JPH02311498A - Functional polypeptide - Google Patents

Abstract

NEW MATERIAL:A functional peptide which is shown by formula I (C277 is 277 amino acid peptide residue corresponding to Pro<1239>-SER<1515> of cell adhesion domain of human fibronectin; H271 is 271 amino acid peptide residue correspond ing to Ala<1690>-Thr<1360> of heparin adhesion domain of human fibronectin; X is peptide residue shown by formula II; (n) is 0 or 2) wherein the cell adhesion domain of human fibronectin is bonded directly or through a linker peptide to the heparin adhesion domain of human fibronectin. USE:A treating agent for wounds. PREPARATION:For example, a DNA to code a polypeptide wherein a cell adhe sion domain of human fibronectrin is bonded to a heparin adhesion domain of human fibronectin is synthesized, bonded to a manifestation vector to prepare a recombinant plasmid. The plasmid is introduced to a host cell, which is transformed and then the transformant is cultured to give a functional peptide shown by formula I.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel polypeptide, and more specifically to a novel functional polypeptide containing a human fibronectin cell adhesion domain peptide and a heparin-binding domain peptide, and a novel functional polypeptide thereof. Regarding the manufacturing method.

[Conventional technology]

Fibronetatin (hereinafter referred to as FN) is a glycoprotein present in plasma and extracellular matrix, and is known to have a variety of functions [Annual Review
Of Biochemistry - (^nnual
Review or old chemisLry)
, Vol. 57, pp. 375-413 (1988)). Attempts have been made to use natural FN for wound healing, medicines such as eye drops, and cosmetics, but since it is collected from blood,
It has not been put into practical use because of limited supply, high cost, and the possibility of contamination with pathogenic bacteria and viruses. Furthermore, extracting and utilizing the functional domain of FN has not been put to practical use for the same reason.

[Problem to be solved by the invention]

It is known that FN has two regions that bind to heparin (heparin-binding domains), one of which is located near the N-terminus, and that Ca ions are required for binding. The other region is located near the C-terminus, and the heparin binding activity of this region is stronger than that of the aforementioned region, and is not affected by Ca ions.

Recent studies have gradually revealed that the heparin-binding domain of FN, like the cell adhesion domain, plays an important role in the adhesion, spreading, and migration of fibroblasts, endothelial cells, and certain cancer cells. It's here. The heparin-binding domain of FN binds to proteoglycans on the surface layer of cells and is thought to contribute to cell adhesion, spreading, migration, etc. by causing interaction between cells and extracellular matrix. Therefore, a polypeptide with both cell-adhesive and pepperin-binding functions is useful for both cell and extracellular proteins.
It binds to both J-Fuchs and contributes to the repair of tissue at the wound site and maintenance of homeostasis, and is expected to be used as a pharmaceutical.

An object of the present invention is to provide a novel functional polypeptide that has both the cell adhesion activity and heparin binding activity of FN, and an advantageous method for producing the same.

[Failure to solve the problem]

To summarize the present invention, the first invention of the present invention is a novel functional polypeptide characterized in that the cell adhesion domain and heparin binding domain of FN are linked directly or via a linker peptide. Regarding this, the second invention contains DNA encoding the polypeptide. il
l J (Regarding the Jj4 plasmid, the third invention is
Regarding the transformant into which the recombinant plasmid has been introduced, the fourth invention is a method for producing a novel functional polypeptide, characterized in that the transformant is cultured and the polypeptide is collected from the culture. Regarding the manufacturing method.

The present inventors researched the construction of a novel polypeptide that has both cell spreading activity and heparin binding activity and its manufacturing method, and found that: We created a novel functional polypeptide using genetic engineering.

As a result of examining the biological activities of this novel functional polypeptide, it was found that it has both cell spreading activity and heparin binding activity, and that the cell spreading activity against BII-I K and NRK cells was determined by the cell adhesion domain alone. It was found that this was enhanced compared to the case of .

The present invention will be explained in detail below.

11:) The genetic structure of FN is described in The[!M[]OJournal, Volume 4, Pages 1755-1759 (1985). In addition, cDNA clones (pLF2, pLF2, pLF2) encoding the cell adhesion domain and heparin binding domain were
t, p3, pLF4 and pLF5) in Biochemistry, No. 25.
Vol., pp. 4936-4941 (1986). The present inventors have developed a cell adhesion active polypeptide and a method for producing the same by extracting the cDNA fragment for the cell adhesion domain from 11 L l' 5, connecting it to an expression vector, and introducing it into Escherichia coli. A patent application was filed (Japanese Patent Application No. 63-31820). The cDN^ of the cell adhesion domain required in the present invention is disclosed in Japanese Patent Application No. 63-3.
The recombinant plasmid ρTF7021 described in No. 1820 can be used. pTF7021
pTF7021 is a plasmid expressing FN Pro12”-Met” (279 amino acid residues).
By introducing a cloning site, e.g. Nco [site, immediately before the stop codon at the C-terminus of the translated region of
Eight can be connected.

