JPH0931099A - Peptide bonding to sulfated polysaccharide and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new peptide having affinity to sulfated polysaccharides and effective for modifying the pharmacological action of a protein factor having hepatocyte growth activity (HGF) by the restricted decomposition of HGF with a protein decomposition enzyme. SOLUTION: This new peptide is included in an amino acid sequence essentially the same as the amino acid sequence expressed by formula (Xaa is pyroglutaric acid) and has affinity to sulfated polysaccharides. It competitively inhibits the bonding of the surface layer of hepatocyte growth factor(HGF) to heparan sulfate and suppresses the capture and decomposition of the factor. It is useful e.g. as an agent for modifying the physiological action of HGF. The peptide can be produced by subjecting a protein factor having hepatocyte growth activity (HGF) to restricted decomposition with a protein decomposition enzyme (e.g. Staphylococcal serine proteinase) under non-reducing condition and purifying by passing the decomposition product through a reversed phase partition column, a heparin affinity column, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention originates from a protein factor having hepatocyte proliferation activity (hereinafter abbreviated as "HGF") and has a strong affinity for sulfated polysaccharides such as heparin. The present invention relates to a peptide and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION The liver is a highly differentiated glandular organ that plays a central role in in-vivo intermediate metabolism, and its function is carried by the liver parenchymal cells that make up the liver. . HGF is a proteinaceous growth factor discovered by research focusing on the mechanism of liver parenchymal cell proliferation (liver regeneration). Furthermore, it is known that this HGF not only has hepatic parenchymal cell proliferating activity but also plays an important role in the development, differentiation, and tissue construction of the living body, and the cancer cytotoxic factor (Tumor Cytotoxic Fac).
tor: TCF) and the same molecule.

By the way, since HGF is purified by a heparin affinity column, it is known as a growth factor having an affinity for heparin. On the other hand, this HGF synthesized in the liver has a very short blood half-life. The reason is that HGF is known to be captured and decomposed by heparan sulfate, which is a kind of glycosaminoglycan existing in large numbers on the cell surface (Naka et al.,
FEBS LETTERS, 329 , 147-152
(1993)). That is, it is considered that this heparan sulfate having a structure similar to that of heparin functions as a kind of low affinity receptor for HGF.

As described above, the property that HGF binds to sulfated polysaccharides such as heparin and heparan sulfate is extremely important in the mechanism in which the growth factor exerts a pharmacological effect. However, there are few reports of studies focusing on this property of HGF. Report of Okigaki et al. (Biochemistr
y, 31 , 9555-9561 (1992)) describes a mutant of HGF in which amino acids of several tens of residues in the amino terminal region are deleted, and this mutant loses its ability to adsorb to a heparin affinity column. However, the heparin binding site of HGF has not been identified yet. In addition, there is no report that the peptide corresponding to this site was protein-chemically isolated to examine the affinity for heparin, and that this peptide was used for the purpose of inhibiting the binding between HGF and heparan sulfate on the cell surface. .

The object of the present invention is to identify a sulfated polysaccharide binding site of HGF, which has not been investigated in detail so far, and then obtain a peptide corresponding to the site to use it as a modifier of the pharmacological action of HGF. To do.

[0006]

Means for Solving the Problems The present inventors have comparatively analyzed the affinities for heparin of various peptides produced by the limited degradation of HGF with a proteolytic enzyme using a heparin affinity column. As a result, HG
We succeeded in finding a peptide having a heparin affinity equivalent to that of F, and further completed the present invention by examining the properties of the peptide.

[0007] That is, the gist of the present invention is a peptide which is contained in an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and has an affinity for sulfated polysaccharides, and a method for producing the same. Exist in. Hereinafter, the present invention will be described in detail.

