JPH08333400A - Fusion protein having antithrombotic activity and method for producing the same - Google Patents

Abstract

(57) [Summary] [Objective] To provide a highly stable antithrombotic fragment derived from human von Willebrand factor. [Structure] A glycoprotein Ib binding site of human von Willebrand factor on the surface of platelets (provided that one or more amino acid residues that do not substantially affect the activity of inhibiting the binding between human von Willebrand factor and platelets). Which may have a mutation, a deletion or an insertion of), and a peptide having at least a part thereof, and a highly stable protein,
A fusion protein having high stability and having an activity of inhibiting the binding between human von Willebrand factor and platelets is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fusion protein having antithrombotic activity, and more specifically, to a highly stable fusion protein containing a fragment derived from human von Willebrand factor, a method for producing the fusion protein, and the fusion protein. It relates to a pharmaceutical composition. Furthermore, the present invention provides a method for examining platelet function, which comprises measuring the platelet function using the fusion protein.

[0002]

2. Description of the Related Art It is widely known that platelets are deeply involved in the development of so-called thrombosis such as myocardial infarction and cerebral thrombosis ("platelet"; Yamanaka, Yamazaki ed .; Medical Institute, p158-). 163 (1991)). In recent years, von Willebrand factor, which is one of blood proteins, and glycoprotein Ib (glycoprotein) on the surface of platelets are involved in adhesion of platelets to vascular endothelial tissue, which is considered to be an early reaction leading to thrombosis. Ib) binding has been shown to be important (JPCean et al., J. Lab. Clin. Med.,
87, 586-596 (1976)).

It is known that the binding of these two types of proteins does not occur under normal conditions, but only under the condition that high shear stress is applied to platelets in vivo (TT Vi.
ncent et al., Blood, 65, 823-831 (1985)). Furthermore, as a method for observing this binding in vitro, an antibiotic ristocetin (MA Howard, BG Firkin, Th
romb. Haemastasis, 26, 362-369 (1971)), Botrocetin, a protein derived from snake venom (MS Read et al., Pro.
c. Natl. Acad. Sci. USA, 75, 4514-4518 (1978))
Methods using such substances are widely known. Aggregation of platelets occurs by adding these substances to the platelet suspension, and this aggregation depends on the binding between von Willebrand factor and glycoprotein Ib.

Several compounds have already been reported as substances exhibiting an inhibitory effect on platelet aggregation by ristocetin and botrocetin. For example, Olin
Aurin tricarboxylic acid (MD
Phillips et al., Blood, 72, 1989-1903 (1988)), pigment substances such as aromatic amidino compounds (JD Geratz eta
l., Thromb. Haemostasis, 39, 411-425 (1978)), as well as the snake Crotalus Holidas, which produces hemorrhagic toxins.
Peptides having a molecular weight of 25 kilodaltons have been reported by Holidas (Crotalus horridus horridus) and Cerastes cerastes (WO920847).
No. 2 international publication pamphlet). It has also been reported that a platelet aggregation inhibitory peptide similar to the above peptide was isolated from the venom of the snake extract, Echis carinatus (M. Peng et al., Blood, 81, 2321-2328 (1993)).

Further, a partial fragment of von Willebrand factor or a partial fragment of glycoprotein Ib is also
It is known to have platelet aggregation inhibitory activity (Y. F.
ujimura et al., J. Biol. Chem., 261, 381-385 (198
6), K. Titani. Et al., Proc. Natl. Acad. Sci. US
A., 84, 5610-5614 (1987)).

