JP2829397B2 - Fibrin binding active polypeptide - Google Patents

Description

The present invention relates to a fibrin-binding activity polypeptide of fibronectin acid, and more particularly, to a fibrin-binding activity polypeptide containing a C-terminal fibrin-binding domain polypeptide of human fibronectin. And a nucleic acid encoding the same, and a method for producing the DNA using the DNA by genetic engineering.

[Conventional technology]

Fibronectin (hereinafter referred to as FN) is a glycoprotein present in plasma and extracellular matrix, and is known to have various functions [Annual Review of Biochemis
try), Vol. 57, pp. 375-413 (1988)]. Attempts have been made to use natural FN for wound healing, medicines and cosmetics such as eye drops, but the supply is limited because it is collected from blood, the cost is high, and pathogenic bacteria and It has not been put to practical use because of the possibility of contamination by viruses and the like. Extraction and use of a functional domain of natural FN has not been put to practical use for the same reason.

[Problems to be solved by the invention]

FN has two regions that bind to fibrin (fibrin binding domains), one at the amino (N) terminus and the other at the carboxy (C) terminus. The fibrin-binding domain polypeptide is expected to be used as an anticoagulant by inhibiting the binding of platelets to fibrin. Also, recent knowledge suggests that FN
It has been clarified that the C-terminal fibrin-binding domain has an insulin-binding activity (the 46th Annual Meeting of the Cancer Society of Japan, page 181), and its use as a drug delivery system is also conceivable.

An object of the present invention is to provide a fibrin-binding polypeptide having FN-fibrin-binding activity, and an advantageous method for producing the same.

[Means for solving the problem]

In summary, the first invention of the present invention relates to a fibrin-binding polypeptide, and has the following general formula I: (W) _l- (X) _m- (Y) _n -Z _} [ I] [wherein, W represents a methionine residue (Met), X represents an alanine residue (Ala), and Y is a polypeptide residue of human fibronectin, corresponding to Asn ²¹³³ -Ser ²¹⁴³ .
Denotes an 11 amino acid polypeptide residue and has the following Formula II: Wherein Z is the C of human fibronectin.
Represents a 132 amino acid polypeptide residue corresponding to Cys ²¹⁴⁴ -Gly ²²⁷⁵ of the terminal fibrin binding domain, and has the following formula II
I: And l, m, and n each represent a number of 1 or zero.]

The second invention of the present invention relates to a nucleic acid encoding the fibrin-binding activity polypeptide of the first invention.

Further, a third invention of the present invention relates to a recombinant plasmid containing a DNA encoding the fibrin-binding activity polypeptide represented by the above general formula I, and a fourth invention relates to the introduction of the recombinant plasmid. A fifth aspect of the present invention relates to a method for producing these polypeptides, which comprises culturing the transformant and collecting the fibrin-binding activity polypeptide represented by the general formula I from the culture. About the method.

For the C-terminal fibrin binding domain, fragments obtained by digestion with trypsin, thermolysin, plasmin and the like have been reported (fibronectin, D.
F. Mosher, Academic Press
Inc., p. 73 (1988)], starting at 20 kD
It reaches 34kD. Although the details of this domain have not been specified in detail, a fragment containing three type I analogous sequences generally consisting of about 44 amino acids, and adjacent sequences having no homology with them before and after them is known. . The present inventors have
The construction of a polypeptide having the fibrin-binding activity of FN and the method for producing the same were studied, and the fibrin-binding activity and insulin-binding activity were determined by comparing the type I analogous sequence portion (Cys ²¹⁴⁴ -Gly ²²⁷⁵ , 13
2 amino acid residues) is sufficient, and the presence or absence of the peptide sequence existing before and after the type I analogous sequence portion significantly affects the expression level in E. coli cells. Was found. The present invention has been achieved based on the above findings. In this specification, the superscripts attached to the amino acids represent EMBL DATA BANK.
NK) shows the number of amino acid residues from the N-terminal added to the amino acid sequence obtained by translating the cDNA sequence of FN in N. Hereinafter, the present invention will be described specifically.

