JP2004339189A - Extraction and utilization of cell growth peptide from silk protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide that has excellent cell growth promotion action with high safety and high stability because of a relatively small molecular weight, that is different from the cell growth factor produced by other abnormal cells, for example, tumor cells. <P>SOLUTION: The peptide composition having excellent cell growth promotion action includes one or more partial peptide chains selected from the amorphous peptide chains constituting the silk proteins and the partial peptides have specific amino acid sequences comprising 4-40 amino acids residues. Peptides having a specific amino acid sequences with a molecular weights of less than 10,000 or 4,000-400 are separated from the amorphous part of the silk protein and fractionated and simultaneously similar peptides are synthesized whereby new peptides having excellent cell growth properties can be provided. These peptides are useful as a cell adhesion agent, a cell proliferation agent, wound healing enhancing agent, as a skin care material, for example, cosmetics or as a base material for cell culture. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a peptide derived from silk protein having excellent cell growth promoting properties and a method for producing the same, as well as its use in the fields of pharmaceuticals, quasi-drugs, cosmetics and the like as a material for skin care, and a cell culture substrate as a biomaterial About the use to.

Since silk thread has been used as a surgical thread for a long time, silk protein is considered to be a biocompatible material, and new uses have been actively developed in various fields by paying attention to its properties.
For example, after dissolving a silk thread into an aqueous solution of silk protein, pulverizing the solution by coagulation, drying, pulverization, etc., as a cosmetic additive, or casting the aqueous solution of silk protein on a flat plate to form a film, and culturing cells. Development for floor and wound covering materials and coating materials, and for using silk protein aqueous solutions as gels for use as foods and cosmetics has been promoted.

  Examples of such developments include, for example, Japanese Unexamined Patent Publication No. Sho 62-000415, Japanese Patent Publication No. 01-04320, Japanese Patent Laid-Open Publication No. H01-254164, Japanese Patent Publication No. 06-004679, Japanese Patent Laid-Open Publication No. Japanese Unexamined Patent Application Publication No. 11-276876, Japanese Patent No. 29997758, Japanese Patent No. 2990239, Japanese Patent Application Laid-Open No. 11-253155, Japanese Patent Application No. 2002-230656, Japanese Patent Application No. 2002-148849, and Japanese Patent Application Laid-Open No. 2001-163899. (See Patent Documents 1 to 12).

In the process of developing these new silk materials, it was revealed that silk proteins have various functions such as cell growth, antioxidant properties, antibacterial properties, alcohol digestibility, and anticoagulant properties.
However, it is not yet clear what part or structure of the silk protein is responsible for their function.
The present inventors have paid attention to the cell growth function of the silk protein, and after dissolving the cocoon thread or the silk thread, convert the cocoon thread or the silk thread into powder, film, gel, and the like, and use these as skin care materials to modify wound dressing materials, cosmetics, and synthetic fibers. The development and the research of the function for using as quality have been advanced (for example, see Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11, Patent Document 12).
In the process, it was found that the H-chain and L-chain of fibroin constituting the silk protein and the a component of sericin have an action of promoting the growth of human normal skin-derived fibroblasts (for example, Patent Document 13, Patent Document 13). 14, Patent Document 15).

On the other hand, cocoons are used as fibers in fields other than clothes, for example, cocoons are processed into raw silk and silk fabric through various processing steps, and are used in medical (surgical suture) and makeup (puff) fields. However, recently, it has been found that the molecular weight of silk protein is reduced by such a cocoon or raw silk processing step.
In addition, it has been found that the molecular weight of the silk protein also decreases in the processing step (particularly, the step of dissolving the cocoon thread or silk thread) of converting the cocoon thread or silk thread into powder, film, gel, or the like (H. Yamada et al .: Materials Science & Engineering C, 14, P. 41-46 (2001))
[Kozo Tsubouchi, Hiroo Yamada, Yoko Takasu: Journal of the Japanese Society of Silk Science, Vol. 71, No. 1, P.1-5 (2002)] (see Non-Patent Documents and Non-Patent Document 2).

The silk protein, whose molecular weight has been reduced by such processing, shows only one broad band with one smear between the molecular weights of about 1 to 200,000 in the electrophoresis image.
Although a molecular weight of about 10,000 or less is almost excluded in a step such as dialysis, the molecular weight is actually reduced to about an amino acid or a peptide.
It has been found that the silk protein having a reduced molecular weight has a reduced ability to promote human cell growth or inhibits cell growth (see Patent Document 11).

In other words, although undegraded fibroin and undegraded sericin have excellent cell growth promoting properties, the processing of acid, alkali, light, heat, etc. during the processing of cocoons causes complicated or uneven cleavage of peptide bonds. As a result, cell growth is inhibited with a decrease in molecular weight.
Therefore, in order to utilize the cell growth promoting function of silk protein, undegraded silk fibroin, undegraded silk sericin or fibroin H chain (molecular weight of about 350,000), L chain or sericin a (molecular weight of about 400,000) and the like are not used. It is preferable to use it in a decomposed state.

However, these three components (especially H chain and sericin a) have a very large molecular weight and low stability for long-term storage with certain properties.
Further, the L chain is contained only in a weight ratio of 10% or less in fibroin, and is small in quantity.
Furthermore, no report has been made yet which clarifies which of these three components has a cell growth promoting action.

JP-A-62-000415 Japanese Patent Publication No. 01-044320 Japanese Unexamined Patent Publication No. Hei 01-254164 JP-A-04-202435 Japanese Patent Publication No. 06-004679 JP-A-11-139986 JP-A-11-276876 Patent No. 2997758 Patent No. 2990239 JP-A-11-253155 Japanese Patent Application No. 2002-230656 Japanese Patent Application No. 2002-148849 JP 2001-163899 A Japanese Patent Application No. 2001-180169 JP-A-2002-128691 H. Yamada et al .: Materials Science & Engineering C, 14, P. 41-46 (2001) Kozo Tsubouchi, Hiroo Yamada, Yoko Takasu: Journal of the Japanese Society of Silk Science, Vol. 71, No. 1, P.1-5 (2002) Tashiro Yutaka and Otsuki Eiichi, Journal of Cell Biology, Vol, 46, P1 (1970)

The H chain of fibroin and the a component of sericin are excellent in promoting cell growth, but have a high molecular weight of 300,000 or more and are easily crystallized.
On the other hand, the light chain of fibroin has a molecular weight of 250,000, and has a molecular weight smaller than that of the H chain and the component a, but has a property of easily forming crystals as compared with ordinary proteins.
Further, the weight ratio of the H chain and the L chain of fibroin is approximately 13 (H chain) to 1 (L chain), and the ratio of the L chain is very small.
Such high molecular weight, crystalline proteins have low stability in aqueous solutions.
In addition, when a pharmaceutical or cosmetic additive such as an oil component is added, the gel is easily formed, the properties are unstable, and the stability for long-term (one year or more) storage is low.

