JP2805384B2 - Functional polypeptide - Google Patents

Description

The present invention relates to a novel polypeptide derived from human vitronectin (hereinafter, vitronectin is abbreviated as VN) which inhibits cell adhesion, and a novel polypeptide derived from human VN. The present invention relates to a novel artificial functional polypeptide to which an insulin-binding activity polypeptide is directly or indirectly bound, a nucleic acid encoding the same, and a method for producing the peptide using the DNA thereof by genetic engineering.

[Conventional technology]

Human VN is a glycoprotein present in plasma and extracellular matrix, and is known to have various functions. Natural human VN may be used in wound healing and other pharmaceuticals and cosmetics, but its supply is limited because it is collected from the blood, it is expensive, and pathogenic bacteria, viruses, etc. It has not been put to practical use because of the possibility of contamination by ash.

In addition, extracting and utilizing a natural human VN fragment peptide has not been put to practical use for the same reason.

[Problems to be solved by the invention]

One of the physiological functions of human VN is cell adhesion activity. The center of human VN cell adhesion activity is its amino acid sequence.
Arg ^45- Asp ⁴⁷ has already been identified [Chemistry and Biology, Vol. 24, p. 312 (1986)]. If a polypeptide containing this sequence can be prepared, it will competitively inhibit the cell adhesion activity of human VN, and proteins or polypeptides that inhibit such cell adhesion will promote cell motility at low concentrations, thereby It is thought to be effective for healing and to inhibit cancer cell metastasis by inhibiting the adhesion of cancer cells at a high concentration.

As a new physiological function of VN, the present inventors
It has been found that VN has insulin binding activity (JP-A-1-85934 and JP-A-1-86000). If VN or an insulin-binding active site polypeptide of VN is used as an insulin-binding carrier, these insulin-binding carriers can regulate the homeostasis of insulin having a growth factor activity and a hormonal activity. When administering insulin to a variety of illnesses, it is possible to create a long-lasting, safe, non-immunogenic drug with excellent targeting to the site of action of insulin, and to improve the effects of insulin. It is possible to construct a new formulation and a drug delivery system that is maximized and has no side effects. For example, a transdermal drug that provides a growth-promoting agent 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.

Furthermore, if an artificial polypeptide in which such an insulin-binding carrier is bound to a cell adhesion-inhibiting polypeptide can be prepared, the wound healing effect and the like can be further enhanced.

An object of the present invention is to provide a novel polypeptide that inhibits the cell adhesion activity of human VN, and an artificial functional polypeptide that binds the polypeptide to the insulin-binding active site polypeptide of human VN and has both functions in one molecule. An object of the present invention is to provide peptides and a method for producing these polypeptides.

[Means for solving the problem]

According to an overview of the present invention, the first invention of the present invention relates to an artificially functional polypeptide, and has the following general formula I: X- (Y) _n -Z (I) wherein X is a human vitronectin molecule 2 shows the amino acid sequence Ile ³³⁵ -Ser ³⁸⁸ shown in FIG. 2 derived from the polypeptide in FIG. 2, Y represents the spacer represented by the formula Lys-Asn-Ser, and Z represents the polypeptide derived from the human vitronectin molecule. 1 represents the amino acid sequence Lys ⁴⁰ -Pro ¹²⁵ shown in FIG. 1, and n represents 1 or 0), and has an insulin binding activity and a cell adhesion inhibitory activity.

The second invention of the present invention relates to a nucleic acid encoding the artificially functional polypeptide of the first invention. The third invention of the present invention relates to a plasmid incorporating a DNA encoding the artificially functional polypeptide of the first invention. The fourth invention of the present invention relates to a transformant into which the plasmid of the third invention has been introduced. The fifth invention of the present invention relates to a method for producing the artificially functional polypeptide of the first invention, wherein the transformant of the fourth invention is cultured, and the artificially functional polypeptide of the first invention is cultured from the culture. Is collected.

The spacer in the formula [I] is also an insertion sequence for adjusting the intermolecular distance between the insulin-binding active site polypeptide and the cell adhesion-inhibiting active polypeptide, and any amino acid or polypeptide can be used. Can also be removed according to. In one example of the present invention, Lys-Asn-Ser- is inserted between the insulin-binding active site polypeptide and the cell adhesion-inhibiting active polypeptide. The additional amino acid in the formula (I) is
A plasmid derived from the plasmid used, which is added to the polypeptide of the present invention when the polypeptide for inhibiting cell adhesion of the present invention is produced by genetic engineering. Ala-Met-Ile-Thr -Asn-Ser-, which can be removed if necessary.

