JPH1042878A - Production of functional polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new transformant which is transformed with a recombinant plasmid containing a gene coding a functional polypeptide having a specific amino acid sequence, useful for producing a functional polypeptide, especially a human fibronectin, etc. SOLUTION: This new transformant is transformed with a recombinant plasmid containing a gene coding a functional polypeptide having an amino acid sequence of the formula and is useful for producing the functional polypeptide participating especially in physiological actions of adhesion, spreading, movement, differentiation, proliferation, phagocytosis, etc., of cell and taking an important role in construction and restoration of tissue, blood coagulation, biophylaxis, etc. The transformant is obtained by cloning a cDNA coding a cell adherent domain of a human fibronectin, incorporating the obtained cDNA fragment into a secretion type vector, inserting the obtained recombinant vector into a host such as Escherichia coli, etc., and transforming.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a functional polypeptide, particularly a functional polypeptide of human fibronectin (hereinafter abbreviated as FN) by genetic engineering.

[0002]

2. Description of the Related Art Fibronectin is a multifunctional glycoprotein widely distributed in the blood and tissues of humans and animals, and is involved in physiological functions such as cell adhesion, spreading, migration, differentiation, proliferation, phagocytosis, etc. It is known to play an important role in repair, blood coagulation, biological defense and the like. Recent advances in molecular biology have elucidated the entire amino acid sequence and gene structure of FN [The EMBO Journal, Volume 4, 1755-175.
9 (1985)]. FN has a polypeptide of up to 2327 amino acids with a molecular weight of about 250,000,
Two SS bonds form a dimer. The intramolecular amino acid sequence has type I, type II, and type III repeating structures, and further, binding regions (domains) for cells, collagen, heparin, fibrin, and the like are independently present.
These functions of FN are industrially useful. For example, the cell adhesion function can be used as a coating agent for a cell culture substrate or as a wound treatment agent. Fibrin binding ability
It can be used as an anticoagulant by inhibiting the binding of platelets to fibrin. Conversely, a peptide having both functions of cell adhesion activity and fibrin binding activity promotes binding of platelets to fibrin and enhances the wound healing effect. Furthermore, recent findings have revealed that the fibrin-binding domain of FN has insulin-binding activity (46th Annual Meeting of the Japanese Cancer Society, page 181),
A polypeptide having both functions of cell adhesion activity and insulin binding activity may be used as a drug delivery system.

[0003]

As described above, FN is useful as itself or as a functional fragment thereof, but is expensive and has a limited amount to be collected from blood.
In addition, their use has been greatly restricted due to problems such as virus infection. Further, as a method for extracting a region having a specific function from FN, limited decomposition with an enzyme or limited decomposition with bromocyanide or the like is known. However, any of these methods is complicated and the yield is extremely low. Hard to say. An object of the present invention is to construct a novel functional polypeptide having fibrin-binding activity, provide a gene encoding the same, and provide a method for producing the polypeptide by genetic engineering using the gene.

[0004]

SUMMARY OF THE INVENTION In summary, the first invention of the present invention relates to a transformant,
It is a transformant transformed with a recombinant plasmid containing a gene encoding a functional polypeptide represented by an amino acid sequence represented by the following formula (Formula 1).

[0005]

Embedded image Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gly Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu

[0006] The second invention of the present invention relates to a method for producing a functional polypeptide, wherein the transformant of the first invention is cultured, and the desired functional polypeptide is obtained from the culture. It is characterized by being collected.