For the primary structure of human FN protein, see The ENBO Journal, Volume 4, 1755.
~ 1759 (1985). The DNA fragment encoding the C-terminal fibrin binding domain is pLF5, pLF5,
LF3, pLF4 and pLF2435 constructed by joining the FN cDNA portions of pLF2 [Biochemistry,
Vol. 25, pp. 4936-4494 (1986)]. A necessary cDNA fragment was excised from pLF2435 with a restriction enzyme, and a synthetic DNA containing a start codon on the 5 ′ side and a synthetic DNA containing a stop codon on the 3 ′ side were ligated with DNA ligase, respectively, and then added to an appropriate expression vector. By the connection, a plasmid encoding the polypeptide of interest can be obtained (see FIGS. 1 and 2). That is, FIG. 1 shows the W-
FIG. 2 is a flow chart for constructing a plasmid pFD705 encoding XYZ, and FIG. 2 is a flow chart for constructing a plasmid pFD905 encoding WXX.

At the N-terminus of the polypeptide expressed by the plasmid thus obtained, an initiation codon derived from a synthetic DNA containing an NcoI linker or an NcoI site existing at the site where the 5 'end of the cDNA is linked to the vector. (In the above formula I, represented by W) and Ala residue (in the above formula I, represented by X), which do not affect the effects of the present invention. However, these additional sequences can be removed if necessary. For example, for Met, methionine aminopeptidase contained in the recombinant [Journal of Bacterology (J. Bacter
iol.) Vol. 169, p. 751, (1987)] by culturing the recombinant under conditions that facilitate the action or by allowing methionine aminopeptidase to act on the partially purified polypeptide. Met can be removed. Ala can be removed by site-directed mutagenesis.

As the expression vector, any of the existing vectors can be used. For example, pUC118N / pUC119N [Febus
FEBS Letters, Volume 223, Pages 174-180 (1
987)] and its derivatives can be used to obtain good results. By introducing these plasmids into E. coli and culturing them under appropriate conditions, the C-terminal fibrin-binding domain polypeptide is accumulated in E. coli. Immunoblotting is used to confirm expression. After separating the whole cell proteins of the recombinant E. coli by SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
Transfer the migration pattern to a nitrocellulose membrane. FN C
The band detected by the monoclonal antibody (HFN-11F6, Serotech) recognizing the terminal fibrin binding domain is the target polypeptide.

The target polypeptide is insolubilized in Escherichia coli cells to form a so-called inclusion body. For this reason, the purification of the target polypeptide is performed, for example, as follows.

After culturing the recombinant Escherichia coli in a medium such as L-broth and collecting the cells, a lysate of the cells is obtained by sonication, and this is centrifuged to reduce the precipitation of the inclusion body containing the target polypeptide.
This precipitate is washed with various surfactants [eg, Triton
n) The precipitate is washed by repeating suspension and centrifugation in a buffer containing X-100 or the like. This precipitate is dissolved in a buffer containing urea and dithiothreitol, and the solution is subjected to gel filtration column chromatography. Next, the target polypeptide fraction is refolded by a method such as dialysis. By the above operation, the target polypeptide can be purified.

The obtained polypeptide is used for measurement of fibrin binding activity and insulin binding activity.

For fibrin binding activity, human fibrin
-A fixed amount of sample is adsorbed to a fibrin-immobilized column prepared by binding to an activated carrier for affinity chromatography such as Tresyl Toyopearl 650 (Tosoh), eluted with an arginine solution, and passed through this column. The ability to bind to fibrin can be shown by comparing the amount of sample obtained and the amount of sample eluted from this column.

Insulin binding activity can be determined according to the immunoblotting technique. That is, the sample is
-After separation by PAGE, transfer to a nitrocellulose filter and allow enzyme-labeled insulin to act. After washing the filter, the bound insulin can be determined by enzyme activity.

The above measurement proves that the obtained polypeptide has a strong affinity for fibrin and insulin.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 C-terminal fibrin binding domain of FN Asn ²¹³³ -Gly
Cloning of cDNA fragment encoding ²²⁷⁵ (143 amino acid residues, hereinafter abbreviated as F-143) (see FIG. 1) (1-1) Preparation of synthetic DNA adapter Type I repeat present in C-terminal fibrin binding domain The 3′-side adapter (chain length 29 mer and 37 mer, see FIG. 1) for connecting the sequence and the 11 amino acid sequence adjacent upstream to the vector was synthesized using a DNA synthesizer manufactured by Applied Biosystems. 300pmol chain length 29mer
After phosphorylating the 5 'end of the product, an equal amount of DNA having a chain length of 37 mer was added, and an annealing operation was performed to form a double strand.

