JP2022160015A - Adhesive for synthetic leather, and synthetic leather and production method thereof - Google Patents

Abstract

An object of the present invention is to provide a biodegradable synthetic leather adhesive.
A synthetic leather adhesive containing spider silk fibroin.
[Selection figure] None

Description

TECHNICAL FIELD The present invention relates to an adhesive for synthetic leather, synthetic leather, and a method for producing the same.

BACKGROUND ART Conventionally, synthetic leather such as artificial leather has been used as a substitute for natural leather. Unlike natural leather, which is limited in supply, synthetic leather can be artificially mass-produced. It has come into use.

For example, Patent Document 1 discloses a method for producing artificial leather by impregnating a non-woven fabric pretreated with a sizing macromolecular substance with a polyurethane resin solution, wet-coagulating the non-woven fabric, and then removing the sizing macromolecular substance. into a mixed emulsion of reactive methyl H silicon and a metal catalyst, followed by heating at 100-150°C.

JP-A-10-131059

In recent years, due to the heightened awareness of the environment, synthetic leather is also required to reduce the environmental load. Synthetic resins such as polyvinyl alcohol (PVA), which are generally used as adhesives for synthetic leather, are not biodegradable and have a large environmental impact.

Accordingly, an object of the present invention is to provide a biodegradable adhesive for synthetic leather.

The present invention provides each of the following inventions.
[1] An adhesive for synthetic leather containing spider silk fibroin.
[2] The adhesive for synthetic leather according to [1], which further contains an organic solvent.
[3] The adhesive for synthetic leather according to [2], wherein the organic solvent is formic acid.
[4] The synthetic leather adhesive according to any one of [1] to [3], further comprising glycerol.
[5] A synthetic leather comprising a skin layer, a base material layer containing a fiber base material, and an adhesive layer for bonding the skin layer and the base material layer to each other, wherein the adhesive layer contains spider silk fibroin.
[6] The synthetic leather according to [5], wherein the adhesive layer further contains glycerol.
[7] A method for producing a synthetic leather, wherein the skin layer, the base layer containing the fiber base material, and the skin layer and the base layer are adhered to each other, wherein the skin layer contains spider silk fibroin A method comprising forming an adhesive layer and adhering a skin layer and a substrate layer via the adhesive layer.
[8] The method according to [7], wherein at least one of the base layer and the skin layer contains a biodegradable material.
[9] The method of [8], wherein at least one of the base layer and the skin layer contains spider silk fibroin.

According to the present invention, a biodegradable synthetic leather adhesive can be provided.

1 is a schematic cross-sectional view of a synthetic leather according to one embodiment; FIG. BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram of the manufacturing method of the synthetic leather which concerns on one Embodiment. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified spider silk fibroin. FIG. 4 is a diagram showing the distribution of z/w (%) values of naturally occurring fibroin. FIG. 4 is a diagram showing the distribution of x/y (%) values of naturally occurring fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified spider silk fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified spider silk fibroin. 1 is a photograph showing a synthetic leather produced in an example.

DETAILED DESCRIPTION OF THE INVENTION Embodiments for carrying out the present invention will be described in detail below. The present invention is not limited to the following embodiments.

<Adhesive for synthetic leather>
The synthetic leather adhesive according to the present embodiment contains spider silk fibroin. Since the adhesive for synthetic leather according to the present embodiment contains the biodegradable spider silk fibroin as an active ingredient, it is possible to provide synthetic leather with reduced environmental load. According to the synthetic leather adhesive according to the present embodiment, for example, in the synthetic leather adhesive comprising a base material layer containing a fiber base material such as woven fabric, knitted fabric, or non-woven fabric and a skin layer, the base layer and The skin layers can be well adhered to each other.

Synthetic leather has the appearance and functions of natural leather, and in this specification, synthetic leather includes so-called artificial leather (a special non-woven fabric (a fiber layer having a random three-dimensional three-dimensional structure as a base material) The base material is impregnated with polyurethane or similar flexible polymeric material). That is, the synthetic leather adhesive according to the present embodiment is also suitably used for adhering a base material layer containing a special nonwoven fabric and a skin layer.

The content of spider silk fibroin in the synthetic leather adhesive according to the present embodiment is 1 to 90% by mass, 1 to 80% by mass, 1 to 70% by mass, based on the total mass of the synthetic leather adhesive. 1-60% by mass, 1-50% by mass, 1-40% by mass, 1-30% by mass, 1-20% by mass, 1-10% by mass, 1-5% by mass, 2-20% by mass, 2- 15% by weight, 2 to 10% by weight, 2 to 8% by weight, 2 to 5% by weight, 3 to 15% by weight, 3 to 12% by weight, 3 to 10% by weight, or 4 to 8% by weight good.

The content of spider silk fibroin is 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, based on the total mass of the synthetic leather adhesive. , 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass or more, or 15% by mass or more, and 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less , 20% by mass or less, 18% by mass or less, 15% by mass or less, 12% by mass or less, 10% by mass or less, 9% by mass or less, 8% by mass or less, 7% by mass or less, 6% by mass or less, or 5% by mass It may be below.

When the skin layer contains spider silk fibroin, the content of spider silk fibroin in the adhesive for synthetic leather is preferably less than or equal to the spider silk fibroin content in the skin layer.

[Liquid medium]
The synthetic leather adhesive according to the present embodiment may further contain a liquid medium. When a liquid medium is included, the spider silk fibroin may be dissolved or dispersed in the liquid medium in the adhesive for synthetic leather. The liquid medium may be water, organic solvents or combinations thereof. As used herein, "organic solvent" means an organic compound that is liquid at 25°C and has a boiling point of less than 250°C at atmospheric pressure.

The type of organic solvent can be appropriately selected according to the type and concentration of spider silk fibroin. That is, the adhesive for synthetic leather according to one embodiment may contain spider silk fibroin and an organic solvent in which the spider silk fibroin is dissolved or dispersed.

Organic solvents include, for example, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid and alcohols. The alcohol may be, for example, a lower alcohol having 1-5 carbon atoms. Lower alcohols having 1 to 5 carbon atoms include, for example, methanol, ethanol and 2-propanol. The alcohol may be an alcohol in which at least part of the hydrogen atoms are substituted with halogen atoms such as fluorine atoms, such as hexafluoroisopropanol (HFIP).

The content of the liquid medium in the synthetic leather adhesive according to the present embodiment is, based on the total mass of the synthetic leather adhesive, 10% by mass or more, 30% by mass, 50% by mass or more, 60% by mass or more, or It may be 70% by mass or more, and may be 95% by mass or less, 90% by mass or less, 85% by mass or less, or 80% by mass or less.

[Plasticizer]
The synthetic leather adhesive according to the present embodiment may further contain a plasticizer. Examples of plasticizers include polyhydric alcohols and carboxyl-terminated oligo-alkylene glycols (carboxyl-terminated poly(ethylene glycol), etc.).

Examples of polyhydric alcohols include polyalkylene glycols such as glycerol and polyethylene glycol (PEG). The polyalkylene glycol preferably has a number average molecular weight of 200-600. Polyethylene glycol 400, for example, can be used as the polyalkylene glycol having a number average molecular weight of 200-600. The number average molecular weight is a polystyrene-equivalent value obtained by gel permeation chromatography (GPC) using a standard polystyrene calibration curve.