One specific example of the novel functional polypeptide according to the present invention has the following general formula: Cz, t+ Me, hH2t+
J [wherein C2ff7 is p, o12311. of the cell adhesion domain of human FN. , -2 corresponding to 3er1515
It shows 77 amino acid peptide residues, and the following formula ■: Pro Asp Thr Met 8rg
Val Thr Trp 8 la Pr.

Pro Pro Ser lie 8sp
Leu Thr 8sn Phe LeuVa
l Arg Tyr Ser Pro Val Lys
Asn Glu Glu eights Val Ala
Glu Leu Ser Ile Ser
Pro Ser^sp Asn Ala
Val Val Leu Thr 8sn
Leu LeuPro Gly Thr Glu T
yr Val Val Ser Vat ScrSer
Val Tyr Glu Gin l1i
s Glu Ser Thr Pr.

Lau Arg Gly Arg Gln
Lys Thr Gly Leu AspSe
r Pro Thr Gly Ile As
p Phe Ser Asp IIeThr
Ala Asn Sar Phe Thr Val H
is Trp l1eAla Pro Arg Al
a Thr lie Thr Gly Tyr
Arg11e＾rg His His Pra G
lu 1lis Phe Ser Gly^rg P
ro Arg Glu Asp Arg V
al Pro l1is Ser＾rg As
Ser lie Thr Leu Th
r Asn Leu ThrPro Gly
Thr Glu Tyr Val Val
Ser Ile Val^1a Leu A
sn G, 1y 8rg Gtu Glu
Ser r'ro LeuLeu IIeGfy
Gln Gln Ser 'rhr Val Ser^
5pVal Pro Arg Asp Leu
Glu Val Vat 8la 81a
Thr Pro Thr Ser Leu
Lau lie Ser Trp Asp^1a
Pro Ala Val Thr Val
Arg Tyr Tyr ^[glle Th
r Tyr Gly Glu Thr Gl
y G'ly Asn 5erPro Val
Gln Glu Phe Thr Vat
Pra Gly 5erLys Ser T
hr Ala Thr lie Ser G
ly Leu LysPro Gly Vat
Asp Tyr Thr lle Thr
Vat Tyr^1a Val Thr
Gly Arg Gly Asp Ser
Pro AlaSer Ser Lys Pr
o lie Ser lie Asn Ty
r ArgThr Glu Ile Asp Ly
s Pro Ser ---[II], 1lzt+ is Ala Ig 90-T hr 19 B of the heparin-binding domain of human) I''N
271 amino acid peptide residues corresponding to ' are shown,
The following formula ■: 8 la Ile Pro 8 la Pro
Thr 8sp Leu Lys PheTh
r Gin Val Thr Pro Thr Ser
Leu Ser AlaGfn Trp Thr P
ro Pro^sn Val Gin Leu Thr
Gly Tyr Arg Val Arg
Val Thr Pro Lys GluLy
s Thr Gly Pro Met Ly
s Glu lie 8sn Leu^1a
Pro Asp Ser Ser Ser Ser Val V
al Val 5erGly Leu Met Val
^la Thr Lys Tyr Glu ValSe
r Val Tyr Ala Leu Lys Asp
Thr Leu Thr Ser Arg Pro A
la Gln Gly Vat Vat Thr Th
rLeu Glu 8sn Vat Ser
Pro Pro 8rg Arg 8la^
rg Val Thr^sp Ala Thr GIu
Thr Thr 1ieThr lie Ser
Trp Arg Thr Lys Thr
Old u Thrlle Thr Gly P
he Gln Val 8sp 8la V
al Pr.

Ala Asn Gly Gin Thr Pro I
le Gin^rg Thrlle Lys Pr
o Asp Vat Arg Ser Ty
r Thr rleThr Gly Leu
Gln Pro Gly Thr 8sp
Tyr Lys IIs Tyr Leu T
yr Thr Leu Asn 8sp A
sn ＾1a8r Ser Ser Pro
Val Val lie Asp 8 la
5erThr Ala Ile Asp
Ala Pro Ser Asn Leu
ArgPhe Leu Ala Thr Th
r Pro 8sn Ser Leu Le
uVal Ser Trp Gin Pro
Pro 8rg Ala Arg 1leTh
r Gly Tyr! IClie Lys
Tyr Glu Lys Pr.