(1-2) Preparation of NcoI-HindIII Fragment A 5.9 kb plasmid pLF2435 containing a cDNA fragment encoding F-143 of FN [Biochemistry Vol. 25, Nos. 4936-4
941 (1986)] 100 μg was digested with Sau3A I and subjected to agarose gel electrophoresis to recover a 0.96 kb fragment. This fragment was further cut with Hinc II and Hae II, and subjected to agarose gel electrophoresis to recover the 405b fragment.
2 μg of this fragment and 150 pmol of NcoI linker [Takara Shuzo Co., Ltd., d (pA-G-C-C-C-A-T-G-G-C-
T)] and 150 pmo of the 3′-side adapter obtained in (1-1).
l Buffer for T4 DNA ligase, 0.5 mM ATP, 10 mM DT
Incubate at 15 ° C. overnight in a 125 μl reaction containing 300 units of T4 DNA ligase. Reaction solution at 70 ° C,
After incubation for 10 minutes, T4 polynucleotide kinase was added, and the mixture was incubated at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end of the 3 ′ adapter with a chain length of 37. The reaction mixture was extracted with phenol / chloroform, and then precipitated with ethanol to recover DNA.
d III, agarose gel electrophoresis, 0.44 k
About 1 μg of the NcoI to HindIII fragment of b was recovered.

(1-3) Construction of pUC119NT 12 μg of secretory expression vector pIN III-ompAl [Diembo Journal, Vol. 3, pp. 2437-2442 (1984)] was used.
Digested with Hind III and Sal I, subjected to agarose gel electrophoresis, 0.95 kb Hind II containing 1 pp terminator sequence
The I-Sal I fragment was recovered. A buffer for T4 DNA ligase was prepared together with 0.5 μg of plasmid pUC119N which had been obtained by previously digesting 0.75 μg of this fragment with Hind III and Sal I and dephosphorizing it,
Incubated overnight at 16 ° C. in a 20 μl reaction containing 0.5 mM ATP, 10 mM DTT and 2.8 units of T4 DNA ligase. Escherichia coli JM109 was transformed with 2 µ of the reaction solution,
A plasmid having a pp terminator sequence was obtained and pUC119NT
It was named.

Note that pUC119N was prepared by introducing an NcoI site into the translation initiation codon site of a commercially available pUC119 vector (available from Takara Shuzo Co., Ltd.) and further increasing the distance between the ribosome binding site and the initiation codon by 8
It is a base.

(1-4) Cloning of NcoI to HindIII Fragment into pUC119NT 1 μg of the plasmid pUC119NT obtained in (1-3) was
And dephosphorylated after digestion with Hind III. This plasmid
0.5 μg of the NcoI to HindIII fragment obtained in (1-2) 0.2 μg
g4 with T4 DNA ligase buffer, 0.5mM ATP, 10m
50 μl containing MDTT and 5 units of T4 DNA ligase
Was incubated at 16 ° C. for 5 hours. 2 µ of the reaction solution was used for transformation of Escherichia coli JM109.

(1-5) Transformation of Escherichia coli and confirmation of plasmid Using 2 µ of the reaction solution obtained in (1-4), Escherichia coli JM10
9 were transformed. Plasmid analysis was performed on 6 clones among the obtained transformants. That is, the plasmid prepared by the rapid method was digested with Nco I and Hind III, subjected to agarose gel electrophoresis, and the expected Nco I
The generation of a band of the ~ Hind III fragment (0.44 kb) was examined. As a result, generation of a target band was observed in one clone. In addition, the nucleotide sequence of the integrated cDNA fragment region was determined by the dideoxy method, and it was confirmed that the target sequence was contained. This recombinant plasmid was named pFD705.

In addition, Escherichia coli JM109 carrying this plasmid was
Richia coli JM109 / pFD705 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology
MP-11037)].

