activity), mutation such as deletion, substitution, or addition occurs in at least one, for example, one or several amino acids in the amino acid sequence. Is also good.

For example, at least one of the core region (amino acid sequence represented by SEQ ID NO: 2), preferably 1 to 10
About 1 amino acid, more preferably 1 to 5 amino acids may be deleted, and the amino acid sequence represented by SEQ ID NO: 1 has at least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids. May be added, or at least one of the amino acid sequences represented by SEQ ID NO: 2,
Preferably, about 1 to 10, more preferably 1 to 5 amino acids may be substituted with another amino acid.

The amino acid sequence contains the amino acid sequence of the core region, and is 579-800 or more, preferably 5
The amino acid length is not limited as long as the amino acids 79 to 734 and thereafter are truncated. For example, the amino acids 224 to 604 of the amino acid sequence represented by SEQ ID NO: 4 (referred to as G224) and the gene encoding the amino acid are also included in the IP3 sponge and the IP3 sponge gene of the present invention.

Furthermore, in addition to the amino acid sequence of G224, the mutation may occur in at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids of the amino acid sequence. Accordingly, in the amino acid sequence of G224, the lysine at position 508 was substituted with alanine (m
30) or those obtained by replacing arginine at position 441 with glutamine (referred to as m49) are also included in the IP3 sponge of the present invention (see FIG. 1). The number of the amino acid position is designated (first) with reference to the first position of the amino acid sequence represented by SEQ ID NO: 4.

Furthermore, it has an amino acid sequence having a homology of 70% or more with the core region (amino acid sequence represented by SEQ ID NO: 2) and has a high affinity binding activity to inositol 1,4,5-triphosphate. Polypeptides are also included in the polypeptides of the present invention.

Accordingly, the gene of the present invention includes a gene having the amino acid sequence into which the above mutation has been introduced and encoding a polypeptide having high affinity binding activity to IP3. Further, in addition to the nucleotide sequence encoding the amino acid contained in the IP3 sponge of the present invention, degenerate isomers encoding the same polypeptide that differs only in degenerate codons are included in the gene of the present invention. Furthermore, DNAs having a homology of 70% or more with the nucleotide sequence of the above genes, for example, DNAs of other types belonging to the IP3 receptor gene family and encoding the corresponding regions of the polypeptide of the present invention, are also included in the present invention. Included in genes.

Once the nucleotide sequence of the gene of the present invention is determined, the gene of the present invention can be obtained by chemical synthesis or by PCR using primers synthesized from the determined nucleotide sequence. .

3. Preparation of Recombinant Vector and Transformant Containing IP3 Sponge Gene of the Present Invention (1) Preparation of Recombinant Vector The recombinant vector of the present invention
It can be obtained by ligating (inserting) the IP3 sponge gene. The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in the host,
For example, plasmid DNA, phage DNA and the like can be mentioned.

As the plasmid DNA, plasmids derived from Escherichia coli (for example, pET-3a, pBR322, pBR325, pUC118, pUC118)
119), Bacillus subtilis-derived plasmids (eg, pUB110, pTP
5), yeast-derived plasmids (eg, YEp13, YEp24, YC
p50), and phage DNA includes λ phage. Also, animal viruses such as retrovirus, adenovirus or vaccinia virus, or insect virus vectors such as baculovirus can be used. Furthermore, GST, GFP, His-tag, Myc-t
ag etc. are linked (for example, pGEX-2T, pE
GFP-N3) can also be used.

To insert the gene of the present invention into a vector, first, the purified DNA is cut with an appropriate restriction enzyme,
For example, a method of inserting into an appropriate restriction enzyme site or a multiple cloning site of vector DNA and ligating to the vector is employed.

The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the vector of the present invention includes, in addition to the promoter and the gene of the present invention, cis elements such as enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), if desired.
And the like can be linked. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

(2) Preparation of Transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the gene of the present invention. For example, Escherichia c.
oli), Escherichia, Bacillus subtilis (Ba
cillus subtilis), bacteria belonging to the genus Pseudomonas such as Pseudomonas putida (Pseudomonas putida), and Saccharomyces cerevisiae (Sacch
aromyces cerevisiae), Schizosaccharomyces pombe (S
yeasts such as chizosaccharomyces pombe); animal cells such as COS cells, CHO cells, HEK293 cells, and PC12 cells; and insect cells such as Sf9 and Sf21.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and comprises a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Further, a gene controlling a promoter may be included.

As Escherichia coli, for example, Escherichia
Coli (Escherichia coli) BL21, JM109, HB101, and the like.Examples of Bacillus subtilis include Bacillus subtilis (B
acillus subtilis) MI 114, 207-21 and the like.

Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example trp
Promoter, lac promoter, P _L promoter, P _R
A T7 promoter derived from Escherichia coli phage, such as a promoter, is used. An artificially designed and modified promoter such as a tac promoter may be used.

The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions [Cohen,
SNet al .: Proc. Natl. Acad. Sci., USA, 69: 2110-
2114 (1972)], electroporation and the like.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe,
e), Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast.For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. Is mentioned.

The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, an electroporation method [Becker, DM et.
al .: Methods. Enzymol., 194: 182-187 (1990)], spheroplast method [Hinnen, A. et al .: Proc. Natl. Aca
d. Sci., USA, 75: 1929-1933 (1978)] and the lithium acetate method [Itoh, H .: J. Bacteriol., 153: 163-168 (1983)].

When an animal cell is used as a host, monkey cell CO
S-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, PC12 or NG108-15
Cells or human FL, HEK293, HeLa or Jurkat cells, etc. are used. As the promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a β-actin promoter and the like are used, and an early gene promoter of human cytomegalovirus may be used.

Examples of a method for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method. When an insect cell is used as a host, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method,
An electroporation method or the like is used.

4. Production of IP3 sponge The IP3 sponge of the present invention can be obtained by culturing the transformant and collecting the transformant from the culture.
The term “culture” means any of a culture supernatant, a cultured cell or a cultured cell, or a crushed cell or a cell. The method for culturing the transformant of the present invention is performed according to a usual method used for culturing a host.

A culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be assimilated by the microorganism. Any of a natural medium and a synthetic medium may be used as long as the medium can be performed in a controlled manner. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate and other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like. No. Examples of the inorganic substance include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

The cultivation is usually performed at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, the pH is maintained at 7.0-7.5. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. During culture, antibiotics such as ampicillin and tetracycline may be added to the medium as needed.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, the expression vector pET-3a is isopropyl-
Since it has a T7 promoter inducible with β-D-thiogalactopyranoside (IPTG), when culturing a microorganism transformed with the expression vector, IPTG or the like can be added to the medium. When culturing a microorganism transformed with an expression vector using a trp promoter inducible with indoleacrylic acid (IAA), IAA or the like can be added to the medium.

As a medium for culturing a transformant obtained using animal cells as a host, generally used RPMI16
40 media, DMEM media, or media obtained by adding fetal bovine serum or the like to these media. Culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.

After culturing, when the IP3 sponge of the present invention is produced in the cells or cells, the IP3 sponge is subjected to sonication, repeated freeze-thawing, homogenizer treatment and the like to disrupt the cells or cells. Collect sponge. When the IP3 sponge of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or the like, alone or in appropriate combination, in the culture. Can be isolated and purified from the IP3 sponge of the present invention.

5. Calcium Antagonist, etc. Since the protein and gene of the present invention have a function of inhibiting IP3-induced calcium release, they are useful as calcium antagonists, therapeutic agents for diseases associated with calcium production, and gene therapeutic agents for diseases associated with calcium production. Therefore, the therapeutic agent or gene therapeutic agent of the present invention can be administered orally or parenterally systemically or locally.

When the protein or gene of the present invention is used as a therapeutic agent for a disease associated with calcium production or a gene therapeutic agent, the subject to be used is not particularly limited. For example, nervous system (brain, spinal cord, etc.), vascular system (artery, vein, heart, etc.), respiratory system (trachea, lung, etc.), digestive system (salivary gland, stomach, intestine, liver, pancreas, etc.), lymphatic system (Lymph nodes,
Spleen, thymus, etc.), urinary system (kidney, etc.), reproductive system (testis,
Treatment or prevention can be used for a specific purpose for diseases that occur in tissues such as ovaries and uterus. These diseases may be used alone, in combination, or in combination with other diseases other than those described above, and all of them are subject to use of the protein or gene of the present invention. Can be.

When the therapeutic agent of the present invention is orally administered, it may be any of tablets, capsules, granules, powders, pills, troches, solutions for internal use, suspensions, emulsions, syrups and the like. Or a dry product that is redissolved when used. In addition, when the therapeutic agent of the present invention is administered parenterally, a pharmaceutical form such as intravenous injection (including intravenous drip), intramuscular injection, intraperitoneal injection, subcutaneous injection, and suppository can be selected. In the case of preparations, they are provided in unit dose ampules or multidose containers.

These various preparations can be prepared by using excipients, extenders, binders, wetting agents, disintegrants, lubricants,
Surfactants, dispersants, buffers, preservatives, solubilizing agents, preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents, etc. are appropriately selected and can be produced by a conventional method. it can.

The above-mentioned various preparations may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like can be mentioned. The additives to be used are appropriately or in combination selected from the above depending on the dosage form of the present invention.

The dose of the therapeutic agent of the present invention varies depending on the age of the subject to be administered, the route of administration, and the number of administrations, and can be varied over a wide range. In this case, the effective amount administered as a combination of the effective amount of the protein of the present invention and a suitable diluent and a pharmacologically usable carrier is in the range of 0.0001 to 1000 mg / kg body weight per administration. The amount can be selected and it is administered once a day or several times a day.

When the gene of the present invention is used as a gene therapy agent, the method of directly administering the gene of the present invention by injection and the method of administering a vector into which the gene has been incorporated can be used. Examples of the above-mentioned vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, and the like. Use of these virus vectors enables efficient administration. Alternatively, a method of introducing the gene of the present invention into a phospholipid vesicle such as a liposome and administering the liposome may be employed. That is, since the liposome is a closed vesicle containing a biodegradable material, the gene of the present invention is mixed in the aqueous layer or lipid bilayer inside the liposome by mixing the liposome with the gene of the present invention. Retain (liposome-gene complex). Next, when the complex is cultured with cells, the genes in the complex are incorporated into the cells (lipofection method). Then, the obtained cells may be administered by the following administration method.

The gene therapy agent of the present invention may be administered in the form of a normal intravenous or intraarterial systemic administration, a central nervous system tissue (brain, spinal cord, etc.), a vascular system (artery, vein, heart, etc.) , Respiratory system (trachea, lungs, etc.), digestive system (salivary glands,
Stomach, intestine, liver, pancreas, etc.), lymphatic system (lymph nodes, spleen,
Thymus), urinary system (kidney), reproductive system (testis, ovary,
Local administration can be performed to the uterus and the like. further,
Dosage forms in combination with catheterization, surgery and the like can also be employed.

The dosage of the gene therapy agent of the present invention depends on the age,
Although it depends on gender, symptoms, administration route, administration frequency, and dosage form, usually, the weight of the gene of the present invention is suitably in the range of 0.01 to 100 mg / body per day for an adult.

[0063]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. Example 1 Construction of Expression Plasmid for High-Affinity IP3 Binding Polypeptide (IP3 Sponge) Mouse Type 1 7 at the amino-terminal side of IP3 receptor (mIP3R1)
34 amino acids (T734) have specific IP3 binding activity. Plasmid p obtained by cloning the cDNA portion encoding T734 into the E. coli expression vector pET-3a (the expression of which is controlled by the T7 promoter inducible by adding IPTG)
ET-T734 (Yoshikawa F, et al., J. Biol Chem. 271: 182)
77-18284, 1996.) was used as a parent plasmid to construct an expression plasmid for the following IP3-binding polypeptide (hereinafter, high-affinity polypeptide is also referred to as IP3 sponge).

1-1. High-affinity IP3 sponge T604 expression plasmid T734 encoding the polypeptide (T604) from the first methionine (M-1) to the 604th lysine (K-604) using a complementary oligonucleotide as a primer Site-directed mutagenesis using PCR (Yoshikawa
F, et al., J. Biol Chem. 271: 18277-18284, 1996.), and a stop codon (TAA) at position 605 of T734 followed by Ba.
An mHI recognition site (GGATCC) was introduced.

[0065] The sense primer contains the first methionine codon.
An NdeI cleavage recognition sequence (CATATG) was introduced into the sequence immediately before (ATG) (underlined site).

The PCR reaction solution was composed of 100 ng of template DNA, 10 mM KCl,
6 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.2), 2 mM MgCl ₂ , 0.
1% TritonX-100, 10 μg / ml BSA, 200 μM dNTPs, 1 μM
Sense primer, 1 μM antisense primer, Pfu
2.5 units of DNA polymerase was used in a total volume of 100 μl. PCR was performed at 95 ° C for 1 minute, followed by
3 minutes at 72 ° C and 3 minutes at 72 ° C as one cycle.
Performed 0 cycles.

The obtained amplified fragment was subjected to
NdeI, the 3 'end was treated with BamHI, and the 6th
Deletion mutant pET-T604 containing a DNA encoding an amino acid sequence in which the amino acid sequence at the 05th position (carboxyl terminal side) was deleted was prepared.