Gly Ser Pro Pro Arg
Glu Val Vat Pro
Arg Pro Gly Val Thr
Glu Ala Thr l1eThr
Gly Leu Glu Pro Gly
Thr Glu Tyr Thrlle Tyr
Val lie eight la Leu Lys
8sn 8sn GlnLys Ser
Glu Pro Leu lie Gly
Arg Lys Lys Thr
... has a sequence represented by [III], and X is the following formula IV: 85p-Glu-Leu-Pro-Gln-L
eu-Va I-Thr-Leu-t'ro-Old 5-P
ro-^5n-Leu-old 5-Gly-Pro-Glu
-11e-Leu-^5p-Val-Pro-Ser-
A peptide residue represented by Thr-(IV) or a group in which part or all of it is deleted, Net represents a methionine residue, and n represents l or the number of zero] Examples include functional polypeptides characterized by:

Regarding the heparin-binding domain, fragments obtained by digestion with trypsin, thermolysin, cathepsin D, etc. have been reported, and their sizes range from 29 kO to 38 kO.
It has reached the mouth. Although the domain has not been specified in detail, fragments are known that generally include three m-type similar sequences consisting of about 90 amino acids, followed by a type region sequence of the Illcs-type sequence. The X described in formula ■ corresponds to a part of the 1IIcs type sequence.The idea is that mcs is not required for heparin binding activity, but the 11lcs sequence is required for adhesion of certain lymphoid cells. The present inventors have developed a fragment containing three m-type similar sequences of the heparin-binding domain (corresponding to j1□7. described in the formula I of the present invention) and a fragment further containing a part of n1cs. (112tl-X of formula I) was expressed in Escherichia coli and the heparin binding activity and cell adhesion activity were measured. As a result, both had heparin binding and oil-retaining properties as well as adhesion activity to BHK cells, and ++ ,,1.-X,
It was found that adhesion, spreading, and activity were enhanced.

cDN8 encoding the heparin binding domain is pLF
2435. ρL F 2435
is a plasmid reconstructed from the aforementioned pLF2, pLF3, pLF4, and pLF5, and contains cDN^ encoding the heparin-binding domain of FN. However, lll
Since it does not contain cDN^ corresponding to the cs portion, the DNA sequence corresponding to X must be constructed by chemical synthesis. Excise the necessary c[lN^ fragment from pLF2435 with a restriction enzyme, add a synthetic NA containing the start codon on the 5' side, and a synthetic NA containing the stop codon on the 3' side.
After ligation with N A IJ gauze, a plasmid expressing a peptide having a sequence consisting of three similar sequences can be obtained by connecting to an appropriate expression vector (see Figure 1). In other words, Figure 1 shows If
FIG. 2 is a process diagram for constructing a plasmid puotoi expressing February.

By combining this plasmid with a chemically synthesized NA corresponding to part (X) of [Ics, a plasmid expressing a peptide containing llIc5 can be obtained (see FIG. 2). That is, FIG. 2 is a process diagram for constructing plasmid plasmid No. 02 expressing I2,6.

All existing expression vectors can be used, but for example, pU[: 11BN/pUc11
9N [FBBS Letters
s), Volume 223, Pages 174-iao (19B?)
Good results can be obtained by using , and its derivatives. By introducing these plasmids into E. coli, it is possible to express the heparin-binding domain polypeptide and examine its properties.

Next, cDN^ fragments were extracted from these plasmids and used as a plasmid derived from the pTF7021 (pT
By connecting to the 3'-terminal Nco 1 site of the translated region of F7520), a recombinant plasmid expressing a polypeptide in which the FN cell adhesion domain and heparin binding domain are linked can be obtained (Figs. 3 and 4). (see figure). That is, in FIG. 3, C277-Met-H2ff+
Fig. 4 is a process diagram for constructing plasmid p[')1101 expressing Catt-Met-11a.
It is a process diagram for constructing plasmid pc11102 expressing ss.

The junction in the plasmid contains a methionine residue derived from the Nco I site as a linker.

The presence or absence of a linker does not affect the effectiveness of the present invention, but if necessary, it can be easily removed by site-specific mutagenesis.

By introducing the obtained plasmid into E. coli and culturing it under appropriate conditions, the target peptide is accumulated in E. coli. Immunoblotting is used for expression (dl MJI).Total cell proteins of recombinant E. coli were collected at 5O
After separation by 8-polyacrylamide electrophoresis, the migration pattern is transferred to a nitrocellulose membrane. A monoclonal antibody that recognizes the cell adhesion domain of FN (1'N-1
The polypeptide of interest is the band detected by both FN 0, Takara Shuzo) and a monochrome antibody (I5T-1 or I5T-2, Boehringer) that recognizes the heparin domain of FN.

Purification of the target polypeptide is performed, for example, as follows. After culturing the recombinant Escherichia coli in a medium such as L-gloss and collecting the cells, a bacterial cell disruption solution is obtained by ultrasonication, and this is centrifuged to obtain a supernatant.