(1-6) Purification of Polypeptide from Recombinant Escherichia coli JM109 / pFD705 obtained in (1-5)
Shaking culture was carried out at 37 ° C. overnight in a test tube containing 5 ml of L-broth supplemented with 0 μg / ml of ampicillin. This was inoculated into two Erlenmeyer flasks containing 500 ml of the same medium, and 100 rpm.
The culturing was continued. When the absorbance at 660 nm was 0.3, IPTG (isopropyl-β-D-thiogalactoside) was added to 2 mM, and the cells were collected 16 hours later. Immunoblotting was performed using a part of the cells. That is, whole cell proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis), and the electrophoresis pattern was transferred to a nitrocellulose membrane. Then, a monoclonal antibody [HFN-11F6, Serotec) and then a peroxidase-labeled second antibody. The peroxidase activity of the bound second antibody was developed in the presence of 4-chloro-1-naphthol and hydrogen peroxide, and it was confirmed that the target polypeptide was produced at around 16.5 kD. next,
The whole cell pelt (wet weight 4.4 g) was suspended in 21 ml of a buffer [50 mM Tris-HCl, pH 8.0, 25% sucrose, 1 mM EDTA], and 1 ml of a 2 mg / ml lysozyme solution was added. 0 ℃, 3
After standing for 0 minutes, the cells were disrupted by sonication. 66 μl of 1 M MgCl ₂ and 1 M MnCl ₂ were added to the cell lysate.
55 μl of 22 μl and 60,000 unit / ml deoxyribonuclease I (DNase I) were added, respectively.
Incubated for 0 minutes. Then, 4.4 ml of 20 mM Tris-HCl, pH 7.5 buffer containing 0.2 M NaCl, 1% deoxycholic acid, 1% Nonidet P-40 was added, and 12,000 were added.
After centrifugation at rpm for 10 minutes, the supernatant was discarded. Precipitation buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 0.1 mM
The suspension was suspended in 22 ml of 5% Triton X-100), centrifuged at 14,000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was then suspended in 22 ml of a 20% aqueous ethylene glycol solution and centrifuged at 14,000 for 10 minutes. The obtained precipitate was further subjected to 2% Nonidet P-
The suspension was suspended in 22 ml of 40 aqueous solution, centrifuged at 14,000 rpm for 10 minutes, and the supernatant was discarded to obtain a 0.5 g (wet weight) pellet of the purified inclusion body. This inclusion body was added to 20 mM Tris-HCl,
It was dissolved in 10 ml of a pH 8.3, 50 mM DTT, 7 M urea solution. After removing insoluble matter by centrifugation, the lysate was equilibrated with 20 mM Tris-HCl, pH 8.3 buffer containing 1 mM EDTA, 5 M urea, and 5 mM DTT, Sephacryl S-100HR (manufactured by Pharmacia). Gel filtration chromatography was performed using a column containing 750 ml. Elution and fractionation were carried out with the same buffer, and a part of each fraction was subjected to SDS-PAGE to collect 90 ml of the target fraction. This fraction was dialyzed in a 20 mM Tris-HCl, pH 9.0 buffer containing 1 mM oxidized glutathione at 4 ° C. for two days and nights to refold the protein. The dialysis solution is subsequently desalted by dialysis in distilled water,
Lyophilize to electrophoretically isolate single polypeptide for 21.5
mg was obtained. When the amino acid sequence from the N-terminal of the polypeptide was examined using a peptide sequencer 477A / 120A of Applied Biosystems, Ala-Asn-Glu-G
The sequence of ly-Leu-Asn was recognized, and Ala was added to the N-terminal of F-143.
(Hereinafter, the polypeptide is abbreviated as Ala-F-143).

(1-7) Removal of Intervening Sequence (GCT) from pFD705 The polypeptide (Ala-F-143) expressed by the plasmid pFD705 obtained in (1-5) has an Ala derived from an NcoI linker at the N-terminus. Is added. The sequence (GCT) corresponding to Ala was removed by site-directed mutagenesis. That is, oligonucleotide d [AGCCTTCGTTCATG
GTCTGT] was synthesized using Site-Directed Mutagenesis System Mutan-K (Takara Shuzo Co., Ltd.). As a result, a plasmid expressing F-143 from which the terminal Ala had been removed was obtained and named pFD708.

Example 2 C-terminal fibrin binding domain of FN Cys ²¹⁴⁴ -Gly
Cloning of cDNA fragment encoding ²²⁷⁵ (132 amino acid residues, hereinafter abbreviated as F-132) (see FIG. 2) (2-1) Preparation of synthetic DNA adapter Type I repeat present in C-terminal fibrin binding domain The 5'-side adapter (chain length 11mer # 1 and # 2, see FIG. 2) for connecting the sequence to the vector was synthesized using a DNA synthesizer manufactured by Applied Biosystems. 300
After phosphorylation of the 5 'end of 11mol # 2 of pmol, the chain length was 11m.
An equivalent amount of the synthetic DNA of er # 1 was added to form a double strand by an annealing operation. On the 3 'side, the adapter (29mer and 37mer) prepared in (1-1) was used.
A double chain was obtained in the same manner as described above.