1-2. Expression of high-affinity IP3 sponge G224 Plasmid T734, the first methionine (M-1) to the 604th lysine (K-
PC using a complementary oligonucleotide as a primer to encode the polypeptide (T604) up to
Site-directed mutagenesis with R (Yoshikawa F, et a
l., J. Biol Chem. 271: 18277-18284, 1996.)
A stop codon (TAA) was introduced at position 605 of 34, followed by an EcoRI recognition site (GAATCC). .

[0069] PCR was performed under the same conditions as described in 1-1.

The obtained amplified fragment was subjected to
NdeI, the 3 'end was treated with EcoRI, and 6th of T734
A deletion mutant pET-T604e containing a DNA encoding an amino acid sequence in which the amino acid sequence at the 05th position (carboxyl terminal side) was deleted was prepared.

Next, PCR was performed using pET-T604e as a template.
Site-directed mutagenesis, and a BamHI recognition site (GGATCC) immediately before the 224th methione (M-224) of T604.
Was introduced. PCR was performed under the same conditions as described in 1-1.

The obtained amplified fragment (mutation-introduced plasmid)
Was digested with BamHI and EcoRI to obtain a cDNA fragment encoding the 224th to 604th amino acid sequences. This cDNA fragment is
This was ligated in frame with the BamHI-EcoRI site of the T fusion plasmid pGEX-2T to obtain pGEX-G224. Plasmid pGEX-G22
4 expresses a fusion polypeptide G224 (FIG. 1) in which GST is followed by a series of polypeptides from M-224 to K-604.

1-3. Low-affinity IP3-binding polypeptide G224-
Site-directed mutagenesis was performed by sequential PCR using the m30 expression plasmid pGEX-G224 as a template. To introduce a substitution mutation (K508A) that makes the 508th lysine (K-508) of T604 an alanine, the following two types of mismatched oligonucleotides were synthesized.

5′-GAGAGCGGCAG GCA CTGATGAGGG-3 ′ (SEQ ID NO: 9) 5′-CCCTCATCAG TGC CTGCCGCTCTC-3 ′ (SEQ ID NO: 10) Site-specific mutagenesis was performed by continuous PCR using the above primers. The PCR conditions and the composition of the reaction solution are as follows.

First reaction 1 PCR was performed under the same conditions as described in section 1-1.

First reaction 2 PCR conditions are the same as those described in section 1-1.

Second reaction PCR was performed under the same conditions as in the first reaction, using 10 μl of the PCR reaction products of the first reactions 1 and 2 and 1 μM of each of the primers (SEQ ID NOs: 7 and 8). BamHI and Ec
This fragment was digested with oRI, and this fragment was ligated to the GST fusion plasmid pGEX-2T B
Ligation in frame to the amHI-EcoRI site, plasmid p
GEX-G224-m30 was obtained. This mutant plasmid expresses a polypeptide G224-m30 having a point mutation K508A in G224 (FIG. 1, m30).

1-4. Site-directed mutagenesis was performed by continuous PCR using the expression plasmid pGEX-G224 of the high-affinity IP3 sponge G224-m49 as a template. In order to introduce a substitution mutation (R441Q) that makes the 441st arginine (R-441) of T604 a glutamine, the following two types of mismatched oligonucleotides were synthesized. 5'-GCTGAGGTT CAA GACCTGGACTTTG-3 '(SEQ ID NO: 11) 5'-AAAGTCCAGGTC TTG AACCTCAGC-3' (SEQ ID NO: 12)

First reaction 1 PCR conditions are the same as those described in section 1-3.

First reaction 2 PCR conditions are the same as those described in section 1-3.

Second reaction PCR was carried out under the same conditions as in the first reaction, using 10 μl of each of the PCR reaction products of the first reactions 1 and 2 and 1 μM of each of the primers (SEQ ID NOs: 6 and 8). BamHI-Eco
After digestion with RI, the fragment was ligated in frame to the BamHI-EcoRI site of pGEX-2T. Obtained plasmid pGEX-G224
-m49 is a polypeptide G234-m having a point mutation R441Q in G224
It expresses 49 (FIG. 1, m49).

[Example 2] Expression and preparation of high-affinity IP3-binding polypeptide in Escherichia coli The IP3-binding core is composed mainly of an insoluble inclusion body (inclusion bod).
y) and the expression level is low. The present inventor, by genetically modifying the IP3 binding region, that it is a smaller molecule, that it can be stably expressed in large quantities, that it can be recovered as a soluble protein, that it has a higher affinity,
Then, an attempt was made to produce a high-affinity IP3 polypeptide characterized in that its specificity was almost the same as that of a conventional IP3 receptor.

T734 can be stably expressed in large amounts and has a relatively good recovery rate in the soluble fraction by employing a low-temperature culture method (16 to 22 ° C.) (Kd = 50 ± 2.4 nM, Bmax = 46pmpl
/ mg protein per liter of E. coli culture
1.85 mg (corresponding to about 0.5 g of wet weight of E. coli)), and several tens of times this becomes an inclusion body (Yoshikawa F, et al., J. Biol Chem. 27).
1: 18277-18284, 1996). First, an attempt was made to produce a polypeptide as small as possible while retaining these characteristics.

Of the expression plasmids obtained in Example 1, the pET system was used for E. coli BL21 (DE3), the pGEX system was used for JM109,
Each was introduced by the transformation method. IPTG
The expression induction by E. coli and the preparation of an expressed protein from E. coli are mainly performed by the method of Yoshikawa et al. (Yoshikawa F, et al.,
J. Biol Chem. 271: 18277-18284, 1996).

That is, the E.co.
The li and shaking culture at 22 ° C. in L-broth (100 [mu] g / ml ampicillin), IP when absorbance OD _{600 of} was about 1.5
TG was added to 0.5 mM. After further shaking culture at 16 ° C. for several hours, E. coli was recovered by centrifugation and suspended in PBS containing a protease inhibitor (1 mM PMSF, 10 μM leupeptin, 1 μM pepstatin A, 2 μg / ml aprotinin).
After crushing E. coli by sonication, ultracentrifugation (Be
The supernatant (soluble fraction) containing the expressed polypeptide was collected using a ckman Ti35 rotor, 25,000 rpm, 1 hour, 4 ° C.).

To purify the GST fusion polypeptide from the soluble fraction, a glutathione-Sepharose column (Pharmaci
a LKB) according to the affinity purification method.
That is, the GST fusion polypeptide was eluted from the column with 10 mM glutathione / 50 mM Tris-HCl (pH 8.0) mainly according to the manual provided by the manufacturer. Then, using a PD10 desalting column (Pharmacia LKB), the polypeptide solution was equilibrated to 10 mM HEPES-KOH (pH 7.2), 88 mM NaCl, 1 mM KCl, and dispensed to obtain each IP3 sponge (FIG. 1; G224, m30, m4
9, GST). IP3 sponges were stored at -80 ° C until use.

The results are shown below. As a result of preparing and analyzing a series of deletion mutants in which T734 was gradually shortened from the carboxyl terminus, T705 and T699 did not differ much from the characteristics of T734. In T569, T572, and T576, the expression level as a soluble protein decreased. As a result,
By producing and developing T604 deleted to the 604th lysine (K-604), we succeeded in stably expressing it as a soluble protein in large quantities (FIG. 1).

In FIG. 1, the top (IP3R1) indicates the amino-terminal side of the IP3 receptor including the IP3-binding core region (core; amino acids 226-578). Below that are T604 (1 to 604 amino acids), G224 (GST + 224 to 604 amino acids), and G224m30
(K508A mutation in G224), G224m49 (R441Q mutation in G224)
, GST (derived from pGEX-2T).

The [ ³ H] IP3 binding activity of T604 was almost the same as that of T734 (Kd = 45 nM), but the yield was high as a soluble protein (Bmax = 690 pmol / mg protein). That is, the yield was as high as 19 mg per liter of E. coli culture (FIG. 2, Table 1).

[0090]

[Table 1]

Although the expression level of T604 is almost the same as that of T734, the yield as a soluble protein is significantly improved. In addition, T604 had almost the same high yield as a soluble protein even when cultured at 30 ° C. or 37 ° C., and reached a peak within only 2 hours after the start of expression induction.

In FIG. 2, a is the result of Western blot analysis of 0.1 μg of the protein of E. coli extract (soluble fraction) expressing T604 (66 kDa). As a control, T6
A cell extract transformed with only a vector (pET-3a) not containing 04 was used. (b) The total amount of specific IP3 binding contained in 0.7 μl of the soluble fraction was compared between T734, T604 and the vector. c: 3 μg of the T604 soluble fraction and 9.6
Binding of nM [ ³ H] IP3 to unlabeled IP3 (cold IP3
This is a Schatchard plot analysis by competitive inhibition. The results are: Kd = 45 ± 7.6, Bma
x = 690 ± 64 pmol / mg protein.

Next, an attempt was made to shorten the amino terminal side. The amino terminus of T734 loses IP3 binding activity, even outside the core region, or even with a small deletion (eg, deletion of only 31 amino acids), but close to the amino terminus of the core region. Activity is reduced by shortening to amino acid residues (Yoshikawa et al., 1996). The reason for this is unknown, but is probably due to the degree of deletion as a steric hindrance when the core region forms an active conformation.
The active form in which the amino terminus is shortened to 200 to 225 amino acids generally has high affinity, but in any case, the expression level as a soluble protein is reduced.

In the same manner as described above, a protein deleted from position T223 to position 223 (N4-T604; amino acid residues 224 to 224)
604) is more active (about 3 times) than the original T604, but a large amount of soluble protein was not obtained. Therefore, it was found that T604 is most suitable for expressing only proteins in the IP3 binding region having high binding affinity as stable and soluble proteins in large amounts.

[Example 3] Expression of IP3 sponge (1) Inhibition experiment of [ ³ H] -IP3 binding (IP3 binding) Based on the results obtained in Example 2, higher affinity was obtained.
An attempt was made to produce an IP3-binding polypeptide. As mentioned above, T
High when the amino terminal side of 604 or T734 is deleted up to the 223rd position
[ ³ H] IP3 binding activity is shown. Only 356 amino acid residues in the 224 to 579 amino acid region closest to the core show a high affinity of Kd = 2.3 nM (Yoshikawa et al., 1996 (supra)). But,
All of these have reduced expression levels as soluble proteins. That is, although a large deletion of the amino terminus leads to high affinity, it becomes impossible to stably express a large amount of a soluble protein, and most of them become insoluble inclusion bodies.

In general, it is known that the stability, solubility, expression level and the like of a foreign polypeptide are generally improved when a GST fusion is used. Therefore, in this embodiment, IP3
GS instead of the amino-terminal region of the receptor (positions 1-223)
T was ligated to prepare a fusion protein G224 of GST and an IP3 binding site (224 to 604 amino acid region) (FIG. 1). The measurement of the IP3 binding activity of these fusion proteins was mainly performed by Yoshi.
According to the method of Kawakawa et al. (1996).

0.2 μg of fusion protein (IP3 sponge)
With 9.6 nM D-myo- [ ³ H] (1,4,5) IP3 (777 GBq / mmol; DuPo
nt NEN) (hereinafter abbreviated as “[ ³ H] IP3”) and unlabeled at each concentration
D-myo- (1,4,5) IP3 (Dojindo) (hereinafter abbreviated as "cold IP3")
100 μl of binding buffer α (50 mM Tris-HCl (4 ° C
PH 8.0), 1mM EDTA, 1mM β-mercaptoethanol)
And left on ice for 10 minutes. 4 μl of 50 mg / ml γ-
Globulin (Sigma) (final concentration: 1 mg / ml) and 100 μl of 30% PEG
6000 (Sigma) / binding buffer α solution (final concentration: 15%)
And were mixed. After standing still on ice for 5 minutes, 10,000
Xg was centrifuged at 2 ° C for 5 minutes to recover the polypeptide / PEG complex. The PEG precipitated [ ³ H] IP3-binding polypeptide was well solubilized by adding 180 μl of solubilizer Solvable (DuPont NEN). After neutralizing with 18μl glacial acetic acid, 5m
l liquid scintillation counter (Atomlight [DuPo
nt NEN]) and the radioactivity was measured. Non-specific binding was determined by measuring radioactivity in the presence of 2 μM or 10 μM cold IP3, and specific binding was calculated by subtracting the non-specific binding value from each measured value.

On the other hand, Scatchard plot analysis was performed under the following conditions. For low affinity (G224-m30 and control GST)
Is 9.6 nM [ ³ H] IP3 (DuPont NEN) and 1 μg against 2 μg IP3 binding polypeptide in 100 μl binding buffer α.
0-20nM cold IP3 added condition and 0.01μg IP3
9.6 nM of [ ³ H] IP3 (DuPont NE
A binding experiment was performed under the conditions to which N) and 50 nM-2 μM cold IP3 were added. In case of high affinity IP3 sponge (G224 and G224-
m49) was obtained by adding cold IP3 to 0.02 μg of IP3 sponge and increasing the concentration of [ ³ H] -IP3 to 0.15, 0.3, 0.6, 1.2, 2.4, and 4.
A binding experiment was performed on the samples sorted to 8, 9.6 nM.

The inhibitory effect of the IP3 binding polypeptide (IP3 sponge) on [ ³ H] IP3 binding activity of cerebellar microsomes was analyzed as follows. Preparation of the microsomal fraction from the mouse ddY (Nippon SLC) cerebellum was mainly performed by the method of Nakada et al. (Nakada S. et al., Biochem. J. 277: 125-131, 1991).
Followed. IP at each concentration in 100 μl binding buffer α
40 μg of cerebellar microsomes with the addition of 3 sponges and 9.
The above method the change in the binding of ^[3 H] -IP3 of 6 nM (Scatchard
(See plot analysis).