On the other hand, substances having a lower molecular weight than silk fibroin H chain or sericin a and having excellent cell growth properties include growth factors of various cells, for example, fibroblast growth factor (FGF) having a molecular weight of 1.77 to 19,000. .
These are secreted by tumors, cancerous cells, or rapidly proliferating cells, some of which are used as skin ulcer treatments (Fiblast Spray, Kaken Pharmaceutical Co., Ltd.) There is a problem with sex.
The present invention has been made to solve such a problem.
That is, unlike cell growth factors produced by abnormal cells such as tumor cells, the objective is to obtain peptides that are excellent in safety, relatively low in molecular weight, excellent in stability, and excellent in cell growth promotion. And

The present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in finding that the peptide chain at a specific site of silk fibroin has a cell growth promoting function, and completed the present invention. That is what led to it.
That is, the present invention comprises (1) a partial peptide of one or more peptide chains selected from peptide chains in an amorphous portion constituting a silk protein, and the partial peptide has 4 amino acid residues. The present invention relates to a peptide composition having an excellent cell growth promoting property, which is a peptide having a specific amino acid sequence consisting of
More specifically, the peptide chains of the N-terminal part (I), the non-crystalline part (A), and the C-terminal part (a) constituting the H chain of silk fibroin of silkworm, the peptide chain of the L chain, and the natural silkworm A partial peptide of one or more peptide chains selected from the N-terminal part (I), the non-crystalline part (A), and the C-terminal part (a) of each of the peptide chains constituting the silk fibroin of wild silkworm belonging to Antheraea Wherein the partial peptide is a peptide having a specific amino acid sequence consisting of 4 to 40 amino acid residues and is present in a peptide composition having excellent cell growth promoting properties.

And (2) the peptide chain having the specific amino acid sequence is present in the peptide composition having any one of the following amino acid sequences (1) to (8).
(1) A-6-2 VITTDSDGNE
(2) A-6-6 NINDFDED
(3) SfHE AASSVSSASSRSYDYSRRNVRKN
(4) SfHA GSSGFGPYVAHGGYSGYEYAWSSESDFGT
(5) AfH1 YGWGDGGYGSDS
(6) AfH5 DEYVDN
(7) AfH6 VETIVLEEDPYGHEDIYEED
(8) AfH7 DDGFVLDGGYDSE

Further, (3) a peptide having an amino acid sequence of any of the following (1) to (8), which is excellent in promoting cell growth.
(1) A-6-2 VITTDSDGNE
(2) A-6-2 NINDFDED
(3) SfHE AASSVSSASSRSYDYSRRNVRKN
(4) SfHA GSSGFGPYVAHGGYSGYEYAWSSESDFGT
(5) AfH1 YGWGDGGYGSDS
(6) AfH5 DEYVDN
(7) AfH6 VETIVLEEDPYGHEDIYEED
(8) AfH7 DDGFVLDGGYDSE

  Further, (4), after hydrolyzing undegraded silk protein of the silkworm or undegraded silk fibroin of the wild silkworm belonging to Antheraea, separating and obtaining the peptide in the non-crystalline part having excellent cell growth promoting properties by molecular weight fractionation. Lies in the way you do.

  And (5) a method for separating and obtaining a peptide in the non-crystalline portion having excellent cell growth promoting ability according to the fourth aspect of the present invention, wherein the hydrolysis is performed with a dilute acid, hydroxylamine or a protease. Exists.

  Further, the present invention resides in (6) and (7), the peptide compositions of (1) and (2) and the cell growth promoter containing the peptide of (3), respectively.

Further, the present invention resides in the cell adhesive containing (8) and (9), the peptide composition of (1) and (2), and the peptide of (3), respectively.

  Further, the present invention resides in (10) and (11), a wound healing promoter containing the peptide composition of (1) and (2) and the peptide of (3), respectively.

  Further, the present invention resides in cosmetics containing (12) and (13), the peptide composition of (1) and (2), and the peptide of (3), respectively.

  Further, (14) and (15) exist in a cell culture substrate containing the peptide composition of (1) and (2) and the peptide of (3), respectively.

According to the present invention, a peptide having a specific amino acid sequence having a molecular weight of 10,000 or less, preferably from 4,000 to 400, is separated and separated from an amorphous portion of a silk protein, and a peptide similar thereto is synthesized. As a result, a novel peptide having excellent cell viability could be provided.
These peptides can be useful as cell adhesives, cell growth promoters, wound healing promoters, skin care materials such as cosmetics, and biomaterials such as cell culture substrates.

  The peptide of the non-crystalline portion of the silk protein having excellent cell growth promoting properties of the present invention or the peptide composition thereof was obtained by the following operation, and the amino acid sequence thereof was determined.

(1) A silkworm cocoon layer belonging to Antheraea such as a silkworm or a silkworm is scoured, and the scoured cocoon layer is dissolved in an aqueous LiSCN solution, centrifuged, and the supernatant is separated and collected. After dialysis, the dialysate is centrifuged again to separate and collect the supernatant.
(2) Hydrolysis treatment is performed by adding a protease such as chymotrypsin to the separated and collected purified supernatant.
(3) The supernatant (non-crystal part) of the hydrolyzed liquid is separated and collected from the precipitate layer (crystal part).
(4) The supernatant (non-crystal part) is fractionated by reverse phase chromatography.
(5) Cell culture is performed for each fraction fractionated by reverse phase chromatography.
(6) The fraction having excellent cell growth is subjected to molecular weight fractionation by gel filtration chromatography, and the amino acid sequence is determined for the fraction (peptide chain) having a cell growth rate twice or more that of the control.
(7) Designing and synthesizing a peptide having excellent cell growth promoting properties based on the obtained peptide.

  In addition, the present invention separates and separates peptides having a molecular weight of 10,000 or less, preferably 4,000 to 400, having excellent cell growth promoting properties from the non-crystal part of silk protein, and synthesizes peptides similar thereto. The peptide can be synthesized and provided as a cell adhesive, a cell growth promoter, a wound healing promoter, a skin care material such as cosmetics, or a biomaterial such as a cell culture substrate.