The content of the plasticizer is 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, based on the total mass of the synthetic leather adhesive. 8% by mass or more, 9% by mass or more, 10% by mass or more, or 15% by mass or more, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20 % by mass or less, 18% by mass or less, 15% by mass or less, 12% by mass or less, 10% by mass or less, 9% by mass or less, 8% by mass or less, 7% by mass or less, 6% by mass or less, or 5% by mass or less There may be.

In the adhesive for synthetic leather, the ratio of the mass of the plasticizer to the mass of spider silk fibroin (plasticizer/spider silk fibroin) is 0.2 or more, 0.25 or more, 0.3 or more, 0.4 or more, 0 .5 or more, 0.8 or more, 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1 .8 or greater, 1.9 or greater, 2.0 or greater, 2.5 or greater, 3.0 or greater, 3.5 or greater, 4.0 or greater, or 4.5 or greater, 4.5 or greater, 4 .0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less, 1.9 or less, 1.8 or less, 1.7 or less, or 1.6 or less.

[Other additives]
The adhesive for synthetic leather may further contain other additives (except those corresponding to spider silk fibroin, liquid medium and plasticizer). Other additives include, for example, dissolution accelerators such as inorganic salts, coloring agents, smoothing agents, antioxidants, ultraviolet absorbers, dyes, fillers, cross-linking agents, matting agents, leveling agents, flame retardants, foaming agents, agents.

Other additives can be blended in an appropriate amount depending on the type or concentration of spider silk fibroin. The total content of other additives is 0.1% by mass or more, 1% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or It may be 15% by mass or more, and may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less. The total content of other additives is the total content of components other than spider silk fibroin, liquid medium and plasticizer in the adhesive for synthetic leather.

<Synthetic leather>
The synthetic leather according to this embodiment includes a base material layer containing a fiber base material, a skin layer, and an adhesive layer that bonds the base material layer and the skin layer to each other. The adhesive layer contains spider silk fibroin. In the synthetic leather according to the present embodiment, since the synthetic leather is adhered via the adhesive layer containing the biodegradable spider silk fibroin, the environmental load is reduced.

FIG. 1 is a schematic cross-sectional view of a synthetic leather according to one embodiment. In the synthetic leather 20 shown in FIG. 1, the base material layer 2 and the skin layer 1 are adhered via the adhesive layer 3 . In the synthetic leather 20 shown in FIG. 1, the skin layer 1 also functions as a shape retaining layer for retaining the shape of the synthetic leather.

At least one of the base layer and the skin layer may contain a biodegradable material, and both the base layer and the skin layer may contain a biodegradable material. Biodegradable materials are materials that are completely consumed by microorganisms and produce only natural by-products (carbon dioxide, methane, water, biomass, etc.). A biodegradable material may be, for example, a biodegradable polymer. Examples of biodegradable materials include plant-derived polysaccharides such as cellulose and starch; animal-derived polysaccharides such as chitin and hyaluronic acid; and proteins. The protein may be, for example, an animal-derived protein such as collagen, albuminic acid, or spider silk fibroin.

[Adhesive layer]
The adhesive layer contains spider silk fibroin. The adhesive layer can be formed, for example, from the synthetic leather adhesive described above.

The adhesive layer may consist only of spider silk fibroin, or may contain components other than spider silk fibroin. The content of spider silk fibroin in the adhesive layer may be, for example, 10% by mass or more, 20% by mass or more, 30% by mass or more, or 35% by mass or more, based on the total mass of the adhesive layer, and 90% by mass. % or less, 80 mass % or less, 70 mass % or less, 60 mass % or less, 50 mass % or less, or 45 mass % or less.

Components other than spider silk fibroin include, for example, the components described above as plasticizers and additives. The adhesive layer preferably contains a plasticizer, and more preferably contains a polyhydric alcohol.

The content of the plasticizer in the adhesive layer may be, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, or 55% by mass or more, based on the total mass of the adhesive layer, and 90% by mass. 80% by mass or less, 70% by mass or less, or 65% by mass or less.

The ratio of the mass of plasticizer to the mass of spider silk fibroin (plasticizer/spider silk fibroin) in the adhesive layer is 0.2 or more, 0.25 or more, 0.3 or more, 0.4 or more, 0.5 0.8 or more, 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 1.9 or more, 2.0 or more, 2.5 or more, 3.0 or more, 3.5 or more, 4.0 or more, or 4.5 or more, 4.5 or less, 4.0 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less, 1.9 or less, 1.8 or less, 1.7 or less, or 1.6 or less.

The adhesive layer can be formed, for example, by drying a coating film of the adhesive for synthetic leather described above, if necessary. The drying temperature may be, for example, 90°C to 150°C. Drying times may be, for example, from 5 minutes to 24 hours.

The thickness of the adhesive layer is not particularly limited, but may be, for example, 1 to 50 μm, 1 to 40 μm, 5 to 30 μm, or 5 to 20 μm.

[Base material layer]
The substrate layer includes a fibrous substrate. The base material layer may contain other components as long as the function as the base material layer is not impaired. For example, the base material layer may contain a fiber base material and a polymer substance. .

The substrate layer may be a single layer, or may have a multilayer structure composed of a plurality of layers of fiber substrates. The thickness of the base material layer can be arbitrarily selected depending on the use of the obtained synthetic leather, and is not particularly limited, but from the viewpoint of the balance between the adhesive layer and the skin layer, for example, 100 to 2000 μm. Well, it can be 100-1000 μm, it can be 500-1000 μm, it can be 500-750 μm.

The substrate layer may be raised on one side or both sides. Raised fabric may be raised on one side or raised on both sides. From the viewpoint of adhesiveness to the adjacent adhesive layer, it is preferable that at least the surface on the side in contact with the adhesive layer is raised.

(fiber base material)
As the fiber base material, for example, a sheet having a suitable thickness and a feeling of fullness and having a soft texture can be used. As the fiber base material, for example, various fiber base materials used in conventional methods for producing leather-like sheets can be used as they are.

The fibrous substrate may be, for example, a woven or non-woven fabric. Knitted or nonwoven fabrics as fiber base materials include synthetic fibers such as polyester, polyamide, polyacrylonitrile, polyolefin and polyvinyl alcohol, natural fibers such as cotton and hemp, regenerated fibers such as rayon, staple fiber, acetate and collagen, artificial casein, It consists of single or blended fibers such as artificial protein fibers such as collagen and fibroin, or multicomponent fibers denatured to ultrafine fibers by dissolving at least one component or splitting bicomponent fibers. good.

The fiber base material preferably contains protein from the viewpoint of biodegradability, hygroscopicity and strength. The protein may be a structural protein, preferably fibroin, more preferably spider silk fibroin. By including fibroin (preferably, spider silk fibroin), it is possible to impart the functionality of heat retention, hygroscopic heat generation and/or flame retardancy to the fiber base material. As a result, the synthetic leather according to the present embodiment can be imparted with the functionality of heat retention, moisture absorption heat generation and/or flame retardancy, and the value as a material is further increased. Proteins may be included in the fiber base material as protein fibers. Preferred embodiments of modified fibroin are described below.