As a result, G224 had 500 times or more higher affinity than T734 (Kd = 83 pM, Bmax = 1.6 pmol / μg protein) (FIG. 3 a). And G224 is (1,4,5) IP3 and (2,4,5) IP3
3 well combined, yielding about 30 per liter of E. coli culture
mg IP3 binding protein. After purification with a glutathione column and subsequent PD10 column,
It could be recovered at 24 mg / l (Table 1). R441Q to T734
Introduction of mutation increases binding activity (Yoshikawa
Et al., 1996 (supra)), G224-m in which the R441Q mutation was introduced into G224.
At 49, the affinity was further increased 2-fold, about 1,000-fold over T734 (Kd = about 43 pM, Bmax = 1.7 pmol / μg protein)
(FIG. 3, Table 1). On the other hand, when the K508A mutation was introduced into T734, the binding activity was significantly reduced (Yoshikawa et al., 1996 (supra)).
G224-m30 in which the K508A mutation was introduced into 224 also significantly reduced the binding activity (Kd = about 330 nM, Bmax = 3.0 pmol / μg protein), about 1 / 4,000 of G224, and about 1/700 of G224-m49. (FIG. 3, Table 1).

(2) Inhibition of Absorption of IP3 Binding by the New IP3 Sponge The IP3 binding polypeptides G224 and G224-m49 exhibit strong IP3 binding activity showing 500 to 1,000 times higher affinity than the original IP3 receptor. With. Therefore, whether G224 and G224-m49 can be used as specific IP3 absorbers (sponges) (IP3 sponges), that is, competitive binding of the IP3 receptor to the IP3 receptor by specifically binding and absorbing IP3 in solution (Fig. 4).

The mouse cerebellum is a tissue in which the microsomal fraction has at least a 50-fold higher [ ³ H] IP3 binding activity than other tissues, and is the tissue rich in the IP3 receptor. (Maeda et al., 1990, supra). 40 μg of cerebellar microsomes in a 100 μl solution (Kd = 21 nM, Bmax = 23 pmo
the binding of the l / mg protein) and of ^[3 H] 9.6 nM IP3, the percentage (% of IP3 sponge is competitively inhibited at each concentration), i.e. is 100% activity at IP3 sponge absence Was analyzed. About 0.92 pmol in this 100 μl solution
IP3 binding site of cerebellar IP3 receptor and 0.96 pmol of [ ³ H] IP3
Is the calculation that exists.

As a result, 100 μg / ml of control GST alone was used.
However, no inhibitory effect was observed (FIG. 4). On the other hand, G224 and G224-m49 with high affinity exhibited strong IP3 binding inhibitory activity and had an IC ₅₀ of about 10 μg / ml (FIG. 4).
On the other hand, G224-m30 with low affinity has low inhibitory activity and IC ₅₀
It was 100 μg / ml. In this in vitro experimental system, IP3
When the sponge concentration is about 25 μg / ml or more, it tends to sediment with the microsomal membrane, and the concentration-dependent curve tends to be disturbed (FIG. 4). The apparent inhibition at G224-m30 observed from concentrations of 25 μg / ml and higher may also be due to the main component being the effect of centrifugation at this high concentration.

[0104] These results for ^[3 H] IP3 binding more efficiently IP3 receptor in response to binding affinity shown to inhibit in a concentration-dependent manner. Therefore, the high affinity IP3 binding polypeptide of the present invention is a novel IP3 sponge as an IP3 neutralizing agent,
It can be applied to IP3 inducible calcium antagonists.

[Example 4] IP3-induced Ca ²⁺ release (IP3-induce
d Ca ²⁺ release; IICR) Inhibition Experiment The microsomal fraction of the mouse cerebellum used for IP3-induced Ca ²⁺ release was prepared as in Example 3 and finally suspended in buffer B before dispensing. And stored at -80 ° C until use. Buffer B composition: 110 mM KCl, 10 mM NaCl, 5 mM KH ₂ PO
₄ , 1 mM DTT, 50 mM HEPES-KOH, pH 7.2 (Protease inhibitor cocktail [0.1 mM PMSF, 10 μM leupeptin, 10 mM
μM pepstatin A, 10 μM E-64] and 2 mM MgCl ₂

Regarding the IP3-induced Ca ²⁺ release activity using cerebellar microsomes, two-wavelength excitation (340 nm and 380 nm) upon addition of IP3 was performed using fura-2 (Molecular Probe) as a Ca ²⁺ fluorescent indicator.
The change in the ratio of fluorescence intensity at 500 nm (F340 / F380) due to the change in fluorescence intensity at 500 nm was measured using a fluorescence spectrophotometer CAF110 (Nihon Bunko).

The IP3-induced Ca ²⁺ release from cerebellar microsomes is generally EC ₅₀ = 100-200 nM IP3. For measuring 100 μg of cerebellar microsomes together with 500 μl of release buffer (buffer B contains 1 mM MgCl ₂ , 2 μM fura2, 1 mM DTT, 10 mM creatine phosphate, 40 U / ml creatine kinase, 1 μg / ml oligomycin, protease inhibitor cocktail) Mix in a stir bar in the cuvette. The following reaction was carried out at 30 ° C. under the condition of constantly stirring and mixing with a stir bar.

1 mM ATP was added to the mixture in the cuvette to activate a Ca ²⁺ pump (Ca ²⁺ -ATPase), thereby incorporating Ca ²⁺ into the microsomal lumen (Ca ²⁺ loading). Ca ²⁺ loading was confirmed by continuing to monitor until the decrease in fura2 fluorescence level was constant. Then, a change in the fura2 fluorescence intensity ratio (F340 / F380) when IP3 was added at a subthreshold level was measured.

On the other hand, the analysis of the inhibitory effect of the IP3 sponge on the IP3-induced Ca ²⁺ release activity of the cerebellar microsomes revealed that the curve of the decrease in the fluorescence intensity of fura2 due to the addition of ATP (due to the incorporation of Ca ^{2+ into} the microsomes) was almost constant. And then mix each concentration of IP3 sponge, and after about 1 minute,
The measurement was performed by adding μM IP3 to the reaction mixture and measuring the change in the fluorescence intensity of fura2 induced.

The concentration dependency of the IP3 sponge using G224 with high affinity was 3.125, 6.25, 12.5, 25, 50, 100, 200 μg.
/ ml at each concentration and add 100 nM
It was determined by measuring the Ca ²⁺ release activity induced by the addition of IP3. Also, the concentration dependency of low affinity G224-m30 is
G224-m30 was added at a concentration of 200, 400, 500 μg / ml, and 1 minute later, 100 nM IP3 was added to induce Ca ²⁺ release activity, and G
One minute after adding 224-m30 at 500 μg / ml, 50 nM IP3 was added to determine the induced Ca ²⁺ releasing activity.

As a result, the IP3 sponge absorbs IP3.
And IP3 binding to the mouse cerebellar microsomal IP3 receptor
Are shown to competitively and specifically inhibit
7). 5 and 6, the vertical axis represents the fluorescence intensity ratio of fura2 (F3
40 / F380) (i.e., Ca^{Two +}Change in amount), the horizontal axis is time
(Seconds).

As shown in FIGS. 5a, 5b and 5c, in the absence of the IP3 sponge (control), IP3 concentration-dependent IP3-induced Ca ²⁺ release activity was observed. The low-affinity G224-m30 had no inhibitory effect on Ca ²⁺ release by 100 nM IP3 even at a concentration of 500 μg / ml, and there was almost no change in 50 nM IP3 compared to the control (FIGS. 5d, e). . GST alone showed no change in 50 nM IP3-induced Ca ²⁺ release activity even at the high concentration of 632 μg / ml (FIG. 5f). Therefore, in each case, no remarkable change was observed as compared with the control.

In contrast to these results, the high affinity G224
, A remarkable inhibitory effect of concentration-dependent IP3-induced Ca ²⁺ release was observed (FIG. 6). In addition, high affinity G224 is 100 μg
The maximum inhibitory effect was obtained at / ml, and almost completely inhibited IP3-induced Ca ²⁺ release (FIG. 6f).

Here, the peak value of Ca ²⁺ release when each concentration of G224 shown in FIG. 6 was added was plotted assuming 100% in the absence of G224 (FIG. 7). The horizontal axis shows G224 of each concentration, and the vertical axis shows the peak value of Ca ²⁺ release. As is clear from the results in FIG. 7, the concentration of G224 required for 50% inhibition of IP3-induced Ca ²⁺ release is about 20 μg / ml.

From the above, it is clear that a high-affinity IP3-binding polypeptide, as an IP3 sponge, specifically inhibits IP3-induced Ca ²⁺ release by the IP3 receptor on cerebellar microsomes in a concentration-dependent manner. became.

[0116]

Industrial Applicability According to the present invention, a polypeptide having a high affinity binding to inositol 1,4,5-triphosphate, a gene encoding the polypeptide, a recombinant vector containing the gene, and a transformation containing the vector A body and a method for producing the polypeptide are provided.

The polypeptide of the present invention can be used for inhibiting and controlling specific cell functions dependent on IP3-induced calcium signaling (IP3 neutralizing agent, IP3-induced calcium antagonist, etc.). In addition, the polypeptides and genes of the present invention are useful as IP3 signal detection agents capable of capturing activation of IP3-induced calcium signaling. Furthermore, the gene of the present invention is useful as a therapeutic drug for diseases associated with calcium production.

[0118]