The region of von Willebrand factor binding to the platelet glycoprotein Ib was studied by von Willebrand factor fragmented by protease (Y. ujimura e
t al., J. Biol. Chem., 261, 381-385 (1986), M. Hon.
da et al., J. Biol. Chem. 261, 12579-12585 (1986))
And research on genetically modified von Willebrand factor fragment (M. Sugimoto et al., J. Biol. Chem., 268, 1
2185-12192 (1993)) reported that the region called A1 loop consisting of the cysteine residue at the 509th amino acid to the cysteine residue at the 695th amino acid of the natural von Willebrand factor is important. (H. Moh
ri et al., J. Biol. Chem., 263,17901-17904 (198
7), JE Sadler et al., J. Biol. Chem. 266, 22777.
-22780 (1991)). It is known that a von Willebrand factor fragment in which a part of amino acids in this region is deleted does not bind to the platelet glycoprotein Ib (M. Sugimot.
o et al., J. Biol. Chem. 268, 12185-12192 (199
3)). In addition, type IIb type von Willebrand's disease (L. Holmberg et al., N. Engl.) In which plasma von Willebrand factor disappears due to abnormally enhanced binding of von Willebrand factor to platelet glycoprotein Ib. J. Med. 30
9, 816-821 (1983)) is also present in the A1 loop (D. Ginsburg et al., Blood, 7).
9, 2507-2519 (1992)), platelet glycoprotein Ib in this region
The importance of binding to is shown.

[0007]

The shortest peptide currently reported as a von Willebrand factor-derived fragment that inhibits the binding between von Willebrand factor and platelet glycoprotein Ib at a low concentration of 1 μmol / L or less is a natural peptide. From tyrosine residue 508 of type von Willebrand factor 7
It is the 04th proline residue (CP Prior et al., Bi
o / technology, 11, 709-713 (1993)). However, when the A1 loop fragment of von Willebrand factor is produced by gene recombination technology, its solubility in aqueous solution is known to be poor (MA Cruz et al., J. Bi.
ol. Chem. 268, 21238-21245 (1993), M. Sugimoto et.
al., Biochemistry, 30, 5202-5209 (1991), C. Prior
et al., Bio / tecnology 10, 66-73 (1992)). Also, this fragment is rapidly degraded by protease.
Therefore, in order to use the fragment derived from von Willebrand factor as an antithrombotic drug, it was essential to improve the stability.

The present invention has been made from the above viewpoint, and an object of the present invention is to provide a highly stable antithrombotic fragment derived from von Willebrand factor.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that von Willebrand factor fragment is expressed and produced in the form of a stable protein and a fusion protein to obtain platelet sugars. The present invention has been completed by finding out that the protein can be obtained in a state of having a binding activity with the protein Ib.

That is, the present invention provides a protein having an activity of inhibiting the binding between human von Willebrand factor and platelets, which is a glycoprotein Ib binding site on the surface of human von Willebrand factor (provided that human von Willebrand factor is present). A peptide having at least a part of one or more amino acid residue mutations, deletions or insertions that do not substantially affect the activity of inhibiting the binding between the factor and platelets), A fusion protein with a highly stable protein; and a DNA encoding the fusion protein.

The present invention also provides a D encoding a fusion protein.
Culturing a microbial cell or an animal cell into which NA is introduced and expressing the fusion protein, producing and accumulating the recombinant protein in the cell or in a medium, and collecting the fusion protein from the culture. A method for producing a fusion protein is provided.

The present invention further provides a pharmaceutical composition containing the fusion protein and / or a pharmaceutically acceptable salt thereof as an active ingredient. Furthermore, the present invention provides a method for examining platelet function, which comprises measuring the platelet function using the fusion protein.

Hereinafter, the present invention will be described in detail. The fusion protein of the present invention is a protein having an activity of inhibiting the binding between human von Willebrand factor and platelets, and is a glycoprotein Ib binding site (hereinafter simply referred to as “glycoprotein Ib” on the platelet surface of human von Willebrand factor. Also referred to as the "binding site")
Is a fusion protein of a peptide having at least a part of the above (hereinafter also referred to as "human von Willebrand factor fragment") and a highly stable protein.

The fragment peptide having the amino acid sequence of the glycoprotein Ib binding site is highly sensitive to protease and is extremely unstable by itself, and it is difficult to produce it by cell engineering. Even if it can be produced, it is expected that when a pharmaceutical composition containing this peptide is administered to a patient, it will be rapidly degraded in the body. The present inventor can enhance stability by fusing such an unstable glycoprotein Ib binding site with a highly stable protein,
It was found that the obtained fusion protein has an activity of inhibiting the binding between human von Willebrand factor and platelets.

