JP2854445B2 - Functional polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to novel polypeptides, and more particularly to artificial polypeptides having insulin-binding activity, nucleic acids encoding them, and the same.
The present invention relates to a genetic engineering production method using DNA .

[0002]

2. Description of the Related Art Human collagen type V (hereinafter referred to as h-ColV)
Is an extracellular matrix widely present in various tissues, has binding properties to thrombospondin, DNA and heparin, and is known to be synthesized by epidermal cells migrating at the wound site. Have been. The present inventors have previously found that h-ColV has a strong insulin binding activity (Japanese Patent Application Laid-Open No. 2-209899).

[0003]

[0005] h-ColV is useful as an insulin-binding carrier, and can construct a new drug delivery system for insulin having growth factor activity and hormonal activity. However, h-ColV
Is a substance in the living body and is difficult to prepare in large quantities. In addition, the incorporation of contaminants derived from living organisms is inevitable. It is an object of the present invention to provide h useful as an insulin-binding carrier.
-To create an artificial polypeptide corresponding to ColV.

[0004]

SUMMARY OF THE INVENTION In general , the present invention relates to an artificially functional polypeptide,
It is represented by the following general formula (Formula 1).

[0005]

(X) _m- (Y) _n -Z

(Wherein X is a cell adhesion domain polypeptide of human fibronectin, Y is a spacer, Z is a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 in its sequence and having an insulin binding activity , (m and n are 1 or 0) The second invention of the present invention relates to a nucleic acid encoding the artificial functional polypeptide of the first invention. Further, the third invention of the present invention relates to a recombinant plasmid containing a DNA encoding the artificial functional polypeptide of the first invention. The fourth invention of the present invention relates to a transformant into which the recombinant plasmid of the third invention has been introduced. Further, the fifth invention of the present invention provides an artificial functional plant, comprising culturing the transformant of the fourth invention and collecting the artificial functional polypeptide of the first invention from the culture. The present invention relates to a method for producing a polypeptide.

[0007] The insulin-binding active polypeptide of the present invention may be any polypeptide having an amino acid sequence containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is disclosed in Japanese Patent Laid-Open Publication No. Hei 2-209899, Biochimica et Biophys.
icaActa), Vol. 1035, pp. 139-154 (19
90) Hyp at positions 6, 9, and 33 of the insulin and heparin binding site polypeptide in the α1 chain of h-ColV represented by SEQ ID NO: 2 in the sequence listing described in P.
ro has been substituted.

[0009] The polypeptide represented by SEQ ID NO: 1 in the sequence listing can be chemically synthesized, for example. The polypeptide containing the polypeptide in its sequence includes a polypeptide represented by SEQ ID NO: 3 in the sequence listing (hereinafter referred to as ColV-I).
n), and the polypeptide can be produced, for example, by genetic engineering.

The gene sequence of the α-chain of h-ColV has been previously elucidated by the present inventors [The Journal of Biologi.
calChemistry), Vol. 266, Nos. 13124-131
29 (1991)], and the amino acid sequence of ColV-In represented by SEQ ID NO: 3 in the sequence listing is 79th described in the literature.
It is identical to the amino acid sequence from the 9th to the 984th.

[0011] A DNA fragment corresponding to this site can be obtained by, for example, PCR [P] from cDNA synthesized from human placenta mRNA.
olymerase Chain Reaction: Saiki et al., Science, 230, 1350-135.
4 (1985)].
Next, the amplified DNA fragment is treated with NcoI and BamHI and, for example, pTV118N [Febs Letters (F
EBS Letters), Volume 223, Page 174 (198
5)], a plasmid pTVColV containing the DNA fragment can be prepared.

In order to obtain a plasmid with high expression of the target polypeptide, DNA encoding the target polypeptide is excised from, for example, pTVColV with NcoI and BamHI, and then a plasmid with high expression, for example, pET8c
To create the plasmid pETColV. The plasmid is shown in FIG. In FIG. 1, A is ColV.
The DNA fragment site encoding -In is shown.

[0013] The pET-type plasmid is a study (Studi
e) A plasmid having a T7 RNA polymerase promoter prepared by the present inventors. By introducing this plasmid into Escherichia coli BL-21 expressing T7 RNA polymerase, proteins can be expressed effectively. pETColV
To transform E. coli BL-21 strain, and culturing the transformed strain, to obtain the Col represented by SEQ ID NO: 3 in the sequence listing.
V-In can be expressed, and SDS-
PAGE confirms the insulin binding activity of the polypeptide having a molecular weight of about 18 kd.