A knitted fabric is a general term for knitted fabrics and woven fabrics. Knitted fabrics include knitted fabrics with weft knitting structures such as weft knitting and circular knitting (also referred to simply as "weft knitted fabrics"), knitted fabrics with warp knitting structures such as tricot and raschel (simply referred to as "warp knitted fabrics"). ) can be either. The fabric may be a fabric having any of a plain weave, a twill weave, or a satin weave. The knitted or woven body may be an unprocessed knitted or woven body itself obtained by knitting or weaving, or may be a knitted or woven body that has undergone a water-repellent finish or the like after knitting or weaving.

A knitted or woven body can be obtained by knitting or weaving raw material yarns. Known methods can be used as the knitting method and the weaving method. As the knitting machine to be used, for example, a circular knitting machine, a warp knitting machine, a flat knitting machine, etc. can be used, and from the viewpoint of productivity, use of a circular knitting machine is preferable. The flat knitting machine includes a forming knitting machine, a seamless knitting machine, and the like, but the use of a seamless knitting machine is more preferable because it can produce a knitted fabric in the form of a final product. The looms used include shuttle looms and shuttleless looms such as gripper looms, rapier looms and air jet looms.

The raw yarn may be a single yarn, a composite yarn (for example, a blended yarn, a mixed yarn, a covering yarn, etc.), or a combination thereof. The single yarn and composite yarn may be a spun yarn obtained by twisting short fibers, or a filament yarn obtained by twisting long fibers. Examples of fibers contained in the raw yarn include synthetic fibers such as nylon, polyester, polyamide, polyacrylonitrile, polyolefin, polyvinyl alcohol, polyethylene terephthalate and polytetrafluoroethylene; regenerated fibers such as cupra, rayon and lyocell; and natural fibers such as silk, and protein fibers.

A nonwoven fabric can be manufactured by a well-known manufacturing method using the fiber mentioned above, for example. Specifically, for example, after forming a web (including a single-layer web and a laminated web) from the above-described fibers by a dry method, a wet method, an airlaid method, etc., a chemical bond method (immersion method, spray method) etc.) and by needle punching or the like to bond the fibers of the web to obtain a nonwoven fabric.

Protein-containing nonwoven fabrics, for example, proteins are dissolved in solvents such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP) as a dissolution accelerator, if necessary. It can also be obtained by adding it together with an inorganic salt of and dissolving it to prepare a dope solution, and then spinning it by an electrospinning method (electrospinning method) using the dope solution. In the electrospinning method (electrostatic spinning method), a voltage is applied between a supply side electrode (which can also be used as a spinneret) and a collection side electrode (for example, a metal roll or a metal net), and the dope liquid is extruded from the spinneret. is given an electric charge and blown away to the collection side electrode. At this time, the dope is stretched to form fibers. The applied voltage is usually 5-100 kV, preferably 10-50 kV. The distance between the electrodes is usually 1-25 cm, preferably 2-20 cm.

Numerical ranges of the fiber density (basis weight), porosity, bulk density, etc. of the non-woven fabric are appropriately set so that, for example, waterproofness and moisture permeability can be sufficiently ensured. The basis weight, porosity, bulk density, etc. can be adjusted, for example, by increasing or decreasing the amount of fibers constituting the web, or by increasing or decreasing the number of laminations in the case of a laminated web.

(Polymer substance)
The base layer may further contain polymeric substances. The molecular substance may be present in the interstices between the fibers of the fiber substrate in a state of being fixed to the fibers. The polymeric substance is obtained by, for example, impregnating the fiber base material with an impregnating liquid containing the polymeric substance and a medium in which the solvent is dissolved or dispersed, and then solidifying (solidifying) the fiber base material by removing the solvent. It can be attached to the material. The polymeric substance may constitute the base layer of the synthetic leather together with the fiber base material as a so-called impregnated body integrated with the fiber base material. By using a polymer substance, the fibers of the fiber base material are connected to each other, making it easier to maintain the shape of the base material layer and to easily impart a predetermined strength to the base material layer.

As the polymeric substance, for example, resins conventionally used for manufacturing leather-like sheets can also be used. Resins as polymeric substances include, for example, polyurethane-based resins, polyvinyl chloride-based resins, polyvinyl butyral-based resins, polyacrylic acid-based resins, polyamino acid-based resins, silicone-based resins, and mixtures of these resins. The resin may be a polymer obtained by polymerizing one type of monomer, or a copolymer obtained by polymerizing two or more types of monomers.

The polymer substance preferably contains protein from the viewpoint of biodegradability, hygroscopicity and strength. The protein may be a structural protein, preferably fibroin, more preferably spider silk fibroin. By containing fibroin (preferably, spider silk fibroin), it is possible to impart the functionality of heat retention, moisture absorption heat generation and/or flame retardancy to the substrate layer. As a result, the synthetic leather according to the present embodiment can be imparted with the functionality of heat retention, moisture absorption heat generation and/or flame retardancy, and the value as a material is further increased. Preferred embodiments of fibroin are described below.

[Skin layer]
A skin layer is a layer mainly formed of polymers. The skin layer may be a porous layer made of a porous resin, or a non-porous layer made of a non-porous resin.

Polymers forming the skin layer include, for example, synthetic resins such as polyvinyl chloride, polyolefin, polystyrene, polyurethane, polyester resin, epoxy resin, acrylic polymer, acrylonitrile polymer, and protein. A composite material of synthetic resin and protein may be used as the polymer forming the skin layer. Examples of polyurethane include polyether-based polyurethane, polyester-based polyurethane, and polycarbonate-based polyurethane, and these may be used alone or in combination of two or more.

The epidermal layer preferably contains protein. The protein preferably contains collagen, casein and fibroin, more preferably spider silk fibroin.

The skin layer may contain known additives as necessary. Additives include, for example, colorants (e.g., dyes, pigments), smoothing agents, antioxidants, ultraviolet absorbers, fillers, cross-linking agents, plasticizers, matting agents, leveling agents, flame retardants, foaming agents, etc. is mentioned.

The thickness of the skin layer is not particularly limited, but may be, for example, 1 to 500 μm, 1 to 400 μm, 5 to 300 μm, or 50 to 200 μm.

The surface of the skin layer (the surface opposite to the surface in contact with the adhesive layer) may be surface-treated. Surface processing can apply a well-known method. Examples of surface processing include embossing, buff suede processing, film lamination processing, and gravure processing (gravure printing).

<Manufacturing method of synthetic leather>
The synthetic leather according to the present embodiment includes, for example, a step of forming an adhesive layer containing spider silk fibroin on the skin layer (adhesive layer forming step), and the skin layer and the base layer through the adhesive layer. and a step of adhering (adhesion step). The skin layer, the adhesive layer and the base material layer can be applied in the same manner as described above.

The synthetic leather manufacturing method according to the present embodiment may include a step of forming a skin layer on the release paper (skin layer forming step) before the adhesive layer forming step. When the skin layer forming step is provided, the adhesive layer is formed on the surface of the skin layer opposite to the surface in contact with the release liner in the adhesive layer forming step.

Hereinafter, as an example of the synthetic leather manufacturing method according to the present embodiment, a manufacturing method including a skin layer forming step, an adhesive layer forming step, and a bonding step will be described.

