WO2002102845A1

WO2002102845A1 - Process for producing silk fibroin-origin functional polypeptide and utilization thereof

Info

Publication number: WO2002102845A1
Application number: PCT/JP2002/005663
Authority: WO
Inventors: Kozo Tsubouchi; Hiroo Yamada; Yoko Takasu; Hiroshi Nakao
Original assignee: National Institute Of Agrobiological Sciences; Kowa Co., Ltd.
Priority date: 2001-06-14
Filing date: 2002-06-07
Publication date: 2002-12-27
Also published as: JPWO2002102845A1

Abstract

Cell proliferation promoters, wound healing promoters, cell culture media and cosmetics containing as the active ingredient a silk fibroin L-chain polypeptide; and a process for producing the L-chain polypeptide. Because of having an excellent effect of promoting cell proliferation and a high safety, the silk fibroin L-chain polypeptide is useful as drugs such as wound healing promoters and skin care cosmetics.

Description

Detail

Method for producing functional polypeptide derived from silk fiproin and its use

The present invention relates to a method for producing silk fiber mouth-in L-chain polypeptide having an excellent cell growth promoting action and its application to the fields of medicine and cosmetics. Background art

Hitherto, silk fiber mouth-in has been studied as a wound covering material in the form of a powder or a film, as it has an effect of promoting the healing of skin tissue that has been lost due to wounds, burns, etc. Nos. 2 5 4 1 6 4, 8-1 9 8 7 0 7, 9 1 9 12 9 10, 11 1 10 4 2 2 8). In addition, silk fiber mouth-in has been applied as a cosmetic because it has such an effect.

However, the healing promoting effect of these conventional wound dressings using silk fibroin is not sufficiently satisfactory, and it is desired to provide a more effective wound healing promoter.

An object of the present invention is to provide a naturally derived component excellent in the ability to regenerate defective skin tissue, that is, a cell growth promoting effect, and to provide the use thereof. Disclosure of the invention

From such a viewpoint, the present inventors have found that undegraded silk fiber mouth-in, that is, an H chain having a molecular weight of about 350,000 has an excellent fibroblast proliferation activity, and has filed a patent application earlier (Japanese Patent Application No. Hei. 1 _ 1 9 8 2 3 3)).

However, since the silk-fiber mouth H chain has a large molecular weight, gelation in aqueous solution It wakes up within a day and is not easy to handle.

Therefore, as a result of further study to obtain a component having a cell growth promoting action that is less likely to cause gelation, the inventors succeeded in isolating a silk fibroin H chain and a low molecular weight silk fibrous in L chain different from its degradation product. Further, they have found that the silk-fiber mouth-in L chain has an excellent skin fibroblast proliferation promoting action, and is useful as a wound healing promoter, a cell culture bed, and a cosmetic. Reached.

That is, the present invention provides a method for scouring a cocoon layer or a cocoon thread of a raw cocoon, a dried cocoon or a cooked cocoon or a cocoon thread, a raw silk, a silk fabric, or a remnant thereof, and dissolving the obtained fiber-in-fiber in a neutral salt aqueous solution. An object of the present invention is to provide a method for producing a silk-fiber mouth-in L-chain polypeptide characterized by fractional precipitation.

The present invention also provides a cell growth promoter comprising a silk-fiber opening in L chain polypeptide as an active ingredient.

The present invention also provides a wound healing promoting agent containing a silk-fiber opening in L chain polypeptide as an active ingredient.

The present invention also provides a cell culture bed containing the silk-fiber opening in L chain polypeptide.

The present invention also provides a cosmetic containing the silk-fiber in L chain polypeptide.

The present invention also provides use of silk fibrous in-L chain polypeptide for producing a cell growth promoter or a wound healing promoter.

Further, the present invention provides a method for promoting wound healing, which comprises administering a silk-fiber opening in L chain polypeptide. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a view showing an electrophoresis (SDS-PAGE) image of each precipitate.