  The silk protein-derived cell growth-promoting peptide in the present invention is at least twice as high as the control in which cells are cultured without adding the peptide in a 3-day cell culture test as described in Examples 1 to 3. Refers to a peptide having a cell growth rate of

Primary structure of silkworm fibroin In the case of silkworm, silkworm spits silk protein during cocoon production to make cocoons (consisting of cocoons and pupae).
It is known that cocoons have fibroin in the center and sericin in the periphery, and the abundance is (70 to 80% fibroin): 20 to 30% (sericin).
Fibroin of cocoon thread (collectively, fibroin and sericin, or alone as silk protein) has a molecular weight of about 370,000.
[Tasiro Yutaka and Otsuki Eiichi, Journal of Cell Biology, Vol, 46, P1 (1970)] (see Non-Patent Document 3).

Fibroin with a molecular weight of about 370,000 has an SS bond of about 350,000 (H chain) and about 250,000 (L chain).
The fibroin H chain has a very high molecular weight, but is composed of repeating amino acid residue sequences.

According to GenBank (Gen Bank accession no. AF 226688), the precursor protein of the H chain is composed of the N-terminal part (I), the repeating part of the crystalline part (R) and the non-crystalline part (A), It consists of a terminal part (a).
In the repeating part, twelve crystal parts (R01 to R12) are repeated through eleven non-crystal parts (A01 to A11).

The crystalline portion is composed of long repeats of highly crystalline (Gly-X) dipeptide.
As X, Ser (S) is mainly used in addition to Ala (A) having high crystallinity, and Tyr (Y), Val (V), etc. are present as minor components.
In particular, GAGAGS or GA is often repeated.
As a result, in the crystal part, the sum of G and A exceeds 50% and is about 70% or more.

The peptides A01 to A11 in the non-crystal part have similar amino acid sequences, but there are slight differences in the amino acid sequences of the peptides.
The sum of G and A in the non-crystal parts (A01 to A11) is 50% or less.
The N-terminal portion and the C-terminal portion are considered to be non-crystalline portions based on the amino acid sequence and amino acid composition, and the sum of G and A is 50% or less as in A01 to A11.
However, in the non-crystal part, as a partial peptide of about 6 to 10 residues, there is a part where the sum of G and A exceeds 50%.

<Fibroin H chain precursor>
I-R01A01R02A02R03A03R04A04R05A05R06A06R07A07R08A08R09A09R10A10R11A11R12-a

N-terminal: I
The N-terminal part (I) is a part of the initial peptide, and the amino acid sequence is as follows.
MRVKTFVILCCALQYVAYTNANINDFDEDYFGSDVTVQSSNTTDEIIRDASGAVIEEQITTKKMQRKNKNHGILGKNEKMIKTFVITTDSDGNESIVEEDVLMKTLSDGTVAQSYVAADAGAYSQSGPYVSNSGYSTHQGYTSDFSTSAAV

Crystal part: R01, R02, ..., R12
Each of R01, R02,..., R12 is a site called a crystal part, and the number of each amino acid residue is 300 or more. However, the number of amino acid residues of R12 is 54.
(A01 to A11) In all the crystal parts, the sum of G and A is about 70% or more.

Amorphous part: A01, A02, ..., A11
It is composed of 28 to 32 amino acid residues and is called an amorphous part (A).
Their amino acid sequences are as follows:
A01 GSSGFGPYVANGGYSRSDGYEYAWSSDFGT
A02 GSSGFGPYVAHGGYSGYEYAWSSESDFGT
A03 GSSGFGPYVANGGYSGYEYAWSSESDFGT
A04 GSSGFGPYVAHGGYSGYEYAWSSESDFGT
A05 GSSGFGPYVAHGGYSGYEYAWSSESDFGT
A06 GSSGFGPYVANGGYSGYEYAWSSESDFGT
A07 GSSGFGPYVANGGYSGYEYAWSSESDFGT
A08 GSSGFGPYVANGGYSGYEYAWSSESDFGT
A09 GSSGFGPYVNGGYSGYEYAWSSESDFGT
A10 GSSGFGPYVANGGYSGYEYAWSSESDFGT
A11 GSSGFGPYVANGGYSRREGYEYAWSSKSDFET

C terminal: a
The amino acid sequence at the non-crystal site on the C-terminal side is as follows.
AASSVSSASSRSYDYSRRNVRKNCGIPRRQLVVKFRALPCVNC

For convenience of understanding of the present invention and for reference, Table 10 shows notations of amino acids or amino acid residues in proteins, charge characteristics of amino acids, and the like.

Amino acids are classified into four groups, acidic amino acids, polar uncharged amino acids, nonpolar amino acids, and basic amino acids, as follows, based on the characteristics of the side chains.
(1) Acidic amino acids: E, D
(2) Polar uncharged amino acids: N, S, Q, Y, T, C, F
(3) Non-polar amino acids: A, G, V, P, L, I, W
(4) Basic amino acids: H, K, R

The fibroin L chain shown below is a non-crystal part when compared with the amino acid sequences of the crystal part and the non-crystal part of the H chain.
<Amino acid sequence of fibroin L chain>
MKPIFLVLLVATSAYAAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYSNVASGRNGESHANAVSIGG

On the other hand, the amino acid sequence of the silk protein of the wild silkworm is different from that of the domestic silkworm.
In the silkworm fibroin, a portion consisting of only repeating alanine (A) of 10 residues or more is defined as a crystalline portion, and the other portion is defined as a non-crystalline portion.
Wild silk fibroin belonging to Antheraea has a smaller number of residues in each of the crystal part and the non-crystal part compared to the silkworm fibroin.
The amino acid sequence of the non-crystal part of the silkworm fibroin except for the crystal part where alanine is continued for 10 residues or more is shown below.
From the amino acid composition, the N-terminal part and the C-terminal part are also non-crystal parts.

