JP2021054989A - Heat compression molding body and production method therefor - Google Patents

Abstract

To provide a heat compression molding body that does not lose translucency even when a non-translucent substance is added and a production method therefor.SOLUTION: A method for producing a heat compression molding body has a first step of dispersing a non-translucent substance in a solution with protein dissolved in a solvent to obtain a dispersion, a second step of removing the solvent from the obtained dispersion to obtain a protein mixture, and a third step of heating and compressing the obtained protein mixture.SELECTED DRAWING: None

Description

The present invention relates to a protein molded product and a method for producing the same.

The metal material used as a material for molding processing may be replaced with an organic material (for example, phenol resin) for the purpose of weight reduction, cost reduction, facilitation of molding processing, and the like. Further, in order to further improve the flexural modulus and bending strength, a method of adding reinforcing fibers such as phenol resin fibers to a matrix resin containing a phenol resin is known (see, for example, Patent Document 1).

In recent years, bioplastics, which are non-petroleum materials, have been attracting attention for the purpose of reducing the environmental load. For example, a molded product obtained by resinifying silk powder is known to have a flexural modulus of 4.5 GPa (see, for example, Non-Patent Document 1). Further, the present inventors have found that a molded product obtained by heat-pressing a composition containing a specific polypeptide shown in Patent Document 1 exhibits excellent bending strength and translucency.

International Publication No. 2017/047503 International Publication No. 2016/067898

Shinji Hirai, Monthly Functional Materials Magazine, June 2014, "Environmentally Friendly Silk and Wool Resin Using Waste-Derived Animal Proteins"

Various additives may be added to impart additional properties to such molded articles. However, when the additive does not have translucency, there is a problem that the translucency of the material itself for molding processing cannot be maintained.

For example, in Patent Document 2, when silver-containing powder is added in order to impart antibacterial properties to phosphate glass powder, the mass ratio thereof and the BET specific surface area of the silver-containing powder are set in a specific range. Is being considered.

Therefore, an object of the present invention is to provide a heat-press molded article which does not lose its translucency even when a substance having no translucency (non-transmissive substance) is added, and a method for producing the same.

The present invention provides the following [1] to [13].
[1] The first step of obtaining a dispersion liquid by dispersing a translucent substance in a solution in which a protein is dissolved in a solvent.
The second step of removing the solvent from the obtained dispersion to obtain a protein mixture, and
A method for producing a heat-pressed molded article, which comprises a third step of heating and pressurizing the obtained protein mixture.
[2] The method for producing a heat-press molded product according to [1], wherein the solution in the first step further contains a solubilizer.
[3] A group in which the solubilizer comprises dimethyl sulfoxide, 1,1,1,3,3,3-hexafluoro-2-propanol, urea, guanidine hydrochloride, guanidine thiocyanate, sodium iodide and perchlorate. The method for producing a heat-press molded product according to [2], which comprises at least one selected from the above.
[4] The method for producing a heat-pressurized molded product according to [2], wherein the solubilizer is an aqueous solution containing at least one selected from the group consisting of urea, guanidine hydrochloride and guanidine thiocyanate.
[5] The method for producing a heat-press molded product according to any one of [1] to [4], wherein the protein is fibroin.
[6] The method for producing a heat-press molded product according to [5], wherein the fibroin is a modified spider silk fibroin.
[7] The heat and pressure molding according to any one of [1] to [6], wherein the translucent substance contains at least one selected from the group consisting of an antibacterial agent, a strengthening agent, a flame retardant and a nucleating agent. How to make a body.
[8] The method for producing a heat-press molded product according to any one of [1] to [6], wherein the translucent substance contains an antibacterial metal compound.
[9] The method for producing a heat-press molded product according to [8], wherein the antibacterial metal compound is silver nanoparticles.
[10] A heat-press molded product containing a protein and a translucent substance and having a light transmittance of 40% or more.
[11] The heat-press molded product according to [10], wherein the protein is fibroin.
[12] The heat-press molded product according to [11], wherein the fibroin is a modified spider silk fibroin.
[13] A composition containing a protein, a solvent, and a translucent substance, wherein the protein is a solvent in the solvent and the translucent substance is dispersed in the solvent.
[14] The composition according to [13], wherein the protein is modified spider silk fibroin.

According to the present invention, it is possible to provide a new heat-pressurized molded article (material for molding process) and a method for producing the same, which does not lose the translucency even if a translucent substance is added. The heat-press molded body produced by the present invention retains translucency and imparts desired properties (for example, antibacterial property and flame retardancy), and for example, automobile interior materials, furniture, etc. It can be used for various purposes. In addition, the material can also be obtained as a molded product having a desired shape by selecting and trimming a mold at the time of manufacturing.