As the glycoprotein Ib binding site, specifically, the A1 loop, that is, the 509th amino acid (cysteine) residue to the 695th amino acid (cysteine) residue from the N-terminal of natural human von Willebrand factor Areas including up to. Glycoprotein I used in the present invention
The b-binding site may be a part of the fusion protein of this and a highly stable protein described below, as long as it has an activity of inhibiting the binding between human von Willebrand factor and platelets, and As long as the fusion protein does not retain the platelet aggregation activity of von Willebrand factor, it may include a region adjacent to the site. For example, the natural human von Willebrand factor has 508th amino acid (tyrosine) to 704th amino acid (proline) from the N-terminus.
A region containing up to residues can be preferably used in the present invention. The amino acid sequence of this region is shown in SEQ ID NO: 3.
In addition, the human von Willebrand factor fragment
Although it may be a natural type, it may have one or more amino acid residue mutations, deletions or insertions that do not substantially affect the activity of inhibiting the binding between human von Willebrand factor and platelets. Good. Examples of such mutations include amino acid mutations represented by mutation points of type IIb von Willebrand disease.

Examples of highly stable proteins used in the present invention include maltose binding protein, thioredoxin,
It may be a highly soluble protein such as glutathione S transferase, or may be serum albumin or various globulins that stably exist in blood. It may also be a part of these proteins. Further, synthetic peptides and other natural peptides may be used as long as they can enhance the stability of human von Willebrand factor fragment.

The mode of fusion of the human von Willebrand factor fragment and the highly stable protein is not particularly limited as long as the fusion protein is stable and has an activity of inhibiting the binding between human von Willebrand factor and platelets. However, if the highly stable protein is a maltose-binding protein or a part thereof, it is preferable to arrange the maltose-binding protein at the N-terminal side and the human von Willebrand factor fragment at the C-terminal side of the fusion protein. . Also,
A peptide derived from another protein or a synthetic peptide may be interposed at the fusion site. Further, another protein or the like may be bound to the end opposite to the end to which the highly stable protein is bound to the human von Willebrand factor fragment.

DNA encoding the fusion protein of the present invention
Is a DNA in which a first DNA fragment encoding a peptide having at least a part of a glycoprotein Ib binding site and a second DNA fragment encoding the above highly stable protein are linked. In addition, if necessary, a DNA encoding a peptide derived from another protein or a synthetic peptide, etc.
It may contain fragments.

DN encoding the above fusion protein
Microbial cells or animal cells into which A is introduced and expressing the fusion protein are cultured in a suitable medium, the recombinant protein is produced and accumulated in the cell or in the medium, and the fusion protein is collected from the culture. As a result, the fusion protein of the present invention can be obtained.

In order to introduce the DNA encoding the fusion protein into microbial cells or animal cells, a usual gene recombination technique is used, this DNA is incorporated into an expression vector suitable for the host cell, and the host cell is transformed with this expression vector. By transforming, or alternatively, the DNA is transformed into host chromosome D
This can be done by incorporating it in the NA. When introducing the DNA of the present invention into a host cell, a promoter and terminator suitable for the host cell may be appropriately linked. Also, a drug resistance marker gene suitable for selection of transformed cells may be linked.

Examples of microorganisms used as hosts include bacteria such as Escherichia coli (E. coli), Bacillus subtilis (Bacillus subtilis) and actinomycetes, and eukaryotic microorganisms such as yeast and filamentous fungi. . Examples of animal cells include animal-derived cultured cells and insect cultured cells.

When the fusion protein is expressed in a host cell, it may be expressed as a soluble protein in the cells of Escherichia coli, or may be secreted and produced in the periplasm. Alternatively, Gram-positive bacteria such as Bacillus subtilis and actinomycetes may be secreted and produced extracellularly as a host. When the sugar chain is involved in the stability of the protein fused to the human von Willebrand factor fragment, yeast, filamentous fungi, or animal or insect cultured cells are used as the host, and the form having the sugar chain is used. The expression product may be secreted and produced extracellularly. The expressed and produced fusion protein is preferably accumulated in the cell or medium as a soluble protein.