After the supernatant is dialyzed, it is passed through a column of DBAll ion exchanger, followed by affinity chromatography such as CM ion exchanger and/or heparin-agarose. By the above operations, the polypeptide of interest can be purified.

The obtained polypeptide is used to measure cell spreading activity and heparin binding activity for BHK and NRK cells. Cell spreading activity can be measured using, for example, the method of Ruoslti et al. [Methods in Enzymology].
ymology), Vol. 82, pp. 803-831 (1981)]. That is, after coating the sample, a suspension of BHK or NRK cells is added to a microtiter plate blocked with BSA,
After incubation at 37° C. for about 1 hour, unadsorbed cells are washed, fixed with formalin, and the percentage of cells that have spread can be measured under a microscope to determine the strength of cell spread. On the other hand, heparin binding activity can be determined by adsorbing a sample on a column of a heparin-bound carrier, such as AF-Heparintoyobal (Tosoh), eluating it by increasing the NaCl salt concentration, and determining the concentration of the eluted salt.
Can exhibit the ability to bind to heparin.

The above measurements demonstrate that the obtained polypeptide exhibits strong cell spreading activity against BHK and NRK cells, and also exhibits strong affinity for heparin.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The invention is not limited to these examples.

Example 1 1a16911-Thr to the heparin binding domain of FN
191+11 (271 amino acid residues, hereinafter 11-271
(1-1) Preparation of synthetic ON^adapter The cDNA fragment encoding the heparin-binding domain CD island fragment to the vector (5' side (chain length 63 and 55, see Figure 1) and the 3' side (11 lengths 25 and 33, see Figure 1) adapters were synthesized using an Applied 7N/Iosystems ON8 forming machine.

After phosphorylating 2 μg of each of the 5' ends, an annealing operation was performed to obtain a double chain.

(1-2) Preparation of Nco I-3ac I fragment F
5. Contains the cDNA octane encoding 11-271 of N.
9kb plasmid pLF2435 [Biochemistry Vol. 25, pp. 4936-4941 (1ri 8G)
3100 μg was digested with Oamtll and 3ac I,
A 1.2 kb fragment was recovered by agarose gel electrophoresis. This fragment was further digested with 1laeII, subjected to agarose gel electrophoresis, and the 0.46 kb 1laeII
-5ac I fragment was recovered. 700 ng of this fragment and 120 g of the 5' side adapter obtained in (l-1) were transferred to T4.
Bar for 0sho ligase/77-10,5mM ATP
, 10mM DTT and 2.8 units of T4 ON^
Incubate overnight at 16° C. in 20 μl of reaction solution containing ligase. After treating the reaction solution at 65°C for 10 minutes,
Digested with Nco I and Sac I, subjected to agarose gel electrophoresis, 0.52 kb (7) Ncol-5
Approximately 120 ng of acl fragment was recovered.

(1-3) Sac I-〇, amH! Of fragments? 100 μg of the above plasmid pLF2435 manufactured by A was placed in the mouth [
] 109 I and Sac I, subjected to agarose gel electrophoresis, and a 0.52 kb fragment was recovered.

This fragment was further digested with [Ian II and subjected to agarose gel electrophoresis to generate a 0.27 kb Sac I-Roa
B) Fragments were recovered. This fragment 400n'g and (1-1
80 ng of the 3' side adapter obtained in ) was added to T4 ON^ ligase buffer, 0.5 mM ATI', 10
The mixture was incubated overnight at 16 DEG C. in a 20 .mu.l reaction mixture containing mM IITT and 2.8 units of T40N aryase. After treating the reaction solution at 65°C for 10 minutes, Da
Digested with Sac I and Sac I, subjected to agarose gel electrophoresis, and a 0.30 kb Sac I-Roamll
Approximately 65 ng of fragment was recovered.

(1-4) Nco I-3ac I fragment 12 obtained in preparation of Nco I-Bam1l I fragment (1-2)
0 ng and 65 ng of the Sac I-low amll fragment obtained in (1-3) were mixed with T4 DNA lysate buffer and 0.0 ng of the Sac I-low amll fragment obtained in (1-3).
The mixture was incubated overnight at 16°C in a 20 μl reaction containing 5mM HTP, 110mM DTT, and 2.8 units of T4ON Hgose. The reaction solution was heated to 65°C.
After treatment for 10 minutes, [1Gmtl I and Nco I digestion, agarose gel electrophoresis, and
About 28 ng of the Nco I-Bam11 I fragment was recovered.