 Hereinafter, the present invention will be described specifically.

Examples of the polypeptide that inhibits the cell adhesion activity of human VN include Lys ^{40 to} Pro ¹²⁵ polypeptide shown in FIG. 1. The polypeptide can be prepared by, for example, a genetic engineering method. That is, FIG. 1 is a view showing the amino acid sequence of the above-mentioned cell adhesion inhibitory activity polypeptide.

The insulin binding active site polypeptide in the human VN molecule in the present invention is shown in, for example, FIG.
There is a polypeptide of Ile ^335- Ser ³⁸⁸ , which can be made, for example, by genetic engineering. That is, FIG. 2 is a view showing the amino acid sequence of the insulin binding active site polypeptide.

The polypeptide containing the amino acid sequence shown in FIG. 1 can be prepared, for example, as follows.

From human placenta m-RNA (manufactured by Clonetech), a conventional method [Methods in Enz
Single-stranded cDNA is synthesized in accordance with the method described in Vol. 152, pp. 316-324 (1987)]. Using this as a template, Lys ⁴⁰
-A DNA region encoding a region containing Pro ¹²⁵ was subjected to PCR [Polymerase Chain Reaction].
eaction): Saiki et al., Science (Scienc)
e), Vol. 230, pp. 1350-1354 (1985)]. The amplified DNA was treated with EcoR I and BamHI, and the digested DNA fragment was treated with plasmid pTV118N [FEBS Letters (FEBS Le
223, p. 174 (1987)] to create plasmid pTVVN- (RGD). To obtain a highly expressing plasmid for the polypeptide of interest, pTV VN
-From (RGD), the DNA encoding the target polypeptide was
o I, excise with BamHI, then high expressing plasmid,
For example, introduced to the same site of pET8c (Novagen),
Create plasmid pET VN- (RGD). The plasmid is shown in FIG. That is, FIG. 3 shows the plasmid pET VN-
It is a figure showing the composition of (RGD).

This pET-type plasmid is a plasmid having a T7 RNA polymerase promoter created by Study et al.
By introducing the protein into the BL-21 strain, the protein can be expressed effectively, and the pET VN- (RGD) of the present invention is used to transform E. coli B
By transforming the L-21 strain and culturing the transformed strain, the target polypeptide is efficiently expressed. As a method for purifying the expressed polypeptide, for example, Al ₂ O ₃ is added to cultured cells, mixed and crushed, and a soluble fraction is collected.
This is, for example, DEAE-Sephacel (Pharmacia),
Purification is performed by reverse phase-ODS column HPLC (manufactured by Tokyo Chemical Industry) or the like. Purified polypeptide was analyzed for its amino acid composition, N
Perform terminal analysis and confirm the expression of the target polypeptide.

Cell adhesion inhibition activity of the obtained polypeptide is BHK or NRK.
It is expressed by measuring the inhibitory activity of the polypeptide on the adhesive activity of VN to these cells using cells. The measurement of cell spreading inhibitory activity can be performed, for example, by using Ruoslati (Ruoslati).
hti) et al. [Methods in Enzymology,
Vol. 82, pp. 803-831 (1981)]. That is, after coating human VN, a suspension of the present polypeptide and BHK or NRK cells is added to a microtiter plate blocked with BSA and incubated at 37 ° C. for about 1 hour. After washing, the cells are fixed with formalin, and the percentage of cells that have spread is measured under a microscope, whereby the strength of the inhibitory activity of the present polypeptide on the cell spreading activity of VN can be determined.

The polypeptide containing the amino acid sequence shown in FIG. 2 can be prepared, for example, as follows.

 First, the insulin binding active site of human VN is determined.

That is, human VN or, for example, chemically degraded polypeptide is separated by SDS-polyacrylamide gel electrophoresis at an appropriate concentration, and this is blotted onto 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 insulin-binding active site of human VN is in a 5 kd polypeptide obtained by fragmenting the human VN molecule with bromocyanide, and this polypeptide is purified by reverse phase-ODS column HPLC (0.01% TFA, acetonitrile system) to obtain the N-terminal amino acid. The sequence can be determined. The polypeptide is Ala-Pro-Arg-P from its N-terminus.
It has the amino acid sequence of ro. This sequence is the previously reported human VNcD
NA sequence [The EMBO Journa
l), Vol. 4, pp. 3153-3157 (1985)]
Sequence corresponding to ^341- Pro ³⁴⁴ .