[Sequence List] SEQUENCE LISTING <110> The Institute of Physical and Chemical Research; Katsuhiko Mikoshi ba <120> High affinity IP3 binding polypeptide <130> RJH11-054N <140> <141> <150> JP98 / 242207 <151> 1998-08-27 <160> 12 <170> PatentIn Ver. 2.0 <210> 1 <211> 1812 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (1812) <400> 1 atg tct gac aaa atg tcg agt ttc cta cat att gga gac att tgt tct 48 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 ctg tat gcg gag gga tct acg aat gga ttt atc agc acc tta ggc ttg 96 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 gtt gat gac cgt tgt gtt gta cag cca gaa gcc ggg gac ctt aac aat 144 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 cca ccc aag aaa ttc aga gac tgc ctc ttt aag cta tgt cct atg aat 192 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 cga tac tcc gca cag aaa cag ttc tgg aaa gct gct aag ccc ggg gcc 240 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 aac agc act aca gat gca gtg ctg ctc aac aaa ttg cat cat gct gca 288 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 gac ttg gaa aag aag cag aat gag aca gaa aac agg aaa ttg ttg ggg 336 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 acc gtc atc caa tat ggc aac gtg atc cag ctc ctg cat ttg aaa agc 384 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 aat aaa tac ctg act gtg aat aag agg ctc cca gccttg gag aag 432 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 aat gcc atg agg gtg acg ttg gac gag gct gga aat gaa ggg tcc tgg 480 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 ttt tac att caa cca ttt tac aag ctt cgc tcc atc gga gac agt gtg 528 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 gtc ata ggc gac aag gta gtt ttg aat cct gtc aat gct ggc cag cct 576 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 cta cat gcc agc agt cat cag ctg gtg gat aac cca ggc tgc aat gag 624 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 gtc aac tcc gtc aac tgt aat aca agc tgg aag ata gtg ctt ttc atg 672 Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 aaa tgg agt gat aac aaa gac gac att ctc aaa gga ggt gat gtg gtg 720 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 agg ctc ttc cat gcc gag caa gag aag ttt ctc acc tgt gat gag cac 768 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 cgg aag aag cag cat gtg ttc ctg agg acc acc ggc agg cag tca gcc 816 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 270 acg tcg gcc acc agt tct aaa gcc ctg tgg gaa gtg gag gta gtc cag 864 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 cac gac cca tgt cgg ggt gga gct ggg tac tgg aat agc ctc ttc cgg 912 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 ttc aag cac ctg gct aca ggg cat tac ttg gct gca gag gta 960 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 gac ttt gag gaa gaa tgc ctg gag ttt cag ccc tca gtg gac cct gat 1008 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Se r Val Asp Pro Asp 325 330 335 cag gat gca tct cgg agt agg ttg aga aac gcg caa gaa aaa atg gta 1056 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 tac tct ctg gtc tccgt cct gaa ggc aac gac atc tcc tcc atc ttt 1104 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 gag cta gac ccc acg act ctg cgt gga ggt gac agc ctt gtc cca agg 1152 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 aac tcc tat gtc cgt ctc aga cac ctg tgc acc aac acc tgg gta cac 1200 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 agc aca aac atc ccc atc gac aag gaa gag gag aag cct gtg atg ctg 1248 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Glu Lys Pro Val Met Leu 405 410 415 aaa aaa att ggt acc tct ccc ctg aag gag gag ag gaa gca ttt gcc ata 1296 Lys Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 gtt cct gtt tcc cct gct gag gtt cgg gac ctg gac ttt gcc aat gat 1344 Val Pro Val Ser Pro Ala Gl u Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 gcc agc aag gtg ctg ggc tcc atc gct ggg aag ttg gaa aag ggc acc 1392 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thrc 450 455 460 acc cag aat gag aga agg tct gtc acg aag ctt ttg gaa gac ttg 1440 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 475 480 gtt tac ttt gtc acg ggt gga act aac tct ggc cag ctt gaa 1488 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 gtt gtc ttc tct aag ccc aat cga gag cgg cag aag ctg atg agg gaa 1536 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 cag aat att ctc aag cag atc ttc aag ctg ttg cag gcc ccc ttc acg 1584 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 gac tgc ggg atg ctt cgg ctg gag gag ctg ggg gat cag 1632 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 cgc cat gct cct ttc aga cat att tgc cga ctc tgc tac agg gtcg Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 cga cac tca cag caa gac tac agg aag aac cag gag tac ata gcc aag 1728 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 cag ttt ggc ttc atg cag aag cag att ggc tat gac gtg ctg gcc gaa 1776 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 590 gac acc atc act gcc ac act cc cgg aaa 1812 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys 595 600 <210> 2 <211> 604 <212> PRT <213> Mus musculus <400> 2 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 Val Asn Ser V al Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp 325 330 335 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Glu Lys Pro Val Met Leu 405 410 415 Lys Ile Gly T hr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr 450 455 460 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 480 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 Arg His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys 595 600 <210> 3 <211> 9848 <212> DNA <213> Mus musculus <220> <221> CDS <222> (329) .. (8575) <400> 3 gctgaagcgt ttcctcaagc ctgccggggt gggaggagag gaggaggtgg tggtggtgga 60 ggaggtggag gcagagggtg gagagagaga aagcgcacgc cgagaggagg tgtgggtgtt 120 ccgctcccat cctaacggaa cgagctccct cttcgcggac atgggattgc ccagcggctg 180 ctaacccctc tcctggtcct gatcccccaa accggcgtgg ctccccggtc accaaggagc 240 tgattacaag ggaccaggat ttgcatcctt ggctgggcgt ccattggcta cagagtgcct 300 gacctgggtc aggctttcca acacggac atg tct gac aaa atg tcg agt ttc 352 Met Ser Asp Lys Met Ser Ser Phe 1 5 cta cat att gga gac att tgt tct ctg tat gcg gag gga tct acg aat 400 Leu His Ile Gly Asp Ile Cys Ser Leu Tyr Ala Glu Gly Ser Thr Asn 10 15 20 gga ttt atc agc acc tta ggc ttg gtt gat gac cgt tgt gtt gta cag 448 Gly Phe Ile Ser Thr Leu Gly Leu Val Asp Asp Arg Cys Val Val Gln 25 30 35 40 cca gaa gcc ggg gac ctt aac aat cca ccc aag aaa ttc aga gac tgc 496 Pro Glu Ala Gly Asp Leu Asn Asn Pro Pro Lys Lys Phe Arg Asp Cys 45 50 55 ctc ttt aag cta tgt cct atg aat cga tac tcc gca cag aaa cag ttc 544 Leu Phe Lys Leu Cys Pro Met Asn Arg Tyr Ser Al a Gln Lys Gln Phe 60 65 70 tgg aaa gct gct aag ccc ggg gcc aac agc act aca gat gca gtg ctg 592 Trp Lys Ala Ala Lys Pro Gly Ala Asn Ser Thr Thr Thr Asp Ala Val Leu 75 80 85 ctc aac aaa ttg cat cat gct gca gac ttg gaa aag aag cag aat gag 640 Leu Asn Lys Leu His His Ala Ala Asp Leu Glu Lys Lys Gln Asn Glu 90 95 100 aca gaa aac agg aaa ttg ttg ggg acc gtc atc caa tat ggc aac gtg 688 Thr Arg Lys Leu Leu Gly Thr Val Ile Gln Tyr Gly Asn Val 105 110 115 120 atc cag ctc ctg cat ttg aaa agc aat aaa tac ctg act gtg aat aag 736 Ile Gln Leu Leu His Leu Lys Ser Asn Lys Tyr Leu Thr Val Asn Lys 125 130 135 agg ctc cca gcc ttg cta gag aag aat gcc atg agg gtg acg ttg gac 784 Arg Leu Pro Ala Leu Leu Glu Lys Asn Ala Met Arg Val Thr Leu Asp 140 145 150 gag gct gga aat gaa ggg tcc tgg ttt tac at caa cca ttt tac aag 832 Glu Ala Gly Asn Glu Gly Ser Trp Phe Tyr Ile Gln Pro Phe Tyr Lys 155 160 165 ctt cgc tcc atc gga gac agt gtg gtc ata ggc gac aag gta gtt ttg 880 Leu Arg Ser Ile Gly Asp Ser Val Ile G ly Asp Lys Val Val Leu 170 175 180 aat cct gtc aat gct ggc cag cct cta cat gcc agc agt cat cag ctg 928 Asn Pro Val Asn Ala Gly Gln Pro Leu His Ala Ser Ser His Gln Leu 185 190 195 200 gtg gat aac cca ggc tgc aat gag gtc aac tcc gtc aac tgt aat aca 976 Val Asp Asn Pro Gly Cys Asn Glu Val Asn Ser Val Asn Cys Asn Thr 205 210 215 agc tgg aag ata gtg ctt ttc atg aaa tgg agt gat aac aaa gac gac 1024 Ser Trp Lys Ile Val Leu Phe Met Lys Trp Ser Asp Asn Lys Asp Asp 220 225 230 att ctc aaa gga ggt gat gtg gtg agg ctc ttc cat gcc gag caa gag 1072 Ile Leu Lys Gly Gly Asp Val Val Arg Leu Phe His Ala Glu Gln Glu 235 240 245 aag ttt ctc acc tgt gat gag cac cgg aag aag cag cat gtg ttc ctg 1120 Lys Phe Leu Thr Cys Asp Glu His Arg Lys Lys Gln His Val Phe Leu 250 255 260 agg acc acc ggc agg cag tca gcc acg tcg gcc acc agt tct aaa gcc 1168 Arg Thr Thr Gly Arg Gln Ser Ala Thr Ser Ala Thr Ser Ser Lys Ala 265 270 275 280 ctg tgg gaa gtg gag gta gtc cag cac gac cca tgt cgg ggt gga gct 1216 Leu Trp Glu Val Glu Val Val Gln His Asp Pro Cys Arg Gly Gly Ala 285 290 295 ggg tac tgg aat agc ctc ttc cgg ttc aag cac ctg gct aca ggg cat 1264 Gly Tyr Trp Asn Ser Leu Phe Arg Phe Lys His Leu Ala Thr Gly His 300 305 310 tac ttg gct gca gag gta gac cct gac ttt gag gaa gaa tgc ctg gag 1312 Tyr Leu Ala Ala Glu Val Asp Pro Asp Phe Glu Glu Glu Cys Leu Glu 315 320 325 ttt cag ccc tca gtg gac cct gat cag gatgt agg ttg 1360 Phe Gln Pro Ser Val Asp Pro Asp Gln Asp Ala Ser Arg Ser Arg Leu 330 335 340 aga aac gcg caa gaa aaa atg gta tac tct ctg gtc tcc gtg cct gaa 1408 Arg Asn Ala Gln Glu Lys Met Val Tyr Ser Leu Val Ser Val Pro Glu 345 350 355 360 ggc aac gac atc tcc tcc atc ttt gag cta gac ccc acg act ctg cgt 1456 Gly Asn Asp Ile Ser Ser Ile Phe Glu Leu Asp Pro Thr Thr Leu Arg 365 370 375 gga ggt gac agc ctt gtc cca agg aac tcc tat gtc cgt ctc aga cac 1504 Gly Gly Asp Ser Leu Val Pro Arg Asn Ser Tyr Val Arg Leu Arg His 380 385 390 ctg tgc acc aac acc tgg gta cac agc aca aac atc ccc atc gac aag 1552 Leu Cys Thr Asn Thr Trp Val His Ser Thr Asn Ile Pro Ile Asp Lys 395 400 405 gaa gag gag aag cct gtg atg ctg aaa att ggt acc tct ccc ctg aag 1600 Glu Glu Glu Lys Pro Val Met Leu Lys Ile Gly Thr Ser Pro Leu Lys 410 415 420 gag gac aag gaa gca ttt gcc ata gtt cct gtt tcc cct gct gag gtt 1648 Glu Asp Lys Glu Ala Phe Ala Ile Val Pro Val Ser Pro Ala Glu Val 425 430 435 435 440 cgg gac ctg gac ttt gcc aat gat gcc agc aag gtg ctg ggc tcc atc 1696 Arg Asp Leu Asp Phe Ala Asn Asp Ala Ser Lys Val Leu Gly Ser Ile 445 450 455 gct ggg aag ttg gaa aag ggc acc atc acc cag aat gag aga agg tct 1744 Ala Gly Lys Lys Gly Thr Ile Thr Gln Asn Glu Arg Arg Ser 460 465 470 gtc acg aag ctt ttg gaa gac ttg gtt tac ttt gtc acg ggt gga act 1792 Val Thr Lys Leu Leu Glu Asp Leu Val Tyr Phe Val Thr Gly Gly Thr 475 480 480 485 aac tct ggc caa gac gtg ctt gaa gtt gtc ttc tct aag ccc aat cga 1840 Asn Ser Gly Gln Asp Val Leu Glu Val Val Phe Ser Lys Pro Asn Arg 490 495 500 gag cgg cag aag ctg atg agg gaa cag aat att ctc g cag atc ttc 1888 Glu Arg Gln Lys Leu Met Arg Glu Gln Asn Ile Leu Lys Gln Ile Phe 505 510 515 515 520 aag ctg ttg cag gcc ccc ttc acg gac tgc ggg gat ggc ccg atg ctt 1936 Lys Leu Leu Gln Thr Asp Cys Gly Asp Gly Pro Met Leu 525 530 535 cgg ctg gag gag ctg ggg gat cag cgc cat gct cct ttc aga cat att 1984 Arg Leu Glu Glu Leu Gly Asp Gln Arg His Ala Pro Phe Arg His Ile 540 545 550 tgc cga tgc tac agg gtc