The transformed cells are cultivated in a medium suitable for the host cells, and if necessary, an inducer and a drug suitable for inducing the promoter are added. The fused protein accumulated in cells or medium after culturing is subjected to gel filtration, ion exchange, adsorption, various column chromatography such as reverse phase, ultrafiltration, electrophoresis, and countercurrent distribution in the same manner as for purification of ordinary proteins. And the like can be combined to purify. When a maltose-binding protein is used as a highly stable protein, the fusion protein can be purified by affinity column chromatography using amylose resin or the like.

The pharmaceutical composition of the present invention contains the fusion protein of the present invention and / or a pharmaceutically acceptable salt thereof as an active ingredient. The fusion protein of the present invention has an activity of greatly inhibiting the binding between von Willebrand factor and platelets, particularly the binding of von Willebrand factor caused by botrocetin to platelets, and is expected to have an antithrombotic effect. It The fusion protein and its salt may be used alone or as a mixture of two or more kinds. Further, a substance having an antithrombotic effect other than the fusion protein of the present invention may be contained. In this case, the fusion protein of the present invention need not be the main component in the pharmaceutical composition. Further, other materials usually used in preparations, for example, proteins such as serum albumin, salts for buffering action and adjusting osmotic pressure, carriers, excipients and other components may be added.

Examples of the dosage form include tablets, capsules, fine granules, syrups, suppositories, ointments, injections and eye drops. The administration method may be intravenous administration, subcutaneous administration, oral administration, eyedrops, enteral administration, or the like.

When the pharmaceutical composition of the present invention is administered as an antithrombotic agent to animals or humans, the dose is usually 0.1 μg / amount of the fusion protein of the present invention and / or a pharmaceutically acceptable salt thereof. A desired effect can be expected in the range of kg to 100 mg / kg, and an amount that provides the best medicinal effect can be selected from this range.

The diagnostic agent of the present invention contains the fusion protein of the present invention. For example, by measuring the binding ability to platelets using the labeled fusion protein, it is possible to test platelet function, especially to diagnose thrombosis. In this case, a radioisotope such as iodine 125 ( ¹²⁵ I) can be used for labeling the fusion protein, or a coloring reagent can be used. When a coloring reagent is used, for example, the fusion protein of the present invention is labeled with biotin, and avidin or an anti-avidin antibody to which alkaline phosphatase is bound is used to measure the color development by the phosphatase reaction to bind the fusion protein to platelets. Can be detected. Further, a labeled fusion protein premixed with a factor that activates von Willebrand factor such as botrocetin or ristocetin is used to measure the binding ability to platelets, whereby a more detailed examination of platelet function can be performed.

[0028]

EXAMPLES The present invention will be described in more detail below with reference to examples. In the following examples, the human von Willebrand factor may be referred to as “hvWF”.

[0029]

Example 1 Human von Willebrand factor (hvWF)⁵⁰⁸
Tyr-⁷⁰⁴Construction of Pro fragment expression system <1> hvWF⁵⁰⁸Tyr-⁷⁰⁴Code the Pro fragment
Acquisition of DNA Tyrosine residue 508 of natural hvWF (⁵⁰⁸Tyr) to 704
Th proline residue ( ⁷⁰⁴(Pro) fragment
Polymerase chain reaction (PCR) method
From a human cDNA library by amplification with
I got it. The primers used for PCR are
Two kinds of synthetic ores that were outsourced to Io Service Co., Ltd.
Gonucleotide (VWP1: Sequence Listing SEQ ID NO: 1, VWP2: Sequence
Table SEQ ID NO: 2) was used. At this time, VWP1 has a restriction enzyme
BamHI recognition sequence (nucleotide number in SEQ ID NO: 1
4-9) and a cleavage recognition peptide for blood coagulation factor X
Isoleucine-glutamic acid-glycine-arginine
Encoding DNA sequence (Nucleotide in SEQ ID NO: 1)
No. 10 to 21) are included. In addition, VWP2 has a limited fermentation
Elementary SalI recognition sequence (nucleotide number in SEQ ID NO: 2
4-9) are included.