If a polypeptide having an insulin-binding activity is combined with a cell-adhesive activity, the affinity for various cells can be further increased, and the function of the insulin-binding carrier increases.

Such a polypeptide is, for example, the aforementioned ColV
-In and a cell adhesion active polypeptide can be prepared by binding them by a protein engineering technique or a genetic engineering technique. It can also be prepared, for example, by grafting a cell adhesion active polypeptide into the sequence of ColV-In.

The cell adhesion-active polypeptide is not particularly limited as long as it can impart cell adhesion. For example, a cell adhesion domain polypeptide of human fibronectin (hereinafter abbreviated as h-FN) can be used.

The primary structure of h-FN protein is described in TheEMBO Journal
), Vol. 4, pp. 1755-1759 (1985). For a cDNA clone (pLF5) encoding the cell adhesion domain, see Biochemistry, Vol. 25, No. 4936-
P.4941 (1986). We found that from pLF5, the cDNA for the cell adhesion domain
By taking out the fragment, connecting it to an expression vector and introducing it into Escherichia coli, a cell adhesion-active polypeptide and a method for producing the same have been developed and a patent application has been filed (Japanese Patent Application Laid-Open No. 1-2069).
No. 98). Cell adhesion domain c required in the present invention
DNA can be prepared, for example, using a recombinant plasmid pTF7021 described in JP-A-1-206998. Note that pTF7021 is the P of FN.
This plasmid expresses ro ¹²³⁹ -Met ¹⁵¹⁷ (279 amino acid residues: polypeptide represented by SEQ ID NO: 4 in the sequence listing). Immediately before the C-terminal stop codon of the translation region of pTF7021, a cloning site such as Nco
By introducing the I site, c of the cell adhesion domain
DNA can be ligated to DNA encoding other polypeptides.

In the present specification, the superscript number given to the amino acid of the cell adhesion active polypeptide is EMBL.
FN cDNA in the data bank (EMBL DATA BANK)
Shows the number of amino acid residues from the N-terminal added to the amino acid sequence obtained by translating the sequence.

The above pTVColV was replaced with NcoI and Hind II.
I to prepare a DNA fragment encoding ColV-In, followed by NcoI, Hin at the 3 'end of the translation region of plasmid pTF7520 derived from pTF7021.
By connecting to the dIII site, the polypeptide represented by SEQ ID NO: 5 in the sequence listing in which the cell adhesion domain of h-FN is linked to the insulin-binding activity polypeptide (hereinafter, referred to as “the polypeptide”).
C277-ColV) (recombinant plasmid pTF7520ColV). FIG.
Shown in In FIG. 2, A represents D encoding CoV-In.
NA fragment site, B: DN encoding cell adhesion domain
The A fragment site is shown.

[0020] The linker in pTF7520ColV contains DNA encoding the Met spacer as a linker. The presence or absence of a linker does not affect the effect of the present invention, but can be easily removed by site-specific mutagenesis if necessary. That is, the spacer in the above formula (Formula 1) is also an insertion sequence for adjusting the intermolecular distance between the cell adhesion active polypeptide and the insulin binding active polypeptide, and any amino acid or polypeptide can be used. , And can be removed as needed. In addition, the amino acid sequence of this spacer can be added to the C-terminal of the cell-adhesive polypeptide and the N-terminal of the insulin-binding polypeptide according to the purpose, or can be removed.

The resulting plasmid pTF7520ColV
Is introduced into Escherichia coli and cultured under appropriate conditions, whereby the target polypeptide is accumulated in Escherichia coli. Immunoblotting is used to confirm expression. After separating the whole cell proteins of the recombinant Escherichia coli by SDS-PAGE, the migration pattern is transferred to a nitrocellulose membrane. h
The target polypeptide is a monoclonal antibody (FN-10, Takara Shuzo) recognizing the cell adhesion domain of FN and a band detected by the method for measuring insulin binding activity.

The objective polypeptide is purified, 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,
This is centrifuged to obtain a supernatant. After dialysis of the supernatant, affinity chromatography such as heparin-agarose is performed. By the above operation, the target polypeptide can be purified.