(1-5) Construction of pUc118NT Secretory expression vector plNII[-ompA+
Embo Journal, Vol. 3, pp. 2437-2442 (1984)) l μg [lamll I and 5a
Digested with lt, subjected to agarose gel electrophoresis, lpp
0.95kb tlamll including terminator sequence
The I-3al I fragment was recovered. 30ng of this fragment
T with 30Mg of plasmid pUc118N, which had been previously digested with []amHI and 5alI and dephosphorylated.
4 DNA ligase buffer, 0.5 mM AT
P, 10 mM old T and 2°8 units of T40N
The cells were incubated overnight at 16° C. in 20 μl of reaction solution containing A ligase.

Reaction solution 10μ! was used to transform E. coli 11 old 01 to obtain a plasmid with Ipp terminator sequence,
It was named Uc118NT.

In addition, an Nco I site was introduced into the translation start codon site of pUcl18N Ha, commercially available (7) pUc118/tar [sold by Takara Shuzo Co., Ltd.], and the distance between the liposome binding site and the start codon was made 8 bases. .

(1-6) Nco I-Bam1l I fragment p
Cloning into Uc118NT (1-5) pUc118NT
After decomposing 1 μg of O, with Nco I and lamHI, it was dephosphorylated.

20 ng of conoplasmid was obtained from (1-4).
T41) with 20 ng of loam111 fragment) N^Ligase Nokpuffer, 0.5mM ATP, 10mM
[Incubated overnight at 16° C. in a 20 μl reaction solution containing fTT and 2.8 units of T4 ON^ligase. 10 μβ of this reaction mixture was used to transform E. coli II 8101.

(1-7) Transformation of E. coli and confirmation of plasmid (1-
10μ of the reaction solution obtained in 6)! Escherichia coli ] 1 old was transformed using Plasmid analysis was performed on 18 clones among the obtained transformants. That is, a plasmid prepared by the Ravid method was transformed into Bamll and Nco I.
and subjected to agarose gel electrophoresis to obtain the predicted Nc.

The generation of a band of the 1-BamHI fragment (0.82 kb) was examined. As a result, the desired band was observed in one clone. In addition, the base sequence was determined by the dideoxy method,
Confirmed that it contains the desired sequence. This recombinant plasmid was named pH[1101.

In addition, E. coli 118101 carrying this plasmid was injected into 13 scherichia coli 11 mouths 101/
It was designated as pHDI01 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology [Feikoken Jyoki No. 2264 (former RM'-2264)].

<1-8) Bscherichia coli If obtained by purification of peptide from recombinant (door) 101/
pH old low with 50 Mg/m ampicillin added 5
The cells were cultured in test tubes containing 1 ml of Shiichi broth at 37° C. with shaking overnight. This is 2! containing 500ml of the same medium! The cells were inoculated into an Erlenmeyer flask, and the culture was continued at ioorpm. 66,
When the Onm absorbance was 0.3, 2mM IPTG (
isopropyl-β-thiogalacside) was added, and the bacteria were collected 20 hours later. Immunoblotting was performed using a portion of the bacterial cells. In other words, all bacterial protein is converted to 5O3-
PAGE! After separation and transfer of the electrophoresis pattern to nitrocellulose membrane, a monoclonal antibody [15T-1,
sold by 5era-Lab],
Next, a peroxidase-labeled second antibody was applied. The peroxidase activity of the bound second antibody was determined by 4-chloro-
1 = Color developed in the presence of naphthol and hydrogen peroxide, 29
The desired peptide is produced near kD! I confirmed that there is. Next, the whole bacterial pellet was added to 20mM
K, IIPO, (pH 7,0), 1 mM
suspended in a solution containing BDTA, 5 mM meltcaptoethanol, and 3 μl of paraamidinophenyl methanesulfonyl fluoride (p-APMSF).
Ultrasonic treatment was performed. Centrifuge at 1200 rpm for 20 minutes and remove 25ml supernatant! I got it. This was mixed with 20mM K2
It was passed through a column (15 mg) of C:M-) Yopar 650M equilibrated with 11PO-(r+lI 7.0) buffer. After removing the non-adsorbed fraction with the same buffer, 0.
20mMK containing 15M NaCl, 1lPO4 (ptl
7,0) bar/77- and fractionated. The eluate was subjected to immunoblotting, and the target fractions were collected. −
Next, these two aliquots were added to a heparin-Toyovar 650M column (80 ml
).

Coat the column with 20 mM K211 containing 0.2 M NaCl.
After washing with P port 4 (pH 7.0) buffer, 20 m
M K211PO-(pif7.0) in buffer, 0
．． Fractionation was performed by elution with a linear concentration gradient from 2 M NaCl to 0.45 M NaCl. The target fractions were collected by immunoblotting, desalted, and lyophilized to obtain approximately 20 mg of an electrophoretically homogeneous peptide. When the amino acid sequence from the N-terminus of this peptide was investigated using AB1's peptide sequencer-477A/1208, it was found to be ^la-11e-Pro-^1a-Pro-Th.
The sequence of r-Asp-Leu was observed and matched the N-terminal sequence of the peptide of interest. Further, by carboxypeptidase P (Zen Shuzo) digestion method, it was confirmed that the C-terminus was Thr.