The present inventors have clarified the sequence of a polypeptide having an activity substantially equivalent to that of FN with respect to the cell adhesion-active polypeptide of FN, developed a method for producing the polypeptide by genetic engineering, and filed a patent application [ Japanese Patent Application Nos. 63-148 (JP-A-1-180900) and 63-31820 (JP-A-1-206998)]. After that, further research was carried out, and as a result of repeated research on functional polypeptides having both cell adhesion activity and other functions, a hybrid polypeptide of cell adhesion activity polypeptide and fibrin binding domain peptide was produced by genetic engineering. Succeeded. As a result of isolating this hybrid polypeptide and examining its biological activity, it was found that the hybrid polypeptide had biological activity as a multifunctional polypeptide having both cell adhesion activity and fibrin binding activity. The present invention has been completed based on the above findings.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Means for preparing a hybrid peptide of a cell adhesion activity polypeptide and a fibrin binding domain peptide of FN by genetic engineering include a plasmid containing DNA encoding the cell adhesion activity polypeptide and a DNA encoding the fibrin binding domain peptide This is accomplished by removing the necessary fragments from the plasmid, joining both DNA fragments, and connecting them to an appropriate expression vector so that the reading frames match. Various fragments have already been cloned as a DNA fragment encoding a cell adhesion active polypeptide (Japanese Patent Laid-Open No. 1-180).
Nos. 900 and 1-2206998). Necessary parts can be cut out from these plasmids. On the other hand, the DNA fragment encoding the fibrin domain is pLF5,
pLF2435 constructed by joining the FN cDNA portions of pLF3, pLF4 and pLF2 [Biochemistry, Vol. 25, Nos. 4936-4941.
(1986)]. A hybrid peptide can be expressed in E. coli by connecting a DNA fragment encoding a hybrid peptide of a cell adhesion active polypeptide and a fibrin domain polypeptide to an appropriate expression vector and introducing the resultant into E. coli. As the expression vector, all existing vectors can be used.

Confirmation of the hybrid peptide by Escherichia coli is checked by immunoblotting. After separating all bacterial proteins by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), the migration pattern was transferred to a nitrocellulose filter, and a monoclonal antibody recognizing the cell adhesion domain of FN and a monoclonal antibody recognizing the fibrin domain Can be detected. Conventional chromatography techniques are used for purification of the expressed peptide.
For example, the cell pellet is suspended in a buffer, and a soluble fraction is obtained by sonication. This is applied to a column of Sepharose 4B to which the antibody used for immunoblotting has been bound, and affinity purification is performed. By collecting target fractions by immunoblotting, a functional polypeptide having both functions of cell adhesion activity and fibrin binding activity can be obtained. If necessary, it can be further purified by FPLC or HPLC.

The functional polypeptide thus obtained is used for measurement of biological activities such as cell adhesion activity, fibrin binding activity and insulin binding activity. For the measurement of cell adhesion activity, NRK cells are used. For example, a sample is dissolved in a buffer and adsorbed to a microplate.
Add RK cells and incubate at 37 ° C. for a certain time. By observing the extension of the cells under a microscope and comparing the minimum amount required for the expression of the extension with the natural FN, the strength of the cell adhesion activity can be expressed. For the measurement of fibrin binding activity, Sepharose 4B to which fibrin has been bound [Literature Thrombosis Research, Vol. 2, pp. 137-154 (1973)] can be used. That is, after passing the sample through the fibrin-binding column, the presence or absence of fibrin-binding activity is determined by performing SDS-PAGE of the flow-through solution. Insulin binding activity can be determined according to the immunoblotting technique. That is, the sample is separated by SDS-PAGE, transferred to a nitrocellulose filter, and allowed to act with enzyme-labeled insulin. After washing the filter,
Bound insulin can be determined by enzyme activity.

Preferred examples of the peptide thus obtained include Ala ^1235- having the cell adhesion activity of FN.
Gln ¹⁵¹⁶ (282 amino acid residue peptide) or Ile
^1410- Gln ¹⁵¹⁶ (107 amino acid residue peptide) and fibrin binding domain peptide Asn ²¹³³ -Glu ²³²⁴ (1
(92 amino acid residues). The amino acid sequence of the polypeptide having the cell adhesion activity of FN is as shown in the following formula (Formula 2):

[0012]

Embedded image ₁₂₃₅ Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser ₁₄₁₀ Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys P ro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser ₁₅₁₆ Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln

The amino acid sequence of the fibrin-binding domain peptide of FN is as shown in the above-mentioned formula (Formula 1).