The insulin binding activity of the polypeptide of the present invention is detected by the following method. That is, the polypeptide of the present invention is separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at an appropriate concentration, and this is blotted on a nitrocellulose membrane. This is reacted with peroxidase-labeled insulin in PBS, washed sufficiently, and labeled peroxidase activity is detected using 4-chloro-1-naphthol and hydrogen peroxide as substrates.

The cell adhesion activity can be measured, for example, by the method of Ruoslahti (Methods in Enzymology), Vol.
803-831 (1981)]. That is, after coating a sample, a suspension of Swiss mouse 3T3 or B16-F10 or MTD cells was added to a microtiter plate blocked with BSA.
After incubating for about 1 hour at ℃, wash the unadsorbed cells, collect the adsorbed cells by trypsin treatment, and measure the number of cells with a coulter counter to measure the strength of cell adhesion. Can be.

Table 1 shows the insulin binding activity and cell adhesion activity of the polypeptide prepared according to the present invention.

[0026]

[Table 1] Table 1 ────────────────────────────────── Insulin binding activity Cell adhesion activity ──── ────────────────────────────── ColV-In ++-C277-ColV ++ ++ ────────── ──────────────────────── (++: strong binding activity,-: no activity)

As shown in Table 1, the polypeptide having the polypeptide represented by SEQ ID NO: 1 in the sequence listing of the present invention in the molecule has a different structure from the natural α-chain of h-ColV, but has a strong insulin binding activity. Is shown.

The insulin-binding carrier and the insulin-binding carrier having cell adhesion activity can regulate homeostasis of insulin having growth factor activity and hormonal activity, and can be used for various diseases represented by diabetic patients. When you administer insulin to people, you can make a long-lasting, safe, non-immunogenic drug with excellent targeting to the site of action of insulin,
It is possible to construct a new formulation and a drug delivery system that maximizes the effect of insulin and has no side effects. For example, a transdermal drug that provides a growth-promoting reagent that is effective for biochemical research, provides useful cosmetics that increase the proliferation of epidermal cells, and prevents skin aging, and that promotes wound healing after trauma or surgery And a therapeutic agent are provided, and an insulin preparation which is used for the treatment of diabetes and has high durability and no side effects is provided.

[0029]

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

Example 1 Preparation of ColV-In (1-1) Cloning and Expression of DNA Encoding Insulin Binding Site Single-stranded cD from human placenta m-RNA (Clontech) using oligo dT primer
Synthesized NA [Methods in Enzymology
152, 316-324 (1987)]. That is, the reaction solution 25 mM Tris-HCl, pH
8.3, 100 mM KCl, 10 mM MgCl ₂ ,
500 μM dNTP (dATP, dCTP, dGT
P, TTP) oligo d (T) _12-18 0.5 μg (20 in total)
μl) is heated at 95 ° C. for 5 minutes, then 0.5 μg
m-RNA was added. The mixture was further heated at 95 ° C. for 3 minutes and rapidly cooled in ice. To this was added β-mercaptoethanol at a final concentration of 5.76 μM, and 25 units of reverse transcriptase (TAV: RAV-2) were added (total 25 μl).
The reaction was performed at 60 ° C. for 60 minutes. PCR of this product under the following conditions
To That is, primers represented by SEQ ID NOs: 6 and 7 in the sequence listing were synthesized by a DNA synthesizer,
Purified and used. Nucleotide numbers 9 to 27 of the primer are a DNA sequence encoding the 799th to 804th amino acid sequence of h-ColV described in The Journal of Biological Chemistry, supra, and base numbers 4 to 9 are introduced at the ends for cloning. NcoI restriction enzyme site, and base numbers 14 to 31 of the primer
The antisense sequence of the DNA encoding the 984th amino acid sequence, base numbers 5 to 10 are BamHI restriction enzyme sites introduced at the ends for cloning, base numbers 11 to 11.
13 is a newly introduced stop codon. For PCR, use a gene amplifier kit manufactured by Takara Shuzo Co., Ltd.
C., 30 seconds → 55 ° C., 1 minute → 72 ° C., 1 minute for 30 cycles. One-tenth of the reaction solution was subjected to 4% agarose gel electrophoresis, and as a result, amplification of a 580 bp target DNA fragment was confirmed. This product was treated with NcoI and BamHI, and placed at the same site of plasmid pTV118N at 16 ° C.
Ligation was performed for 30 minutes (using a ligation kit manufactured by Takara Shuzo). The plasmid thus constructed is called p
This was named TVColV, and was further named NcoI, BamHI.
After the treatment, a DNA fragment of about 580 bp was recovered using agarose gel electrophoresis. Next, this DNA fragment was ligated to the same site of the above-mentioned plasmid pET8c under the above-mentioned conditions. The plasmid constructed in this way is called pE
It was named TColV and introduced into the aforementioned E. coli BL-21 strain to obtain a transformant. Next, 18 of the resulting transformants were
Plasmid analysis was performed on the clones. That is, plasmids prepared by the rapid method were
The fragment was digested with amHI and subjected to agarose gel electrophoresis to examine the generation of a band of the expected NcoI-BamHI fragment (0.58 kb). As a result, a target band was observed in one clone. In addition, the nucleotide sequence was determined by the dideoxy method, and it was confirmed that the target sequence was contained. Escherichia coli BL-21 carrying this plasmid is Escherichia
coli BL-21 / pETColV, designated and designated by the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.
No. 8 (FERM P-12558). This strain was cultured in L-medium containing 50 μg / ml ampicillin.
The cells were cultured to D ₆₀₀ ≒ 0.5, and 1 mM IPTG was further added thereto, followed by culturing overnight. Cultured cells were transformed into 4 → 20% SDS-PAG
Analysis by E confirmed the expression of the polypeptide at a molecular weight of about 18 kd. Further, when the insulin binding property was checked by the Western blotting method described above, the polypeptide had insulin binding activity.