The origin of the peptides described in the present invention is "HG
“F” is derived from mammals including human and is not particularly limited as long as it is a proteinaceous factor having an activity of promoting the proliferation of hepatocytes. For example, HGF can be obtained by purifying from plasma or the like or by expressing the cDNA by a gene recombination method. Specifically, JP-A-63-225
According to the method described in JP-A No. 26-26, a protein library is chemically separated and purified from plasma of a patient with fulminant hepatitis, or from a cDNA library derived from human placenta according to the method described in JP-A-3-285693. Got H
It can also be obtained by constructing an expression vector containing a cDNA encoding GF and expressing it in a host such as CHO cells. In addition to the above-mentioned natural or recombinant HGF, HGF may be a natural or recombinant HG as long as it does not impair its precursor protein or the activity of growing hepatocytes.
It is also possible to use one in which some amino acids of F have been altered, such as substituted, deleted, inserted, deleted or modified. Specifically, JP-A-2-288899, WO90 / 106
51, JP 3-130091 A, 3-2.
No. 55096, No. 4-30000, Natu
Re, 342 , 440-443 (1989), etc. can be mentioned as a modified substance.

Human-derived HGF is a non-reducing SDS.
-Molecular weight of about 76,000-9 by PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
Give a band to 2,000. Further, the HGF has a strong affinity for heparin, and when it is adsorbed on a heparin affinity column in a sodium phosphate buffer of pH 7.5, the concentration of NaCl required for desorption is about 1.3 M. Is.

The peptide of the present invention can be chemically synthesized based on a conventional peptide synthesis method, but it is possible to limit the decomposition of HGF by using an appropriate proteolytic enzyme (hereinafter referred to as "protease"). Can be easily manufactured. The protease is not particularly limited as long as it is an enzyme that cleaves a peptide bond of a protein. For example, both highly specific proteases with limited cleavable bonds and relatively less specific proteases can be used. Specifically, S
The peptide of the present invention can be obtained by using taphylococcal serine proteinase (hereinafter abbreviated as "V8 protease") or endoproteinase Asp-N.

In the present invention, the "sulfated polysaccharides" are heparin and heparan sulfate, which are N or O-sulfated ester compounds having a repeating structure of a disaccharide unit of a uronic acid residue and a glucosamine residue. It also includes dextran sulfate (average molecular weight 5,000), which is a sulfated ester of α-1,6-glucan known as an analog molecule of.

The peptide of the present invention can be obtained by the following procedure from the peptide mixture produced by reacting HGF with the above protease under non-reducing conditions. For example, it can be carried out by adding the peptide mixture to a heparin affinity column, adsorbing it on the ligand heparin, and selecting peptides having a high affinity for heparin. The term "affinity" as used herein means the ability to bind (adsorb) to sulfated polysaccharides such as heparin, and in the peptide of the present invention, the NaCl necessary to eliminate such binding (adsorption). Those with a concentration of about 0.4 to 1.3 M are judged to have high affinity. The peptides of the present invention can also be selected by separating them by reverse phase partition chromatography using a volatile eluent. This method is advantageous in that it does not require an operation for removing salts such as NaCl after purification.

Further, the peptide of the present invention can be obtained as a partial peptide having an affinity for heparin by subjecting the sulfated polysaccharide-binding peptide obtained by the above method to limited degradation with another protease. . That is,
The peptide of the present invention is specified by having an affinity for heparin and can be efficiently obtained, for example, by the above-mentioned production method, but its structure (amino acid sequence) and production method are not particularly limited as long as it has the above-mentioned characteristics. Not done.

The peptide of the present invention thus obtained is contained in the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing or in an amino acid sequence substantially the same as the amino acid sequence. The peptide may have 4 or more amino acid residues, and the longer the length, the more preferable.