Figure 2 is a place where dissolved when the L i SCN was dissolved Fuibu opening in with C a C 1 ₂ It is a figure which shows the electrophoresis image of the fibroin of the case. BEST MODE FOR CARRYING OUT THE INVENTION

Silkworms secrete liquid silk into the silk gland cavity of the body and are called liquid silk. Liquid silk consists of silk fiber mouth and silk sericin, and liquid silk fiber mouth has a molecular weight of about 370,000 (Tasi ro Yutaka and Otsuki Eiichi, Jounal of Cell Biology, Vol. 46, PI (1970) ). Silk fiber mouth with a molecular weight of about 370,000 is divided into molecular weights of about 350,000 and 25,000 by reduction.Here, silk fiber mouth with a molecular weight of about 250,000 is called silk fiber mouth in L chain, A silk fiber mouth having a molecular weight of about 350,000 is called a silk fiber mouth heavy chain.

Silkworms spin liquid silk during cocoon production to make cocoons (consisting of cocoons and pupae). It is known that cocoon silk has silk fibrous mouth in the center and silk sericin around it, and the abundance ratio is 70-80 (fibrous mouth in): 20-30 (sericin). Raw silk is made by collecting several to several ten cocoons (reeling). The process of removing silk sericin from cocoon, raw silk or raw weave is called scouring, and the fiber after scouring is called silk thread or silk fiber mouth. The scouring does not always completely remove silk sericin. In 5-minute kneading and 7-minute kneading, half or about 70% of sericin may be removed. However, in this case, silk thread is used because it has undergone a scouring process. Raw weave refers to unrefined silk fabric. In general, silk refers to fiber mouth-in alone, sericin alone, or coexistence of fiber mouth-in and sericin at the same time.For silk fiber-in and sericin, any of film, powder, fiber, etc. It is also called silk for its form. Thus, silk fiber mouth and fibroin and silk sericin and sericin have the same meaning. Silk fibroin refers to silk fibroin film, silk fiber mouth powder, silk fiber mouth, etc.The silk fiber mouth is obtained by scouring cocoon thread, raw silk, raw woven fabric, or their residual yarn. Say fiber.

The conventional method for producing silk thread is to start with raw cocoons produced by sericulture farmers. Untreated cocoons) are dried cocoons, boiled cocoons, and reeled into raw silk to make raw weave. Next, the raw silk or raw weave is refined to obtain silk yarn or silk fabric. Debris generated in these processes is called residual yarn. Dry cocoons are prepared by gradually lowering the raw cocoons from a temperature of 115 to 12 O: to a temperature of about 80 ° C in 5 to 6 hours. Boiled cocoons are treated with steam and hot water at 100 to 105 ° C for about 10 minutes.

On the other hand, silk fiber mouths can also be obtained from liquid silk. For example, a gel-like content (liquid silk) is taken out from the middle and posterior glands of the silk gland of the silkworm and washed with an aqueous solution to remove a small amount of attached silk sericin. You can get five mouths. However, this method requires dissection of the silkworm, removal of the silk gland from the body of the silkworm, and removal of the silk fiber mouth from the silk gland cavity. The maximum amount of silk-fiber mouth obtained from one silkworm is about 0.4 g.Since it is easy to contain impurities such as silk fluid and silk gland cells, and it takes time to obtain silk fibroin, It is not an industrial production method.

In the present invention, an industrially advantageous raw cocoon, a cocoon layer or a cocoon thread of a dried or boiled cocoon, or a raw silk, a silk fabric, or a residual thread thereof is used as a material. Since silk fibroin is easily decomposed during storage, it is most desirable to use fresh raw cocoons as a raw material. Raw cocoons, dry cocoons, raw silk, silk threads, and their remaining threads are preferably stored at a temperature of room temperature or less to block light.