In bromocyan treatment, the 5 kd polypeptide corresponds to Ala ³⁴¹ -Met ³⁸¹ since the Met residue is cleaved off.
Therefore, a DNA fragment corresponding to this site is amplified by PCR from human genomic DNA, and then the amplified DNA
The fragment was treated with NcoI and EcoRI, and ligated to the same site of pTV118N to obtain a human insulin-binding active site polypeptide and 1acZ '[gene (Gen (Gen.
e) The plasmid pTV VN-In. containing the gene encoding the fusion protein of Vol. 33, pp. 103-119 (1985)].
Can be produced. The plasmid is shown in FIG. FIG. 4 shows the structure of the plasmid pTV VN-In. Microorganisms such as E. coli
By transforming the JM109 strain and culturing the transformant, a fusion polypeptide with 1acZ 'containing the insulin-binding active site polypeptide of human VN can be expressed. The insulin binding activity of the polypeptide is confirmed by the method described above.

Next, the polypeptide of the present invention in which the human VN insulin-binding active site polypeptide is bound to the human VN-derived cell adhesion-inhibiting activity polypeptide can be prepared, for example, as follows.

From the plasmid pTV VN-In. Described above, a DNA fragment encoding the insulin binding active site polypeptide was
It is excised with oRI and introduced into the same site of plasmid pTV VN- (RGD) to prepare plasmid pTV VN-In. + (RGD). The plasmid is then treated with NcoI, BamHI and the insulin binding active site polypeptide and human VN
A gene fragment encoding the fusion polypeptide of the cell adhesion-inhibiting activity polypeptide derived therefrom was cut out and introduced into the same site of the above-described high expression plasmid pET 8c, and the plasmid, pE
Create TVN-In. + (RGD). The plasmid is shown in FIG. That is, FIG. 5 shows the plasmid pET VN-In. + (R
FIG. 3 is a diagram showing a configuration of GD).

The linking part in pET VN-In. + (RGD) contains a DNA encoding the spacer Lys-Asn-Ser 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.

Each of the obtained plasmids is introduced into Escherichia coli and cultured under appropriate conditions, whereby the target polypeptide is accumulated in Escherichia coli. For confirmation of expression, for example, immunoblotting is used. After the whole cell protein of the recombinant Escherichia coli is separated by SDS-polyacrylamide gel electrophoresis, the migration pattern is transferred to a nitrocellulose membrane.
The target polypeptide is a monoclonal antibody recognizing human VN and a band detected by the method for measuring insulin binding activity described above.

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 obtain a supernatant. After dialysis of the supernatant, it is passed through a column of a DEAE ion exchanger, followed by affinity chromatography such as a CM ion exchanger and / or heparin-agarose.
By the above operation, the target polypeptide can be purified.

The obtained polypeptide can be measured for its cell spreading inhibitory activity by the method described above, and its insulin binding activity can also be confirmed.

As described above, an artificially functional polypeptide having both insulin binding activity and cell adhesion inhibitory activity is provided.

The cell adhesion inhibitory activity polypeptide of the present invention blocks a receptor involved in cell adhesion, thereby promoting its motility or causing adhesion inhibition. By these effects, for example, the motility of cells at a wound site can be controlled and contribute to healing.

In addition, cancer metastasis or the like can be suppressed by inhibiting readhesion of cancer cells to tissues.

Further, the fusion polypeptide of the insulin binding active site polypeptide of the present invention and the cell adhesion inhibitory activity polypeptide is a novel polypeptide having both functions in one molecule, while promoting cell motility, and Insulin can activate cells with this polypeptide, which can heal small wounds, for example, minor wounds on the face, effectively and quickly.

In this way, the novel polypeptide can effectively exert the various effects of insulin on cells, and controls the use and action of insulin, such as the construction of a local drug delivery system and the development of sustained-release drugs. It is also useful.

Reference Example 1 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 Human VN Cell Adhesion Inhibitory Activity Polypeptide (1-1) Construction of Human VN Cell Adhesion Inhibitory Activity Polypeptide Expression System Human placenta-derived m-RNA (manufactured by Clontech) using oligo dT primers Single-stranded cDNA was synthesized.