ctg cga cac tca cag caa gac tac agg 2032 Cys Arg Leu Cys Tyr Arg Val Leu Arg His Ser Gln Gln Asp Tyr Arg 555 560 565 aag aac cag gag tac ata gcc aag cag ttt ggc ttc atg cag ag aag ag Lys Asn Gln Glu Tyr Ile Ala Lys Gln Phe Gly Phe Met Gln Lys Gln 570 575 580 att ggc tat gac gtg ctg gcc gaa gac acc atc act gcc ctg ctc cac 2128 Ile Gly Tyr Asp Val Leu Ala Glu Asp Thr Ile Thr Leu His 585 590 595 600 600 aac aac cgg aaa ctc ctg gag aag cac atc acc gcg gca gag att gac 2176 Asn Asn Arg Lys Leu Leu Glu Lys His Ile Thr Ala Ala Glu Ile Asp 605 610 615 acg ttt gtc agc cg gtg gtg a aag aac agg gag ccc agg ttc ttg gat 2224 Thr Phe Val Ser Leu Val Arg Lys Asn Arg Glu Pro Arg Phe Leu Asp 620 625 630 tac ctc tct gac ctc tgc gta tcc atg aac aag tca atc cct gtg aca 2272 Tyru Asp Leu Cys Val Ser Met Asn Lys Ser Ile Pro Val Thr 635 640 645 cag gag ctc atc tgt aaa gct gtg ctc aat ccc acc aat gct gac atc 2320 Gln Glu Leu Ile Cys Lys Ala Val Leu Asn Pro Thr Asn Ala Asp Ile 650 655 660 ctg att gag acc aag ctg gtt ctt tct cgt ttt gag ttt gaa ggc gtt 2368 Leu Ile Glu Thr Lys Leu Val Leu Ser Arg Phe Glu Phe Glu Gly Val 665 670 675 680 tcc act gga gag aat gct ctg gag gcc gat gag gaa gag gtg 2416 Ser Thr Gly Glu Asn Ala Leu Glu Ala Gly Glu Asp Glu Glu Glu Val 685 690 695 tgg ctg ttc tgg agg gac agc aac aaa gag atc cgt agt aag agt gtc 2464 Trp Leu Phe Asrp Arg Lys Glu Ile Arg Ser Lys Ser Val 700 705 710 cgg gaa ttg gcg caa gat gct aaa gag gga cag aag gaa gac agg gac 2512 Arg Glu Leu Ala Gln Asp Ala Lys Glu Gly Gln Lys Glu Asp Arg Asp 715 720 725 atc ct agc tac tac aga tat cag ctg aac ctc ttt gca agg atg tgt 2560 Ile Leu Ser Tyr Tyr Arg Tyr Gln Leu Asn Leu Phe Ala Arg Met Cys 730 735 740 740 ctg gac cgc cag tac ctg gcc atc aat gga atc tcc 2608 Leu Asp Arg Gln Tyr Leu Ala Ile Asn Glu Ile Ser Gly Gln Leu Asp 745 750 755 760 gtt gat ctc att ctc cgc tgc atg tct gac gag aac ctc ccc tac gac 2656 Val Asp Leu Ile Leu Arg Cyslu Met Asp Leu Pro Tyr Asp 765 770 775 ctc agg gca tcc ttt tgc cgc ctc atg ctt cac atg cat gtg gac cga 2704 Leu Arg Ala Ser Phe Cys Arg Leu Met Leu His Met His Val Asp Arg 780 785 790 790 gat ccc caa gag cag gt cct gtg aaa tat gcc cga ctg tgg tca 2752 Asp Pro Gln Glu Gln Val Thr Pro Val Lys Tyr Ala Arg Leu Trp Ser 795 800 805 gaa att ccc tct gag atc gcc att gat gac tat gac agc agt gga aca 2800 Glu Ile Pro Ser Glu Ile Ala Ile Asp Asp Tyr Asp Ser Ser Gly Thr 810 815 820 tcc aaa gat gaa att aag gag agg ttt gca cag acg atg gag ttt gtg 2848 Ser Lys Asp Glu Ile Lys Glu Arg Phe Ala Gln Thr Met Glu Phe Val 825 830 835 840 gag gag tac cta aga gat gtg gtt tgt caa aga ttc ccc ttc tct gat 2896 Glu Glu Tyr Leu Arg Asp Val Val Cys Gln Arg Phe Pro Phe Ser Asp 845 850 855 aag gag aaa aat aag ctc acg ttt g gtg aac tta gcc agg aat 2944 Lys Glu Lys Asn Lys Leu Thr Phe Glu Val Val Asn Leu Ala Arg Asn 860 865 870 ctc ata tac ttt ggt ttc tac aac ttt tct gac ctt ctc cga tta acc 2992 Leu Ile Tyr Phe Gly Asn Phe Ser Asp Leu Leu Arg Leu Thr 875 880 880 885 aag atc ctc ttg gca atc tta gac tgt gtc cat gtg acc act atc ttc 3040 Lys Ile Leu Leu Ala Ile Leu Asp Cys Val His Val Thr Thr Ile Phe 890 895 900 ccc att agc aag atg aca aaa gga gaa gag aat aaa ggc agt aac gtg 3088 Pro Ile Ser Lys Met Thr Lys Gly Glu Glu Asn Lys Gly Ser Asn Val 905 910 915 915 920 atg agg tct atc cat ggc gtt ggg gag ctg atg acc cag gt ctg 3136 Met Arg Ser Ile His Gly Val Gly Glu Leu Met Thr Gln Val Val Leu 925 930 935 cgg gga gga ggc ttc ttg ccc atg act ccc atg gct gcg gcc cct gaa 3184 Arg Gly Gly Gly Phe Leu Pro Met Thr Pro M et Ala Ala Ala Pro Glu 940 945 950 gga aat gtg aag cag gca gag cca gag aaa gag gac atc atg gtc atg 3232 Gly Asn Val Lys Gln Ala Glu Pro Glu Lys Glu Asp Ile Met Val Met 955 960 965 gac acc aag ttg aag atc att gaa ata ctc cag ttt att ttg aat gtg 3280 Asp Thr Lys Leu Lys Ile Ile Gle Ile Leu Gln Phe Ile Leu Asn Val 970 975 980 aga ttg gat tat agg atc tcc tgc ctc ctg tgt ata ttt aag c Asp Tyr Arg Ile Ser Cys Leu Leu Cys Ile Phe Lys Arg Glu 985 990 995 1000 ttt gat gaa agc aat tcc cag tca tca gaa aca tcc tcc gga aac agc 3376 Phe Asp Glu Ser Asn Ser Gln Ser Ser Glu Thr Ser Ser Gly Asn Ser 1005 1010 1015 agc cag gaa ggg cca agt aat gtg cca ggt gct ctt gac ttt gaa cac 3424 Ser Gln Glu Gly Pro Ser Asn Val Pro Gly Ala Leu Asp Phe Glu His 1020 1025 1030 att gaa gaa caa gcg gaa ggc ata gga agt gag gag aac aca 3472 Ile Glu Glu Gln Ala Glu Gly Ile Phe Gly Gly Ser Glu Glu Asn Thr 1035 1040 1045 cct ttg gac ctg gat gac cat ggt ggc aga acc ttc ctc agg gtc ctg 3520 Pro Leu Asp Leu Asp Asp His Gly Gly Arg Thr Phe Leu Arg Val Leu 1050 1055 1060 ctc cac ttg aca atg cat gac tac cca ccc ctg gtg tct ggg gcc ctg 3568 Leu His Leu Thr Met His Asp Tyr Pro Pro Leu Val Ser Gly Ala Leu 1065 1070 1075 1080 cag ctc ctc ttt cgg cac ttc agc cag agg cag gag gtc ctt cag gcc 3616 Gln Leu Leu Phe Arg His Phe Ser Gln Arg Gln Glu Val Leu Gln Ala 1085 1090 1095 ttc aaa cag gtt caa ctg gcg gtt cag gat gtg gac aac tac 3664 Phe Lys Gln Val Gln Leu Leu Val Thr Ser Gln Asp Val Asp Asn Tyr 1100 1105 1110 aaa cag atc aag caa gac ttg gac caa cta agg tcc att gtg gag aag 3712 Lys Gln Ile Lys Gln Asp Lep Asp Gln Leu Arg Ser Ile Val Glu Lys 1115 1120 1125 tct gag ctc tgg gtg tac aaa ggc caa ggt ccc gat gag cct atg gac 3760 Ser Glu Leu Trp Val Tyr Lys Gly Gln Gly Pro Asp Glu Pro Met Asp 1130 1135 1140 gga gcc tcc ggt gaa aat gag cat aag aaa acc gag gag ggg acg agc 3808 Gly Ala Ser Gly Glu Asn Glu His Lys Lys Thr Glu Glu Gly Thr Ser 1145 1150 1155 1160 aag cca ctg aag cac gag agc acc ag c agc tac aac tac cga gtg gtg 3856 Lys Pro Leu Lys His Glu Ser Thr Ser Ser Tyr Asn Tyr Arg Val Val 1165 1170 1175 aaa gag att ttg att cga ctt agc aag ctc tgc gtg cag gag agc gcg 3904 Lys Glu Ile Arg Leu Ser Lys Leu Cys Val Gln Glu Ser Ala 1180 1185 1190 tcg gtg agg aag agc cgg aag cag cag caa cga ctg ctg agg aac atg 3952 Ser Val Arg Lys Ser Arg Lys Gln Gln Gln Arg Leu Leu Arg Asn Met 1195 1200 120 ggc gca cac gct gtg gtg ctg gag ctg ctg cag atc ccc tac gag aag 4000 Gly Ala His Ala Val Val Leu Glu Leu Leu Gln Ile Pro Tyr Glu Lys 1210 1215 1220 gcc gaa gac aca aag atg caa gag atc atg cgg gaa ttt 4048 Ala Glu Asp Thr Lys Met Gln Glu Ile Met Arg Leu Ala His Glu Phe 1225 1230 1235 1240 ttg cag aat ttc tgt gca ggc aac cag cag aat caa gct ttg ctg cat 4096 Leu Gln Asn Phe Cys Ala Gly Asn Gln Ala Leu Leu His 1245 1250 1255 aaa cac ata aac ctg ttt ctc aag cca ggg atc ctg gag gca gtg acg 4144 Lys His Ile Asn Leu Phe Leu Lys Pro Gly Ile Leu Glu Ala Val Thr 1260 126 5 1270 atg cag cac atc ttc atg aac aac ttc cag ctg tgc agt gag atc aac 4192 Met Gln His Ile Phe Met Asn Asn Phe Gln Leu Cys Ser Glu Ile Asn 1275 1280 1285 gag aga gtg gtc cag cac ttt gtt cag acc cac ggt cga 4240 Glu Arg Val Val Gln His Phe Val His Cys Ile Glu Thr His Gly Arg 1290 1295 1300 aac gtc cag tat atc aag ttt ctc cag acg att gtc aag gca gaa ggg 4288 Asn Val Gln Tyr Ile Lys Phe Leu Gln Thr Ile Val Lys Ala Glu Gly 1305 1310 1315 1320 aaa ttc att aaa aag tgc caa gac atg gtc atg gct gag ctt gtc aac 4336 Lys Phe Ile Lys Lys Cys Gln Asp Met Val Met Ala Glu Leu Val Asn 1325 1330 1335 tctg gac gtc ctc gtg ttc tac aat gac aga gcc tct ttc cag 4384 Ser Gly Glu Asp Val Leu Val Phe Tyr Asn Asp Arg Ala Ser Phe Gln 1340 1345 1350 act ctg atc cag atg atg cgg tcc gag cgt gac cgg atg 432 gag Thr Leu Ile Gln Met Met Arg Ser Glu Arg Asp Arg Met Asp Glu Asn 1355 1360 1365 agc cct ctc atg tac cac atc cat ctg gtg gag ctc ttg gcc gtg tgc 4480 Ser Pro Leu Met Tyr His Ile His Leu Val Glu Leu Leu Ala Val Cys 1370 1375 1380 aca gag ggc aag aat gtg tac acg gag atc aag tgc aac tcc ttg ctc 4528 Thr Glu Gly Lys Asn Val Tyr Thr Glu Ile Lys Cys Asn Ser Leu Leu 1385 1390 1395 1400 cc gat gac atc gtt cgt gtg gtc act cat gaa gac tgc atc ccc 4576 Pro Leu Asp Asp Ile Val Arg Val Val Thr His Glu Asp Cys Ile Pro 1405 1410 1415 gag gtt aag atc gct tac att aac ttc ctg aat cac tgc tat g 4624 Glu Val Lys Ile Ala Tyr Ile Asn Phe Leu Asn His Cys Tyr Val Asp 1420 1425 1430 acg gag gtg gag atg aag gag att tac aca agc aac cac atg tgg aag 4672 Thr Glu Val Glu Met Lys Glu Ile Tyr Thr Ser Asn His Met Trp Lys 1435 1440 1445 ttg ttt gag aat ttc ctc gtg gac atc tgc agg gcc tgt aac aac aca 4720 Leu Phe Glu Asn Phe Leu Val Asp Ile Cys Arg Ala Cys Asn Asn Thr 1450 1455 1460 agc gac gag gacc att ctg gag aag tac gtc act gaa 4768 Ser Asp Arg Lys His Ala Asp Ser Ile Leu Glu Lys Tyr Val Thr Glu 1465 1470 1475 1480 atc gtg atg agc atc gtc acc acc ttc ttc agc tct ccc t tc tca gac 4816 Ile Val Met Ser Ile Val Thr Thr Phe Phe Ser Ser Pro Phe Ser Asp 1485 1490 1495 cag agc acc act ctg cag acc cgc cag cct gtc ttt gtg caa ctc ctg 4864 Gln Ser Thr Thr Lele Gln Thr Arg Gln Pro Val Phe Val Gln Leu Leu 1500 1505 1510 caa ggc gtc ttc cga gtt tac cac tgc aac tgg ctg atg ccg agc caa 4912 Gln Gly Val Phe Arg Val Tyr His Cys Asn Trp Leu Met Pro Ser Gln 1515 1520 1525 aaa gcc gcg gt agc tgc atc cgg gtg ctc tct gac gta gcc aag 4960 Lys Ala Ser Val Glu Ser Cys Ile Arg Val Leu Ser Asp Val Ala Lys 1530 1535 1540 agc cgg gcc ata gcc att cct gtt gac ctg gac agc caa gtc aac aac 500 Ala Ile Ala Ile Pro Val Asp Leu Asp Ser Gln Val Asn Asn 1545 1550 1555 1560 ctc ttc ctg aag tcc cac aac att gtg cag aaa aca gcc ctg aac tgg 5056 Leu Phe Leu Lys Ser His Asn Ile Val Gln Lys Thr Ala Leu Asn Trp 1565 1570 1575 cgg tta tca gcc cga aac gcc gct cgc aga gac tct gta ctg gca gca 5104 Arg Leu Ser Ala Arg Asn Ala Ala Arg Arg Asp Ser Val Leu Ala Ala 1580 1585 1590 tcc aga gac tac cga aat atc att gag agg tta cag gac atc gtg tct 5152 Ser Arg Asp Tyr Arg Asn Ile Ile Glu Arg Leu Gln Asp Ile Val Ser 1595 1600 1605 gcc cta gag gac cgg ctc agg ccc ctg gtg cag gct gag ctg tct Ala Leu Glu Asp Arg Leu Arg Pro Leu Val Gln Ala Glu Leu Ser Val 1610 1615 1620 ctc gtg gat gtt cta cac aga cca gaa ctg ctc ttc ccc gag aac acg 5248 Leu Val Asp Val Leu His Arg Pro Glu Leu Leu Phe Pro Glu Asn Thr 1625 1630 1635 1640 gat gcc agg agg aaa tgt gag agt gga ggt ttc atc tgc aag cta ata 5296 Asp Ala Arg Arg Lys Cys Glu Ser Gly Gly Phe Ile Cys Lys Leu Ile 1645 1650 1655 aaa cat acc aag caa ggg ctg gag aat gaa gag aaa cta tgc att 5344 Lys His Thr Lys Gln Leu Leu Glu Glu Asn Glu Glu Lys Leu Cys Ile 1660 1665 1670 aaa gtc tta cag acc ctc agg gaa atg atg acc aaa gac aga ggc tat 5392 Lys Val Leu Leu Arg Glu Met Met Thr Lys Asp Arg Gly Tyr 1675 1680 1685 gga gag aag caa att tcc att gat gaa tcg gaa aat gcc gag ctg cca 5440 Gly Glu Lys Gln Ile Ser Ile Asp Glu Ser Glu Asn Al a Glu Leu Pro 1690 1695 1700 cag gca ccg gaa gct gag aac tcc aca gag cag gag ctt gaa cca agt 5488 Gln Ala Pro Glu Ala Glu Asn Ser Thr Glu Gln Glu Leu Glu Pro Ser 1705 1710 1715 1720 cca ccc ctg agg caact gaa gac cat aaa agg ggt gag gca ctc cga 5536 Pro Pro Leu Arg Gln Leu Glu Asp His Lys Arg Gly Glu Ala Leu Arg 1725 1730 1735 caa att ttg gtc aac cgt tac tat gga aac atc aga cct tca gga Leu 5584 Gln Val Asn Arg Tyr Tyr Gly Asn Ile Arg Pro Ser Gly Arg 1740 1745 1750 aga gag agc ctt acc agc ttt ggc aat ggc cca cta tca cca gga gga 5632 Arg Glu Ser Leu Thr Ser Phe Gly Asn Gly Pro Leu Ser Pro Gly Gly 1755 1760 1765 ccc agc aag cct ggt gga gga ggg gga ggt cct gga tct agt tcc aca 5680 Pro Ser Lys Pro Gly Gly Gly