[0015]

EFFECTS OF THE INVENTION Since the peptide of the present invention has a property of competitively binding with HGF to sulfated polysaccharides such as heparin, it is considered that it also binds to heparan sulfate on the cell surface. . On the other hand, one of the reasons why HGF alone cannot sufficiently exert its pharmacological effect is the above-mentioned capture / decomposition by heparan sulfate on the cell surface, and therefore HGF is the original receptor c-M.
It is considered to have low binding ability to et. Therefore,
By using the peptide of the present invention, the effect of competitively inhibiting the binding of HGF to heparan sulfate on the cell surface and suppressing the above-mentioned capture and decomposition (the effect of modifying the pharmacological action of HGF) can be expected. It should be noted that this effect can also be expected as an effect-modifying substance for a sulfated polysaccharide-binding protein factor other than HGF (for example, fibroblast growth factor).

Another use of the peptide of the present invention is as an affinity ligand for purifying a sulfated polysaccharide having an affinity for a sulfated polysaccharide-binding proteinaceous factor such as HGF. Thought to be available.

[0017]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the invention. In the following examples, H
GF was prepared by a gene recombination method in accordance with the method described in Japanese Patent Laid-Open No. 3-285693 "Recombinant Human HG.
F "was used. In addition, when evaluating the binding properties of the peptides isolated from HGF to sulfated polysaccharides, an affinity column with heparin, which is one of the sulfated polysaccharides, as a ligand was used, and the adsorptivity to this column was determined by its affinity to heparin. It was used as an index of sex. A peptide having an affinity for heparin is considered to have an affinity for the above-mentioned other sulfated polysaccharides as well.

Example 1 Identification of sulfated polysaccharide binding site of HGF and isolation and purification of sulfated polysaccharide binding peptide (1) Identification of sulfated polysaccharide binding site of HGF Recombinant human HGF (hereinafter referred to as "r Abbreviated as "-hHGF") in 50 mM ammonium acetate buffer (pH 4.
0) (1.3 mg / ml), V8 protease (0.016 mg / ml) was added thereto, and the mixture was reacted under non-reducing conditions at 37 ° C. overnight. After the reaction, the enzyme was inactivated by heat treatment at 90 ° C for 2 minutes. S in a part of this enzyme reaction solution
DS treatment buffer (10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and 2.5% SDS) was added and heat treated at 95 ° C for 5 minutes, and then SDS- under non-reducing condition.
PAGE was performed. As a result, the HGF band completely disappeared, and a new band appeared at a position smaller than HGF by a molecular weight of 1 to tens of thousands. At the same time, a new band appeared in a molecular weight range smaller than that of α-lactalbumin, which is a molecular weight marker protein having a molecular weight of 14,000.

A heparin affinity column, TSKgel Hepari, in which a part of the above-mentioned enzyme reaction solution is equilibrated with a 10 mM sodium phosphate buffer (pH 7.5).
Add to n-5PW (Tosoh), 0M to 2M NaC
Elution was performed with a linear gradient of 1. The resulting chromatogram has a retention time of 10 as shown in FIG.
In addition to the non-adsorbed peak that elutes within minutes, the presence of heparin-adsorbed components with long retention times was confirmed. Therefore, this heparin-adsorbed component was isolated and purified by reverse phase partition chromatography, which has a high separation ability. Reversed-phase partitioning column equilibrated with 0.1% trifluoroacetic acid aqueous solution, Bekamond W
The above-mentioned enzyme reaction solution was added to P-C8 (JT Baker), and elution was carried out with a linear gradient of acetonitrile. Then, the main peaks of the chromatogram shown in FIG. 2 were isolated and each was eluted from the heparin affinity column, and as a result, only the peptides of peak A and peak B shown in FIG. 2 exhibited adsorptivity. FIG. 3 shows the elution pattern of the peptide of peak B from the heparin affinity column. From the retention time of the peak, the NaCl concentration at elution was 1.0 M, and HGF (NaCl concentration at elution was about 1.3 M). It was found to have a heparin affinity of about the same strength as. The peptide of peak A was eluted from the column at 0.7M. Furthermore, FIG.
Peak X of the chromatogram corresponds to the rest of the above-mentioned heparin-affinity peptide cleaved from HGF, and the molecular weight in SDS-PAGE was 1 to 1 as compared with HGF.
It is thought that it is a tens of thousands smaller polypeptide chain, but as a result of eluting it from the heparin affinity column,
The chromatogram of FIG. 4 was obtained. That is, it was revealed that this polypeptide chain lost the affinity for heparin almost completely. From the above results, it was shown that the above-mentioned heparin affinity (sulfated polysaccharide binding) peptide corresponds to the sulfated polysaccharide binding site of HGF.