(1-2) Purification of expressed polypeptide Escherichia coli BL-21 / pE obtained in (1-1)
TColV was cultured overnight in 10 ml of L-medium containing 50 μg / ml ampicillin. 0.2 ml of this preculture was
100 ml of L-medium containing g / ml ampicillin was inoculated and cultured at 37 ° C. When the absorbance at 600 nm was 0.4, IPTG (isopropyl-β-D-thiogalactoside) was added to 1 mM, cultured overnight, and then collected. The obtained cells were cultured in 1 mM EDTA, 0.05%
Noridet P-40, 10 μg / ml aprotinin, 10 μg / ml leupeptin (l
eupeptin), 5 ml of PBS (-) containing 2 mM PMSF
And sonicated for 2 minutes to disrupt the cells. The supernatant obtained by centrifuging the cell lysate was equilibrated with PBS (-) and heparin-5PW HPLC column (Tosoh).
And eluted with PBS (-) containing 0.5M NaCl. When the SDS-polyacrylamide gel electrophoresis was performed on the eluted fraction, an almost single 18 kd target polypeptide was confirmed.

(1-3) Action of heparin on expressed polypeptide Since the insulin-binding site of the α1 chain of h-ColV also exhibits heparin-binding activity, heparin and insulin were applied to the polypeptide obtained in (1-2). Was examined for its binding properties. That is, in the western blotting method described above, after the blotting, when reacting 0.5 μg / ml of peroxidase-labeled insulin, 0.1 μg,
Changes in peroxidase color development were observed in the presence of 1.0 μg, 10 μg, 100 μg, and 1 mg of heparin. As a result, it was shown that the insulin binding property of the present polypeptide was strongly inhibited by the added heparin (Table 2).

[0033]

Table 2 Table 2 量 Amount of heparin added (μg / ml) Peroxidase Color development ０．１ 0.1 ++ 1 + 10 -100-1000-── ──────────────────────────────── (++: strong coloring, +: weak coloring,-: no coloring)

Example 2 Preparation of C277-ColV (2-1) Cloning of DNA Encoding Fusion Polypeptide The pTVColV of (1-1) was replaced with NcoI and HindI.
II, which was treated with NcoI of pTF7520,
The ligation was carried out at the HindIII site under the conditions described above. The plasmid thus constructed was replaced with pTF752
The plasmid was designated as 0 ColV, and Escherichia coli JM109 was transformed with the plasmid. 1 of the obtained transformants
Eight clones were analyzed for plasmid. That is, the plasmid prepared by the rapid method was replaced with HindIII.
And NcoI, subjected to agarose gel electrophoresis, and the expected NcoI-HindIII fragment (0.5
The generation of an 8 kb) band was examined. As a result, a target band was observed in one clone. In addition, the nucleotide sequence was determined by the dideoxy method, and it was confirmed that the target sequence was contained. E. coli JM109 carrying this plasmid was
scherichia coli JM109 / pTF7
No. 520ColV, designated and indicated by FERM BP-5277 at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.
Deposited as The above strain is cultured in an L-medium containing 50 μg / ml ampicillin, 1 mM (7) IPTG,
Next, the cultured bacterial cell protein was added to 4 → 20% SDS-PAG.
Analysis by E confirmed the expression of the polypeptide at a molecular weight of 48 kd. Furthermore, the binding property of this polypeptide to insulin and FN-10 was confirmed.