Example 2 Part of the llIc5 region of FN (85pI91'1-Th
cD encoding a heparin-binding domain (^Ia"l1o-Thr"", 296 amino acid residues, hereinafter abbreviated as 1to296) containing rI985.25 amino acid residues)
N^DNA cloning (see Figure 2) (2-1) Preparation of Ban It -Bam1l I fragment C3I region of l1lcs of FN [Journal of Cell Biology (J, Cell Port 100) No. 10
3, pp. 2637-2647 (1986)) containing synthetic NAs (chain lengths 77 and 7)
8, see Figure 2) by Applied Biosystems' D
It was synthesized using an NA synthesizer. After phosphorylating 2 μg of each of the 5' ends, an annealing operation was performed to form complementary sequence portions into double chains. This DNA was added to 7mM)
ris)-II(:I (pi17.5 > , 0.1
mM BDTA, 20mM NaCl, 7mM
MgCIi, O, 1mM dATP, dGTP,
The cells were incubated for 30 minutes at 37°C in a 100 μl reaction solution containing dCTP, dT'l'P, and 2 units of Flenow enzyme.

After stopping the reaction at 70°C for 5 minutes, Bam1ll and B
Digested with an■, subjected to agarose gel electrophoresis,
Approximately 400 ng of the 1lkb(7) Ban11-Bam11 I fragment was recovered.

(2-2) Preparation of Sac I-flamll I fragment (2-1) Loan11- Loamll obtained in (2-1)
200ng of l fragment and 0.27k obtained in (1-3)
490 ng of the Sac I-loanlo fragment of T4
LON heligase buffer, 0.5mM ATP,
10mM DTT and 2.8 units of T4 DNA
20μ including U gauze! The reaction mixture was incubated overnight at 16°C. After treating the reaction solution at 65°C for 1 minute, it was digested with Bam111 and Sac I, and subjected to agarose gel electrophoresis to generate 0.38 kb Sac I-Roam.
Approximately 1100n of lll fragments were recovered.

(2-3) Sac l-Bam1lI with pHD101
Preparation of fragment (vector fragment) Plasmid encoding p271 18 of 10101
After decomposing G with Sac I and BamHI and dephosphorylating it, it was subjected to agarose gel electrophoresis, and 4 to 6 kb of Sa
Approximately 280 ng of c l -namtl I vector fragment was recovered.

(2-4) Ligation of Sac 1-loarnHI fragment and vector (0.38 kb Sac I obtained in 2-2)
-[1aITrll I fragment 50ng and (2-3)
20 ng of the 4 to 6 kb Sac I + amH1 vector fragment obtained in 1 was added to T40N^ligase buffer, 0
．． 5mM ATP, 10mM DTT and 2.8
The cells were incubated overnight at 16° C. in a 20 μE reaction solution containing Unit's T4 ON^ligase. This reaction solution l
Oμl was used to transform E. coli t+oiot.

(2-5) Transformation of E. coli and confirmation of plasmid. Using 10 μm of the reaction solution obtained in (2-4), E. coli HDI
The OI was transformed. Plasmid analysis was performed on 12 clones among the obtained transformants. That is, a plasmid prepared by the rabbit method was transformed into amllI and Nco
It was digested with I and subjected to agarose gel electrophoresis to examine the formation of the expected band of Nco 1-Oamt (I fragment (0.9 kb). As a result, the desired band was observed in the I clone. Also, The base sequence was determined by the dideoxy method and confirmed to contain the desired sequence.This recombinant plasmid was named p II 0102.

In addition, Escherichia coli 110101 carrying this plasmid was transformed into 5cherihia coli II old 01/p
It was designated as HD102 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.
-10721)].

(2-6) Bscherichia coli 11 mouths 10 obtained in (2-5) purification of peptide from recombinant
1/pt10102 was cultured and purified in the same manner as in (1-8), and approximately 5 mg of an electrophoretically homogeneous peptide was obtained from the 500-culture solution. The N of this peptide was determined using AB1's peptide sequencer 4778/120A.
When the amino acid sequence from the end was examined, it matched the N-terminal sequence of the target peptide.

Further, by carboxypeptidase P digestion method, it was confirmed that the C-terminus was Thr.