On the other hand, as the human cDNA library used as the template DNA, a human vascular endothelial cell cDNA library (manufactured by Clontech) was used. The PCR reaction was carried out in the following reaction solution composition using a DNA thermal cycler (manufactured by Perkin Elmer) at 95 ° C for 1 minute, 37 ° C for 2 minutes and 7
It was carried out by repeating a reaction cycle consisting of 3 minutes at 4 ° C. for 30 cycles.

(Composition of reaction solution) cDNA library DNA: 5 μg PCR reaction buffer: 10 μl 2 mM dNTP: 10 μl Primer: 50 pmol each of Taq polymerase: 10 units (total solution volume 100 μl)

D amplified by PCR as described above
NA was treated with phenol to inactivate Taq polymerase, and then purified by ethanol precipitation. This DNA and vector plasmid pUC18 were double-digested with restriction enzymes BamHI and SalI (Takara Shuzo Co., Ltd.), respectively. Each digested fragment was subjected to agarose gel electrophoresis,
DNA fragments of about 2700 base pairs were recovered from the agarose gel using a DNA recovery kit (EASYTRAP: manufactured by Takara Shuzo Co., Ltd.), and each fragment was recovered using a ligation kit (Takara Shuzo Co., Ltd.). Were ligated to form a cyclized product.

E. coli JM109 was transformed with the obtained recombinant plasmid by the competent cell method, and L agar medium containing 100 μg / ml ampicillin (1% bactotryptone, 0.5% yeast extract, Sodium chloride 0.5
%, Agar 2%), and transformants were selected.

The transformant thus obtained was liquid-cultured, and then the plasmid DNA was extracted by the alkaline SDS method. The nucleotide sequence of the insert fragment contained in the obtained plasmid DNA was determined using two kinds of primers M13M4 and M13reverse (manufactured by Takara Shuzo Co., Ltd.) and a DNA sequencer A373 (manufactured by Perkin Elmer Co., Ltd.). It was confirmed that the DNA having the sequence of was obtained. The amino acid sequence corresponding to the nucleotide sequence of the insert fragment is shown in SEQ ID NO: 4. Further, only the amino acid sequence is shown in SEQ ID NO: 5. The plasmid thus obtained was designated as pUCvWF.

<2> Construction of Plasmid Expressing Maltose Binding Protein-hvWF Fragment Fusion Protein The expression vector pMal expressing the maltose binding protein under the control of the tac promoter is used for the DNA encoding the hvWF fragment obtained as described above. -c2 (manufactured by New England Biolab) was constructed to construct a vector expressing a fusion protein of maltose binding protein and hvWF fragment. Ie pUCvWF and pMal
-c2 was double-digested with restriction enzymes BamHI and SalI (Takara Shuzo Co., Ltd.), and each digested fragment was subjected to agarose gel electrophoresis to obtain a DNA fragment of about 200 base pairs and about 600 base pairs, respectively. Was recovered using a DNA recovery kit (EASYTRAP: manufactured by Takara Shuzo Co., Ltd.). Recombinant DN obtained by ligating these DNA fragments using a ligation kit (manufactured by Takara Shuzo Co., Ltd.)
E. coli JM109 was transformed with A by the competent cell method and grown on L agar medium containing 100 μg / ml of ampicillin to select transformants.

The transformant thus obtained was cultivated in liquid, and the plasmid was extracted by the alkaline SDS method. The recombinant plasmid thus obtained was named pMalVWF. The structure of pMalVWF is shown in FIG. pMalVWF has a tac promoter, a DNA encoding a maltose binding protein-hvWF fragment fusion protein, a rrnB terminator, and an ampicillin resistance gene.

[0037]

Example 2 Production of Maltose-Binding Protein-hvWF Fragment Fusion Protein <1> Expression of Maltose-Binding Protein-hvWF Fragment Fusion Protein The trait retaining the maltose-binding protein-hvWF fragment fusion protein expression plasmid pMalVWF obtained in Example 1. The transformant was inoculated into 10 flasks containing 100 ml of L broth (1% of bactotryptone, 0.5% of yeast extract, 0.5% of sodium chloride, pH 7.2), and cultured by shaking at 37 ° C for 16 hours, followed by IPTG. (Isopropyl-β-D-thiogalactopyranoside, final concentration 0.5 mM) was added, and the mixture was further cultured for 4 hours.