That is, the reaction solution 25 mM Tris-HC1 pH 8.3,
100 mM KCl, 10 mM MgCl ₂ , 500 μM dNTP (dATP, dCTP, dGT
P, TTP), oligo d (T) _12-18 0.5μg (total 20μ)
Heat treatment at 95 ° C. for 5 minutes, and then add 0.5 μg of m-RNA. Further heat-treat at 95 ° C for 3 minutes and quench in ice. To this, β-mercaptoethanol was added to a final concentration of 5.76 μM, and 25 units of reverse transcriptase (RAV-2 Takara Shuzo) were added (total 25 μ), and reacted at 42 ° C. for 1 hour. This product was directly subjected to PCR under the following conditions using a gene amplifier kit and a thermal cycler (both manufactured by Takara Shuzo).

That is, the following primers were synthesized by a DNA synthesizer using the above cDNA as a template, and then purified by HPLC.

Here, the underline is the DNA sequence of human VN.

Is the corresponding amino acid number, Indicates a restriction enzyme site introduced at the end for cloning.

Next, 0.1 μl of the cDNA product and 0.5 μl of Tac polymerase (Takara Shuzo) were added to the primers and 50 pmol each, × 10 PCR buffer, 500 μM dNTP (99.4 μm in total), and 94 ° C. for 1 minute and 58 ° C. for 1 minute using a thermal cycler. , 72 ° C 1.
The 40-cycle reaction was repeated with a temperature cycle of 5 minutes.

As a result, a partial cDNA (279 bp) containing a DNA sequence encoding amino acids No. 40 to No. 125 of human VN was obtained.

This 279 bp cDNA was treated with restriction enzymes EcoR I and BamH I, and dephosphorylated EcoRI-BamH of plasmid pTV118N.
The plasmid pTV VN- (RGD) (6th
Figure). FIG. 6 shows the structure of the plasmid.

Further, the plasmid pTVVN- (RGD) was treated with restriction enzymes NcoI and BamHI, and a target 286 bp DNA fragment was recovered by agarose gel electrophoresis. This was transformed into a plasmid pET8c (7th
The plasmid introduced into the NcoI-BamHI site shown in the figure) and constructed was named pET VN- (RGD). That is, the seventh
The figure shows the structure of plasmid pET8c. Escherichia coli B having T7 RNA polymerase on its chromosome under the control of this plasmid pET VN- (RGD) under the 1ac promoter.
It was introduced into L21 strain to obtain a transformant. Next, plasmid analysis was performed on 20 clones in the obtained transformants. That is, the plasmid prepared by the rapid method was
After digestion with I and Nco I, the resultant was subjected to agarose gel electrophoresis, and the formation of a band of the expected Nco I-BamHI fragment (2.9 kb) 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. Escherichia coli BL21 / pET
It was named VN- (RGD).

(1-2) Expression and Purification of VN Cell Adhesion Inhibitory Activity Polypeptide The above strain was cultured in an L-medium in the presence of 150 μg / ml ampicillin and 1 mM IPTG (isopropylthio-β-D-galactoside), and the cells were 12.5% Analysis was performed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). as a result,
50-60% of the total bacterial mass was the target polypeptide.

 Next, the target polypeptide was purified by the following method.

An appropriate amount of Al ₂ O ₃ was added to the cells collected by centrifugation, and the cells were mixed and crushed. 50 mM Tris-HC1 pH 7.3 containing 1 mM PMSF (phenyl methanesulfonyl fluoride),
A buffer of 50 mM NaCl was added, and a soluble fraction was collected. The extract was added to a DEAE Sephacell (Pharmacia) column equilibrated with the same buffer, washed sufficiently, and the target peptide was recovered with the same buffer containing 125 mM NaCl.

 Next, this fraction was dialyzed against distilled water and freeze-dried.

0.1% using a reverse phase column (ODS column Tokyo Kasei)
Acetonitrile gradient in the presence of trifluoroacetic acid 0
Chromatography was performed under conditions of ％ 30% to obtain a final purified product.

N-terminal amino acid sequence analysis of the polypeptide, and performs the amino acid composition analysis, the polypeptide is Lys ⁴⁰ -Pro ¹²⁵
Was confirmed to be a polypeptide (hereinafter, referred to as polypeptide VN- (RGD)) in which an additional polypeptide derived from an Ala-Met-Ile-Thr-Asn-Ser- plasmid was bound to the N-terminus.