(2-2) Purification of expressed polypeptide Escherichia coli JM109 / pT obtained in (2-1)
F7520ColV was cultured overnight in 10 ml of L-medium containing 50 μg / ml ampicillin. 0.2 ml of this preculture
100 ml of L-medium containing 50 μg / ml ampicillin
And cultured at 37 ° C. The absorbance at 600 nm is 0.
At time point 4, IPTG was added to a concentration of 1 mM, cultured overnight, and then collected. The obtained cells were treated with 1 mM EDT.
A, 0.05% Noridet P-40, 10 μg / ml aprotinin, 10 μg / ml leupeptin, 2 mM PMS
The cells were suspended in 5 ml of PBS (-) containing F, and sonicated for 2 minutes to disrupt the cells. The supernatant obtained by centrifuging the cell lysate was equilibrated with PBS (-) and heparin-5PW
Apply to HPLC column (Tosoh), 0.5M NaC
and eluted with PBS (-). When the SDS-polyacrylamide gel electrophoresis was performed on the eluted fraction, an almost single 48 kd target polypeptide was confirmed.

Example 3 Using the polypeptides obtained in Examples 1 and 2,
Insulin binding activity, cell adhesion activity and cell growth promoting activity were measured.

(3-1) Measurement of Insulin Binding Activity ColV-In and C277-ColV were each subjected to SDS
Polyacrylamide gel electrophoresis (SDS-PAGE)
And transferred to a nitrocellulose membrane by Western blotting, and then incubated in a 0.05% Tween 20-PBS (-) solution supplemented with 10 μg / ml peroxidase-labeled insulin (manufactured by Sigma) at room temperature. And reacted for 2 hours. After washing the nitrocellulose membrane three times with 0.05% Tween 20-PBS (-), 0.3 mg of peroxidase activity bound to the nitrocellulose membrane was measured.
The detection was performed using 4-chloro-1-naphthol and 0.015% hydrogen peroxide dissolved in PBS (-) at a concentration of 0.15 / ml as substrates.
As a result, the peroxidase-labeled insulin was found to contain 18 kd molecular weight of ColV-In and 48 kd molecular weight of C277.
Each bound to -ColV.

(3-2) Measurement of Cell Adhesion Activity Cell adhesion activity was measured according to the method of Luos Lati et al. [Methods in Enzymology, Vol. 82, pp. 803-831 (1981)]. Sample is distilled water, PB
It was dissolved in S (phosphate buffered saline) or the like and injected into a 24-well microplate. After incubating at room temperature for 2 hours, the sample was adsorbed on the plate (400 μl / well). PBS containing 2% BSA (bovine serum albumin)
The solution was added at 500 μl / well and incubated at 37 ° C. for 2 hours to block the plate. After washing the plate with PBS, and advance Dulbecco ^(Dulbecco, S) Eagle's Minimal Swiss mice were suspended in nutrient medium (DMEM) 3T3 or B16-F10 or MTD cells 1 × 10
⁵ cells / well were dispensed and incubated at 37 ° C. for 1 hour. The cells used were those obtained by subculturing the cryopreserved strain and then treating with trypsin (37 ° C., 5 minutes). After washing the plate with PBS, the adsorbed cells were collected by trypsin treatment, and the cell adhesion activity was measured by counting the number of cells with a coulter counter. C277-
Table 3 shows the results of ColV and ColV-In.

[0039]

Table 3 ───────────────────────────── Sample (0.5 μM) Cell adhesion activity ───── ──────────────────────── C277-ColV ++ ColV-In − ─────────── (++: strong activity,-: no activity)

The numerical values indicate the concentrations when the sample was adsorbed on the plate. C277-ColV showed adhesive activity in all cells.