[0014]

EXAMPLES 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 Cell adhesion domain of FN Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ (108 amino acid residues) and fibrin binding domain Asn ²¹³³ -Gl
u ²³²⁴ Cloning of cDNA fragment (192 amino acid residues) code (see FIG. 1) FIG. 1 is a process diagram of a PTF 1301 constructed according to the present invention. (1-1) Preparation of cDNA fragment 7.8 k including cDNA fragment encoding Fle cell adhesion domain Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ (108 amino acid residues)
b) Plasmid pTF201 (Japanese Patent Laid-Open No. 1-180900)
100 μg of a buffer containing the restriction enzyme XbaI, 100 units of XbaI and 100 units of EcoRI
The mixture was incubated at 37 ° C. for 2 hours in μl of the reaction solution.
The reaction solution was applied to an HPLC column manufactured by DIAGEN and Nucleogen DEAE-400.
0 (6 × 125 mm) to give 2 μg of a 0.41 kb fragment. Next, this fragment was digested with 20 units of FokI at 37 ° C.
After treating for 1 hour, the XbaI-FokI fragment (1
80 bp) and 250 ng of each FokI-FokI fragment (203 bp). On the other hand, a 5.9 kb plasmid pLF 2 containing a cDNA fragment encoding the fibrin binding domain Asn ²¹³³ -Glu ²³²⁴ (192 amino acid residues)
435 [Biochemistry, Vol. 25, No. 4936-4
941 (1986)] in a 100 μl reaction mixture containing a buffer for Hind III, 100 units of Hind III and 100 units of HincII at 37 ° C., 2
Incubated for hours. This was subjected to agarose electrophoresis to cut out a 1.2 kb fragment (yield: 2 μg). 1 μg of this fragment was treated with Sau3AI at 37 ° C. for 1 hour, and applied to a Nucleogen DEAE-4000 column.
200 ng of the II-Sau3AI fragment (621 bp) was obtained. (1-2) Preparation of synthetic DNA adapter cDNA of cell adhesion domain and fibrin binding domain
Adapters for connecting fragments (chain lengths 24 and 20,
(See FIG. 1) using an Applied Biosystems DNA synthesizer, and 2.7 μg and 2.3 μg, respectively.
Obtained. 100 ng of each 2 ng by annealing operation
Heavy chain.

(1-3) Binding of cDNA fragment to vector The cDNA fragment obtained in (1-1) is ligated to a secretory expression vector p.
IN III-omp AI [Diembo Journal, Vol. 3, pp. 2437-2442 (1984)]. That is, 10 ng of the XbaI-FokI fragment (180 bp),
FokI-10 ng of FokI fragment (203 bp), Hinc II
-30 ng of the Sau 3AI fragment (621 bp), 10 ng of the synthetic DNA adapter obtained in (1-2), and XbaI
PIN III-ompAI vector 50ng of T ₄ DNA ligase buffer was removed -BamHI insert, 0.5 mM
ATP, 10 mM DTT and 2.8 units of T ₄ D
Incubated overnight at 16 ° C. in a 20 μl reaction containing NA ligase. This reaction solution was used for transformation of E. coli.

(1-4) Transformation of Escherichia coli and confirmation of plasmid Escherichia coli HB1 was used with 20 μl of the reaction solution obtained in (1-3).
01 was transformed. Plasmid analysis was performed on 21 clones of the obtained transformants. That is, 1.5 clones containing 50 μg / ml ampicillin for each clone.
After culturing with shaking overnight in L-broth (ml), a plasmid was prepared by the rapid method, and a part thereof was double-digested with XbaI and SalI. This was separated by agarose electrophoresis and the expected XbaI-XbaI fragment (0.56 kb) and XbaI-
The generation of a band of the SalI fragment (1.5 kb) was examined. As a result, the target band was observed in 12 clones. Furthermore, when its base sequence was determined by the dideoxy method,
It was confirmed that it had the correct nucleotide sequence except that the codon of methionine at the C-terminal of 108 amino acids of the cell adhesion domain was missing. This recombinant plasmid was named pTF1301. Escherichia coli HB1 carrying this plasmid
01 was designated as Escherichia coli HB101 / pTF1301, and deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology [Microtechnical Laboratory No. 9947 (FERM P-9947)].