In addition, this polypeptide has a 12-5% gradient SDS-P
AGE gives a single band at a molecular weight of about 23 kd, and its mobility differs greatly from the calculated molecular weight of about 11 kd. This SDS
The fact that the mobility on -PAGE does not match the molecular weight is a characteristic of the present peptide.

(1-3) Removal of Intervening Sequence from pET VN- (RGD) Polypeptide VN Expressed by pET VN- (RGD)
-(RGD) has Ala-Met-Ile at the N-terminal of Lys ⁴⁰ -Pro ¹²⁵
-Thr-Asn-Ser is added. The DNA sequence corresponding to the additional polypeptide is removed by a technique site-specific mutagenesis, to obtain a plasmid expressing Lys ⁴⁰ -Pro ^125, pET
/ VN- (RGD).

Reference Example 2 Cloning of Insulin Binding Active Site Polypeptide of Human VN (2-1) Identification of Insulin Binding Active Site Human VN (manufactured by Takara Shuzo Co., Ltd.) becomes 5 mg / ml in a 70% formic acid solution containing 20 mg / ml bromocyan. After dissolving in the same manner and blowing N ₂ gas to remove O ₂ in the solution, the mixture was reacted at room temperature for 24 hours to fragment. A 10-fold amount of water was added thereto, and lyophilized. The sample was dissolved in PBS (-) to a concentration of 1 mg / ml, and 10 µl of this solution was subjected to 20-5% gradient SDS-PAGE.
After transfer to a nitrocellulose membrane by Western blotting, the mixture was reacted at room temperature for 2 hours in a 0.05% Tween 20-PBS solution supplemented with 5 μg / ml peroxidase-labeled insulin (manufactured by Sigma). .

After washing the nitrocellulose membrane seven times with 0.05% Tween 20-PBS and three times with PBS, the peroxidase activity bound to the nitrocellulose membrane was adjusted to 0.5 mg / ml with PBS (−).
4-chloro-1-naphthol and 0.01% hydrogen peroxide dissolved in E. coli were detected as substrates.

As a result, peroxidase-labeled insulin was converted to human VN
Among the fragments, it bound to a polypeptide having a molecular weight of about 5 kd.

Next, this 5 column was subjected to reverse phase-ODS column (Tokyo Chemical) HPLC.
The kd polypeptide was purified under the following conditions.

That is, in the presence of 0.1% TFA, acetonitrile 0% → 60
Using a% gradient, the peak showing the absorption at UV 220 nm was analyzed by SDS-PAGE to obtain a polypeptide having a molecular weight of 5 kd.

Next, when the N-terminal amino acid sequence of this polypeptide was determined, the N-terminal amino acid sequence was Ala-Pro-Arg-Pro.
Met. When this amino acid sequence is compared with the previously reported sequence of human VN cDNA (described above), it matches Ala ^341- Pro ³⁴⁴ . The amino acid residue cleaved by bromocyanine is Met, which indicates that the 5 kd polypeptide is converted to Ala ³⁴¹ -Met ^381.
Was identified as the polypeptide corresponding to

(2-2) Cloning and Expression of DNA Encoding Insulin Binding Active Site Polypeptide A human VN DNA fragment corresponding to the polypeptide of (2-1) is cloned as follows, and then cloned into E. coli 1acZ 'polypeptide. Was expressed as a fusion polypeptide.

That is, the following primers were synthesized and purified by a DNA synthesizer using human lung genomic DNA as a template, and PCR was performed using the primers.

The underline is the DNA sequence of human VN, Is the corresponding amino acid number, Indicates a restriction enzyme site introduced at the end for cloning.

PCR was performed using the GeneAmp kit and template D.
NA is 1μg, temperature cycle 94 ° C, 1 minute → 55 ° C, 2 minutes → 72
The cycle was performed at 30 ° C. for 1 minute. One-tenth of the reaction solution was subjected to 4% agarose gel electrophoresis. As a result, the target DNA of 189 bp was obtained.
The amplification of the fragment was confirmed.