(3-3) Measurement of Cell Growth-Promoting Activity The cell growth-promoting activity of C277-ColV, which exhibited cell adhesion activity in (3-2), was examined. C27 dissolved in PBS
7-ColV was added to a 24-well microplate at 400 μl /
The sample was adsorbed on the plate after placing in a well and incubating at 37 ° C. for 2 hours. After washing the plate with PBS, an insulin solution was added at 400 μl / well, and incubated at room temperature overnight to adsorb insulin. After washing the plate with PBS, Dulbecco's minimal nutrient medium (DMEM) containing 10 μg / ml transferrin and 5 mg / ml BSA, ham F12 medium (HamF12) 1: 1.
5 × 10 ⁴ MTD cells / well and 0.1 μCi / well of ³ H-thymidine suspended in a mixed medium were added. 5%
After culturing at 37 ° C. for 48 hours in the presence of CO ₂ , the plate was washed with PBS, 5% TCA was added at 500 μl / well, and the cells were allowed to stand at 4 ° C. for 4 hours to fix the cells. After removing the TCA, 0.1N NaOH containing 2% Na ₂ CO ₃
The immobilized cells are lysed by adding 400 μl / well,
³ H-thymidine incorporation was measured by a liquid scintillation counter. In addition, as a contrast,
No. 998, a cell adhesive polypeptide represented by SEQ ID NO: 8 (abbreviated as C-274) was used. When C277-ColV is used as shown in Table 4,
Compared with C-274, it showed higher ³ H-thymidine incorporation, that is, a cell growth promoting activity.

[0042]

[Table 4] Table 4 ³ Sample ³ Incorporation of H-thymidine (× 10 ³ cpm) ────────────────────────────────── C277-ColV 10.5 C-274 6.6 ──────────────────────────────────

Next, the cell growth promoting activity was examined by measuring the number of cells. As described above, MTD cells were added to the plate on which each sample was adsorbed and on which insulin was further adsorbed. 5
After culturing for 48 hours in the presence of% CO ₂ , the number of cells was measured. As a result, when the present polypeptide was adsorbed, proliferation of more cells was observed as compared with C-274 as a control (Table 5).

[0044]

[Table 5] Table 5 数 Number of sample cells (× 10 ⁵ ) ──── {C277-ColV 1.7 C-274 1.1} ──────────────────

[0045]

As described above, according to the present invention, a novel polypeptide having an insulin binding activity, a novel polypeptide having a cell adhesion activity and an insulin binding activity, a nucleic acid encoding the same, and a DNA thereof are provided by the present invention.
The genetic engineering production method used is provided. These polypeptides can enhance the affinity between insulin and cells and exhibit useful actions and effects in fields where biochemical or medical significance can be found in the use of insulin. And a drag delivery system.

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of plasmid pETColV.

FIG. 2 shows the structure of plasmid pTF7520ColV.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/02 A61K 7/00 J // A61K 7/00 C12N 15/00 A 38/00 ADS A61K 37/02 ADS 38/28 AEE 37/26 AEE (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Akihiro Noda 3-4-1, Seta, Otsu City, Shiga Prefecture Takara Shuzo Co., Ltd. (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu, Shiga Prefecture Inside Takara Shuzo Co., Ltd. (72) Inventor Mika Hatai 1-9-10 Irifune, Chuo-ku, Tokyo (72) Invention The person Yoshihito Yaoi 1-30-1 212, Minowa-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture (56) References JP-A-2-311498 (JP, A) JP-A-2-209899 (JP, A) THE JOURNAL OF BIO LOGICAL CHEMISTRY, Vol. 266, no. 20, p. 13124-13129 (1991. July) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 19/00 C07K 14/47 C07K 14/78 C12N 1/21 C12N 15/00-15/62 C12P 21 / 00-21/02 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ

Claims (5)

(57) [Claims] 1. The following general formula (1): ## STR1 ## (X) _m- (Y) _n -Z (where X is a cell adhesion domain polypeptide of human fibronectin, Y is a spacer, and Z is its sequence) An insulin containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
An artificially functional polypeptide, characterized in that the polypeptide has phosphorus-binding activity , and m and n are 1 or 0). (2) The artificial functional polypeptide of claim 1.
A nucleic acid that encodes a code. (3) The artificial functional polypeptide of claim 1.
Recombinant plasmid containing DNA encoding DNA
De. (4) Introduction of the recombinant plasmid according to claim 3.
Transformed transformant. (5) Culturing the transformant according to claim 4,
2. The artificial functional polypeptide according to claim 1 from a culture.
Of an artificial functional polypeptide characterized by being collected
Production method.