The above culture solution was centrifuged at 4000 × g to collect the bacterial cells. 50 ml of the column buffer solution (20 mM Tris-hydrochloric acid (pH 7.4), 200 mM sodium chloride, 1 mM EDTA)
The cells were resuspended in 20 ml of a buffer solution for columns, after being resuspended in 20 ml, and centrifuged again to wash the cells. Ultrasonic crusher
Sonicate at 200W for 10 minutes using 200M (Kubota),
The cells were crushed. The disrupted solution was centrifuged at 9,000 xg for 20 minutes to remove the precipitated fraction. The crude extract thus obtained was diluted by adding 150 ml of a column buffer.

Maltose binding protein-hvWF fragment fusion protein was purified from the above crude extract by affinity chromatography with amylose resin (New England Biolabs). That is, the amylose resin column (amylose resin
15 ml) was equilibrated, the crude extract prepared as described above was adsorbed to the resin, and the column was washed with a column buffer, and then the elution buffer (20 mM Tris-hydrochloric acid (pH 7.4), 200 mM sodium chloride was added. , 1 mM EDTA, 10 mM maltose) was used to elute the fusion protein at a flow rate of 1 ml / min. An example of this affinity chromatogram is shown in FIG. Moreover, when the protein before and after purification was subjected to SDS-polyacrylamide gel electrophoresis, a molecular weight of 65 estimated as a fusion protein,
It was confirmed that a protein of 000 daltons was produced and accumulated in the bacterial cells in a significant amount, and that an almost single protein could be purified by the affinity chromatogram.

<2> Activity of Maltose-Binding Protein-hvWF Fragment Fusion Protein Inhibition of binding of hvWF to platelets caused by botrocetin or ristocetin by the maltose-binding protein-hvWF fusion protein prepared and purified as described above. It was measured by the following method.

The formalin-immobilized platelets used in the test were prepared as follows. Human platelet-rich plasma (pla obtained by centrifuging fresh blood collected from a healthy person at 900 rpm for 15 minutes)
1/10 volume of 3.8% sodium citrate was added to the telet rich plasma, physiological saline containing the same volume of 2% paraformaldehyde was further added, and the mixture was allowed to stand at 4 ° C overnight for storage. After storage, the platelets were collected by centrifugation and washed twice with a 0.15 M sodium chloride aqueous solution containing 20 mM phosphate buffer (pH 7.4). Thereafter, the immobilized platelets were suspended in the same solution and stored.

For the binding of ¹²⁵ I-labeled hvWF to the immobilized platelets obtained as described above,
Inhibition by a maltose binding protein-hvWF fragment fusion protein is described by Chopek et al. (MW Chopek et al., B
iochemistry, 25, 3146-3155 (1986)). That is, the prepared immobilized platelet suspension, maltose binding protein as a measurement sample or control, and botrocetin or ristocetin,
¹²⁵ I-labeled hvWF was added, and the mixture was reacted at room temperature for 30 minutes. The immobilized platelets were then collected by centrifugation and
After washing with 0.15 M sodium chloride aqueous solution containing mM phosphate buffer (pH 7.4), the suspension was resuspended in the same buffer and the radioactivity was measured by a gamma counter (Packard Multi-Pr
ias, manufactured by Packard) to measure the amount of hvWF bound to the immobilized platelets.

The inhibition of von Willebrand factor-induced binding to platelets caused by botrocetin is shown in FIG. On the other hand, inhibition of hvWF binding to platelets caused by ristocetin is shown in FIG. As a result, the inhibitory activity against the binding of hvWF to platelets caused by botrocetin was IC ₅₀ = 10 μg / ml, but the inhibitory activity against the binding of hvWF to platelets caused by ristocetin induction was up to 300 μg / ml. I was not able to admit. On the other hand, the control maltose-binding protein alone showed no inhibitory activity on the binding of hvWF to platelets caused by botrocetin or ristocetin. From this result, the maltose-binding protein-hvWF fragment fusion protein specifically hvWF only in the presence of botrocetin.
Was determined to have the activity of inhibiting the binding of the to the platelets.