FIG. 2 is a schematic diagram showing an example of a production apparatus for producing the synthetic leather shown in FIG. In the manufacturing method using the manufacturing apparatus 900 shown in FIG. Dry with Thereby, a skin layer is formed on the release paper 91 . Next, an adhesive 94 is applied to the surface of the skin layer (the surface opposite to the surface in contact with the release paper 91 ) and dried with a dryer 93 . Thereby, a laminate having the release paper, the skin layer and the adhesive layer in this order is obtained. Then, the base material layer 96 is pulled out from the roll on which the base material layer 96 is wound, superimposed on the surface of the adhesive layer (the surface opposite to the surface in contact with the skin layer), and thermally pressed by the hot press roll 95 . The synthetic leather can be manufactured by winding the synthetic leather 97 in which the substrate layer, the adhesive layer, the skin layer, and the release paper are laminated in this order on a roll.

(Uses of synthetic leather)
The synthetic leather according to the present embodiment can be used, for example, for the same applications as conventional synthetic leather (for example, synthetic leather made of synthetic resin). The synthetic leather according to the present embodiment can be used for applications such as clothing, accessories such as shoes and bags, various covers and furniture, and automotive interior materials.

<Spider silk fibroin>
As used herein, "spider silk fibroin" is modified spider silk fibroin or naturally occurring spider silk fibroin.

The modified spider silk fibroin according to this embodiment has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The modified spider silk fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeated amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

As used herein, “modified spider silk fibroin” means artificially produced spider silk fibroin (artificial spider silk fibroin). The modified spider silk fibroin may be a spider silk fibroin whose domain sequence differs from the amino acid sequence of the naturally occurring spider silk fibroin, or is a spider silk fibroin whose domain sequence is identical to the amino acid sequence of the naturally occurring spider silk fibroin. may As used herein, "naturally occurring spider silk fibroin" also refers to Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif is a protein containing a domain sequence represented by

"Modified spider silk fibroin" may be one that uses the amino acid sequence of naturally occurring spider silk fibroin as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally occurring spider silk fibroin (e.g., , modified amino acid sequence by modifying the gene sequence of cloned naturally occurring spider silk fibroin), or artificially designed and synthesized without relying on naturally occurring spider silk fibroin (e.g. , having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

As used herein, the term “domain sequence” refers to a crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically, the amino acid sequence REP) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an amino acid sequence that Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in the _n motif may be an integer of 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16 . In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

The modified spider silk fibroin according to the present embodiment is obtained by, for example, replacing, deleting, inserting and/or adding one or more amino acid residues to the gene sequence of the cloned naturally-derived spider silk fibroin. It can be obtained by modifying the corresponding amino acid sequence. Substitution, deletion, insertion and/or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

Naturally occurring spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif It is a protein, and specific examples thereof include fibroin produced by spiders.

Fibroin produced by spiders includes, for example, spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as Araneus spiders, Araneus spiders, red spiders, green spiders, and bean spiders, spiders belonging to the genus Neoscona, such as Araneus spiders, house spiders, loch spiders, and Satsumanomidamashi. Spiders belonging to the genus Pronus, such as the genus Pronus, spiders belonging to the genus Cyrtarachne, such as Torinofundami and the genus Cyrtarachne, and genus Gasteracantha, such as spiny spiders and spiny spiders Spiders belonging to the genus Ordgarius such as spiders belonging to, Ordgarius spiders, and spiders belonging to the genus Ordgarius, spiders belonging to the genus Argiope, such as the argiope spider, the argiope spider, and the argiope spider spiders belonging to the genus), spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders belonging to the genus Cytophora, spiders belonging to the genus Poltys, such as Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the genus Cyclosa, such as the brown spider, the genus Cyclosa, and the genus Chorizopes, such as the brown spider, and the spider silk protein produced by the spider, Spiders belonging to the genus Tetragnatha, such as the genus Tetragnatha, spiders belonging to the genus Leucauge, such as spiders, spiders, and spiders belonging to the genus Leucauge, spiders belonging to the genus Nephila, such as Nephila Spiders belonging to the genus Menosira, such as spiders belonging to the family, spiders belonging to the genus Menosira, spiders belonging to the genus Dyschiriognatha, such as the lesser paper spider, widow spiders, such as black widow spiders, redback spiders, gray widow spiders, and large widow spiders (Latrodectus genus) Spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops and spiders belonging to the genus Euprosthenops be done. Spider silk proteins include, for example, dragline proteins such as MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag, and the like.

More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (derived from Nephila clavata) No. AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidropin 2 [Euprosthenops australispin] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys crentata] (GenBank Accession and Section No. ABR37278.1 (amino acid sequence).

A more specific example of naturally occurring spider silk fibroin is spider silk fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION from among sequences containing INV as DIVISION. It can be confirmed by extracting the specific product character string from the sequence, CDS, and the sequence described with the specific character string from SOURCE to TISSUE TYPE.

Specific examples of modified spider silk fibroin include modified spider silk fibroin (first modified spider silk fibroin) derived from dragline silk protein produced in the major pituitary gland of spiders, content of glycine residues (A) modified spider silk fibroin having a domain sequence with reduced content of _n motifs (third modified spider silk fibroin); ), the content of glycine residues, and (A) a modified spider silk fibroin with reduced content of _n motifs (fourth modified spider silk fibroin), a domain sequence containing a region with a locally high hydrophobicity index. and a modified spider silk fibroin with a reduced content of glutamine residues (sixth modified spider silk fibroin).

A first modified spider silk fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the first modified spider silk fibroin, the number of amino acid residues in the (A) _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and 10 to 20. is even more preferred, integers from 4 to 16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the first modified spider silk fibroin, in Formula 1, the number of amino acid residues constituting REP is preferably 10 to 200 residues, more preferably 10 to 150 residues, 20 to 100 more preferably 20-75 residues, even more preferably 20-75 residues. The first modified spider silk fibroin has the total number of amino acid residues of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number of residues.

The first modified spider silk fibroin comprises an amino acid sequence unit represented by the formula 1: [(A) _n motif-REP] _m , and the amino acid whose C-terminal sequence is shown in any of SEQ ID NOs: 1 to 3 It may be a polypeptide that is an amino acid sequence that has 90% or more homology with any of the sequences or the amino acid sequences shown in SEQ ID NOs: 1-3.

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 has the sequence It is identical to the amino acid sequence shown in No. 1 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 1. Identical to the amino acid sequence.

As a more specific example of the first modified spider silk fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1) or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 Modified spider silk fibroins can include amino acid sequences having greater than 90% sequence identity with. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 4 is an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:4 is identical to the amino acid sequence shown by SEQ ID NO:3.

The modified spider silk fibroin (1-i) may consist of the amino acid sequence shown in SEQ ID NO:4.

The second modified spider silk fibroin has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to the naturally-derived spider silk fibroin. The second modified spider silk fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced with another amino acid residue, compared with the naturally occurring spider silk fibroin. It can be said that

The second modified spider silk fibroin has a domain sequence of GGX and GPGXX (where G is a glycine residue, P is a proline residue, and X is a non-glycine residue) in REP compared to naturally occurring spider silk fibroin. In at least one motif sequence selected from ), an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence being replaced with another amino acid residue may have.

In the second modified spider silk fibroin, the percentage of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more of the entire motif sequence.

The second modified spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence, the (A) _n motif located most C-terminally to the above Let z be the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence, and the above z/w is 30%, where w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side of the domain sequence to the C-terminus of the domain sequence. It may have an amino acid sequence that is 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

The second modified spider silk fibroin preferably has an increased content of the amino acid sequence consisting of XGX by substituting one glycine residue in the GGX motif with another amino acid residue. In the second modified spider silk fibroin, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, and even more preferably 10% or less. , is even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z/w) of the amino acid sequence consisting of XGX below.