Example 2 Cell adhesion domain of FN Ala ¹²³⁵ -Gln ¹⁵¹⁶ (282 amino acid residues) and fibrin binding domain Asn ²¹³³ -G
lu ²³²⁴ Cloning of cDNA fragment (192 amino acid residues) code (see FIG. 2) FIG. 2 is a process diagram of a PTF 1801 constructed according to the present invention. (2-1) Preparation of XbaI-Pvu II fragment of PTF 901 cell adhesion domain Ala ¹²³⁵ - cD encoding Met ¹⁵¹⁷
Plasmid pTF901 containing NA fragment (Japanese Patent Laid-Open No. 1-180)
No. 900) 20 μg of 37 μl of a 200 μl reaction containing 50 units of XbaI and 50 units of Pvu II
Incubated for 1 hour at ° C. The reaction solution was subjected to agarose electrophoresis, and the XbaI-PvuII insert (0.6
1 kb) was cut out (yield 400 ng). (2-2) Preparation of XbaI-PvuII Fragment of pTF1301 Twenty-five μg of the plasmid pTF1301 obtained in Example 1 contains 50 units of XbaI and 50 units of PvuII.
The mixture was incubated at 37 ° C. for 1 hour in 0 μl of the reaction solution. This reaction solution was subjected to agarose electrophoresis, and XbaI-
The Pvu II insert (0.40 kb) was excised (yield 4).
00 ng). 7. Also lacks the XbaI-XbaI insert.
The 0 kb vector was simultaneously excised (yield 2 μg), dephosphorylated, and used for cloning.

(2-3) XbaI-PvuII fragment (0.6
Ligation of 1 kb and 0.40 kb) 400 ng of the 0.61 kb fragment obtained in (2-1) and (2-
400 ng of the 0.40 kb fragment obtained in 2) was used as a buffer for T ₄ DNA ligase, 0.5 mM ATP, and 10 mM DT.
20 containing T and 2.8 units of T ₄ DNA ligase
The mixture was incubated at 16 ° C. for 3 hours in μl of the reaction solution.
Stop the reaction by treatment at 65 ° C for 10 minutes.
Optimize the baI conditions, add 10 units of XbaI and add 3
The reaction was performed at 7 ° C. for 2 hours. The reaction solution was subjected to agarose electrophoresis, and an XbaI-XbaI fragment (1.01 kb) was cut out (yield: 20 ng).

(2-4) XbaI-XbaI fragment (1.01
ligation of XbaI-XbaI fragment (1.01 kb) obtained in (2-3)
20 ng of the 8.0 kb vector 20 obtained in (2-2)
ng of T ₄ DNA ligase buffer, 0.5 mM A
TP, 10 mM DTT and 2.8 units of T ₄ DNA
Incubation was performed at 16 ° C. for 3 hours in a 20 μl reaction solution containing ligase. This reaction solution was used for transformation of E. coli.

(2-5) Transformation of Escherichia coli and confirmation of plasmid Escherichia coli HB1 was prepared using 20 μl of the reaction solution obtained in (2-4).
01 was transformed. Plasmids were confirmed for 12 clones in the obtained transformants. The plasmid prepared by the rapid method was subjected to double digestion with XbaI-PvuII, and subjected to agarose electrophoresis, and the formation of expected inserts (0.61 kb and 0.40 kb) was examined. As a result, a target band was observed in one clone. The clone was further digested with EcoRI, and the orientation of the inserted fragment was examined. As a result, it was confirmed that the clone was inserted in the correct direction. In addition, the nucleotide sequence was determined by the dideoxy method, and it was confirmed that the target sequence was contained. This recombinant plasmid was designated as pTF1801. Further, Escherichia coli HB101 carrying this plasmid was replaced with Escherichia coli HB101.
101 / pTF 1801 and deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology [Microtechnical Laboratory No. 9948 (FERM)
P-9948)].

Example 3 FN chimeric polypeptide Ile ¹⁴¹⁰ -Gln ¹⁵¹⁶ / Asn
Production and purification of ²¹³³ -Glu ²³²⁴ (299 amino acid residues) 50 μg / ml of HB101 / pTF1301 obtained in Example 1
Were inoculated into two test tubes containing 5 ml of L-broth to which ampicillin was added, and cultured with shaking at 37 ° C. overnight. This was inoculated into a 2 liter Erlenmeyer flask containing 500 ml of the same medium, and cultivation was continued.
Then, 2 mM IPTG (isopropyl β-thiogalactoside) was added, and the cells were collected 20 hours later. The whole cell pellet was washed with 10 mM Tris-HCl (pH 7.
5) The suspension was suspended in a solution containing 5 mM EDTA, sonicated, and centrifuged at 12,000 rpm for 30 minutes.
ml was obtained. This was treated with an anti-FN monoclonal antibody (FN-1
Sepharose 4B column (8, Takara Shuzo)
ml). The column was washed with washing buffer A (20 mM Tris-HCl, pH 7.5), and then washed with buffer B (20 mM Tris-HCl, pH 8.0, 0.1
MKCl). Finally, elution buffer (50
mM glycine HCl, pH 2.3, 0.2 M KCl)
Eluted. The eluted fractions were collected, yielding 500 μg of a nearly single 34 kd polypeptide electrophoretically.