<Primary structure of the amorphous part of the silkworm fibroin>
N-terminal: Initial peptide
MRVTAFVILCCALQYATANNLHHHDEYVDNHGQLVERFTTRKHYERNAATRPHLSGNERLVETIVLEEDPYGHEDIYEEDVVINRVPGASSSAAAASSASAGSGQTIIVERQASHGAGGA

Non-crystalline part:
AGAAAGAAAGSSARGG
SGFYETHDSYSSYGSGSSSAAAASSGAGGAGGGYGWGDGGYGSDS
GSGAGGRGDGGYGSGSS
RRAGHDHAAGSSGGGYSWDYSSYGSES
GSGAGGVGGGYGGGDGGYGSGSS
RRAGHDRAAGS
SGAGGSGGGYGWGDGGYGSDS
GSGAGRAG
GDYGWGDGGYGSDS
RQAGHERAAGS
SGAGGSGRGYGWGDGGYGSDS
GSGAGGAGGDYGWGDGGYGSD
GSGAGGAGGDYGWGDGGYGSDS
SGAGGAGGGYGWGDGGYGSDS
SGAGGAGGYGGYGSDS
SGAGGSGGGYGWGDGGYGSGS
GSGAGGVGGGYGWGDGGYGSDS
SGAGGRGDGGYGSGSS
GSGAGGAGGGYGWGDGGYGSDS
RRAGHDRAAGC
SGAGGTGGGYGWGDGGYGSDS
SGAGGSGGGYGWGDGGYGSNS
SGAGRSGGGYGWGDGGYSSDS
SGAGGSGGYGGYGSDS
GSGAGGVGGGYGWGDGGYGGYGSDS
GSGAGGVGGGYGRGDSGYGSGSS
GHGRSSGS
SGAGGSGGGYGWDYGSYGSDS
SSGAGGSGGGYGWDYGGYGSDS
GSGAGGSGGGYGWGDGGYGSDS
SRRAGHDRAYGAGS
GAGASRPVGIYGTDDGFVLDGGYDSEGS

C terminal:
SSSGRSTEGHPLLSICCRPCSHRHSYEASRISVH

By the way, in the present invention, regarding the silk protein component having excellent cell growth promoting property, it has been analyzed which part of each component has the cell growth promoting property, and it is clear that the amorphous portion has the cell growth promoting property. became.
The non-crystal part of fibroin is the amino acid sequence part described above.
Therefore, the non-crystal part includes the N-terminal part and the C-terminal part.

A01 to A11 (peptide of 28 to 32 residues) in the non-crystal part of silkworm fibroin all show the same excellent cell growth, but A01 to A11 do not have exactly the same amino acid sequence.
In the case of A11, the amino acid residue sequence differs from other A01 to A10 within 8 residues out of 32 residues.
Therefore, even if there is a difference in the amino acid residue sequence of about 30% or less, there is no effect on cell viability.
Further, even if the number of amino acid residues increases or decreases within about 20%, there is little effect on cell viability.
However, the difference in amino acid sequence is preferably 50% or less in order to maintain cell growth promoting properties.

On the other hand, a peptide having a very small number of amino acid residues does not exhibit cell growth promoting properties.
In particular, when the number of amino acids is two or less, cell growth may be harmed.
Therefore, the peptide for promoting cell growth is preferably a peptide having 4 to 40 residues, preferably 6 to 32 residues.
Furthermore, even in the non-crystal part, it was found that a partial peptide containing almost no basic amino acid residue and containing many acidic amino acid residues and / or polar uncharged amino acids was excellent in cell growth promoting ability.
In particular, acidic amino acid residues showed extremely excellent cell growth promoting properties.

The non-crystalline portion exhibits excellent cell growth promoting properties as a whole, but as a portion, the sum of G and A exceeds 55%, and since there are many basic amino acid residues, the amino acid sequence portion having a low cell growth rate There is.
There, acidic amino acids are present from the amorphous portion of fibroin, and / or polar uncharged amino acids are present.
In addition, a partial peptide having almost no basic amino acid is separated and recovered.
In the collected partial peptides, the number of amino acid residues was 40 or less, and the cell growth rate was no addition of the peptide as a control. In the cell culture test as in Examples 1 to 3, the cell growth rate was 2 A peptide showing twice or more values is designated as SDFGP.
In addition, even if a mixture of a plurality of peptides having 4 to 40 amino acid residues is used, if the cell growth rate is at least twice as high as that of the control group, SDFGP is used.

As a condition, in the partial peptide in the non-crystal part, (1) the sum of the number of residues of G and A is 55% or less with respect to the total number of residues of the peptide.
This is because if there is a large amount of G or A, it tends to become a crystal.
(2) The number of basic amino acid residues is 25% or less of the total number of residues of the peptide.
(3) There are acidic amino acids and / or polar uncharged amino acids.
Must be satisfied, and this is easily deduced from the points shown in Table 6.
If these are not satisfied, cell viability may be inhibited.

Incidentally, the properties of amino acids or peptides differ depending on the chemical structure of the side chain.
Even though the acidic amino acid is excellent in cell growth promoting properties, E and D have different side chain lengths, and therefore have different flexibility and three-dimensional structure of side chains and main chains, and do not show the same cell growth promoting properties.
The same can be said for various amino acids called polar uncharged amino acids or basic amino acids, and they do not necessarily show the same cell-promoting or inhibiting properties because of their different physicochemical properties.

The cells that make up our bodies can be broadly divided into adherent and planktonic cells.
Adherent cells include skin cells, vascular cells, gland cells, and the like. Suspended cells include blood cells and the like.
The growth process of adherent cells first grows after they adhere, so they can be broadly divided into adhesion and proliferation.
The SDFGP of the present invention has adhesive and proliferative properties, but is superior in adhesiveness when compared.
Excellent proliferative properties cause cells to proliferate abnormally, and there is a problem in safety when used for a long time.
Therefore, the SDFGP of the present invention, which has excellent adhesion, has growth promoting properties, and does not inhibit cell growth, is extremely excellent as a skin care material and a biomaterial.
In fact, when separating and recovering a partial peptide from silk protein, amino acids belonging to the non-crystalline part of the fragment are obtained by cutting the silk protein with a protease that breaks specific peptide bonds or other chemical substances. A peptide having a residue number of 40 or less is recovered.
Further, a peptide excellent in cell growth can be designed and synthesized based on the obtained partial peptide.

1. Separation and Recovery of Peptide from Silk Protein In order to separate and recover the non-crystal part of the silk protein, the characteristics of the non-crystal part may be used.
Since the non-crystalline portion is easily soluble in water, the silk substance may be immersed in water near neutrality (pH 5.0 to 9.0).
However, it is not possible to efficiently obtain a peptide in an amorphous portion simply by immersing a silk substance.
Therefore, the specific peptide bond of the silk protein is actively cleaved.
As a method for cleaving such a peptide bond, it is preferable to use a chemical substance, an enzyme, or the like that actively cuts a specific peptide bond.
This is described below.