In the method of the present invention, first, these raw materials are scoured to remove sericin. Examples of the scouring means include urea aqueous solution treatment, alkali aqueous solution treatment, enzyme aqueous solution treatment, and high-pressure hot water treatment, and urea aqueous solution treatment is particularly preferable because it is mild. In any treatment, scouring is easier and faster as the raw material is separated.

The treatment with the urea aqueous solution is, for example, more than 30% ((weight (g) volume (not more than mlj, simply shown as%)) or more from the viewpoint of preventing decomposition of silk fiber mouth, reaction efficiency and industrial operability. Treat with an aqueous urea solution at 70 to 90 ° C for 60 to 180 minutes, and more preferably with an aqueous urea solution of 45% or more at 75 to 85 ° C and 90 to 150 minutes. Urea Mercaptoethanol or the like may be added to the aqueous solution.

The treatment in this case means that the raw material is immersed in an aqueous urea solution, and stirring may be performed during this.

The treatment with an alkaline aqueous solution is carried out, for example, with an alkaline aqueous solution having a pH of 10 to 12 by appropriately changing the conditions at the boiling temperature under atmospheric pressure and in the range of 2 to 60 minutes. If the pH is less than 10, scouring is not sufficient, and if the pH is more than 12, silk fibroin is rapidly decomposed and control becomes difficult. The preferred pH is from 10.5 to 11.5. Examples of the alkaline aqueous solution to be used include an aqueous solution of an alkaline sodium salt such as sodium carbonate, sodium hydrogen carbonate, sodium silicate, sodium metasilicate, sodium phosphate, and sodium hydroxide. The liquid is particularly preferable because it has an appropriate buffer effect.

In order to refine the silk without decomposing it, the pH, temperature, time, etc. during the refining can be controlled by appropriately changing the processing conditions. For example, temperatures above the boiling temperature (eg, 110 ° and 120 °) When treating with C), adjust the pH toward neutral or shorten the time. When processing at a temperature lower than the boiling temperature, change the conditions appropriately, such as increasing the pH and lengthening the time. Furthermore, when using other sodium salts of sodium carbonate, it is needless to say that conditions are appropriately changed in accordance with sodium carbonate. The treatment in this case means that the raw material is immersed in an aqueous alkaline solution, and stirring may be performed during this. Enzyme aqueous scouring is a method in which evening protein degrading enzyme is applied to the scouring of raw silk and cocoons. In the past, papain was often used, but in recent years Alcalase (Koshindo Chemical Industry) has been used. Pretreatment is required for refining the enzyme aqueous solution with alcalase, and the pretreatment is performed at pH 9 to 10, preferably at pH 9.0 to 9.6. This is performed within 60 minutes, preferably within 10 minutes. Thereafter, as the main treatment, alcalase is added and scouring is performed at 50 to 60 ° C. The scouring time is preferably within 60 minutes, particularly preferably within 20 minutes. In this case, it is preferable to separate the cocoon layer as much as possible. Also, stirring may be performed during this time. Silk scouring is usually performed at temperatures near the boiling point of water at atmospheric pressure. But,

Refining at a high temperature of 10 o ° c or more (also called high-pressure hot water treatment or high-pressure refining) is also possible. The solubility of silk sericin is greatly affected by the temperature of the water, for example, by soaking it in hot water for 30 to 100 minutes at 110 ° C, 20 to 60 minutes at 120 ° C, and 10 to 30 minutes at 130 ° C. Scouring can be performed without using a dissolving agent such as an alkali, and L chains that do not decompose can be obtained.

The fibroin fiber obtained by scouring is dissolved in a neutral salt aqueous solution and subjected to fractional precipitation.

Examples of the neutral salt include lithium thiocyanate, calcium chloride, lithium bromide, sodium thiocyanate, and the like. Although the concentration of the neutral salt varies depending on the type of the neutral salt, a saturated aqueous solution or a concentration of 50% saturation or more is preferable for any neutral salt. Particularly, a concentration of 80% saturated aqueous solution or more is preferable. In the case of lithium thiocyanate, the saturated aqueous solution is about 80%. The pH of these aqueous neutral salt solutions is 5-8.