Gly Gly Gly Gly Pro Gly Ser Ser Ser Thr 1770 1775 1780 agc agg ggt gag atg agc ctg gct gag gtt cag tgt cac ctc gac aag 5728 Ser Arg Gly Glu Met Ser Leu Ala Glu Val Gln Cys His Leu Asp Lys 1785 1790 1795 1800 gag ggg gcc tcc aac ctg gtc atc gat ctc ata atg aat gca tcc agt 5776 Glu Gly Ala Ser Asn Leu Val Ile Asp Leu Ile Met Asn Ala Ser Ser 1805 1810 1815 gac cga gta ttc cat gaa agc att ctg ctg gcc atc gca ctt ctg gaa 5824 Asp Arg Val Phe His Glu Ser Ile Leu Leu Ala Ile Ala Leu Glu 1820 1825 1830 gga ggc aac acc acc atc cag cac tcg ttt ttc tgc cgg ctg aca gaa 5872 Gly Gly Asn Thr Thr Ile Gln His Ser Phe Phe Cys Arg Leu Thr Glu 1835 1840 1845 gat aag aaa tca gag aag ttcttcttc ttt tac gat cga atg aag 5920 Asp Lys Lys Ser Glu Lys Phe Phe Lys Val Phe Tyr Asp Arg Met Lys 1850 1855 1860 gtg gcc cag cag gaa atc aag gcg aca gtg aca gtg aac acc agc gac 5968 Val Ala Gln Gln Glu Ala Thr Val Thr Val Asn Thr Ser Asp 1865 1870 1875 1880 ttg gga aac aaa aag aaa gat gat gaa gtg gac agg gat gcc ccg tct 6016 Leu Gly Asn Lys Lys Lys Asp Asp Glu Val Asp Arg Asp Ala Pro Ser 1885 1890 1895 cgg aag aaa gcc aaa gag ccc aca aca cag ata aca gaa gag gtc cgg 6064 Arg Lys Lys Ala Lys Glu Pro Thr Thr Gln Ile Thr Glu Glu Val Arg 1900 1905 1910 gat cag ctc ctg gaa gca tct gct gcc acc agg aaa gcc ttt acc acc 6112 Asp Gln Leu Leu Glu Ala Ser Ala Ala Thr Arg Lys Ala Phe Thr Thr 1915 1920 1925 ttc cgg agg gag gcc gac cct gat gac cat tac cag tct ggg gag ggc 6160 Phe Arg Arg Glu Ala Asp Pro Asp Asp His Tyr Gln Ser Gly Glu Gly 1930 1935 1940 acc cag gct aca acc gac aaa gcc aag gat gac cta gag atg agc gct 6208 Thr Gln Ala Thr Thr Asp Lys Ala Lys Asp Asp Leu Glu Met Ser Ala 1945 1950 1955 1960 gtc atc acc atc atg cag cct atc ctg cgc ttc ctg cag ctg ctg tgt 6256 Val Ile Thr Ile Met Gln Pro Ile Leu Arg Phe Leu Gln Leu Leu Cys 1965 1970 1975 gaa aac cac aac cga gat ctg cg ag ttg cg ag caa aat aat 6304 Glu Asn His Asn Arg Asp Leu Gln Asn Phe Leu Arg Cys Gln Asn Asn 1980 1985 1990 aag acc aac tac aat ttg gtg tgt gag aca ctg cag ttt ctg gac tgt 6352 Lys Thr Asn Tyr Asn Leu Val Cys Glu Leu Gln Phe Leu Asp Cys 1995 2000 2005 att tgt ggg agc aca acc gga ggc ctt ggt ctt ctt gga ctg tac ata 6400 Ile Cys Gly Ser Thr Thr Gly Gly Leu Gly Leu Leu Gly Leu Tyr Ile 2010 2015 2020 aat gaa aag aat gta gca ctt atc aac caa acc ctg gag agt ctg acg 6448 Asn Glu Lys Asn Val Ala Leu Ile Asn Gln Thr Leu Glu Ser Leu Thr 2025 2030 2035 2040 gag tac tgt caa ggg cct tgc cat gag aac ca aac tgc atc gcc acc 6496 Glu Tyr Cys Gln Gly Pro Cys His Glu Asn Gln Asn Cys Ile Ala Thr 2045 2050 2055 cac gag tcc aat ggc atc gat atc atc aca gcc ctc atc ctc aat gat 6544 His Glu Ser Asn Gly Ile Asp I Ile Thr Ala Leu Ile Leu Asn Asp 2060 2065 2070 atc aac cct ctg gga aag aag cgg atg gac ctg gtg tta gaa ctg aag 6592 Ile Asn Pro Leu Gly Lys Lys Arg Met Asp Leu Val Leu Glu Leu Lys 2075 2080 g aac tcg aag ctg cta ctg gcc atc atg gaa agc aga cac gat 6640 Asn Asn Ala Ser Lys Leu Leu Leu Ala Ile Met Glu Ser Arg His Asp 2090 2095 2100 agt gaa aat gca gag agg atc ctg tac aac atg agg cccagg Ser Glu Asn Ala Glu Arg Ile Leu Tyr Asn Met Arg Pro Lys Glu Leu 2105 2110 2115 2120 gtg gaa gtg atc aag aag gcc tac atg caa ggt gaa gtg gaa ttt gag 6736 Val Glu Val Ile Lys Lys Ala Tyr Met Gln Gly Glu Val Glu Phe Glu 2125 2130 2135 gat ggg gag aac ggt gag gat gga gct gcc tca ccc agg aac gtg ggc 6784 Asp Gly Glu Asn Gly Glu Asp Gly Ala Ala Ser Pro Arg Asn Val Gly 2140 2145 2c aac atc tac atc ctc gct cac cag ttg gct cgg cat aac aaa gaa 6832 His Asn Ile Tyr Ile Leu Ala His Gln Leu Ala Arg His Asn Lys Glu 2155 2160 2165 ctt caa acc atg ctg aaa cct gga ggc cag gtg gat ggg gct 6880 Leu Gln Thr Met Leu Lys Pro Gly Gly Gln Val Asp Gly Asp Glu Ala 2170 2175 2180 ctg gag ttc tac gcg aag cac aca gca caa att gag att gtc aga ctg 6928 Leu Glu Phe Tyr Ala Lys His Thr Ala Gln Ile Val Arg Leu 2185 2190 2195 2200 gac cgg aca atg gaa cag atc gtc ttc cct gtg ccc agc atc tgt gaa 6976 Asp Arg Thr Met Glu Gln Ile Val Phe Pro Val Pro Ser Ile Cys Glu 2205 2210 2215 ttc ctg taga gag aaa ctt cga ata tat tac acc aca gag cgg 7024 Phe Leu Thr Lys Glu Ser Lys Leu Arg Ile Tyr Tyr Thr Thr Glu Arg 2220 2225 2230 gat gag caa ggt agc aag atc aat gac ttc ttc ctg cg c tcc gag gac 7072 Asp Glu Gln Gly Ser Lys Ile Asn Asp Phe Phe Leu Arg Ser Glu Asp 2235 2240 2245 ctc ttt aac gag atg aac tgg cag aag aaa ctt cga gcc cag cct gtc 7120 Leu Phe Asn Glu Met Asn Trp Gln Lys Leu Arg Ala Gln Pro Val 2250 2255 2260 ttg tac tgg tgt gcc cga aac atg tct ttc tgg agc agc atc tcc ttc 7168 Leu Tyr Trp Cys Ala Arg Asn Met Ser Phe Trp Ser Ser Ile Ser Phe 2265 2270 2275 2280 gacac gtc ctg atg aac ctg ctg gtg gcg ttt ttc tat cca ttt 7216 Asn Leu Ala Val Leu Met Asn Leu Leu Val Ala Phe Phe Tyr Pro Phe 2285 2290 2295 aaa gga gtg agg gga gga aca cta gag cca cac tggca Lys Gly Val Arg Gly Gly Thr Leu Glu Pro His Trp Ser Gly Leu Leu 2300 2305 2310 tgg aca gcc atg ctc atc tct ctg gcc att gtc att gct ctg ccc aag 7312 Trp Thr Ala Met Leu Ile Ser Leu Ala Ile Val Ile Ala Leu Pro Lys 2315 2320 2325 ccc cac ggc atc cgg gcc tta att gct tct aca atc cta cga ctg ata 7360 Pro His Gly Ile Arg Ala Leu Ile Ala Ser Thr Ile Leu Arg Leu Ile 2330 2335 2340 ttt tca g tt ggg ttg cag ccc aca ctg ttt ctg ctg gga gct ttc aat 7408 Phe Ser Val Gly Leu Gln Pro Thr Leu Phe Leu Leu Gly Ala Phe Asn 2345 2350 2355 2360 gtc tgc aat aaa atc atc ttc ctg atg gtc ttg atg gtc ttg ag gtc ag gtg ag ggg 7456 Val Cys Asn Lys Ile Ile Phe Leu Met Ser Phe Val Gly Asn Cys Gly 2365 2370 2375 acc ttc acc aga ggc tac cgg gcc atg gtt ctg gat gtg gag ttc ctc 7504 Thr Phe Thr Arg Gly Tyr Arg Ala Met Val Leu As Val Glu Phe Leu 2380 2385 2390 tat cat ttg ctg tat cta ctc atc tgt gcc atg ggc ctc ttc gta cat 7552 Tyr His Leu Leu Tyr Leu Leu Ile Cys Ala Met Gly Leu Phe Val His 2395 2400 2405 gag ttc ttc tt ag ctt ttt gat tta gtg tac aga gag gag 7600 Glu Phe Phe Tyr Ser Leu Leu Leu Phe Asp Leu Val Tyr Arg Glu Glu 2410 2415 2420 act ttg ctt aat gtc att aaa agt gtc acc cgc aat gga cgg tcc atc 7648 Thr Leu Leu Val Ile Lys Ser Val Thr Arg Asn Gly Arg Ser Ile 2425 2430 2435 2440 atc ttg aca gcg gtc ctg gct ctg atc ctg gtt tac ctg ttc tca att 7696 Ile Leu Thr Ala Val Leu Ala Leu Ile Leu Va l Tyr Leu Phe Ser Ile 2445 2450 2455 gtg ggc tat ctg ttc ttc aag gat gac ttt atc ttg gaa gta gat agg 7744 Val Gly Tyr Leu Phe Phe Lys Asp Asp Phe Ile Leu Glu Val Asp Arg 2460 2465 2470 ttgcc gct gtt cca gaa act ggc gag agt ttg gcc aac 7792 Leu Pro Asn Glu Thr Ala Val Pro Glu Thr Gly Glu Ser Leu Ala Asn 2475 2480 2485 gat ttc ctg tac tct gat gtg tgc agg gta gag acg ggg gag aac thec 7840 Leu Tyr Ser Asp Val Cys Arg Val Glu Thr Gly Glu Asn Cys 2490 2495 2500 acc tct cct gca ccc aaa gaa gag ctg ctc cct gcc gaa gaa acg gaa 7888 Thr Ser Pro Ala Pro Lys Glu Glu Leu Leu Pro Ala Glu Glu Thr Glu 2505 2510 2515 2520 cag gat aag gaa cac acg tgt gag acc ctg ctc atg tgc atc gtc act 7936 Gln Asp Lys Glu His Thr Cys Glu Thr Leu Leu Met Cys Ile Val Thr 2525 2530 2535 gtt ctg agt cac ggg ctg cgg agg ggg gta gga gac gtg ctc 7984 Val Leu Ser His Gly Leu Arg Ser Gly Gly Gly Val Gly Asp Val Leu 2540 2545 2550 agg aag cca tcc aaa gag gag cct ctg ttt gct gca agg gtg atc tac 8032 A rg Lys Pro Ser Lys Glu Glu Pro Leu Phe Ala Ala Arg Val Ile Tyr 2555 2560 2565 gac ctc ctc ttc ttc ttc atg gtc atc atc atc gtc ctg aac ctg att 8080 Asp Leu Leu Phe Phe Phe Met Val Ile Ile Ile Leu Ile 2570 2575 2580 ttc ggg gtc atc atc gac acc ttt gct gac ctg agg agt gag aag caa 8128 Phe Gly Val Ile Ile Asp Thr Phe Ala Asp Leu Arg Ser Glu Lys Gln 2585 2590 2595 2600 aag aag gag gag atc ttaaaa acc acg tgc ttc atc tgc ggc ttg gaa 8176 Lys Lys Glu Glu Ile Leu Lys Thr Thr Cys Phe Ile Cys Gly Leu Glu 2605 2610 2615 agg gac aag ttt gac aat aag act gtc acc ttt gaa gag cac atc aag Asp 8224 Arg Asp Asn Lys Thr Val Thr Phe Glu Glu His Ile Lys 2620 2625 2630 gaa gaa cac aac atg tgg cac tat ctg tgc ttc atc gtg ctg gtg aaa 8272 Glu Glu His Asn Met Trp His Tyr Leu Cys Phe Ile Val Leu Val Lys 2635 2640 ggt aag gac tcc aca gag tac acc ggg cct gag agt tac gtg gca gag 8320 Val Lys Asp Ser Thr Glu Tyr Thr Gly Pro Glu Ser Tyr Val Ala Glu 2650 2655 2660 atg atc agg gaa aga aac ctt g at tgg ttc ctc aga atg aga gcc atg 8368 Met Ile Arg Glu Arg Asn Leu Asp Trp Phe Leu Arg Met Arg Ala Met 2665 2670 2675 2680 tcc ctg gtc agc agc gat tct gaa ggg gaa cag aac gag ctg Serg Ser Ser Asp Ser Glu Gly Glu Gln Asn Glu Leu Arg Asn 2685 2690 2695 ctg cag gag aag ctg gag tct acc atg aag ctg gtc acc aat ctt tct 8464 Leu Gln Glu Lys Leu Glu Ser Thr Met Lys Leu Val Thr Asn Leu Ser 2700 2705 2710 ggc cag ctg tca gaa cta aag gac cag atg aca gaa cag agg aag cag 8512 Gly Gln Leu Ser Glu Leu Lys Asp Gln Met Thr Glu Gln Arg Lys Gln 2715 2720 2725 aaa caa aga atc ggc ctt cta gga cct atg aat gtc aac 8560 Lys Gln Arg Ile Gly Leu Leu Gly His Pro Pro His Met Asn Val Asn 2730 2735 2740 cca cag cag ccg gcc taggcaaatg aggcagaggg actctgctca gccttcatgtcatgt gct gcat gct gct gct gct gct gct gct gct gct gct gc gct gc gct gc gct gc gc gct gc gct gtattgtatg tatatgattg ctattctcag 8735 aggtttggac tttcgtattg taattagctc tgttggcatg gtg acttgtc actcctgcca 8795 aaaatattaa aaatgccttt tttggaagga ctacagaaag tacctgattt gcacttgaac 8855 cagattatag atttaaaagt atatgacatg tattttgtat ttaaaactag aatagccagt 8915 atttatgttt tttataaaac tgtgcaatac aaattatgca atcaccataa ctttgtaact 8975 cctgagtgtc ctaagggagt acacatcttt gaagctgatt tgttgatact cgtgtaataa 9035 atggttaaat atcaaatgct gctgctgctg ccaaaattat attaatagcg agtttctggc 9095 ccctgggcaa ttttgtacct tgtaattatc ctatggtgat gctgtttctc gttgctaatg 9155 gcattagtgc ccctgtatcc tagtgataac tccaggtctg tgaaccattc aaacagcatt 9215 cattttgaga aaagcaactt tagtttcaag gataatttta agcttcaaaa ttaatcattt 9275 aaagtgtttc tttaagagag ccatgttaga ggctcacact ttagcttgaa aggagttgat 9335 gaattaattt tttaaaggga actttttaca tgacgtttgg aataacagca tattgctgac 9395 cagtcagtgt catctcccgg gtgaattttg atgtcacgtt atagtcaaat gagttagctg 9455 atggtttcta gattttcttc ctctgaacca tgatgcagta ggtaagaagt tattatgcgt 9515 atatacatat atacattcat atacgacaaa gtaggagctg tccccttagg atgcatagct 9575 gcccctaggg tacgtagctg aacactgaca atggcgttct tctgaaaga g ccacgtttgg 9635 gttttatttc tttgtcacat gatttctttt ctggatgggt gcaaagtatc acaggaagtg 9695 ttttctctct gtcgccttgt tttgtacctg ggtctcgctt tactagaccg tctctgca tacaga cag 9755 aaagttta gacta gag cc <210> 4 <211> 2749 <212> PRT <213> Mus musculus <400> 4 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 Val Asn Ser V al Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp 325 330 335 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Glu Lys Pro Val Met Leu 405 410 415 Lys Ile Gly T hr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr 450 455 460 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 480 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 Arg His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys Leu Leu Glu Lys 595 600 605 His Ile Thr Ala Ala Glu Ile Asp Thr Phe Val Ser Leu Val Arg Lys 610 615 620 Asn Arg Glu ProArg Phe Leu Asp Tyr Leu Ser Asp Leu Cys Val Ser 625 630 635 640 Met Asn Lys Ser Ile Pro Val Thr Gln Glu Leu Ile Cys Lys Ala Val 645 650 655 655 Leu Asn Pro Thr Asn Ala Asp Ile Leu Ile