1) Raw Material In silkworm, silk protein in which the H chain and L chain of fibroin and the a component of sericin remain is used as a raw material.

For wild silkworms (natural silkworms, sac silkworms, eri silkworms, moth silkworms, thasar silkworms, etc.), the raw material is a case where a band as clear as the H chain of the domestic silkworm fibroin is recognized in the electrophoresis image of fibroin.
The raw material may be fibroin or sericin alone, or fibroin and sericin may be present simultaneously.

Therefore, the raw material of the present invention is a cocoon thread, a raw silk, a silk woven or knitted fabric, a silk thread (fibroin fiber), their remnants, or fibers, powders, films, etc. made from them, and silkworms such as silkworms and wild silkworms. All threading protein fiber materials (silk materials) can be targeted.
However, it is necessary that these raw materials include the silkworm fibroin H chain, the a component of sericin, and the wild silkworm corresponding components.
Since the H chain of fibroin is more easily decomposed than the L chain, if only a part of the H chain remains, most of the L chain remains without being decomposed.

2) Dissolution of Raw Material The raw material of the above 1) which is dissolved with a neutral salt may be a refined product, a semi-refined product, an unrefined product, or an intermediate thereof.
Sericin silkworm cocoons may be used.
What is important is that the silk proteins are not degraded, or the H chain of fibroin, the a component of sericin and a part of the corresponding protein remain undegraded even after various processing steps. It is.
Confirmation of the presence of the H chain, the a component, and the like is performed by confirming the presence of bands corresponding to each of the electrophoresis images.

Examples of the neutral salt which is a solubilizer for the raw silk include neutral salts such as calcium chloride, copper ethylenediamine, sodium thiocyanate, lithium thiocyanate, lithium bromide, and magnesium nitrate.
The neutral salt is also preferably a saturated aqueous solution or a concentration of 50% [weight (g) / volume (ml)] saturation or higher.
When fibroin and sericin are contained, for example, cocoons, unrefined materials, semi-refined materials and the like are dissolved by the above-mentioned neutral salt in the same manner as silk fibers.

On the other hand, when sericin is 98% or more, such as sericin silkworm cocoon, it is dissolved in 8M urea at 70 to 90 ° C. within 10 minutes.
Sericin silkworm cocoons can be dissolved at room temperature (20-30 ° C.) within 30 minutes even with 9 MLiBr.
The dissolution conditions are changed according to these conditions.

In the step of dissolving the silk substance in the neutral salt solution, an alcohol such as methyl alcohol, ethyl alcohol, or propyl alcohol may be added to the neutral salt.
When calcium chloride is used as a neutral salt, it is carried out at a temperature of 94 ° C. or less, preferably at a temperature of about 75 to 85 ° C.
When using lithium bromide, the dissolution conditions vary depending on the neutral salt, such as dissolving the raw material at a temperature of about 50 ° C or less. However, the dissolution method is such that the H chain of fibroin and the a component of sericin remain, or the equivalent. I do.

In dissolving silk material,
(1) Dissolution can be promoted by stirring.
(2) It is difficult to dissolve when the dissolution temperature is low.
If the dissolution temperature is high, it is easy to dissolve, but the molecular weight is drastically reduced.
The solution obtained by dissolving silk with a neutral salt contains a neutral salt, an alcohol and the like in addition to fibroin or a mixture of fibroin and sericin.
First, insolubles are removed from the solution, and then low molecular substances having a molecular weight of about 5,000 or less are removed using a dialysis membrane or a dialysis device.
The silk protein aqueous solution is obtained by such dialysis.

3) Degradation and recovery by enzymes Generally, it is said that cleavage of proteins by proteolytic enzymes occurs at specific peptide bonds and cleavage is performed under mild conditions, so that modification of amino acid residue side chains does not occur. I have.
It is also possible to avoid complication of fragments due to non-specific cleavage of proteins.

Such enzymes include lysyl endopeptidase, serine protease, metalloendopeptidase, arginylendopeptidase, metalloprotease, chymotrypsin, papain, alcalase, pepsin, rennin, pancreatin, elastase, carboxypeptidase, aminopeptidase. And a dipeptidase.
Of these, chymotrypsin is particularly preferred for separating the crystalline part from the non-crystalline part.

When a proteolytic enzyme is added to an aqueous silk protein solution, the silk protein is cleaved between specific peptide bonds.
If the cleaved peptide derived from the silk protein is mainly a fragment derived from the crystal part of the silk protein, the peptide easily aggregates, and if aggregated (coagulated or crystallized), it precipitates.
Even when aggregation is difficult, the peptide derived from the crystal part is more likely to aggregate, so if an alcohol (eg, methyl alcohol, ethyl alcohol, etc.) is added as an aggregating agent, the peptide starts to aggregate from the crystal part and precipitate.

If the precipitate is removed, the remainder is a solution of the peptide derived from the non-crystalline part.
To remove the precipitate, the solution containing the precipitate is separated by a centrifuge (1,000 to 10,000 G) to remove the precipitate.
The liquid from which the precipitate has been removed is an aqueous solution of the peptide in the non-crystalline portion separated from the non-crystalline portion.
When this is dried, a peptide in the form of a film or powder is obtained.
However, when alcohol is added to the aqueous peptide solution derived from the non-crystal part, the peptide derived from the non-crystal part also sequentially aggregates and precipitates as the alcohol concentration increases.
The peptide derived from the non-crystal part may contain a partial peptide of the crystal part as long as it is about 50% or less.

However, the sum of the numbers of glycine and alanine residues preferably does not exceed 55% in order to obtain a peptide excellent in cell growth promotion even from a peptide derived from an amorphous portion.
Further, the number of basic amino acid residues is preferably 25% or less of the number of peptide-constituting residues.
On the other hand, the more acidic amino acid residues and nonpolar charged amino acid residues, the better.
In particular, when the number of acidic amino acid residues is large, cell growth promotion is excellent.
Drying is performed by freeze drying or spray drying.
Even if the peptide derived from the non-crystal part is crystallized after drying, it is easily soluble in water because of its low molecular weight.