For the fractional precipitation after the raw materials are dissolved, it is preferable to use acetone-alcohol, particularly ethanol, for crystallizing the amorphous silk fibroin. In this operation, for example, it is preferable to sequentially add ethanol and collect a precipitate. When the concentration of alcohol or acetone is low, the H chain selectively precipitates, and when the concentration is high, the L chain selectively precipitates.Therefore, the L chain is selectively precipitated by adjusting the concentration of these solvents. It can be done. For example, when ethanol is added to a calcium chloride aqueous solution for precipitation, when the ethanol concentration is less than 82.4%, mainly H chains are precipitated, and when the concentration exceeds 82.4%, mainly L chains are precipitated.

The silk-fiber-mouth L chain polypeptide thus obtained exhibits an excellent cell growth promoting action. Therefore, various cell culture additives (especially additives for animal cells including humans), cell culture beds, and wound healing promotion. It is useful as an agent or cosmetic (hereinafter, also referred to as an external preparation). When used as a wound healing promoter, a cell culture bed, or a cosmetic, it is preferable to use silk L chain as an external preparation applied to a wound site or skin in the form of a hide mouth gel, film, powder, or the like. In order to form a film, a silk-fiber mouth-in L chain aqueous solution may be poured on a flat plate and dried. To form a powder, this film is crushed, or the aqueous solution of silk fibroin L chain is sprayed in the air, or the aqueous solution of silk fiber mouth L chain is stirred or the precipitate obtained by adding alcohol to the aqueous solution of silk fiber mouth L chain is washed with water. It may be dried, crushed.

On the other hand, the silk-fiber mouth L chain can be used in the form of a usual external preparation, for example, an ointment, a cream, a cataplasm and the like. In order to prepare such ointments, creams, cataplasms and the like, a silk-fiber mouth L chain may be blended with a usual ointment base, a cream base, a cataplasm and the like.

The amount of the silk-fiber mouth L chain to be added to these external preparations varies depending on the dosage form and the base, but is 0.01 to 30 (weight / weight)%, particularly 0.1 to: L 0 (weight). / Wt)% is preferred. Example

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Function of promoting cell growth when coated on cell culture vessel)

A. Materials and methods

(1) Five mouth in

The cocoon layer of the day-crossed hybrid (13 7 x 14 6) was separated into 5 or 6 cocoons and separated well. Then, the mixture was treated with 30 times the volume (VZW) of a 5 M aqueous urea solution at 100 ° (:, 5 minutes) with good stirring to dissolve and remove the sericin. It was dried and used as a five-mouthed mouth (raw material).

(2) Preparation of sample solution for H-chain and L-chain To a mixture of 26 g of ethanol, 42 g of water, 32 g of calcium chloride and 1.2 g of tris (hydroxyl) aminoaminomethane, add 12.5 DIL of mercaptoethanol 2101, then add 70 to 80 Then, 523 mg of the cocoon layer, which had been de-sericined, was put thereinto and completely dissolved. Ethanol was added to this, and the added ethanol concentration was 0 to 71.8% (1), 71.9 to 72.5% (Π), 72.6 to 78% (ΙΠ), 78 .: obtained at ~ 82.3% (IV), 82.4-84.7% (V), 84.8-89.2% (VI), 89.3-91.7% (VE) The precipitates collected were collected and analyzed for their components. Figure 1 shows the electrophoresis (SDS-PAGE) images of these precipitates. From this image, the I and I fractions were designated as H chains, and the W fraction was designated as L chains. Fractions such as ΠΙ and IV were excluded because the H chain was degraded and the molecular weight was reduced. Next, the precipitates of these H and L chains were dissolved in 9M Li SCN, and dialyzed four times with 50 volumes of water for 30 minutes. Further, the amounts of these proteins were measured by the UV method, and diluted with water to adjust to 0.025% and 0.025%, respectively.