Glu Thr Lys Leu Val Leu 660 665 670 Ser Arg Phe Glu Phe Glu Gly Val Ser Thr Gly Glu Asn Ala Leu Glu 675 680 685 Ala Gly Glu Asp Glu Glu Glu Glu Val Trp Leu Phe Trp Arg Asp Ser Asn 690 695 700 Lys Glu Ile Arg Ser Lys Ser Val Arg Glu Leu Ala Gln Asp Ala Lys 705 710 715 720 Glu Gly Gln Lys Glu Asp Arg Asp Ile Leu Ser Tyr Tyr Arg Tyr Gln 725 730 735 Leu Asn Leu Phe Ala Arg Met Cys Leu Asp Arg Gln Tyr Leu Ala Ile 740 745 Asn Glu Ile Ser Gly Gln Leu Asp Val Asp Leu Ile Leu Arg Cys Met 755 760 765 765 Ser Asp Glu Asn Leu Pro Tyr Asp Leu Arg Ala Ser Phe Cys Arg Leu 770 775 780 Met Leu His Met His Val Asp Arg Asp Pro Gln Glu Gln Val Thr Pro 785 790 795 800 Val Lys Tyr Ala Arg Leu Trp Ser Glu Ile Pro Ser Glu Ile Ala Ile 805 810 815 Asp Asp Tyr Asp Ser Ser Gly Thr Ser Lys Asp Glu Ile Lys Glu Arg 820 825 830 Phe Ala Gln ThrMet Glu Phe Val Glu Glu Tyr Leu Arg Asp Val Val 835 840 845 Cys Gln Arg Phe Pro Phe Ser Asp Lys Glu Lys Asn Lys Leu Thr Phe 850 855 860 Glu Val Val Asn Leu Ala Arg Asn Leu Ile Tyr Phe Gly Phe Tyr Asn 865 870 875 880 Phe Ser Asp Leu Leu Arg Leu Thr Lys Ile Leu Leu Ala Ile Leu Asp 885 890 895 Cys Val His Val Thr Thr Ile Phe Pro Ile Ser Lys Met Thr Lys Gly 900 905 910 Glu Glu Asn Lys Gly Ser Asn Val Met Arg Ser Ile His Gly Val Gly 915 920 925 925 Glu Leu Met Thr Gln Val Val Leu Arg Gly Gly Gly Phe Leu Pro Met 930 935 940 Thr Pro Met Ala Ala Ala Ala Pro Glu Gly Asn Val Lys Gln Ala Glu Pro 945 950 955 960 Glu Lys Glu Asp Ile Met Val Met Asp Thr Lys Leu Lys Ile Ile Glu 965 970 975 Ile Leu Gln Phe Ile Leu Asn Val Arg Leu Asp Tyr Arg Ile Ser Cys 980 985 990 Leu Leu Cys Ile Phe Lys Arg Glu Phe Asp Glu Ser Asn Ser Gln Ser 995 1000 1005 Ser Glu Thr Ser Ser Gly Asn Ser Ser Gln Glu Gly Pro Ser Asn Val 1010 1015 1020 Pro Gly Ala Leu Asp Phe Glu His Ile Glu Glu Gln Ala Glu Gly Ile 025 1030 1035 1040 Phe Gly Gly Ser Glu Glu Asn Thr Pro Leu Asp Leu Asp Asp His Gly 1045 1050 1055 Gly Arg Thr Phe Leu Arg Val Leu Leu His Leu Thr Met His Asp Tyr 1060 1065 1070 Pro Pro Leu Val Ser Gly Ala Leu Gln Leu Leu Phe Arg His Phe Ser 1075 1080 1085 Gln Arg Gln Glu Val Leu Gln Ala Phe Lys Gln Val Gln Leu Leu Val 1090 1095 1100 Thr Ser Gln Asp Val Asp Asn Tyr Lys Gln Ile Lys Gln Asp Leu Asp 105 1110 1115 1120 Gln Leu Arg Ser Ile Val Glu Lys Ser Glu Leu Trp Val Tyr Lys Gly 1125 1130 1135 Gln Gly Pro Asp Glu Pro Met Asp Gly Ala Ser Gly Glu Asn Glu His 1140 1145 1150 Lys Lys Thr Glu Glu Gly Thr Ser Lys Pro Leu Lys His Glu Ser Thr 1155 1160 1165 Ser Ser Tyr Asn Tyr Arg Val Val Lys Glu Ile Leu Ile Arg Leu Ser 1170 1175 1180 Lys Leu Cys Val Gln Glu Ser Ala Ser Val Arg Lys Ser Arg Lys Gln 185 1190 1195 1200 Gln Gln Arg Leu Leu Arg Asn Met Gly Ala His Ala Val Val Leu Glu 1205 1210 1215 Leu Leu Gln Ile Pro Tyr Glu Lys Ala Glu Asp Thr Lys Met Gln Glu 1220 1225 1230 Ile Met Arg Leu Ala His Glu Phe Leu Gln Asn Phe Cys Ala Gly Asn 1235 1240 1245 Gln Gln Asn Gln Ala Leu Leu His Lys His Ile Asn Leu Phe Leu Lys 1250 1255 1260 Pro Gly Ile Leu Glu Ala Val Thr Met Gln His Ile Phe Met Asn Asn 265 1270 1275 1280 Phe Gln Leu Cys Ser Glu Ile Asn Glu Arg Val Val Gln His Phe Val 1285 1290 1295 His Cys Ile Glu Thr His Gly Arg Asn Val Gln Tyr Ile Lys Phe Leu 1300 1305 1310 Gln Thr Ile Val Lys Ala Glu Gly Lys Phe Ile Lys Lys Cys Gln Asp 1315 1320 1325 Met Val Met Ala Glu Leu Val Asn Ser Gly Glu Asp Val Leu Val Phe 1330 1335 1340 Tyr Asn Asp Arg Ala Ser Phe Gln Thr Leu Ile Gln Met Met Arg Ser 345 1350 1355 1360 Glu Arg Asp Arg Met Asp Glu Asn Ser Pro Leu Met Tyr His Ile His 1365 1370 1375 Leu Val Glu Leu Leu Ala Val Cys Thr Glu Gly Lys Asn Val Tyr Thr 1380 1385 1390 Glu Ile Lys Cys Asn Ser Leu Leu Pro Leu Asp Asp Ile Val Arg Val 1395 1400 1405 Val Thr His Glu Asp Cys Ile Pro Glu Val Lys Ile Ala Tyr Ile Asn 1410 1415 1420 Phe Leu Asn His Cys Tyr Val Asp Thr Glu Val Glu Met Lys Glu Ile 425 1430 1435 1440 Tyr Thr Ser Asn His Met Trp Lys Leu Phe Glu Asn Phe Leu Val Asp 1445 1450 1455 Ile Cys Arg Ala Cys Asn Asn Thr Ser Asp Arg Lys His Ala Asp Ser 1460 1465 1470 Ile Leu Glu Lys Tyr Val Thr Glu Ile Val Met Ser Ile Val Thr Thr 1475 1480 1485 Phe Phe Ser Ser Pro Phe Ser Asp Gln Ser Thr Thr Leu Gln Thr Arg 1490 1495 1500 Gln Pro Val Phe Val Gln Leu Leu Gln Gly Val Phe Arg Val Tyr His 505 1510 1515 1520 Cys Asn Trp Leu Met Pro Ser Gln Lys Ala Ser Val Glu Ser Cys Ile 1525 1530 1535 Arg Val Leu Ser Asp Val Ala Lys Ser Arg Ala Ile Ala Ile Pro Val 1540 1545 1550 Asp Leu Asp Ser Gln Val Asn Asn Leu Phe Leu Lys Ser His Asn Ile 1555 1560 1565 Val Gln Lys Thr Ala Leu Asn Trp Arg Leu Ser Ala Arg Asn Ala Ala 1570 1575 1580 Arg Arg Asp Ser Val Leu Ala Ala Ser Arg Asp Tyr Arg Asn Ile Ile 585 1590 1595 1600 Glu Arg Leu Gln Asp Ile Val Ser Ala Leu Glu Asp Arg Leu Arg Pro 1605 1610 1615 Leu Val Gln Ala Glu Leu Ser Val Leu Val Asp Val Leu His Arg Pro 1620 1625 1630 Glu Leu Leu Phe Pro Glu Asn Thr Asp Ala Arg Arg Lys Cys Glu Ser 1635 1640 1645 Gly Gly Phe Ile Cys Lys Leu Ile Lys His Thr Lys Gln Leu Leu Glu 1650 1655 1660 Glu Asn Glu Glu Lys Leu Cys Ile Lys Val Leu Gln Thr Leu Arg Glu 665 1670 1675 1680 Met Met Thr Lys Asp Arg Gly Tyr Gly Glu Lys Gln Ile Ser Ile Asp 1685 1690 1695 Glu Ser Glu Asn Ala Glu Leu Pro Gln Ala Pro Glu Ala Glu Asn Ser 1700 1705 1710 Thr Glu Gln Glu Leu Glu Pro Ser Pro Pro Leu Arg Gln Leu Glu Asp 1715 1720 1725 His Lys Arg Gly Glu Ala Leu Arg Gln Ile Leu Val Asn Arg Tyr Tyr 1730 1735 1740 Gly Asn Ile Arg Pro Ser Gly Arg Arg Glu Ser Leu Thr Ser Phe Gly 745 1750 1755 1760 Asn Gly Pro Leu Ser Pro Gly Gly Pro Ser Lys Pro Gly Gly Gly Gly 1765 1770 1775 Gly Gly Pro Gly Ser Ser Ser Thr Ser Arg Gly Glu Met Ser Leu Ala 1780 1785 1790 Glu Val Gln Cys His Leu Asp Lys Glu Gly Ala Ser Asn Leu Val Ile 1795 1800 1805 Asp Leu Ile Met Asn Ala Ser Ser Asp Arg Val Phe His Glu Ser Ile 1810 1815 1820 Leu Leu Ala Ile Ala Leu Leu Glu Gly Gly Asn Thr Thr Ile Gln His 825 1830 1835 1840 Ser Phe Phe Cys Arg Leu Thr Glu Asp Lys Lys Ser Glu Lys Phe Phe 1845 1850 1855 Lys Val Phe Tyr Asp Arg Met Lys Val Ala Gln Gln Glu Ile Lys Ala 1860 1865 1870 Thr Val Thr Val Val Asn Thr Ser Asp Leu Gly Asn Lys Lys Lys Asp Asp 1875 1880 1885 Glu Val Asp Arg Asp Ala Pro Ser Arg Lys Lys Ala Lys Glu Pro Thr 1890 1895 1900 Thr Gln Ile Thr Glu Glu Val Arg Asp Gln Leu Leu Glu Ala Ser Ala 905 1910 1915 1920 Ala Thr Arg Lys Ala Phe Thr Thr Phe Arg Arg Glu Ala Asp Pro Asp 1925 1930 1935 Asp His Tyr Gln Ser Gly Glu Gly Thr Gln Ala Thr Thr Asp Lys Ala 1940 1945 1950 Lys Asp Asp Leu Glu Met Ser Ala Val Ile Thr Ile Met Gln Pro Ile 1955 1960 1965 Leu Arg Phe Leu Gln Leu Leu Cys Glu Asn His Asn Arg Asp Leu Gln 1970 1975 1980 Asn Phe Leu Arg Cys Gln Asn Asn Lys Thr Asn Tyr Asn Leu Val Cys 985 1990 1995 2000 Glu Thr Leu Gln Phe Leu Asp Cys Ile Cys Gly Ser Thr Thr Gly Gly 2005 2010 2015 Leu Gly Leu Leu Gly Leu Tyr Ile Asn Glu Lys Asn Val Ala Leu Ile 2020 2025 2030 Asn Gln Thr Leu Glu Ser Leu Thr Glu Tyr Cys Gln Gly Pro Cys His 2035 2040 2045 Glu Asn Gln Asn Cys Ile Ala Thr His Glu Ser Asn Gly Ile Asp Ile 2050 2055 2060 Ile Thr Ala Leu Ile Leu Asn Asp Ile Asn Pro Leu Gly Lys Lys Arg 065 2070 2075 2080 Met Asp Leu Val Leu Glu Leu Lys Asn Asn Ala Ser Lys Leu Leu Leu 2085 2090 2095 Ala Ile Met Glu Ser Arg His Asp Ser Glu Asn Ala Glu Arg Ile Leu 2100 2105 2110 Tyr Asn Met Arg Pro Lys Glu Leu Val Glu Val Ile Lys Lys Ala Tyr 2115 2120 2125 Met Gln Gly Glu Val Glu Phe Glu Asp Gly Glu Asn Gly Glu Asp Gly 2130 2135 2140 Ala Ala Ser Pro Arg Asn Val Gly His Asn Ile Tyr Ile Leu Ala His 145 2150 2155 2160 Gln Leu Ala Arg His Asn Lys Glu Leu Gln Thr Met Leu Lys Pro Gly 2165 2170 2175 Gly Gln Val Asp Gly Asp Glu Ala Leu Glu Phe Tyr Ala Lys His Thr 2180 2185 2190 Ala Gln Ile Glu Ile Val Arg Leu Asp Arg Thr Met Glu Gln Ile Val 2195 2200 2205 Phe Pro Val Pro Ser Ile Cys Glu Phe Leu Thr Lys Glu Ser Lys Leu 2210 2215 2220 Arg Ile Tyr Tyr Thr Thr Glu Arg Asp Glu Gln Gly Ser Lys Ile Asn 225 2230 2235 2240 Asp Phe Phe Leu Arg Ser Glu Asp Leu Phe Asn Glu Met Asn Trp Gln 2245 2250 2255 Lys Lys Leu Arg Ala Gln Pro Val Leu Tyr Trp Cys Ala Arg Asn Met 2260 2265 2270 Ser Phe Trp Ser Ser Ile Ser Phe Asn Leu Ala Val Leu Met Asn Leu 2275 2280 2285 Leu Val Ala Phe Phe Tyr Pro Phe Lys Gly Val Arg Gly Gly Thr Leu 2290 2295 2300 Glu Pro His Trp Ser Gly Leu Leu Trp Thr Ala Met Leu Ile Ser Leu 305 2310 2315 2320 Ala Ile Val Ile Ala Leu Pro Lys Pro His Gly Ile Arg Ala Leu Ile 2325 2330 2335 Ala Ser Thr Ile Leu Arg Leu Ile Phe Ser Val Gly Leu Gln Pro Thr 2340 2345 2350 Leu Phe Leu Leu Gly Ala Phe Asn Val Cys Asn Lys Ile Ile Phe Leu 2355 2360 2365 Met Ser Phe Val Gly Asn Cys Gly Thr Phe Thr Arg Gly Tyr Arg Ala 2370 2375 2380 Met Val Leu Asp Val Glu Phe Leu Tyr His Leu Leu Tyr Leu Leu Ile 385 2390 2395 2400 Cys Ala Met Gly Leu Phe Val His Glu Phe Phe Phe Tyr Ser Leu Leu Leu 2405 2410 2415 Phe Asp Leu Val Tyr Arg Glu Glu Thr Leu Leu Asn Val Ile Lys Ser 2420 2425 2430 Val Thr Arg Asn Gly Arg Ser Ile Ile Leu Thr Ala Val Leu Ala Leu 2435 2440 2445 Ile Leu Val Tyr Leu Phe Ser Ile Val Gly Tyr Leu Phe Phe Lys Asp 2450 2455 2460 Asp Phe Ile Leu Glu Val Asp Arg Leu Pro Asn Glu Thr Ala Val Pro 465 2470 2475 2480 Glu Thr Gly Glu Ser Leu Ala Asn Asp Phe Leu Tyr Ser Asp Val Cys 2485 2490 2495 Arg Val Glu Thr Gly Glu Asn Cys Thr Ser Pro Ala Pro Lys Glu Glu 2500 2505 2510 Leu Leu Pro Ala Glu Glu Thr Glu Gln Asp Lys Glu His Thr Cys Glu 2515 2520 2525 Thr Leu Leu Met Cys Ile Val Thr Val Leu Ser His Gly Leu Arg Ser 2530 2535 2540 Gly Gly Gly Val Gly Asp Val Leu Arg Lys Pro Ser Lys Glu Glu Glu Pro 545 2550 2555 2560 Leu Phe Ala Ala Arg Val Ile Tyr Asp Leu Leu Phe Phe Phe Met Val 2565 2570 2575 Ile Ile Ile Val Leu Asn Leu Ile Phe Gly Val Ile Ile Asp Thr Phe 2580 2585 2590 Ala Asp Leu Arg Ser Glu Lys Gln Lys Lys Glu Glu Ile Leu Lys Thr 2595 2600 2605 Thr Cys Phe Ile Cys Gly Leu Glu Arg Asp Lys Phe Asp Asn Lys Thr 2610 2615 2620 Val Thr Phe Glu Glu His Ile Lys Glu Glu His Asn Met Trp His Tyr 625 2630 2635 2640 Leu Cys Phe Ile Val Leu Val Lys Val Lys Asp Ser Thr Glu Tyr Thr 2645 2650 2655 Gly Pro Glu Ser Tyr Val Ala Glu Met Ile Arg Glu Arg Asn Leu Asp 2660 2665 2670 Trp Phe Leu Arg Met Arg Ala Met Ser Leu Val Ser Ser Asp Ser Glu 2675 2680 2685 Gly Glu Gln Asn Glu Leu Arg Asn Leu Gln Glu Lys Leu Glu Ser Thr 2690 2695 2700 Met Lys Leu Val Thr Asn Leu Ser Gly Gln Leu Ser Glu Leu Lys Asp 705 2710 2715 2720 Gln Met Thr Glu Gln Arg Lys Gln Lys Gln Arg Ile Gly Leu Leu Gly 2725 2730 2735 His Pro Pro His Met Asn Val Asn Pro Gln Gln Pro Ala 2740 2745 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 tgtcagacat atgcgtgttg gaa 23 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 cgcgggatcc ttatttccgg ttgttgtgga gcaggg 36 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 ccggaattct tatttccggt tgttgtggag caggg 35 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 cgcggatcca tgaaatggag tgataacaaa gacgaca 37 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 gagagcggca ggcactgatg aggg 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 10 ccctcatcag tgcctgccgc tctc 24 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 11 gctgaggttc aagacctgga ctttg 25 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 12 aaagtccagg tcttgaacct cagc 24