<3> Stability of Maltose Binding Protein-hvWF Fusion Protein In order to evaluate the stability of maltose binding protein-hvWF fusion protein, the fusion protein was digested with blood coagulation factor Xa which is a protease, and maltose binding protein was digested. And the hvWF fragment were cleaved. That is, a buffer solution containing 20 μg each of maltose-binding protein-hvWF fusion protein purified by amylose resin-affinity column chromatography and control maltose-binding protein (elution buffer: 2
0mM Tris-HCl (pH7.4), 200mM sodium chloride, 1mM EDTA, 10m
Blood coagulation factor X (manufactured by New England Biolab) (200 ng (1 μl)) was added to M maltose) and incubated at 32 ° C. for 2, 4, 6, 24 hours, and 5 μl of reaction solution.
Was subjected to SDS-polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue and analyzed.

As a result, the protein having a molecular weight of 65,000 corresponding to the fusion protein decreased with the protease treatment time,
The protein corresponding to 40,000 of the molecular weight of maltose binding protein was increased. On the other hand, a protein having a molecular weight of 25,000 corresponding to the hvWF fragment was not detected. The gel after this electrophoresis was transferred to a nylon filter and subjected to Western blotting using a polyclonal antibody against the anti-maltose binding protein. As a result, a protein having a molecular weight of 65,000 and a protein having a molecular weight of 40,000 were detected and excised by blood coagulation factor Xa. The protein was proved to be a maltose binding protein. From these results, it was expected that the hvWF fragment was decomposed into a protein having a smaller molecular weight by a protease.

When the maltose binding protein-hvWF fusion protein purified by affinity chromatography was stored at 4 ° C., it was not degraded for at least 1 month and inhibited the binding of von Willebrand factor induced by botrocetin to platelets. No decrease in activity was observed and it was stable.

From the above results, it was shown that the ⁵⁰⁸ Tyr- ⁷⁰⁴ Pro fragment of hvWF is very sensitive to proteases, unstable, and stable only when present as a fusion protein.

[0048]

INDUSTRIAL APPLICABILITY According to the present invention, a fusion protein having a fragment derived from human von Willebrand factor, having an activity of inhibiting the binding between human von Willebrand factor and platelets and having high stability is obtained. To be

[0049]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 46 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence type Sequence CTAGGATCCA TCGAAGGTCG TTACTGCAGC AGGCTACTGG ACCTGG 46

SEQ ID NO: 2 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence type Sequence TCTGTCGACT CAAGGAGGAG GGGCTTCAGG GGCAAGG 37

SEQ ID NO: 3 Sequence length: 197 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser 1 5 10 15 Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp 20 25 30 Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val 35 40 45 Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg 50 55 60 Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala 65 70 75 80 Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe 85 90 95 Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Leu 100 105 110 Leu Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val 115 120 125 Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val 130 135 140 Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys 145 150 155 160 Gln Ala Pro Glu Asn lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu 165 170 175 Glu Gln Gln Arg Asp Glu Ile Val ser Tyr Leu Cys Asp Leu Ala Pro 180 185 190 Glu Ala Pro Pro Pro 195