(3) Protein quantificationUV method

The absorbance at 275 mn of the protein solution was measured, and the protein concentration at an absorbance of 1.0 was calculated as 1 mg / mL.

(4) Coating a cell culture petri dish with a fibrous mouth in-polypeptide

Add 1 L of a 0.025% or 0.0025% aqueous solution of the fiber chain into a polystyrene dish (35ππηφ, Falcon), air-dry, and dry 3 times with 2 mL of PBS (phosphate buffered saline). After washing, it was air-dried again and immersed in 70% ethanol for sterilization. Water was added to the control petri dish without the fibrous opening in polypeptide for control, and air-dried instead of the fibrous opening in polypeptide, followed by the same treatment.

(5) Cell

The cells used were frozen human dermal fibroblasts (adult-derived) purchased from Kurabo Industries.

(6) Medium As the medium, a low blood blue medium for human skin fibroblast proliferation purchased from Kurabo Industries was used.

(7) Cell culture

In a cell culture experiment using a petri dish coated with fibroin polypeptide, about 100,000 cells were inoculated with 2 mL of medium per sheet.

(8) Viable cell count

Per dish, add 2 mL of medium and 0.1 mL of IWAK I at a ratio of 0.1 mL. After culturing at 37Ϊ: for 2 hours, calculate the amount of dye reduction calculated from the absorbance at 570 mn and 60 Onm. And

B. Results

After cell culture was performed for 4 days, the viable cell count of human dermal fibroblasts is shown in Table 1 assuming that the case without the addition of the fibrous mouth in polypeptide was 100%.

table 1

Growth of Human Dermal Fibroblasts in Petri Dishes Coated with Five Mouth In-Polypeptides

Viable cell count (%) Confidence rate of significant difference from control group Control group 100 ± 6.5 *

Additive-free)

H chain (25 wg / mL) 183 ± 23.5 99.9%

H chain (2.5 g / mL) 220 soil 30.5 99%

Light chain (25 g / mL) 218 ± 11.5 98%

L chain (2.5 z g / mL) 189 ± 16.8 98%

*:standard deviation

The growth rate of the H chain and the L chain of the Petri dish coated with the in-mouth polypeptide was superior to that of the control.

Example 2 (Cell growth promoting function when added to cell culture medium)

The experimental method is the same as Example 1 except for the following.

The experiment for adding the aqueous solution of the in-chain H chain and L chain to the medium was performed as follows. Ma Put 2niL of medium into a Ruchiwell plate (6 holes, Falcon), inoculate about 100,000 cells, and replace the medium with the aqueous solution (25 ^ gZmL) of the in-chain H chain and L chain one day later. The culture was continued for another 4 days. Table 2 shows the number of viable cells when the fibrous opening in polypeptide was added to the control group in which the number of viable cells when the fibrous opening in polypeptide was not added was 100%.

Table 2

Growth of human dermal fibroblasts in medium supplemented with ribeptide Viable cell count (%) Confidence rate of significant difference from control plot Control plot 100 soil 1.3 »

H chain (25 ^ g / niL) 108 ± 0.8 99%

Light chain (25 / mL) 11 is 1.8 99%

*:standard deviation

As shown in Table 2, the growth rate was better than that of the control when the fibrous mouth in polypeptide was added to the medium.

Example 3 (Cell growth promoting function when undegraded L chain is contained)

It is well known that a neutral salt solution is used as a solvent for silk protein. Among them, a saturated aqueous solution of Li SCN (lithium thiocyanate) dissolves silk protein without reducing the molecular weight of silk protein. However, Li SCN is expensive and commercially available at about 100 times the price of CaCl ₂ (calcium chloride) by weight. C aC 1 ₂ is dissolved well silk protein, it takes a heating tends to reduce the molecular weight of the silk protein. In particular, the molecular weight of the H chain is more likely to be reduced by heat, acid, alkali, light, etc. than the L chain. Therefore, as in Example 1, by dissolving the silk protein below 80 C, the recovery of H chains with C a C 1 ₂ can be. However, when CaCl ₂ is used, part of the H chain is degraded, but L chain is hardly degraded.