[0119]

[Sequence List Free Text]

 SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 8: Synthetic DNA SEQ ID NO: 9: Synthetic DNA SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: Synthetic DNA SEQ ID NO: 12: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of an IP3 sponge.

FIG. 2 shows high expression of T604 and IP3 binding activity.

FIG. 3 shows the IP3 binding activity of an IP3 sponge.

FIG. 4 is a graph showing an IP3 sponge concentration-dependent IP3 binding inhibition curve.

FIG. 5. Low affinity G224m30 and GS in IP3-induced Ca ²⁺ release
It is a figure showing the effect of T.

FIG. 6 shows the inhibitory effect of high affinity IP3 sponge G224 on IP3-induced Ca ²⁺ release.

FIG. 7: Concentration-dependent IP3 of high affinity IP3 sponge G224
FIG. 4 shows an inhibition curve of induced Ca ²⁺ release.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 C12P 21/02 C // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Fumio Yoshikawa 7-13-31 Konandai, Konan-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Tsuyoshi Uchiyama 3-9-25-101, Kami-Osaki, Shinagawa-ku, Tokyo

Claims (11)

[Claims] 1. A recombinant polypeptide of the following (a), (b) or (c): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 wherein at least one amino acid has been deleted, substituted or added; A polypeptide having a high affinity binding activity to 4,5-triphosphate (c) having a homology of 70 to the amino acid sequence represented by SEQ ID NO: 2;
% Or more amino acid sequence, and inositol 1,4,
Polypeptide having high affinity binding activity to 5-triphosphate 2. A gene encoding the following polypeptide (a), (b) or (c). (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2; (b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 in which at least one amino acid has been deleted, substituted or added; A polypeptide having a high affinity binding activity to 4,5-triphosphate (c) having a homology of 70 to the amino acid sequence represented by SEQ ID NO: 2;
% Or more amino acid sequence, and inositol 1,4,
Polypeptide having high affinity binding activity to 5-triphosphate 3. A gene containing the following DNA (d) or (e): (d) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 (e) DNA comprising the nucleotide sequence having a homology of 70% or more with the DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, and inositol
DNA encoding a polypeptide having high affinity binding activity to 1,4,5-triphosphate 4. A DNA comprising a nucleotide sequence having a homology of 70% or more with the gene according to claim 2, and encoding a polypeptide having a high affinity binding activity to inositol 1,4,5-triphosphate. Genes containing. 5. A recombinant vector containing the gene according to any one of claims 2 to 4. A transformant comprising the recombinant vector according to claim 5. 7. A method of culturing the transformant according to claim 6, wherein a polypeptide having a high affinity binding activity to inositol 1,4,5-triphosphate is collected from the obtained culture. A method for producing the polypeptide described above. 8. An IP3 comprising the protein of claim 1.
Inducible calcium antagonist. 9. A therapeutic agent for a disease associated with calcium production, comprising the protein according to claim 1. 10. A gene comprising the gene according to claim 2 or 3.
Gene therapy agent for diseases associated with calcium production. 11. The method according to claim 11, wherein the disease is nervous system, vascular system, respiratory system,
11. The therapeutic agent according to claim 9 or 10, which is at least one selected from the group consisting of those occurring in tissues of the digestive system, lymph system, urinary system and reproductive system.