(2) Structural analysis of the above-mentioned sulfated polysaccharide-binding peptide In order to identify the peptides of the above-mentioned peak A and peak B, the following structural analysis (amino acid composition analysis and mass spectrometry)
Was done. A part of each peptide was hydrolyzed in a gas phase atmosphere of 6N HCl at 110 ° C. for 24 hours using a Picotag workstation (Waters) to completely remove hydrochloric acid, and then a high-speed amino acid analyzer L-8500 type was used. (Hitachi, Ltd.) was used to analyze the amino acid composition. As a result, the amino acid composition of the peptide of peak A is the composition from the amino terminal (hereinafter, abbreviated as “N terminal”) first pyroglutamic acid (PyrGlu) to the 10th residue glutamic acid (Glu) in the amino acid sequence of HGF. Matched with. In addition, the peptide of peak B was in agreement with the amino acid composition from the 11th residue phenylalanine (Phe) to the 80th glutamic acid.

Further, a part of each peptide was dissolved in a sampling solvent (aqueous solution containing 5% acetic acid and 50% methanol), and the molecular weight was measured using an electrospray mass spectrometer TSQ700 type (Finnigan Matt). It was As a result, the measured values of the respective molecular weights were in agreement with the theoretical values of the molecular weights of the amino acid sequence structures having the same composition in the above amino acid composition analysis.

From the above results, the peptides of peak A and peak B corresponding to the heparin binding site of HGF are shown in FIG.
In the amino acid sequence from the N-terminal of the HGF to the first kringle shown by, it was found that there are 10 residues of PyrGlu ¹ -Glu ¹⁰ and 70 residues of Phe ¹¹ -Glu ⁸⁰ , respectively. The structure including the N-terminal region, each basic amino acid of these peptides are concentrated ^{(Arg 2 -Lys-Arg-Arg} 5) As shown in FIG. 5,
And a structure having a basic amino acid of 17 residues (14 residues of lysine and 3 residues of arginine) including a region called a hairpin loop (a region from around residue 35 to around residue 70). On the other hand, sulfated polysaccharides such as heparin have many acidic sulfate groups. Therefore, since electrostatic interaction works between the basic peptide and sulfated polysaccharides such as heparin, these peptides are considered to have high affinity for heparin.

Example 2 Isolation and Purification of Sulfated Polysaccharide-Binding Peptides Other Than Those Above By a method different from that of Example 1, several sulfated polysaccharide-binding peptides were isolated and purified, and each of them has an affinity for heparin. I investigated the sex. (1) A sulfated polysaccharide-binding peptide r-hHGF (1.1 mg / ml) isolated from No. 1 kringle was dissolved in 50 mM ammonium bicarbonate buffer (pH 7.9), and V8 protease was added under non-reducing conditions. (Wako Pure Chemical Industries) substrate / enzyme = 25, mol / mol (ie 0.013 mg /
ml) and reacted at 37 ° C. overnight. 90 degrees after that,
The enzyme was inactivated by heat treatment for 2 minutes. A part of this enzyme reaction solution was eluted from the heparin affinity column by the method shown in Example 1, and as a result, 2 shown in Example 1 was obtained.
The presence of a heparin-adsorbing peptide different from the seed peptide was confirmed. Therefore, the peptide was isolated by reverse phase partition chromatography in the same manner as in Example 1, and structural analysis was performed by the same method as in Example 1, and as a result, HG shown in FIG.
It was found that the peptide was a 39-residue peptide from the 90th residue asparagine (Asn) to the 128th glutamic acid in the amino acid sequence of F. The peptide was also eluted from the heparin affinity column at 0.6M. As shown in FIG. 5, the peptide exists in the kringle structure formed by a disulfide bond (Cys-Cys bond) and should not be isolated under non-reducing conditions, but as described above. It was considered that the peptide could be isolated by dissociating the disulfide bond by allowing the protease to act in a buffer solution having a relatively high pH (about 8).