A method for calculating z/w will be described in more detail. First, in spider silk fibroin (modified spider silk fibroin or naturally occurring spider silk fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , from the domain sequence to the most C-terminal side An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the located (A) _n motif to the C-terminus of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 3, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

The z/w in naturally occurring fibroins, including spider silk fibroin and other fibroins, will now be described. First, as described above, when fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (among them, 415 types of fibroin derived from arachnids (spider silk fibroin)). was extracted. Naturally derived fibroin containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m and having an amino acid sequence consisting of GGX in fibroin content of 6% or less among all the extracted fibroin z/w was calculated from the amino acid sequence of fibroin in the above-described calculation method. The results are shown in FIG. The horizontal axis of FIG. 4 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 4, the z/w for naturally-occurring fibroin is all less than 50.9% (the highest is 50.86%).

In the second modified spider silk fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, and 70 % or more, and even more preferably 80% or more. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

The second modified spider silk fibroin, for example, replaces at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally occurring spider silk fibroin gene sequence to encode another amino acid residue. It can be obtained by modification. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring spider silk fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring spider silk fibroin, one or more amino acid residues are further substituted, Amino acid sequence alterations corresponding to deletions, insertions and/or additions may be made.

The other amino acid residue mentioned above is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( Hydrophobic amino acid residues such as M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, glutamine (Q) residues, asparagine (N) residues, serine (S ) residues, lysine (K) residues and glutamic acid (E) residues are preferred, and hydrophilic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

As more specific examples of the second modified spider silk fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 Modified spider silk fibroin can be mentioned, including:

The modified spider silk fibroin (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring spider silk fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and a His tag sequence are added to the C-terminus of a sequence in which is repeated four times.

The z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%. The z/w values in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x/y in the jagged ratio (described later) of 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8% respectively.

The modified spider silk fibroin (2-i) may consist of the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

The modified spider silk fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified spider silk fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-ii) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is included in REP When the total number of amino acid residues in an amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) is z, and the total number of amino acid residues of REP in the domain sequence is w, then z /w is preferably 50.9% or more.

The second modified spider silk fibroin may contain a tag sequence at either or both the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of the modified spider silk fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity with other molecules. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has the property of specifically binding to metal ions such as nickel. It can be used for isolation. A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence).

Also, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

Furthermore, an "epitope tag" using an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag, and the like. By using an epitope tag, the modified spider silk fibroin can be easily purified with high specificity.

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. The modified spider silk fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

As more specific examples of modified spider silk fibroin containing a tag sequence, (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, modified spider silk fibroin can be mentioned.

The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) was added to the N-terminus of the amino acid sequence shown.

The modified spider silk fibroin (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

(2-iv) modified spider silk fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. (2-iv) modified spider silk fibroin is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is included in REP When the total number of amino acid residues in an amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) is z, and the total number of amino acid residues of REP in the domain sequence is w, then z /w is preferably 50.9% or more.

The second modified spider silk fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A third modified spider silk fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) _n motifs compared to naturally-derived spider silk fibroin. The domain sequence of the third modified spider silk fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) _n motifs compared to the naturally occurring spider silk fibroin. .

A third modified spider silk fibroin may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from naturally occurring spider silk fibroin.

A third modified spider silk fibroin has a domain sequence at least one every 1-3 (A) _n motifs from the N-terminal side to the C-terminal side compared to the naturally-derived spider silk fibroin ( A) It may have an amino acid sequence corresponding to deletion of the _n motif.

The third modified spider silk fibroin has at least two consecutive (A) _n motif deletions from the N-terminal side to the C-terminal side compared to the naturally-derived spider silk fibroin, and It may have an amino acid sequence corresponding to deletion of one (A) _n motif repeated in this order.

The third modified spider silk fibroin has an amino acid sequence corresponding to deletion of at least every two (A) _n motifs from the N-terminal side to the C-terminal side of the domain sequence, good too.

A third modified spider silk fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) The number of amino acid residues of the REP of the _n motif-REP] unit is sequentially compared, and when the number of amino acid residues of the REP with a smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1. Let x be the maximum sum of the sums of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units of 8 to 11.3, and y be the total number of amino acid residues in the domain sequence. It may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 3 shows the domain sequences of the modified spider silk fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called n motif.

Two adjacent [(A) _n -motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 3, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers is 100/50=2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 3, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 for the one with the smaller number of amino acid residues. It is indicated by a solid line. This ratio is referred to herein as the serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 3, the total value of pattern 1 is the largest.

Then x/y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third modified spider silk fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and preferably 70% or more. Even more preferably, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 1.9 to 4.1, x/y is preferably 64.2% or more.

When the third modified spider silk fibroin is a modified spider silk fibroin in which at least 7 of the (A) _n motifs present in the domain sequence are composed only of alanine residues, x/y is 46.4%. is preferably at least 50%, more preferably at least 55%, even more preferably at least 60%, even more preferably at least 70%, 80% or more is particularly preferred. The upper limit of x/y is not particularly limited as long as it is 100% or less.

We now describe x/y in naturally occurring fibroins, including spider silk fibroin and other fibroins. First, as described above, when fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (among them, 415 types of fibroin derived from arachnids (spider silk fibroin)). was extracted. Of all the extracted fibroin, from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , x/y was calculated. FIG. 5 shows the results when the serration ratio is 1:1.9 to 4.1.

The horizontal axis of FIG. 5 indicates x/y (%), and the vertical axis indicates frequency. As is clear from FIG. 5, the x/y ratios for naturally occurring fibroin are all less than 64.2% (the highest is 64.14%).

The third modified spider silk fibroin is obtained, for example, from the cloned naturally occurring spider silk fibroin gene sequence such that x/y is _64.2 % or more. It can be obtained by deleting the Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs such that x/y is 64.2% or more from the amino acid sequence of naturally occurring spider silk fibroin is designed. , can also be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to deletion of the (A) _n motif from the amino acid sequence of naturally occurring spider silk fibroin, one or more amino acid residues are substituted, deleted, inserted and/or Alternatively, modification of the amino acid sequence corresponding to the addition may be performed.

As a more specific example of the third modified spider silk fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 Modified spider silk fibroin can be mentioned, including:

The modified spider silk fibroin (3-i) will be explained. The amino acid sequence shown by SEQ ID NO: 17 is every two (A ) _n motif is deleted and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described for the second modified spider silk fibroin.

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring spider silk fibroin) is 15.0% at a jagged ratio of 1:1.8 to 11.3. The x/y values of the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

The modified spider silk fibroin (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified spider silk fibroin (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified spider silk fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-ii) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared toward the terminal side, and the number of amino acid residues of REP having a smaller number of amino acid residues is set to 1 , two adjacent [(A) _n motif-REP] units in which the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (the jagged ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum value of the total sum of the numbers of amino acid residues, and y is the total number of amino acid residues in the domain sequence.

The third modified spider silk fibroin may contain the tag sequences described above at either or both the N-terminus and the C-terminus.

More specific examples of modified spider silk fibroin containing a tag sequence include (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799). or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, modified spider silk fibroin can be mentioned.