Example 4 FN chimeric polypeptide Ala ¹²³⁵ -Gln ¹⁵¹⁶ / Asn
Production and purification of ²¹³³ -Glu ²³²⁴ (474 amino acid residues) 2 liters of HB101 / pTF1801 obtained in Example 2 was cultured in the same manner as in Example 3, and an anti-FN monoclonal antibody (FN-12) Sepharose 4B column was used. And purified. The fraction eluted with the antibody column contained a polypeptide of 55 kd, which was a degradation product thereof, in addition to the polypeptide of 55 kd of interest. Therefore, it was subsequently purified by a fibrin-Sepharose 4B column. That is, D. L. DL Heene et al. [Trombosis Research,
The eluted fraction of the antibody column was passed through a fibrin-Sepharose 4B column (10 ml) prepared according to Volume 2, pages 137 to 154 (1973)]. Wash the column with wash buffer (10 mM Tris-HCl, pH 7.6, 50 mM Na
After washing with 0.5 mM EDTA (Cl, 0.5 mM), elution buffer (25 mM Tris-HCl, pH 7.6, 6 M urea)
Eluted. The eluted fractions were collected and electrophoretically single 55
100 μg of kd polypeptide was obtained.

Example 5 Measurement of biological activity The chimeric polypeptide Ile ¹⁴¹⁰ obtained in Examples 3 and 4
Gln ¹⁵¹⁶ / Asn ²¹³³ -Glu ²³²⁴ (107CBP / 192
FBP, 299 amino acid residues), Ala ¹²³⁵ - Gln ¹⁵¹⁶ /
Asn ²¹³³ -Glu ²³²⁴ (282CBP / 192FBP, 4
(74 amino acid residues), the following biological activities were measured. (5-1) Cell Adhesion Activity The method of Ruoslahti et al. [Methods
Cell adhesion activity was measured according to Methods in Enzymology, Vol. 82, pp. 803-831 (1981)]. That is, the sample was serially diluted with physiological saline or distilled water, 50 μl of the sample was dispensed into a 96-well microplate, and incubated at 4 ° C. overnight to allow the sample to adhere to the plate. Next, the plate was washed twice with PBS buffer, and 100 μl of 3% BSA was added thereto.
The plate was blocked by incubating at 1 hour at 1 hour. After the plate was washed twice with PBS buffer, rat kidney cells (NRK-4) previously suspended in Eagle's minimal medium (MEM) at 10 ⁶ cells / ml.
9F) was dispensed at a rate of 100 μl / well, at 37 ° C.
Incubated for 2-3 hours. NRK-
The 49F cells were obtained by pre-culturing a cryopreserved strain and then treating with trypsin. The extension of the cells is observed under a microscope, and the minimum amount required for the extension is indicated as the minimum cell adhesion activity. The results are shown in Table 1 below together with other activities. A polypeptide having only a cell adhesion domain of 108 amino acid residues and 283 amino acid residues [108CBP and 283C
BP (Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ and Ala ¹²³⁵ -Met ¹⁵¹⁷ ),
JP-A-1-180900] was simultaneously measured and compared with a chimeric polypeptide. As a result, 107 CBP / 1
The activity of 92FBP was found to be about 52 times or more that of 108CBP. 282CBP / 192FBP is 283
Activity equivalent to that of CBP was observed.