The Fuibu port in showing the C a C 1 ₂ (85) and L i SCN electrophoresis image of Fuibu port in the lysates was dissolved in (room temperature) in Fig. In Fig. 2, CaCl ₂ No H chain can be found in the dissolved fiber mouth. When dissolving the Fuiburoi down with CaC 1 _2, when performing a dissolution temperature of about 85 ° C, H chain mostly decompose, most of the L chain remain without being decomposed. Thus, L chains are more stable than H chains and are easier to handle.

CaC 1 ₂ When dissolving the Fuibu port in using, depending on the melting temperature, most of the H chain is decomposed to about an average molecular weight of 100,000, L chain as hardly decomposed to have a les ^ this An experiment similar to that of Example 1 was performed using the fiber mouth. As a result, the number of viable cells was 133%, and it was found that the presence of the light chain power S had a cell growth promoting action, and that the action could be used.

Example 4

Table 3 shows the results of preparing 0.1% and 0.5% aqueous solutions of the H chain and L chain obtained in Example 1, respectively, and observing the state of the solution at room temperature.

Table 3

1 after 7 after 14

H chain (0.1%) Slightly viscous Loose gel Hard gel

(0.5) Loose gel Hard gel Hard gel

L chain (0.1%) Liquid Liquid Liquid

(0.5%) Liquid Liquid Slightly viscous

As can be seen in Table 3, the H chain aqueous solution started gelling after 1 day at a concentration of 0.5% and completely gelled after 7 days. Even at 0.1%, the viscosity increased as a sign of gelation after 1 day, a loose gel was formed after 7 days, and completely gelled after 14 days.

On the other hand, the L chain aqueous solution was completely liquid until 7 days even at a concentration of 0.5%, and the viscosity slightly increased after 14 days. It was stable for more than 14 days at a concentration of 0.1%. Industrial applicability

INDUSTRIAL APPLICABILITY The silk fiber mouth L chain of the present invention has an excellent cell growth promoting action and high stability, and is therefore useful as a medicine for promoting wound healing and a cosmetic for skin care.

Claims

The scope of the claims

1. Refining the cocoon layer or cocoon thread of raw cocoon, dried cocoon or cooked cocoon or cocoon thread, silk thread or silk thread or the remaining thread, dissolve the obtained fibroin fiber in neutral salt aqueous solution, and then separate and sediment. A method for producing silk fibroin L-chain polypeptide, characterized by the following features.

2. The production method according to claim 1, wherein the scouring is a treatment selected from a urea aqueous solution treatment, an alkaline aqueous solution treatment, an enzyme aqueous solution treatment, and a high-pressure hot water treatment.

3. The method according to claim 1, wherein the aqueous neutral salt solution is an aqueous solution selected from an aqueous solution of calcium chloride, an aqueous solution of lithium thiocyanate, an aqueous solution of lithium bromide, and an aqueous solution of sodium thiocyanate.

4. The production method according to claim 1, wherein the fractional precipitation is caused by a change in the concentration of a solvent selected from alcohol and acetone.

5. A cell growth promoter comprising a silk-fiber mouth in L chain polypeptide as an active ingredient.

6. Wound healing promoter containing silk fiproin L chain polypeptide as an active ingredient.

7. Cell culture bed containing silk-fiber in L chain polypeptide.

8. A cosmetic containing a silk-fiber mouth in L chain polypeptide.

9. Use of silk fibroin L chain polypeptide for producing a cell growth promoter or a wound healing promoter.

10. A method for promoting wound healing, which comprises administering a silk-fiber mouth in L chain polypeptide.