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

The modified spider silk fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

(3-iv) modified spider silk fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified spider silk fibroin (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared toward the terminal side, and the number of amino acid residues of REP having a smaller number of amino acid residues is set to 1 , the maximum sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of the other REP of 1.8 to 11.3. When x is the total number of amino acid residues in the domain sequence and y is the total number of amino acid residues, x/y is preferably 64.2% or more.

The third modified spider silk fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The fourth modified spider silk fibroin has a reduced content of glycine residues in addition to a reduced content of (A) _n motifs in its domain sequence compared to naturally-derived spider silk fibroin. have the same amino acid sequence. The domain sequence of the fourth modified spider silk fibroin has at least one or more (A) _n motifs deleted compared to the naturally-derived spider silk fibroin, and at least one or more of the It can be said to have an amino acid sequence corresponding to substitution of a glycine residue with another amino acid residue. That is, the fourth modified spider silk fibroin is a modified spider silk fibroin having both the characteristics of the second modified spider silk fibroin and the third modified spider silk fibroin. Specific aspects and the like are as described in the second modified spider silk fibroin and the third modified spider silk fibroin.

As more specific examples of the fourth modified spider silk fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), the amino acid sequence shown in SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or sequence A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in number 15 can be mentioned. Specific embodiments of the modified spider silk fibroin comprising the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth modified spider silk fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues with a large hydrophobicity index compared to the naturally-derived spider silk fibroin. and/or may have an amino acid sequence containing regions of high local hydrophobicity index corresponding to the insertion of one or more amino acid residues with high hydrophobicity index into REP.

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). A residue is more preferred.

The fifth modified spider silk fibroin has one or more amino acid residues in REP substituted with amino acid residues with a larger hydrophobicity index than the naturally-derived spider silk fibroin, and / or In addition to the modification corresponding to the insertion of one or more amino acid residues with a large hydrophobicity index, one or more amino acid residues are substituted or deleted compared to the naturally-derived spider silk fibroin There may be alterations in the amino acid sequence corresponding to insertions and/or additions.

The fifth modified spider silk fibroin, for example, removes one or more hydrophilic amino acid residues (eg, amino acid residues with a negative hydrophobicity index) in REP from the cloned naturally occurring spider silk fibroin gene sequence. It can be obtained by substituting a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and/or one or more hydrophobic amino acids in REP It can also be obtained by designing an amino acid sequence corresponding to the inserted residue and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and/or one or more hydrophobic amino acid residues in REP In addition to the modification corresponding to the insertion of an amino acid residue, the amino acid sequence may be modified by substituting, deleting, inserting and/or adding one or more amino acid residues.

A fifth modified spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and the (A) _n motif located most C-terminally to the C-terminal The total number of amino acid residues contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequences excluding the sequences from the domain sequence to p and q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, p/q is 6.2% or more.

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 6, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 6, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 6, 28/170=16.47%.

In the fifth modified spider silk fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, and 20% or more. is even more preferred, and 30% or more is even more preferred. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

The fifth modified spider silk fibroin is obtained by, for example, converting the amino acid sequence of the cloned naturally occurring spider silk fibroin into one or more hydrophilic amino acid residues (eg, REP) in REP so as to satisfy the above p/q conditions. , amino acid residues with a negative hydrophobicity index) with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index), and/or one or more hydrophobic amino acids in REP By inserting residues, it can be obtained by locally modifying the amino acid sequence to include a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring spider silk fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring spider silk fibroin, and/or one or more In addition to the modification corresponding to the insertion of a plurality of amino acid residues with a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is performed. good too.

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

As a more specific example of the fifth modified spider silk fibroin, (5-i) the amino acid represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666) or (5-ii) a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified spider silk fibroin (5-i) will be explained. The amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP except for the terminal domain sequence on the C-terminal side with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). The amino acid sequence (VLI) was inserted at two sites, some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP. be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 8.

The modified spider silk fibroin (5-i) may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified spider silk fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified spider silk fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(5-ii) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located at the most C-terminal side (A ) In all REPs contained in the sequence excluding the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence, contained in the region where the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more Let p be the total number of amino acid residues contained in the domain sequence, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. Sometimes it is preferred that p/q is greater than or equal to 6.2%.

The fifth modified spider silk fibroin may contain a tag sequence at either or both the N-terminus and the C-terminus.

More specific examples of modified spider silk fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666), or (5 -iv) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and hinge sequence) are added.

The modified spider silk fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

(5-iv) modified spider silk fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. The modified spider silk fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(5-iv) modified spider silk fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A ) In all REPs contained in the sequence excluding the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence, contained in the region where the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more Let p be the total number of amino acid residues contained in the domain sequence, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. Sometimes it is preferred that p/q is greater than or equal to 6.2%.

The fifth modified spider silk fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A sixth modified spider silk fibroin has an amino acid sequence with a reduced content of glutamine residues compared to the naturally occurring spider silk fibroin.

The sixth modified spider silk fibroin preferably contains at least one motif selected from a GGX motif and a GPGXX motif in the REP amino acid sequence.

When the sixth modified spider silk fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.

As used herein, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif spider silk fibroin (modified spider silk fibroin or naturally occurring spider silk _fibroin ). The number obtained by multiplying the total number of GPGXX motifs by three (i.e., corresponding to the total number of G and P in the GPGXX motif) is s, and the most C-terminal side (A) From the _n motif to the C-terminus of the domain sequence The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding the sequence of from the domain sequence and excluding the (A) _n motif.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from the _n motif to the C-terminus of the domain sequence” (sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of spider silk fibroin, m This is to eliminate the influence of a small σ (that is, a short domain sequence), which affects the calculation result of the GPGXX motif content. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 7 is a schematic diagram showing the domain sequence of modified spider silk fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the modified spider silk fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. The sequence from the located (A) _n motif to the C-terminus of the domain sequence is excluded from the domain sequence” (sequence shown as “region A” in FIG. 7), so s is calculated. The number of GPGXX motifs for is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (in FIG. 7, the sequence indicated by "region A" .), the total number of amino acid residues t of all REPs further excluding the (A) _n motif from the sequence is 50+40+10+20+30=150. Then s/t (%) can be calculated by dividing s by t, which is 21/150=14.0% for the modified spider silk fibroin in FIG.

The sixth modified spider silk fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and preferably 0%. Especially preferred.

As used herein, "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif spider silk fibroin (modified spider silk fibroin or naturally-derived spider silk fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (sequence corresponding to "region A" in FIG. 7 ), the total number of glutamine residues contained in the region is u, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence Further, (A) The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding the _n motif. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

A sixth modified spider silk fibroin had its domain sequence deleted or substituted with other amino acid residues in one or more glutamine residues in REP compared to the naturally occurring spider silk fibroin. It may have an amino acid sequence corresponding to

The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than that of a glutamine residue. Hydrophobicity indexes of amino acid residues are shown in Table 1.

As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

The sixth modified spider silk fibroin preferably has a REP hydrophobicity of −0.8 or more, more preferably −0.7 or more, still more preferably 0 or more, and 0.8 or more. It is even more preferably 3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

As used herein, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif spider silk fibroin (modified spider silk fibroin or naturally-derived spider silk fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (sequence corresponding to "region A" in FIG. 7 ), the sum of the hydrophobicity index of each amino acid residue in the region is defined as v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is defined as the domain The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs, excluding the (A) _n motif, removed from the sequence. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

A sixth modified spider silk fibroin has a domain sequence lacking one or more glutamine residues in REP and/or one or more In addition to the modification corresponding to the substitution of the glutamine residue with another amino acid residue, the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues There may be.