SEQ ID NO: 4 Sequence length: 618 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Sequence features Characteristic symbols: CDS Location: 7..609 Method of characterization: S sequence GGATCC ATC GAA GGT CGT TAC TGC AGC AGG CTA CTG GAC CTG GTC TTC 48 Ile Glu Gly Arg Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe 1 5 10 CTG CTG GAT GGC TCC TCC AGG CTG TCC GAG GCT GAG TTT GAA GTG CTG 96 Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu 15 20 25 30 AAG GCC TTT GTG GTG GAC ATG ATG GAG CGG CTG CGC ATC TCC CAG AAG 144 Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys 35 40 45 TGG GTC CGC GTG GCC GTG GTG GAG TAC CAC GAC GGC TCC CAC GCC TAC 192 Trp Val Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr 50 55 60 ATC GGG CTC AAG GAC CGG AAG CGA CCG TCA GAG CTG CGG CGC ATT GCC 240 Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala 65 70 75 AGC CAG GTG AAG TAT GCG GGC AGC CAG GTG GCC TCC ACC AGC GAG GTC 288 Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val 80 85 90 TTG AAA TAC ACA CTG TTC CAA ATC TTC AGC AAG ATC GAC CGC CCT GAA 336 Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu 95 100 105 110 GCC TCC CGC ATC GCC CTG CTC CTG ATG GCC AGC CAG GAG CCC CAA CGG 384 Ala Ser Arg Ile Ala Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg 115 120 125 ATG TCC CGG AAC TTT GTC CGC TAC GTC CAG GGC CTG AAG AAG AAG AAG 432 Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys 130 135 140 GTC ATT GTG ATC CCG GTG GGC ATT GGG CCC CAT GCC AAC CTC AAG CAG 480 Val Ile Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln 145 150 155 ATC CGC CTC ATC GAG AAG CAG GCC CCT GAG AAC AAG GCC TTC GTG CTG 528 Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu 160 165 170 AGC AGT GTG GAT GAG CTG GAG CAG CAA AGG GAC GAG ATC GTT AGC TAC 576 Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr 175 180 185 190 CTC TGT GAC CTT GCC CCT GAA GCC CCT CCT CCT TGAGTCGAC 618 Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro 195 200

SEQ ID NO: 5 Sequence length: 201 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Glu Gly Arg Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu 1 5 10 15 Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala 20 25 30 Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val 35 40 45 Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile Gly 50 55 60 Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser Gln 65 70 75 80 Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu Lys 85 90 95 Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser 100 105 110 Arg Ile Ala Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser 115 120 125 Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile 130 135 140 Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg 145 150 155 160 Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser 165 170 175 Val Asp Glu Leu Glu G ln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys 180 185 190 Asp Leu Ala Pro Glu Ala Pro Pro Pro 195 200

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic structure of a fusion peptide expression plasmid pMalVWF in Escherichia coli.

FIG. 2 is a chromatogram for purification of fusion peptide by affinity column.

FIG. 3 is a graph showing the inhibitory activity on the binding of von Willebrand factor induced by the fusion peptide botrocetin to platelets.

FIG. 4 shows the inhibitory activity on the binding of von Willebrand factor to platelets caused by the fusion peptide ristocetin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C12N 1/21 9162-4B C12N 15/00 ZNAA (C12P 21/02 C12R 1:19) ( 72) Inventor, Koichi Ishii, Ajinomoto Co., Ltd., Central Research Laboratory, 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa (72) Inventor, Miki 1-1: 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa ) Inventor Yoshihiro Fujimura 133-1, Sugawara-Higashi-cho, Nara, Nara (72) Inventor Shuji Miura 30-Kaise-cho, Kashihara-shi, Nara (72) Inventor Kosei Nishida 902 Sakurai, Sakurai-shi, Nara

Claims (7)

[Claims] 1. A protein having an activity of inhibiting the binding between human von Willebrand factor and platelets, which is glycoprotein I of human von Willebrand factor on the surface of platelets.
b binding site (however, it does not substantially affect the activity of inhibiting the binding between human von Willebrand factor and platelets 1
Alternatively, a fusion protein of a peptide having at least a part of (mutation, deletion or insertion of 2 or more amino acid residues) and a protein having high stability. 2. A human von Willebrand factor glycoprotein Ib binding site on the platelet surface has a residue from the 508th amino acid (tyrosine) residue to the 704th amino acid (proline) residue of the natural human von Willebrand factor. The fusion protein according to claim 1, which is a site up to the base. 3. The highly stable protein is a maltose binding protein or a part thereof.
The described fusion protein. 4. A DNA encoding the fusion protein according to claim 1. 5. A microbial cell or an animal cell, into which the DNA encoding the fusion protein according to any one of claims 1 to 3 is introduced and which expresses the fusion protein, is cultured, and the microbial cell or animal cell is cultured in a cell or in a medium. Produce and accumulate recombinant proteins,
A method for producing the fusion protein, which comprises collecting the fusion protein from the culture. 6. A pharmaceutical composition containing the fusion protein according to any one of claims 1 to 3 and / or a pharmaceutically acceptable salt thereof as an active ingredient. 7. A method for examining platelet function, which comprises measuring the platelet function using the fusion protein according to claim 1.