2. Synthesis of silk protein-derived fibroblast proliferation peptide (SDFGP) The following peptides are SDFGPs separated and recovered from silk protein.
These are examples of SDFGP.
Needless to say, there are many SDFGPs that satisfy the condition [0047].
A-6-2 VITTDSDGNE
A-6-6 NINDFDED
SfHE AASSVSSASSRSYDYSRRNVRKN
SfHA GSSGFGPYVAHGGYSGYEYAWSSESDFGT
AfH1 YGWGDGGYGSDS
AfH5 DEYVDN
AfH6 VETIVLEEDPYGHEDIYEED
AfH7 DDGFVLDGGYDSE
An excellent cell growth promoting property can be SDFGP even with only a part of the amino acid sequence of these SDFGPs.
Further, these peptides may be repeated within 40 residues or linked to another peptide.

However, the amino acid does not have a cell growth promoting function.
The number of amino acid residues for cell growth having a function must be 4 or more.
However, from the efficiency of peptide synthesis, 40 residues or less are preferable.
The synthesis of SDFGP mimics the amino acid sequence of the amorphous portion of the silk protein and synthesizes a peptide that satisfies the condition [0047].
The amino acid residues of the synthetic peptide are 4 to 40, preferably 6 to 32 residues.
In this case, it may not be completely the same as the amino acid sequence of the silk protein.

For example, the amino acid sequences of the non-crystalline portions (A01 to A11) of the fibroin H chain are not all the same, but the cell growth promoting properties are almost the same, and all are SDFGP.
Therefore, the amino acid sequence may have a difference of about 30% or less, but the difference of the amino acid sequence is preferably 50% or less.
In this case, the more acidic amino acids, the better, and preferably the presence of polar uncharged amino acids.
However, it is preferred that there are few basic amino acids.

That is, the amino acid residue of each SDFGP may be replaced with another amino acid residue.
In this case, the number of basic amino acid residues is at least 25% or less of the number of amino acid residues constituting the peptide.
On the other hand, it is preferable that the number of acidic amino acids in the peptide (SDFGP) is large.
All amino acid residues may be composed of acidic amino acids and / or non-polar charged amino acids.

Accordingly, A-6-2 to DSDGDE, A-6-6 to DEDEDE, EDEDED, SfHA to SSSSE and YGGYEY, AfH1 to DGGYGGD, AfHS to DEYDEY, AfHG to YEEDYEED, etc., and EEEE, EEEEEE EYEYEY, EEYEEY, YYYYYY And many peptides such as EGSEGS can be SDFGP.
It goes without saying that, even if these peptides are repeated or linked to another peptide within the range of the aforementioned condition [0047], the molecular weight should be 10,000 or less, preferably in the range of 4,000 to 400. No.
Further, the amino acid residue may be replaced with another amino acid residue.

4. Utilization Peptide aggregates and SDFGP obtained from the non-crystalline part of silk protein are not only excellent in cell growth promotion but also easily soluble in water.
Further, since the molecular weight is about 4,000 or less, the morphology is stable for a long time even in a dissolved state.
Furthermore, mixing various SDFGPs promotes cell growth more than using SDFGP alone.
Therefore, these are mixed and added to a cell culture solution, lotion, milky lotion, cream, ointment, eye drops, food and the like.
Since the peptide aggregate and SDFGP of the present invention are excellent in promoting the growth of skin cells, they can be used as a skin care material or as a cell culture material in other fields such as clothing fibers, cosmetic powders, resins and the like. Excellent material.

Cell growth activity of enzymatically decomposed product of silk fibroin (1) Non-crystal part of silk protein is decomposed by enzyme, separated and collected. The cocoon of silkworm is cut off to remove pupae. Urea was immersed in 90 ° C. for 10 minutes to extract sericin.
The extraction residue was washed with water and dried to obtain fibroin.

1.0 g of fibroin was immersed in 10 ml of 9M LiSCN to dissolve, 10 ml of distilled water was added thereto, and the mixture was spun down at 3,000 rpm for 10 minutes.
The supernatant was placed in a semi-permeable membrane and dialyzed against 50 volumes of water.
The dialysis was performed four times every 30 minutes, changing the water of the dialysis external solution.
After dialysis, the solution was spun down again, and 0.1 M disodium hydrogen phosphate (pH 8.5) was added to the supernatant to adjust the pH to 7-8.
Chymotrypsin, 1/100 of the amount of fibroin, was added thereto, and the mixture was left at 40 ° C. for 4 hours to precipitate.

At this time, the protein or peptide of the supernatant was obtained from the non-crystal part (A) of fibroin, and the protein or peptide obtained from the precipitate was obtained from the crystal part (C). This was designated as a crystal part (C ′).
The crystal part was washed with water, dissolved in 1 ml of 9M LiSCN, dialyzed against 50 times the volume of water with a semipermeable membrane in the same manner as described above, and the protein amount of the aqueous solution was measured.
The supernatant was directly measured for protein amount.

(2) Coating to cell culture vessel Prepare aqueous solutions of these crystalline part (C ') and non-crystalline part (A') by adding 70% ethanol to concentrations of 0.025% and 0.0025%, respectively. Then, it was put into a polystyrene dish (35 mm ¢, Falcon) in an amount of 1 ml and air-dried.
The control dish was air-dried with 1 ml of 70% ethanol alone.

(3) Cell culture Cells used were (frozen) human skin fibroblasts (derived from normal adult skin) purchased from Sanko Junyaku.
As a medium, a low serum medium for human skin fibroblast proliferation (purified by Kurabo Industries, 10 ml of LSGS (low serum proliferation additive) added to 500 ml of Medium 106S (basic medium of skin fibroblast)) was used.
LSGS has cell proliferation.
2 ml of medium was added to one petri dish, 80,000 cells were inoculated, and cultured for 3 days.

(4) Measurement of viable cell count with alamar blue dye Add 2 ml of culture medium and 0.1 ml of alamar blue (IWAKI) per one petri dish, culture at 37 ° C for 2 hours, and absorbance at 570 nm and 600 nm. The amount of reduction of the alamar blue dye calculated from was used as the number of living cells.
Table 1 shows the growth of human dermal fibroblasts in a petri dish coated with a crystal part and a non-crystal part, with the case where no silk protein component was added as a control (100%).

The cell growth rates in petri dishes coated with the silk protein component were all higher than those in the control group.
In particular, the non-crystalline portion (C ′) has a high growth rate of 0.025% concentration, and is a mixture of SDFGP.
In the crystal part, the growth of the higher concentration (0.025%) was worse than that of the lower concentration (0.0025%).
This is presumably because the silk protein in the crystal part inhibits cell growth.