A sixth modified spider silk fibroin is obtained, for example, by deleting one or more glutamine residues in REP from the cloned naturally occurring spider silk fibroin gene sequence, and/or deleting one or more glutamine residues in REP. It can be obtained by substituting the residue with another amino acid residue. Also, for example, deletion of one or more glutamine residues in REP from the amino acid sequence of naturally occurring spider silk fibroin, and/or one or more glutamine residues in REP to other amino acid residues It can also be obtained by designing an amino acid sequence corresponding to the substitution and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth modified spider silk fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or modified spider silk fibroin comprising the amino acid sequence shown in SEQ ID NO: 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 , SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42, and a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity.

The modified spider silk fibroin (6-i) will be explained. The amino acid sequence shown by SEQ ID NO:25 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 (Met-PRT410) with VL. The amino acid sequence shown by SEQ ID NO: 26 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with TS, and replacing the remaining Q with A. The amino acid sequence shown by SEQ ID NO: 27 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VL, and replacing the remaining Q with I. The amino acid sequence shown by SEQ ID NO:28 is obtained by substituting VI for all QQs in the amino acid sequence shown by SEQ ID NO:7 and L for the remaining Qs. The amino acid sequence shown by SEQ ID NO: 29 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VF, and replacing the remaining Q with I.

The amino acid sequence shown by SEQ ID NO:30 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:8 (Met-PRT525) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 consists of 20 domain sequence regions present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) that are repeated twice, and all QQ in the sequence are replaced with VF, And the remaining Q is replaced with I.

The amino acid sequence shown by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with LI and the remaining Q with V. The amino acid sequence shown by SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 with IF, and replacing the remaining Q with T.

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues The group content is below 9% (Table 2).

(6-i) modified spider silk fibroin is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It may consist of the amino acid sequence shown by 42.

(6-ii) modified spider silk fibroin is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 41 It includes amino acid sequences having 90% or more sequence identity with the amino acid sequence represented by 42. The modified spider silk fibroin of (6-ii) is also represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. It is a protein containing a domain sequence that The sequence identity is preferably 95% or more.

The modified spider silk fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the modified spider silk fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

The sixth modified spider silk fibroin may contain a tag sequence at either or both the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, etc. of the modified spider silk fibroin.

More specific examples of modified spider silk fibroin containing a tag sequence include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), sequence Modified spider silk comprising the amino acid sequence shown in No. 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028) fibroin, or (6-iv) represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence can be mentioned.

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 at the N-terminus of SEQ ID NO: 11 The amino acid sequence (including the His-tag sequence and hinge sequence) is added. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

(6-iii) modified spider silk fibroin is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: It may consist of the amino acid sequence shown by 44.

(6-iv) modified spider silk fibroin is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 43 It includes amino acid sequences having 90% or more sequence identity with the amino acid sequence represented by 44. (6-iv) modified spider silk fibroin is also represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif It is a protein containing a domain sequence that The sequence identity is preferably 95% or more.

The modified spider silk fibroin (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified spider silk fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

The sixth modified spider silk fibroin may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The modified spider silk fibroin includes a first modified spider silk fibroin, a second modified spider silk fibroin, a third modified spider silk fibroin, a fourth modified spider silk fibroin, a fifth modified spider silk fibroin, and a sixth modified spider silk fibroin. The modified spider silk fibroin may have at least two or more of the characteristics of the modified spider silk fibroin.

The modified spider silk fibroin may be hydrophilic modified spider silk fibroin or hydrophobic modified spider silk fibroin. As used herein, the term "hydrophilic modified spider silk fibroin" refers to the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified spider silk fibroin, and then the total number of all amino acid residues. is a modified spider silk fibroin whose value (average HI) divided by 0 is 0 or less. The hydrophobicity index is as shown in Table 1. A "hydrophobic modified spider silk fibroin" is a modified spider silk fibroin with an average HI of greater than zero. Hydrophilic modified spider silk fibroin is particularly excellent in flame retardancy. Hydrophobic modified spider silk fibroin is particularly excellent in hygroscopic heat generation and heat retention.

Hydrophilic modified spider silk fibroin includes, for example, the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, The amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 modified spider silk fibroin comprising the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

Hydrophobic modified spider silk fibroin includes, for example, amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43, SEQ ID NO: 35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:44.

The spider silk fibroin according to this embodiment can be produced by a conventional method using a nucleic acid encoding the spider silk fibroin. The nucleic acid encoding the spider silk fibroin may be chemically synthesized based on base sequence information, or may be synthesized using the PCR method or the like.

Spider silk fibroin fibers can be obtained, for example, by dissolving spider silk fibroin in a soluble solvent to form a dope solution and spinning by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. . Solvents capable of dissolving spider silk fibroin include, for example, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). An inorganic salt may be added to the solvent as a dissolution accelerator.

<Protein>
Proteins contained in the fiber base material, polymer substance, and/or skin layer are not particularly limited, and any protein can be used. Proteins can include naturally occurring proteins and recombinant proteins (artificial proteins). Recombinant proteins include any proteins that can be produced on an industrial scale, and include, for example, proteins that can be used for industrial purposes, proteins that can be used for medical purposes, structural proteins, and the like. Specific examples of proteins of industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccination, vaccines, antigen binding proteins, immunostimulatory proteins, Allergens, full length antibodies or antibody fragments or derivatives may be mentioned. Specific examples of structural proteins include spider silk (spider silk), silkworm silk, keratin, collagen, elastin, resilin, and proteins derived from these. As the protein to be used, fibroin is preferable, and spider silk fibroin is more preferable, because it is excellent in heat retention, hygroscopic heat generation and/or flame retardancy.

Fibroin includes the above spider silk fibroin and other fibroins. Other fibroins are proteins comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif, described above. can be

Other fibroin may be, for example, one or more selected from the group consisting of silk fibroin and hornet silk fibroin. That is, the fibroin may be, for example, one or more selected from the group consisting of spider silk fibroin, silk fibroin, and hornet silk fibroin.

Silk threads are fibers (cocoon threads) obtained from cocoons produced by silkworms, which are larvae of Bombyx mori. In general, one cocoon thread consists of two strands of silk fibroin and a glue substance (sericin) covering them from the outside. Silk fibroin is composed of numerous fibrils. Silk fibroin is covered with four layers of sericin. Practically, silk filaments obtained by dissolving and removing the outer sericin by refining are used for clothing applications. A typical silk thread has a specific gravity of 1.33, an average fineness of 3.3 decitex, and a fiber length of about 1300 to 1500 m. Silk fibroin can be obtained from natural or domestic silkworm cocoons, or from used or discarded silk fabrics.

The silk fibroin may be sericin-removed silk fibroin, non-sericin-removed silk fibroin, or a combination thereof. The sericin-removed silk fibroin is purified by removing sericin covering the silk fibroin and other fats. The silk fibroin thus purified is preferably used as a freeze-dried powder. Sericin-unremoved silk fibroin is unpurified silk fibroin from which sericin and the like have not been removed.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

<Production of spider silk fibroin>
A modified spider silk fibroin (PRT966) having the amino acid sequence shown in SEQ ID NO:40 and a modified spider silk fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO:15 were designed. A nucleic acid encoding the designed modified spider silk fibroin was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) to an OD ₆₀₀ of 0.05. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the spider silk fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of the desired spider silk fibroin-sized band depending on the addition of IPTG confirmed the expression of the desired spider silk fibroin. It was confirmed.