This product was treated with NcoI and EcoRI, and the plasmid pTV11
Ligation was performed at 16 ° C for 30 minutes at the same site of 8N (using a ligation kit manufactured by Takara Shuzo). The plasmid thus constructed was named pTV VN-In.
M109 was transformed (Takara Shuzo Competent Cell JM109
use). Next, plasmid analysis was performed on 18 clones in the obtained transformants. That is, the plasmid prepared by the rapid method was digested with EcoR I and NcoI, subjected to agarose gel electrophoresis, and the expected NcoI-EcoR
The generation of the band of the I fragment (0.18 kb) was examined. as a result,
The desired 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 JM109 carrying this plasmid
Was named Escherichia coli JM109 / pTV VN-In.

Lactic acid strain in the presence of 150 μg / ml ampicillin, 1 mM IPTG
-Cultured in medium and cells were analyzed by 12.5% SDS-PAGE. As a result, a polypeptide having a molecular weight of about 18 kd and fusion of human VN polypeptide and 1acZ 'derived from Escherichia coli as follows was obtained.

Next, as a result of checking the insulin binding activity of the present fusion polypeptide by the method using the aforementioned Western blot, strong insulin binding activity was confirmed.

Example 1 Preparation of Fusion Polypeptide of Human VN Insulin Binding Active Polypeptide and Human VN Cell Adhesion Inhibitory Active Polypeptide (3-1) Construction of Fusion Polypeptide Expression System The above plasmids pTV VN- (RGD) and pTV VN-In. Was used to reconstruct the DNA sequence of the polypeptide they encoded.

That is, plasmid pTV VN-In. Was replaced with restriction enzymes NcoI and EcI.
After treatment with oRI, a DNA fragment (172 bp) encoding the human VN insulin-binding activity polypeptide was obtained. This was introduced into the NcoI-EcoRI site of plasmid pTVVN- (RGD) to prepare plasmid pTVVN-In. + (RGD). This plasmid was then treated with NcoI-BamHI and the excised DNA
The fragment was ligated into the NcoI-BamHI site of plasmid pET8c as described above. The plasmid thus constructed was designated as pET VN-In. + (RGD), and Escherichia coli BL21 was transformed with the plasmid.

Plasmid analysis was performed on 18 clones in the obtained transformants. That is, the plasmid prepared by the rapid method was digested with NcoI and BamHI, subjected to agarose gel electrophoresis, and an expected NcoI-BamHI fragment (4.6 kb) was obtained.
The formation of the 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.

Escherichia coli BL21 carrying this plasmid is
coli BL-21 / pET VN-In. + (RGD), and the Institute of Microorganisms, National Institute of Advanced Industrial Science and Technology
P-11592).

(3-2) Expression and purification of fusion polypeptide Cultured according to the method described in Reference Example 1, and 12.5
% SDS-PAGE was performed to confirm the expression of the expected polypeptide of 27 kd, and the insulin binding activity of the polypeptide was also confirmed by the method described above.

Next, the cells collected by centrifugation were subjected to ultrasonic treatment to obtain a cell lysate. 50mM Tris-HC containing 1mM PMSF
1, a buffer of pH 7.3, 50 mM NaCl was added, and a soluble fraction was collected. This extract was equilibrated with the same buffer.
After adding to the DEAE Sephacel column and washing thoroughly,
The target peptide was recovered with the same buffer containing 125 mM NaCl. Next, the mixture was added to a CM-Toyopearl 650 (manufactured by Tosoh Corporation) column equilibrated with a 20 mM KH ₂ PO ₄ (pH 7.0) buffer, and the non-adsorbed fraction was removed with the same buffer.
The target polypeptide was eluted with the same buffer containing NaCl. The polypeptide was then added to a heparin-Toyopearl 650M (manufactured by Tosoh Corporation) column equilibrated with the same buffer containing 0.15M NaCl, washed thoroughly, and then washed with 0.15-0.4M
Chromatography was performed under NaCl conditions to obtain the desired polypeptide [hereinafter referred to as polypeptide VN-In. + (RGD)].

This polypeptide was single electrophoretically, and amino acid analysis from its N-terminus was consistent with the target sequence.

(3-3) Removal of Intervening Sequence from pET VN-In. + (RGD) The polypeptide VN-In. + (RGD) expressed by pET VN-In. + (RGD) has the following amino acid sequence: , as well as And amino acids and polypeptides derived from the plasmid represented by The DNA sequence corresponding to Lys-Asn-Ser was removed by site-directed mutagenesis and Gly- (Ile ^335- Ser
³⁸⁸ ) and a plasmid expressing a polypeptide in which Lys ⁴⁰ -Pro ¹²⁵ is directly linked, and named pET / VN-In. + (RGD).