(2-2) Preparation of Nco I to Hind III Fragment The Sau3A I fragment (0.96 kb fragment) of pLF2435 obtained in (1-2) was further cut with Fok I and Hae II, and subjected to agarose gel electrophoresis. , A 0.37 kb fragment was recovered.
2 μg of this fragment, 150 pmol of 5 ′ adapter and 150 pm
The 3 'adapter of ol was incubated overnight at 15 ° C in a 125 µl reaction containing 300 units of T4 DNA ligase. After incubating the reaction solution at 70 ° C for 10 minutes,
In the same manner as in (1-2), the 5 'end of the other side of the adapter was phosphorylated, then digested with NcoI and HindIII, subjected to agarose gel electrophoresis, and purified with 0.40 kb of NcoI.
About 0.5 μg of the I to Hind III fragment was recovered.

(2-3) Cloning of NcoI to HindIII fragment into pUC119NT 1 μg of the plasmid pUC119NT obtained in (1-3) was added to NcoI.
And dephosphorylated after digestion with Hind III. This plasmid
0.8 μg and 0.2 μg of the Nco I to Hind III fragment obtained in (2-2)
Ligation kit for g [Sold by Takara Shuzo Co., Ltd.]
Solution A 60μ and Solution B 7.5μ were added and incubated at 16 ° C for 30 minutes. 4 µ of the reaction solution was used for transformation of Escherichia coli JM109.

(2-4) Transformation of Escherichia coli and confirmation of plasmid Using 4 µ of the reaction solution obtained in (2-3), Escherichia coli JM10
9 were transformed. Plasmid analysis was performed on 6 clones among the obtained transformants in the same manner as in (1-5), and the formation of a band of NcoI to HindIII fragment (0.41 kb) was examined. As a result, generation of the target band was observed in three clones. In addition, it was incorporated by the dideoxy method.
The nucleotide sequence of the cDNA fragment region was determined, and it was confirmed that it contained the target sequence. This recombinant plasmid was named pFD905.

In addition, Escherichia coli JM109 carrying this plasmid was
richia coli JM109 / pFD905 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.
MP-11038)].

(2-5) Purification of Polypeptide from Recombinant Escherichia coli JM109 / pFD905 obtained in (2-4) was cultured in the same manner as in (1-6), and purified inclusion bodies were obtained from one cultured cell. 0.5 g (wet weight) was obtained. The target protein was purified from this inclusion body in the same manner as in (1-6) to obtain 12 mg of a single polypeptide by electrophoresis. By subjecting the polypeptide to reductive carboxymethylation [according to the method described in "Biochemistry Experiment Course" I Protein Chemistry II, Tokyo Chemical Dojinsha, pp. 228-230 (1976)], a cysteine residue was previously obtained. As a result of examining the amino sequence at the N-terminus of the protected product, Ala-Cys-Ph
The sequence of e-Asp-Pro-Tyr was recognized, which coincided with the N-terminal sequence in which Ala was added to the N-terminus of F-132 (hereinafter, this polypeptide is abbreviated as Ala-F-132).

(2-6) Removal of Intervening Sequence (GCT) from pFD905 5 ′ having an NcoI site at the N-terminus of the polypeptide (Ala-F-132) expressed by plasmid pFD905 obtained in (2-5) Ala derived from the side adapter (see FIG. 2) is added. Sequence corresponding to this Ala (GC
T) was removed by the technique of site-directed mutagenesis. That is, oligonucleotide d [GGTCAAAGCACATGGTCTGT] was synthesized, and the synthesis was performed using Site-Directed Mutagenesis System Mutan-K (Takara Shuzo Co., Ltd.). As a result, a plasmid expressing F-132 from which the terminal Ala had been removed was obtained and named pFD908.

Reference Example 1 Measurement of Biological Activity Fibrin-binding activity and insulin-binding activity were measured using each of the polypeptides obtained in Examples 1 and 2.