Twenty-four hours after the addition of IPTG, the cells collected were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi).

Modified spider silk fibroin PRT799 was purified by the method described below. First, the cells crushed by the above method were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and heated at 60°C. for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and freeze-dried powder was recovered to obtain spider silk fibroin PRT799.

Modified spider silk fibroin PRT966 was purified by the method described below. First, 300 mL of buffer solution B (50 mM Tris-HCL, 100 mM NaCl, pH 7.0) containing 3 w/v% SDS was added to the disrupted cells, and a mixer (IKA "T18 Basic Ultra Turrax", After being well dispersed in level 2), the mixture was stirred for 60 minutes with a shaker (manufactured by Taitec, 200 rpm, 37°C). Thereafter, the mixture was centrifuged (11,000 g, 30 minutes, room temperature) using a Kubota centrifuge, and the supernatant was discarded to obtain SDS-washed granules (precipitate).

The SDS-washed granules were suspended in a DMSO solution containing 1 M lithium chloride to a concentration of 100 mg/mL and heat-treated at 80° C. for 1 hour. Then, it was centrifuged (11,000 g, 30 minutes, room temperature) and the supernatant was collected.

Three volumes of ethanol were prepared with respect to the recovered supernatant, the recovered supernatant was added to the ethanol, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, centrifugation (11,000 g, 30 minutes, room temperature) was performed to collect aggregated proteins. Next, after repeating the process of washing the aggregated protein with pure water and recovering the aggregated protein by centrifugation three times, water was removed with a freeze dryer to obtain modified spider silk fibroin PRT966 as a freeze-dried powder. rice field.

PRT966 is a hydrophobically modified spider silk fibroin with an average HI greater than zero. PRT799 is a hydrophilic modified spider silk fibroin with an average HI of 0 or less.

<Preparation of base material layer>
[Preparation of nonwoven fabric]
Lyophilized powder of spider silk fibroin PRT966 was added to formic acid to a concentration of 28% by mass and dissolved using a shaker for 3 hours. Insoluble matters and bubbles were removed from the resulting solution to obtain a fibroin solution (spinning dope). The resulting fibroin solution was used as a dope solution (spinning stock solution), and spider fibroin fibers were produced by dry-wet spinning using a known dry-wet spinning apparatus. Next, the obtained spider silk fibroin fibers were cut into staple fibers having a length of 50 mm, and then a sheet (web) was obtained by a known carding process. Thereafter, the obtained sheet was punched with a known needle punch machine to produce a nonwoven fabric composed of spider fibroin fibers. The basis weight (fiber density) of the nonwoven fabric was 100 to 200 g/m ² . The conditions for dry-wet spinning are as follows.
Coagulation bath temperature: 2-15°C
Total draw ratio: 1 to 4 times Drying temperature: 100°C

[Preparation of base material layer]
An impregnation liquid (fibroin impregnation liquid) containing spider silk fibroin PRT799 and a mixed solvent of ethanol and water was prepared. The concentration of spider silk fibroin was 5% by weight with respect to the total weight of the impregnation liquid. The mass ratio (ethanol/water) of ethanol and water in the mixed solvent was 3/7 (w/w).

The nonwoven fabric obtained as described above was impregnated with the fibroin impregnation liquid so that the pick-up rate was 400%. Thereafter, the nonwoven fabric to which the impregnating liquid was attached was immersed in water for 15 minutes to remove the solvent from the impregnating liquid and solidify the modified spider silk fibroin in the impregnating liquid. The nonwoven fabric was then taken out of water and dried at room temperature. As a result, a base layer composed of a nonwoven fabric made of spider fibroin fibers and spider silk fibroin adhered to the nonwoven fabric was produced. The thickness of the base material layer was 1.5 mm.

<Preparation of skin layer>
A freeze-dried powder of spider silk fibroin PRT799 was added to formic acid to a concentration of 20% by mass and dissolved using a shaker at 40° C. for 1 hour or more to obtain a fibroin solution. After that, the pigment was added to the fibroin solution so as to have a concentration of 5% by mass, and then insoluble matter and bubbles were removed. Thus, a skin layer resin was prepared from a fibroin solution containing a pigment. The skin layer resin obtained as described above was coated on release paper pasted on an iron plate so as to have a thickness of 0.5 mm, and then dried at 90° C. for 10 minutes.

<Production of adhesive>
Powdered spider silk fibroin PRT799, which is a freeze-dried product, was added to formic acid to a concentration of 10% by mass, and glycerol was added to a concentration of 15% by mass, and dissolved at 40° C. for 1 hour or more using a shaker. After that, defoaming was performed. This gave an adhesive containing spider silk fibroin, formic acid and glycerol. The concentrations of spider silk fibroin and glycerol are based on the total weight of the adhesive.

<Manufacturing and evaluation of synthetic leather>
The adhesive was coated on one side of the skin layer to a thickness of 0.1-1.0 mm. As a result, a laminate was obtained in which the skin layer and the adhesive layer were laminated in this order. A substrate layer was attached to the surface of the adhesive layer of this laminate opposite to the surface in contact with the skin layer, and then dried at room temperature. As a result, a synthetic leather comprising a skin layer, a base layer, and an adhesive layer for bonding the skin layer and the base layer to each other was obtained. FIG. 8 is a photograph showing the obtained synthetic leather.

In the synthetic leather, it was confirmed that the skin layer and the base material layer were sufficiently adhered via the adhesive layer (Fig. 8). As shown in FIG. 8, the adhesion between the substrate layer and the skin layer via the adhesive layer was sufficiently high. It has also been shown that the skin layer does not impregnate the substrate layer.

REFERENCE SIGNS LIST 1 Skin layer 2 Base material layer 3 Adhesive layer 20 Synthetic leather 91 Release paper 92 Skin layer resin (polymer) 93 Dryer 94 Adhesive 95 Heat Press roll 96 Base material layer 97 Synthetic leather 900 Synthetic leather manufacturing apparatus.

Claims (9)

Adhesive for synthetic leather containing spider silk fibroin. The synthetic leather adhesive according to claim 1, further comprising an organic solvent. 3. The adhesive for synthetic leather according to claim 2, wherein said organic solvent is formic acid. The synthetic leather adhesive according to any one of claims 1 to 3, further comprising glycerol. A synthetic leather comprising a skin layer, a base layer containing a fiber base, and an adhesive layer for bonding the skin layer and the base layer to each other, wherein the adhesive layer contains spider silk fibroin. 6. The synthetic leather of claim 5, wherein said adhesive layer further comprises glycerol. A method for producing synthetic leather, wherein a skin layer, a base layer containing a fiber base material, and the skin layer and the base layer are adhered to each other,
forming an adhesive layer containing spider silk fibroin on the skin layer;
and adhering the skin layer and the base layer via the adhesive layer. 8. The method of claim 7, wherein at least one of the base layer and the skin layer comprises a biodegradable material. 9. The method of claim 8, wherein at least one of the base layer and skin layer comprises spider silk fibroin.

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