Next, the polypeptide Gly expressed by the plasmid
N-terminal G of-(Ile ³³⁵ -Ser ³⁸⁸ )-(Lys ⁴⁰ -Pro ¹²⁵ )
The DNA corresponding to ly was removed by site-directed mutagenesis,
³³⁵ -Ser give ³⁸⁸ and Lys ⁴⁰ -Pro ¹²⁵ is plasmid expressing a binding polypeptide, was designated pETVN-In. + And (RGD).

Reference Example 3 Measurement of Biological Activity Using the respective polypeptides obtained in Example 1 and Reference Example 1, the cell adhesion inhibitory activity was measured.

That is, 1 μg / ml of a human VN PBS solution is added to a 96-well plate (Falcon) at 50 μl / well and left at 37 ° C. for 2 hours. After washing with PBS, 1% BSA in PBS solution 100μ
And block at 37 ° C. for 1 hour. After washing with PBS, DME of each polypeptide containing 3 × 10 ⁴ BHK cells
(Minimum Dulbecco's modification) Add 100μ of medium solution. 37
After standing at 1 ° C. for 1 hour, the percentage of expanded BHK cells was measured under a microscope, and the cell adhesion activity of human VN and the polypeptide VN- (RGD) of the present invention and VN-In. + (RGD) were determined. The cell adhesion inhibitory activity is measured.

Table 1 also shows the cell adhesion inhibitory activity of the polypeptide of the present invention and the insulin binding activity measured by the method described above.

As shown in Table 1, polypeptide VN- (RGD) has cell adhesion inhibitory activity, and polypeptide VN-In. + (RGD) has cell adhesion inhibitory activity and insulin binding activity.

〔The invention's effect〕

The present invention provides a functional polypeptide related to a substance in a living body, a nucleic acid encoding the same, and a method for producing the peptide using the DNA thereof by genetic engineering. This substance is useful as pharmaceuticals such as wound healing agents, cancer metastasis inhibitors, anti-inflammatory agents, and cosmetics, and has a remarkable effect in that it can be supplied in large quantities by genetic engineering.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence and the gene sequence of a human VN-derived polypeptide that inhibits cell adhesion, and FIG.
FIG. 3 shows the amino acid sequence and gene sequence of the insulin binding active site polypeptide of FIG.
FIG. 4 shows the structure of (RGD). FIG. 4 shows the plasmid pTV VN-I.
FIG. 5 shows the structure of plasmid pET VN-In. +
Fig. 6 shows the structure of (RGD), and Fig. 6 shows the plasmid pTV VN-
FIG. 7 is a diagram showing the structure of (RGD), and FIG. 7 is a diagram showing the structure of plasmid pET8c.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 38/00 ABE A61K 37/02 ADS ADP ADU ADS ABE ADU ADU ADP (C12N 1/21 C12R 1:19) (C12P 21 / 02 C12R 1:19) (56) References JP-A-64-86000 (JP, A) Chemistry and Biology 24 (5) 303-313 (1986) 40th Annual Meetin g of the Japan Soc iety for Cell Biol ogy (1987) Cell Stru ct funct 2D 1445 (58) investigated the field (Int.Cl. ^6, DB name) C12N 15/62 C12P 21 / 02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A following general formula _{I: X- (Y) n -Z} ··· [I] (wherein, X is the amino acid sequence Ile ³³⁵ represented by Figure 2 from the polypeptide in human vitronectin molecules -Ser ³⁸⁸ , Y represents a spacer represented by the formula Lys-Asn-Ser, Z represents the amino acid sequence Lys ⁴⁰ -Pro ¹²⁵ represented in FIG. 1 derived from the polypeptide in the human vitronectin molecule, n represents 1 or 0), and has an insulin binding activity and a cell adhesion inhibitory activity. 2. A nucleic acid encoding the artificially functional polypeptide according to claim 1. [3] A plasmid into which a DNA encoding the artificially functional polypeptide according to [1] is incorporated. 4. A transformant into which the plasmid according to claim 3 has been introduced. 5. A method for producing an artificially functional polypeptide, comprising culturing the transformant according to claim 4, and collecting the artificially functional polypeptide according to claim 1 from the culture.