(5-2) Fibrin binding activity Adsorption to a fibrin-Sepharose 4B column according to the method of Sekiguchi et al. [Journal of Biological Chemistry, 256, 6452-6462 (1981)]. Was examined for fibrin binding activity. D. L. Hehn et al.'S method [Trombosis Research, Volume 2, 137-
Fibrin was produced by 154 pages (1973)] - chimeras Sepharose 4B column (1.4 ml) polypeptide ^{Ile 1410 - Gln 1516 / Asn 2133} - Glu 2324 (107C
BP / 192FBP, 299 amino acid residues). 7.5 ml of wash buffer (10 mM Tris-H)
Cl, pH 7.6, 50 mM NaCl, 0.5 mM ED
TA) to remove the non-adsorbed fraction, and then use the elution buffer (25
The adsorbed fraction was eluted with mM Tris-HCl, pH 7.6, 6M urea. 0.5 ml of each fraction
The elution position of 7CBP / 192FBP was examined by an ELISA method using an anti-FN monoclonal antibody (FN-12, Takara Shuzo). As a result, 107CBP / 192FBP
Was observed in the adsorbed fraction, and was confirmed to have fibrin binding activity. A similar result is Ala ¹²³⁵ − Gln ¹⁵¹⁶ / A
sn ²¹³³ -Glu ²³²⁴ (282CBP / 192FBP, 47
(4 amino acid residues). In addition, Ile ¹⁴¹⁰ -Met ¹⁵¹⁶ (108CB
P) and Ala ¹²³⁵ - Met ¹⁵¹⁶ (283CBP) fibrin - adsorption on Sepharose 4B was observed. (See Table 1)

(5-3) Insulin-binding activity It is known that the fibrin-binding domain of FN has insulin-binding activity (the 46th Annual Meeting of the Japanese Cancer Society, page 181). Therefore, the insulin binding activity of the chimeric polypeptide was examined. 107CBP / 192FB
P, 283CBP / 192FBP, 108CBP, 28
3CBP was sequentially diluted by 1/2 from 20 μg, and adsorbed to a nitrocellulose membrane using BIO-DOT ^™ manufactured by Bio-Rad. This nitrocellulose membrane is 3% BS
After performing a blocking operation with a PBS buffer containing A, the mixture was allowed to stand at room temperature for 2 hours in a PBS buffer containing 50 μg / ml of peroxidase-labeled insulin (Sigma). Next, the nitrocellulose membrane was washed twice with PBS for 5 minutes, and the bound peroxidase-insulin was detected using 4-chloro-1-naphthol and hydrogen peroxide as substrates. As a result, 108 CBP and 283
CBP shows no color development, whereas 107C
In BP / 192FBP and 283CBP / 192FBP, color development corresponding to the density was observed. Therefore, 107
CBP / 192FBP and 283CBP / 192FBP
Has an insulin binding activity (Table 1).
reference).

[0027]

Table 1 ─────────────────────────────────── Polypeptide Minimum Cell Adhesion Activity Fibrin Insulin μg / Well pmole / well Binding activity Binding activity ─────────────────────────────────── 108CBP>50> 4400 − − 283CBP 0.03 1.0 − − 107CBP / 192FBP 2.8 84 ++ 282CBP / 192FBP 0.13 2.4 ++ FN 0.18 0.8 ++ ───────────────────────── ────────── 108CBP: Ile ¹⁴¹⁰ -Met ¹⁵¹⁷ 283CBP: Ala ¹²³⁵ -Met ¹⁵¹⁷ 107CBP / 192FBP: Ile ¹⁴¹⁰ -Gln ¹⁵¹⁶ / Asn ²¹³³ -Glu ²³²⁴ 282CBP / 192FBP: Ala ¹²³⁵ -Gln ¹⁵¹⁶ / Asn ²¹³³ − Glu ²³²⁴

[0028]

As described above in detail, according to the present invention, there has been provided a genetic engineering production method which enables mass production of a functional polypeptide having at least fibrin binding activity. The polypeptide is useful for various uses such as wound healing and drug delivery systems.

[Brief description of the drawings]

FIG. 1 is a process chart showing an example of the construction of a plasmid of the present invention.

FIG. 2 is a process chart showing an example of plasmid construction of the present invention.

Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:19) (72) Inventor Yuki Sugawara 3-4-1 Seta, Otsu-shi, Shiga Takara Shuzo Co., Ltd. Central Research Laboratory (72) Inventor Ikunoyuki Kato 3-4-1 Seta, Otsu City, Shiga Prefecture Takara Shuzo Central Research Laboratory Co., Ltd.

Claims (2)

[Claims] 1. A transformant transformed with a recombinant plasmid containing a gene encoding a functional polypeptide represented by an amino acid sequence represented by the following formula (Formula 1). Embedded image Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gly Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2. A functional polypeptide, which comprises culturing the transformant according to claim 1, and collecting a functional polypeptide represented by the amino acid sequence represented by the formula (Formula 1) from the culture. A method for producing a peptide.