Example 1 Amino Acid Sequence of Cell Growth-Promoting Peptide It was found in Example 1 that the non-crystal part of fibroin promoted cell growth more than the crystal part.
However, the non-crystalline portion collected in Example 1 is considered to be a mixture of peptides cleaved by the enzyme.
Therefore, the non-crystalline portion was further separated to identify the cell growth promoting site.
First, the peptides contained in the non-crystalline portion of Example 1 were separated by reverse phase chromatography in order to separate them by polarity.
The column used was 3 ml of RESOURCE® RPCk.
Chromatography was performed using 0.1% TFA (trifluoroacetic acid) for the A pump and 0.1% TFA / 90% acetonitrile for the B pump, with a gradient of 0 to 75% B in 0 to 15 minutes. Was.

As a result, six peaks (A-1,..., A-6) were confirmed.
Each peak was collected, dried with an evaporator, and dissolved with a small amount of buffer (PBS).
Each of A-1 to A-6 was adjusted with 70% ethanol so as to have a concentration of 0.025%, and placed in a polystyrene dish for cell culture (35 mmφ, Falcon) in 1 ml portions and air-dried.
The control petri dish was air-dried with 1 ml of 70% ethanol alone.
Cell culture was performed using these dishes.
The cell culture method was the same as in [Example 1].
Table 2 shows the growth rate of human dermal fibroblasts in a Petri dish coated with a peptide fragment of an amorphous portion.
Among the non-crystal parts, the growth of A-6 was as excellent as about 4 times that of the control.

Next, to separate the peptides contained in A-6 having the highest cell viability by molecular weight, they were separated by Superdex peptide HR10 / 30 (gel filtration chromatography).
As a result, seven peaks from A-6-1 to A-6-7 were confirmed.
Each has a molecular weight of 2,500 or less.
Of the seven peaks, the peptides of five sharp and clear peaks (A-6-2, A-6-3, A-6-4, A-6-6, A-6-7) were obtained. The cells were collected, each peptide was coated on a cell culture vessel, a medium and cells were inoculated therein, cultured for 2 days, and the cell viability was measured.
The experiment on cell viability is the same as in [Example 1].
Table 3 shows the measurement results of the cell growth rate.
A-6-2 and A-6-6 showed a 1.5-fold growth rate in 2-day culture as compared to the control group, and were excellent in cell growth promotion.
After three days of culture, the growth rate was more than twice.

Thus, the amino acid sequences of A-6-2 and A-6-6 were analyzed by LF3000 Protein Sequencor of Beckman Co., Ltd.
A-6-2 VITTDSDGNE
A-6-6 NINDFDED
It became.
Both A-6-2 and A-6-6 were N-terminal peptides of fibroin.
The N-terminal part is a non-crystalline part in view of the amino acid composition.
Therefore, the peptides A-6-2 and A-6-6 were synthesized (consigned to Hokkaido System Science Co., Ltd.), cultured for 3 days in the same manner as described above, and the biological activity of the synthesized peptide was measured. It was confirmed that the extract had a cell growth promoting property similarly to the extract from E. coli.
In addition, when the synthesized peptides A-6-2 and A-6-6 were mixed, cell growth promotion was superior to the case where each was cultured alone.

Cell Growth Rate of Synthetic Peptide Based on the amino acid sequences of the silkworm fibroin H chain and the silkworm fibroin, the cell activity of the peptide synthesized for a total of 12 sites (consigned to Hokkaido System Science Co., Ltd.) was measured.

(1) Peptide synthesis Two partial peptides (SfHC-1, SfHC-2) from the crystal part of silkworm fibroin and two partial peptides (SfHE, SfHA) from the non-crystal part were taken up.
For the silkworm fibroin, a repetitive portion of Ala (A) (AfH0) and a non-crystalline portion of seven partial peptides (AfH1 to AfH) were taken as crystal parts, and they were synthesized.
There are four types of partial peptides of the silkworm fibroin H chain and eight types of partial peptides of the fibroin of the silkworm (AfH0 to AfH7). The amino acid sequences of the respective peptides are shown below.

Silkworm fibroin H chain partial peptide (4 types)
SfHC-1 GAGAGSGAGAGSGAGAGYGAGY
SfHC-2 GAGAGSGAASGAGAGAGAGAGT
SfHE AASSVSSASSRSYDYSRRNVRKN
SfHA GSSGFGPYVAHGGYSGYEYAWSSESDFGT

Partial peptides of silkworm fibroin (8 types)
AfH0 AAAAAAAAAA
AfH1 YGWGDGGYGSDS
AfH2 SGAGGSGGYGGYGSDS
AfH3 GSGAGGRGDGGYGSGSS
AfH4 RRAGHDRAAGS
AfH5 DEYVDN
AfH6 VETIVLEEDPYGHEDIYEED
AfH7 DDGFVLDGGYDSE

(2) Coating of Petri Dish with Synthetic Peptide About 1 mg of each synthetic peptide was taken and dissolved in 200 μl of PBS.
The dissolved peptide solution was diluted with 70% ethanol, adjusted to a concentration of 0.025% and 0.0025%, and placed in a Petri dish by 1 ml and dried.
Since SfHC-1, SfHC-2, and SfHE were difficult to dissolve, 1 ml of 9 M LiSCN was added and dissolved.
The solution was put into a semipermeable membrane, dialyzed with 100 volumes of water every 30 minutes while changing the external solution, and the protein amount was confirmed by absorbance at 275 nm.
Since SfHC-2 did not have tyrosine or the like, the absorbance could not be measured.However, since SfHC-1 and SfHE were present in approximately the same amount as before dissolution, it was assumed that SfHC-2 was also present in the same amount. Similarly to the peptide, it was diluted with 70% ethanol, placed in a petri dish, and dried.
AfH0 was also difficult to dissolve, but because of its small molecular weight when dialyzed, it may come off. Therefore, it was well stirred, diluted with 70% ethanol, and dried in a petri dish.

(3) Culture The cells used were (frozen) human skin fibroblasts (derived from normal adult skin) purchased from Sanko Junyaku.
As the medium, a low serum medium for growing human dermal fibroblasts purchased from Kurabo Industries was used.
2 ml of the medium was added to one petri dish, about 80,000 cells were inoculated, and cultured for 3 days.
Thereafter, 0.1 ml of Alamar Blue (IWAKI) was added, and the mixture was cultured at 37 ° C. for 2 hours, and the amount of reduction of the dye calculated from the absorbance at 570 nm and 600 nm was defined as the number of living cells.
The measurement method for these cell cultures is the same as in Example 1.
The results are shown in Tables 4 and 5.