Example 3 Cell adhesion domain of FN pro+239-8er151
Cloning of cDN^ fragment encoding a fusion protein of 5 (277 amino acid residues) and 11-271 (3rd
(See figure) (3-1) Cell adhesion domain ProI2”-5erIS
Construction of a plasmid encoding Is (277 amino acid residues) NCOI was inserted immediately before the stop codon in the translation region of the recombinant plasmid pTF7021 described in Japanese Patent Application No. 63-31820 by site-directed mutagenesis. A plasmid containing the site was constructed laterally. The Nco I site was introduced into pTF7021 using oligonucleotide d(
p['T8 to T8 A[:CTGGATGGTTTG
] and synthesize Site-Dilefted Mutagienesis System Mikotan-K (5ite-formerly
ecLed mutagenesis system M
utan-K) sold by C Takara Shuzo Co., Ltd.]. With the introduction of this Nco I site, 61nlslli-Me tl S at the C-terminus of the cell adhesion domain
Iff has been replaced with Me (15111-%1a11512 (see Figure 3). The resulting plasmid was
It was named TF7520.

(3-2) Nco l-11incI at pH 0101
! Recombinant plasmid pH obtained in fragment preparation (1-7)
lμg of old and old was digested with Nco I and old nc and subjected to agarose gel electrophoresis, resulting in 1.77 kb of Nco 1
- Hinc [1000 g of fragments were collected.

(3-3) Nco I-Hinc of ptlDlol
[Cloning the fragment into pTF7520 The plasmid pTF7520 obtained in (3-1) was
After decomposition with I and old nc■, dephosphorylation was performed. 50 ng of this plasmid was added to Nco I-11in obtained in (1-2).
50 ng of cH fragment with T4 ON^ ligase buffer, 0.5 mM ATI', 10 mM DTT and 2
．． The cells were incubated overnight at 16° C. in a 20 μl reaction solution containing 8-Lnit T4 DNA ligase. E. coli 118101 was transformed using this reaction solution 1Oμβ,
FN cell adhesion domain Pro12”-8et”” (2
77 amino acid residues) and 11-271 bound via Mat (Ca, t-Met-11B +
) was obtained and named pcllol.

In addition, E. coli ■0101 carrying this plasmid was infected with Bscherichia coli (11 mouths, 10 mouths).
1/pcll 1 old and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology
0RM P-10722) ].

(3-4) The fusion protein (C2tt-Met-f1
2t +) cell adhesion domain pr O+ 239
S et l S + 5 (27? amino acid residues)
Met is added between and 11-271. The sequence (^TG) corresponding to this Met was removed by site-directed mutagenesis. Intervening sequence from pH0101 (^T
G) Removal of oligonucleotide d[pAGG^^T
[GGTTT] to GCGGA was synthesized using Cyto-Dilefted Mikota Genesis System Mutan-K (sold by Takara Shuzo Co., Ltd.). the result,
Cell adhesion domain p, 01239. -3e, l5ls(
A plasmid expressing a fusion protein (C2qt-H2,-') in which 277 amino acid residues) and )I-271 were directly linked was obtained and named pH0101.

(3-5) Purification of peptide from recombinant (3-3) Bscherichia coli II old 01/
Cultivate pc11101 in the same manner as (1-8),
An extract was obtained from 00 cultured bacterial cells. A monoclonal antibody that recognizes the cell adhesion domain of FN (PH-10, Takara Shuzo) and a monoclonal antibody that reacts with both l5T-1 were purified in the same manner as in (1-8).
mg of purified product was obtained.

The N-terminal sequence of this peptide matched the sequence of the target peptide. In addition, by carboxypeptidase P digestion method, C
It was confirmed that the end was Thr.

Example 4 Cell adhesion domain of FN 13,01231j30. +5
Cloning of a cDNA fragment encoding a fusion protein of 15 (277 amino acid residues) and I+-296 (
(See Figure 4) (4-1) p) Nco I of 10102 -) 1in
1 μg of the recombinant plasmid pH0102 obtained in cII fragment preparation (2-5) was digested with Nco I and old ncn, and subjected to agarose gel electrophoresis to obtain 1.84 kb.
Approximately 1100n of the Ncol-11incII fragment was recovered.

(4-2) Nco I-1lincf at pH old 02
Cloning of the f fragment into pTF7520 (3-1) The plasmid pTI+7520 obtained in Nco
After digestion with 1 and 1lincI, dephosphorylation was performed. 50 ng of this plasmid was obtained from (4-1).
-11inc II fragment 50ng with T4 ONN ligase buffer 0.5mM 8TP, 10
The cells were incubated overnight at 16° C. in a 20 μm reaction solution containing mM DTT and 28+−−′−−/t T4ON^ ligase. Using 10 μl of this reaction solution, E. coli 11
0101, and the FN cell adhesion domain Pro
123g-3er15” (277 amino acid residues) and I
A fusion protein (C
A plasmid expressing 27t-Met-1C27t- was obtained and named pcl+102.

In addition, E. coli 110101 carrying this plasmid was transformed into Bscherichia coli IIBIOI/p
cII102 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology
+1-10723)].