(2) Phe, a sulfated polysaccharide-binding peptide
¹¹-Glu⁸⁰Of sulfated polysaccharides obtained by partial decomposition of
Synthetic peptide One of the above-mentioned sulfated polysaccharide-binding peptides (Phe¹¹−
Glu⁸⁰) Is produced by limited decomposition with another protease.
The partial peptide was examined for heparin affinity. sand
That, Phe¹¹-Glu⁸⁰, 20 μg to 50 mM phosphorus
Dissolve in sodium phosphate buffer (pH 7.5) and
And proteinase Asp-N (Boehringer Manha
Im) substrate / enzyme = 100 (weight ratio)
The reaction was carried out at 7 ° C overnight. Fermented by heat treatment at 90 ℃ for 2 minutes
After deactivating the element, reverse phase partition chromatography was performed as in Example 1.
Three peptides were isolated by chromatography. These pep
Structural analysis was performed on Cido by the same method as in Example 1.
As a result, from the 11th phenylalanine to the 22nd
12 residues up to isoleucine (Phe¹¹-Ile^{twenty two}),
From the 23rd aspartic acid to the 36th alanine
14 residues (Asp ^{twenty three}-Ala³⁶) And 37th Ass
44 residues from paraginic acid to 80th glutamic acid
(Asp³⁷-Glu⁸⁰) Peptide
Was. In addition, these peptides were labeled with heparin affinity
As a result of elution from rum, each heparin affinity is eluted
NaCl concentrations of 0.5 M, 0.4 M and 0.8 respectively
It was M.

Regarding the above-mentioned sulfated polysaccharide-binding peptide of the present invention, Na eluted from the heparin affinity column, which is an index of its structure and heparin affinity.
The Cl concentration is shown in Table 1. Among them, the peptide B (Phe ¹¹ -Glu ⁸⁰ ) shown in Example 1 has the strongest affinity for heparin, and is 1.0 M, which is almost the same as HGF. Since the molecular weight of the peptide is about 8,000 and the size is about one tenth of HGF, it is considered that the peptide has a considerably high affinity for heparin. In addition, other peptides also showed a heparin affinity of 0.4 M or more. Therefore,
All peptides having an affinity for heparin in Table 1 are homologous to HGF in that property and therefore HGF
It is considered that it has an action of binding with sulfated polysaccharides such as heparin in competition with.

[0026]

[Table 1] Table 1 ──────────────────────────────────── Structure from the heparin affinity column ( Amino acid sequence) (Number of residues) Eluted NaCl concentration (M) ─────────────────────────────────── ^{── PyrGlu 1 -Glu 10 10 0.7 Phe} 11 -Glu 80 70 1.0 Asn 90 -Glu 128 39 0.6 Phe 11 -Ile 22 12 0.5 Asp 23 -Ala 36 14 0.4 Asp 37 - Glu ⁸⁰ 44 0.8 ────────────────────────────────────

Example 3 Effect of Competitively Inhibiting HGF-Heparin Binding of Sulfated Polysaccharide-Binding Peptide Phe ¹¹ -Glu ⁸⁰ , which has the highest heparin affinity among the sulfated polysaccharide-binding peptides shown in Table 1. It was confirmed using the method described below that the peptide competitively inhibits the binding between HGF and heparin.