(3-1) Fibrin binding activity The method of Sekiguchi et al. [Journal of Biological
Chemistry (Journal of Biological Chemistry) 2
56, pp. 6452-6462 (1981)], the fibrin-binding activity was examined by adsorption onto a fibrin-immobilized column. The method for preparing the fibrin-immobilized column was the method of DL Heene et al. [Trombosis Research (Thr.
ombosis Research), Vol. 2, pp. 137-154 (1973)]. However, AF-Tresyl Toyopearl 650 (Tosoh) was used as the carrier resin. Fibrin-AF Toyopearl column (1 ml) contains various sample polypeptide excess (2
70 pmol). After washing with 5 ml of washing buffer (10 mM Tris-phosphate, pH 7.5, 50 mM NaCl, 1 mM EDTA), 5 ml of elution buffer 1 (0.5 M arginine in PBS)
To elute the adsorbed fraction. Next, elution was carried out with 5 ml of elution buffer 2 (6 M urea, 25 mM Tris-phosphate, pH 7.5). 0.5 ml of each fraction is fractionated, and a monoclonal antibody (HFN-11F6, Serotech) recognizing the C-terminal fibrin binding domain of FN at the elution position of the sample polypeptide
It was examined by ELISA using. As a result, Ala-F-14
3, each of the fibrin-binding polypeptides of F-143, Ala-F-132 and F-132 was observed in the flow-through fraction of this column and the adsorbed fraction eluted with the elution buffer 1, and the fibrin-binding activity was confirmed. It was found to have. In addition, as a result of comparing the binding ability to fibrin in each test based on the ratio of the amount of the passed sample to the amount of the adsorbed sample, there was no significant difference in any of the samples (see Table 1 below).

(3-2) Insulin-binding activity It is known that the fibrin-binding domain of FN has insulin-binding activity (the 46th Annual Meeting of the Cancer Society of Japan, page 181). Therefore, Ala-F-143, F-143, Ala-
The insulin binding activity of F-132 and each of the fibrin binding activity polypeptides of F-132 were examined. Each of these polypeptides, which were sequentially diluted by 1/2 from 20 μg, was adsorbed on a nitrocellulose membrane using BIO-DOT manufactured by Bio-Rad. After blocking the nitrocellulose membrane with a PBS buffer containing 3% BSA, the PBS containing 50 μg / ml peroxidase-labeled insulin (Sigma) was used.
It was left in S buffer at room temperature for 2 hours. Next, the nitrocellulose membrane was washed twice with PBS for 5 minutes, and the bound peroxidase-insulin was washed with 4-chloro-1-
Naphthol and hydrogen peroxide were detected as substrates. As a result, color development corresponding to the concentration was observed in each case, and it was confirmed that there was insulin binding activity (see Table 1).

[Effects of the Invention] As described above, the present invention provides a fibrin-binding activity polypeptide having at least fibrin-binding activity, a nucleic acid encoding the same, and a genetic engineering production method using the DNA. .

The polypeptide is useful for various uses such as a blood coagulation inhibitor and a drug delivery system.

[Brief description of the drawings]

FIG. 1 is a flow chart for constructing a plasmid pFD705 encoding WXXY in the above formula I, and FIG. 2 is a plasmid pF705 encoding WXX in the above formula I.
It is a flowchart for constructing D905.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Fumio Kimizuka Otsu-shi, Shiga 3-4-1 Seta, Takara Shuzo Co., Ltd., Central Research Laboratory (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu-shi, Shiga Takara Shuzo Co., Ltd., Central Research Laboratory (56) References JP 62-89699 (JP, A) The EMBO Journal. Vol. 4, No. 7, p. 1755-1759 (1985) Biochemistry, Vol. 25, p. 4936-4941 (1986) 46th Meeting of the Japanese Society of Cancer Research, page 181 709, amG, 1987 (58) Fields studied (Int. Cl. ⁶ , DB name) C12N 15/00 C07K 14/745 C12N 1 / 21 C12P 21/02 A61K 38/36 CA (STN) REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A following general formula _{I: (W) l - (} X) m - (Y) n -Z ‥‥ (I) wherein, W is shown a methionine residue (Met), and X is alanine Indicates a residue (Ala), and Y is a polypeptide residue of human fibronectin, corresponding to Asn ²¹³³ -Ser ²¹⁴³
Denotes an 11 amino acid polypeptide residue and has the following Formula II: Wherein Z is the C of human fibronectin.
Represents a 132 amino acid polypeptide residue corresponding to Cys ²¹⁴⁴ -Gly ²²⁷⁵ of the terminal fibrin binding domain, and has the following formula II
I: Wherein l, m and n each represent a number of 1 or zero]. 2. A nucleic acid encoding the fibrin-binding activity polypeptide according to claim 1. 3. A recombinant plasmid containing a DNA encoding the fibrin-binding activity polypeptide according to claim 1. 4. A transformant into which the recombinant plasmid according to claim 3 has been introduced. 5. A method for producing a fibrin-binding active polypeptide, comprising culturing the transformant according to claim 4, and collecting the fibrin-binding active polypeptide according to claim 1 from the culture.