  In the silkworm fibroin, the growth rate of SfHE and SfHA, which are partial peptides in the non-crystal part, is higher than the partial peptides SfHC-1 and SfHC-2 in the crystal part of fibroin, showing a value more than twice that of the control group. The partial peptide in the non-crystalline portion, or a mixture thereof, is SDFGP.

It is considered that AfH0, which is a partial peptide of the crystal part of the silkworm fibroin, has almost no activity, and the higher concentration (0.025%) has a lower growth rate, thus inhibiting cell growth.
The non-crystalline partial peptide compounds AfH1 to AfH7 exhibited cell growth promotion, but showed a difference in growth rate.
For example, AfH3 and AfH6 both contain one basic amino acid, but AfH3 has fewer acidic amino acid residues and AfH6 has more acidic amino acid residues, so that AfH6 showed higher cell growth promoting ability.
In particular, AfH1, AfH5, AfH6, and AFH7 show excellent cell growth promoting properties, and all are SDFGPs obtained by synthesis.

As shown in Table 5, the cell growth rate of all the partial peptides obtained from the non-crystal part was not high and did not result in SDFGP.
This is due to the difference in the chemical structure of the amino acid side chains constituting each peptide as shown in Table 6.

Cell growth activity of synthetic peptides
Fibroblast Growth of Synthetic Peptides Based on Acid Amino Acids and Polar Uncharged Amino Acids Mainly Considered to Show Cell Growth Activity Based on the Results of Examples 1 to 3 (Synthesis Outsourced to Hokkaido System Science Co., Ltd.) Activity was measured.
The cell growth activity was measured in the same manner as in [Example 1].
The cell culture was performed for 3 days, and the results are shown in Table 7 when the peptide concentration was 0.025 μg / cm ² .
Glutamic acid had excellent cell growth activity when it had a sequence of 4 residues or more.

Cell adhesion and growth promoting properties of synthetic peptides The cell growth activity of synthetic peptides derived from silk proteins and synthetic peptides mainly composed of acidic amino acids or polar uncharged amino acids, etc. Measured separately.
This measuring method partially differs from the method of (3) cell culture in [Example 1].
In the cell culture here, the cells used were (frozen) human dermal fibroblasts (derived from normal adult skin) purchased from Sanko Junyaku.
The medium used was a low serum medium (Medium 106S 500 ml) for human skin blast cell proliferation purchased from Kurabo Industries.
Medium 106S is a skin fibroblast basal medium.
Here, LSGS (low serum growth additive) is not used.
When measuring the adhesiveness, the cells floating on the medium were removed 5 hours after the cells were inoculated into the medium, and the number of viable cells adhering to the bottom of the petri dish was measured.
When the proliferation was measured, the number of viable cells after three days of culture from the time when the cells were inoculated into the medium was measured.
Other than the cell culture method, the coating of the peptide on the cell culture vessel, the measurement of the number of viable cells using Alamar Blue dye, and the like were performed in the same manner as in [Example 1].
Table 8 shows the results of the obtained adhesiveness, and Table 9 shows the results of the proliferative property, with the number of viable cells when the peptide was not coated on the cell culture vessel as a control (100%).

That is, Table 8 shows the adhesiveness after culturing human skin fibroblasts for 5 hours in a petri dish coated with various peptides.
Here, the synthetic peptide is represented by a symbol or an amino acid sequence.
In this case, the concentration of each peptide when coated on each petri dish is 0.025 μg / cm ² .
The adhesion rate (%) when the peptide was not coated was set to 100.

Table 9 shows the proliferative activity of human dermal fibroblasts after culturing for 3 days in petri dishes coated with various peptides.
Here, the synthetic peptide is represented by a symbol or an amino acid sequence.
In this case, the concentration of each peptide when coated on each petri dish is 0.025 μg / cm ² .
The growth rate (%) when the peptide was not coated was set to 100.
However, the number of viable cells in the control group after culturing for 3 days increased to about 150% during the culturing, but the value (100%) in the control section was based on the number of viable cells after culturing for 3 days. I have.
Therefore, the growth rate when the number of cells does not increase or decrease during the three-day culture is about 70%.

Claims (15)

  It comprises a partial peptide of one or more peptide chains selected from non-crystalline peptide chains constituting the silk protein, and the partial peptide has a specific amino acid sequence consisting of 4 to 40 amino acid residues. A peptide composition having excellent cell growth promoting properties, which is a peptide. The peptide composition according to claim 1, wherein the peptide chain having a specific amino acid sequence has any one of the following amino acid sequences (1) to (8).
(1) VITTDSDGNE
(2) NINDFDED
(3) AASSVSSASSRSYDYSRRNVRKN
(4) GSSGFGPYVAHGGYSGYEYAWSSESDFGT
(5) YGWGDGGYGSDS
(6) DEYVDN
(7) VETIVLEEDPYGHEDIYEED
(8) DDGFVLDGGYDSE A peptide having an excellent cell growth promoting property, comprising the amino acid sequence of any one of the following (1) to (8):
(1) VITTDSDGNE
(2) NINDFDED
(3) AASSVSSASSRSYDYSRRNVRKN
(4) GSSGFGPYVAHGGYSGYEYAWSSESDFGT
(5) YGWGDGGYGSDS
(6) DEYVDN
(7) VETIVLEEDPYGHEDIYEED
(8) DDGFVLDGGYDSE   A method for hydrolyzing undegraded silk protein of Bombyx mori or undegraded silk fibroin of wild silkworm belonging to Antheraea, and then separating and obtaining a peptide having excellent cell growth promoting properties by molecular weight fractionation.   The method according to claim 4, wherein the hydrolysis is carried out with a dilute acid, hydroxylamine or a protease.   A cell growth promoter comprising the peptide composition according to claim 1.   A cell growth promoter comprising the peptide according to claim 3.   A cell adhesive containing the peptide composition according to claim 1.   A cell adhesive containing the peptide according to claim 3.   A wound healing promoter comprising the peptide composition according to claim 1.   A wound healing promoter comprising the peptide according to claim 3.   A cosmetic comprising the peptide composition according to claim 1.   A cosmetic comprising the peptide according to claim 3.   A cell culture substrate comprising the peptide composition according to claim 1.   A cell culture substrate containing the peptide according to claim 3.