(4-3) Intervening sequence (ATG) from pc11102
The fusion protein (C2tt-Met-I) expressed by plasmid pc11102 obtained in removal (4-2)
Cell adhesion domain P r o + 239− of Las)
3 e r l S I S (277 amino acid residues)
Net is added between and H-296. This M
The sequence (A'rG) corresponding to et was removed in the same manner as in (3-4). As a result, the cell adhesion domain pr
A fusion protein (C
277-112ss) was obtained, and p
It was named CH202.

(4-4) Purification of peptide from recombinant (4-2) Bscherichia coti It old 01/
p[:If102 was cultured and purified in the same manner as in (3-5), and approximately 6 mg of an electrophoretically homogeneous peptide was obtained from the 500rd culture solution. The results of N-terminal sequence analysis and C-terminal analysis were consistent with those of the target peptide.

Example 5 Measurement of biological activity Cell adhesion activity and heparin binding activity were measured using each of the polypeptides obtained in Examples 1 to 4 above.

Cell adhesion activity was measured according to the method of Ruoslati et al. [Methods in Enzymology, Vol. 82, pp. 803-831 (1981)]. 1. Pour the sample into distilled water. P
It was dissolved in BS (IJ acid buffered saline) etc. and diluted stepwise on a 96-well microplate. Samples were adsorbed onto the plate (50 μβ/well) at 4° C. for 2 hours; 1-cubate. Add 100 μl/well of PBS solution containing 3% BSA (bovine serum albumin) and
Plates were blocked by incubating for 1 hour at 7°C. After washing the plate with PBS, add Dulbecco's Eagle Minimal Nutrient Medium (DM) in advance.
BM) was incubated with 100 U-turbidated baby hamster kidney cells (formerly 1 to 21) so as to occupy 5× to 7 cells.
Dispense μl/well and incubate at 37° C. for 1 hour. The old IK-21 cells used were cryopreserved strains that had been subcultured and then treated with trypsin (37°C, 5 minutes). After washing the plate with PBS, cells were fixed on the plate with a 3% formalin solution.

Observe the spreading of 1-21 cells under a microscope, and determine the concentration of the sample (BD, ) at which the number of spreading cells is 50% of the number of spreading cells at a high concentration of n-FN, an indicator of cell adhesion activity. And so.

Heparin binding activity was measured as follows.

The sample was loaded onto a column (1.5 ml) of LtaA F heparin-Toyopearl 650M equilibrated with 20 mM phosphate buffer 7 (pH 7.0), and NaCl in the buffer was
The binding strength to heparin was expressed by the salt concentration eluted by increasing the salt concentration stepwise.

The results of measuring the biological activity of each sample using the above method are
Shown in the table.

No. 1 Table H-271 300 I+-296413QO C2tt-Met-112t+ o, 176 3
00 [Effects of the Invention] As described above, the present invention provides a novel polypeptide having both cell adhesion activity and heparin binding activity, and a method for producing the same. This polypeptide is a useful protein that mediates the binding of IJ hooks between cells and extracellular polymers such as heparan sulfate, and is useful for wound healing.

[Brief explanation of drawings]

Figure 1 shows plasmid pHD101 expressing H-271.
Figure 2 is a process diagram for constructing plasmid pII 0102 expressing 11-296, Figure 3 is a process diagram for constructing plasmid pcH101 expressing C2tt-Met-H2v+. Figure, 4th
The figure is a process diagram for constructing plasmid pci+102 expressing C2t, -Met-Ilzss.

Claims (1)

[Scope of Claims] 1. A functional polypeptide characterized in that a cell adhesion domain of human fibronectin and a heparin-binding domain are linked directly or via a linker peptide. 2. General formula I: C_2_7_7-(Met)-_nH_2_7_1-X
...[I] [In the formula, C_2_7_7 is Pro^1^2^3^9 of the cell adhesion domain of human fibronectin.
- It shows the 277 amino acid peptide residues corresponding to Ser^1^5^1^5, and has the sequence represented by the following formula II: [There is an amino acid sequence] [There is an amino acid sequence] ...[II] H_2_7_1 is Ala^1^■^■ of the heparin-binding domain of human fibronectin.
It shows 271 amino acid peptide residues corresponding to ^■-Thr^1^■^■^■, and has a sequence represented by the following formula III: [There is an amino acid sequence] ...[III], and X is The following formula IV: [There is an amino acid sequence] ... [IV] represents a peptide residue represented by, or a group in which part or all of it is deleted, Met represents a methionine residue, and n
represents a number of 1 or zero. 3. D encoding the functional polypeptide according to claim 1
Recombinant plasmid containing NA. 4. A transformant into which the recombinant plasmid according to claim 3 is introduced. 5. A method for producing a functional polypeptide, which comprises culturing the transformant according to claim 4 and collecting the functional polypeptide according to claim 1 from the culture.