The above peptides, 16,60 and 280μ
Heparin Sepharos was dissolved in 10 mM sodium phosphate (pH 7.5) containing 0.15 M NaCl and made into a gel slurry with the same buffer.
e CL-6B (Pharmacia), 20 μl was added and mixed well. Further, HGF was added thereto (mixing amount which does not saturate the gel, several to several tens μg), and the mixture was filtered with a filter to separate into a fraction adsorbed on the gel and a non-adsorbed fraction not adsorbed on the gel. That is, a non-adsorbed fraction that did not adsorb to the gel having heparin as a ligand was recovered in the filtrate. As a control, the same operation was carried out for the sample to which the above peptide was not added. After concentrating the above filtrate, HGF in the filtrate was quantified by reverse phase partition chromatography. As a result, HGF was prepared in the absence of the sulfated polysaccharide-binding peptide.
Was adsorbed on the gel, and no non-adsorbed substances were detected in the filtrate (the lower limit of detection was about 0.02 μg). On the other hand, for the sample solution containing the sulfated polysaccharide-binding peptide, the peak of HGF was detected in the filtrate. From the peak area, non-adsorbed HGF (adsorption-inhibited HGF)
Of the amount of HGF added at the beginning (%)
As shown in FIG. 6, it was found that the inhibitory effect increased as the ratio (degree of excess) of the sulfated polysaccharide-binding peptide to HGF increased.

From the above results, it was shown that the sulfated polysaccharide-binding peptide competitively inhibits the binding between heparin and HGF and that the effect depends on the added amount of the peptide. In addition, the other sulfated polysaccharide-binding peptides shown in Table 1 were also considered to have similar effects, although with varying degrees. The strength of this inhibition was presumed to depend on the strength of the peptide's heparin affinity. That is, since the heparin affinity of the sulfated polysaccharide-binding peptide is weaker than that of HGF, the amount of the peptide added to HGF is sufficient to inhibit the adsorption of HGF on the gel as shown in FIG. It was thought that it would be necessary to do much. That is, if a peptide having a higher affinity for heparin (for example, a peptide linked from the N-terminus to the first kringle) can be obtained, the inhibitory effect can be expected to be further improved.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 177 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Xaa Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys 1 5 10 15 Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys 20 25 30 Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly 35 40 45 Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln 50 55 60 Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu 65 70 75 80 Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn 85 90 95 Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr 100 105 110 Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu 115 120 125 His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn 130 135 140 Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr 145 150 155 160 Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln C ys Ser 165 170 175 Glu Xaa: Pyroglutamic acid

[Brief description of drawings]

FIG. 1 is a drawing showing a chromatogram of non-reduced recombinant human HGF subjected to limited decomposition with V8 protease and eluted with a heparin affinity column.

FIG. 2 is a drawing showing a chromatogram obtained by subjecting non-reduced recombinant human HGF to limited digestion with V8 protease and eluting with a reverse phase partition column.

FIG. 3 is a drawing showing a chromatogram obtained by eluting the peptide of peak B in FIG. 2 with a heparin affinity column.

FIG. 4 is a drawing showing a chromatogram obtained by eluting peak X in FIG. 2 with a heparin affinity column.

FIG. 5 is a drawing showing the amino acid sequence from the N-terminus of HGF to the first kringle through the hairpin loop region.

FIG. 6 is a drawing showing the competitive inhibition effect of HGF-heparin binding using the peptide Phe ¹¹ -Glu ⁸⁰ of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12P 21/06 C12P 21/06 // A61K 38/46 A61K 37/54 (72) Inventor Ken Ishii Hisa, Tokyo 2-5-2 Marunouchi, Chiyoda-ku, Tokyo Mitsubishi Chemical Corporation Pharmaceutical Company (72) Inventor Tetsuro Hayase 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Chemical Co., Ltd. Yokohama Research Institute

Claims (2)

[Claims] 1. A peptide contained in an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing and having an affinity for sulfated polysaccharides. 2. The method for producing a peptide according to claim 1, wherein the proteinaceous factor having hepatocyte proliferation activity is limitedly decomposed by a protease.