In the present specification, "translucency" means the property of transmitting light to the opposite side surface when light (for example, visible light) is irradiated from a certain direction to the object, and the appearance is not necessarily transparent. It does not matter whether it is opaque or opaque. The term "translucent" means that, for example, when observed from one side of a heat-pressed molded product, the presence of an object on the opposite side can be recognized. It is more preferable that the transmittance is extremely high and the shape of the object on the opposite side can be clearly seen (that is, transparent), but the outline of the object is blurred and cannot be clearly recognized like frosted glass (that is, transparent). It may be translucent). Further, the term "having translucency" also includes a case where the shape of an object on the opposite side looks distorted due to a difference in refractive index or the like. The translucency can be visually determined, but when an optical transmittance measuring device is used and the integration time is 0.1 seconds in the wavelength range of 220 to 800 nm, the transmittance is 40% or more. Suitable. On the other hand, "impermeable" means a property that does not transmit light (for example, visible light). The transmittance can be calculated, for example, based on the value of the brightness of the white light measured by irradiating the material according to the present invention with white light and using a spectrophotometer.

It is a schematic cross-sectional view of a pressure molding machine. (A) is a schematic cross-sectional view of a pressure molding machine before the introduction of the composition, (b) immediately after the introduction of the composition, and (c) a state in which the composition is heated and pressed. It is a schematic diagram which shows an example of the domain sequence of spider silk fibroin. It is a schematic diagram which shows an example of the domain sequence of spider silk fibroin. It is a schematic diagram which shows an example of the domain sequence of spider silk fibroin. (A) is a photograph of the heat-pressed molded product of Comparative Example 1, (b) is a photograph of the heat-pressed molded product of Example 1, and (c) is a photograph of the heated and pressurized molded product of Comparative Example 2. It is a photograph of a pressure compact.

Hereinafter, embodiments of the present invention will be described.

One embodiment of the present invention comprises the first step of dispersing a translucent substance in a solution containing a protein and a solvent to obtain a dispersion, and removing the solvent from the obtained dispersion to obtain a protein composition. A method for producing a heat-pressurized molded product, which comprises a second step and a third step of heating and pressurizing the obtained protein composition. The molded product of the present invention is suitable as a material for molding processing.

(First step)
The first step is a step of dispersing a translucent substance in a solution containing a protein and a solvent to obtain a dispersion liquid.

The proteins used in this embodiment will be described later. The protein used may be one type of protein or a combination of a plurality of types of proteins. Proteins typically have a powdery form (lyophilized powder, etc.).

The solvent is not limited as long as it can dissolve the protein or can dissolve the protein in the presence of a solubilizer. Examples of the solvent include water, dimethyl sulfoxide, formic acid, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP).

In the first step, the protein is dissolved in a solvent. If the protein is difficult to dissolve in the solvent, the solvent may be heated as appropriate. The heating temperature may not exceed the temperature at which the protein is denatured or the boiling point of the solvent.

In the first step, if the protein is difficult to dissolve in the solvent, a solubilizer may be further added. The addition of the solubilizer facilitates the dissolution of the protein in the solvent at room temperature. A solubilizer is a component that assists in dissolving a protein in a solvent. Examples of the solubilizer include urea, guanidine hydrochloride (GuHCl), guanidine thiocyanate, sodium iodide, and perchlorate. As the solubilizer, these may be used as they are, or may be used in the form of an aqueous solution. When the solubilizer is an aqueous solution, the concentration of the solubilizer may be 1-10 mol / L, 3-9 mol / L or 5-7 mol / L. The amount of the solubilizer can be appropriately adjusted in consideration of the solubility of the protein in the solvent used. The dissolution of the protein in the solvent facilitates the dispersion of the translucent substances to be added thereafter. Preferred solubilizers are aqueous solutions of urea, guanidine hydrochloride and guanidine thiocyanate. These solubilizers denature proteins and make them more soluble in solvents. If the solubilizer is an aqueous solution of these, it can be recovered and reused after use.

By adding the impermeable substance to the solution in which the protein is dissolved in the solvent, a highly dispersible protein solution of the impermeable substance can be obtained. After obtaining this, by going through the second step and the third step in the present invention, a heat-pressurized molded product in a state in which the impermeable substance is well dispersed can be obtained.

The translucent substance in the present invention is a substance that does not have translucency, and is usually not limited as long as it can impart further properties to the heat-pressed molded article. Examples of the translucent substance include antibacterial agents (for example, silver nanoparticles), fortifiers (for example, carbon black), flame retardant agents (for example, polyphosphoric acid salts, polynucleotide salts), and nucleating agents (for example, for example. Metal oxides, clay minerals). The upper limit of the amount of the translucent substance is, for example, 50 parts by mass or less, 40 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, 10 parts by mass or less, and 9 parts by mass or less with respect to 100 parts by mass of the protein. , 8 parts by mass or less, 7 parts by mass or less, 6 parts by mass or less, 5 parts by mass or less, 4 parts by mass or less, 3 parts by mass or less, 2 parts by mass or less, 1 part by mass or less, or 0.5 parts by mass or less. .. The lower limit of the amount of the translucent substance may be an amount that can impart desired properties, for example, 0.1 part by mass or more with respect to 100 parts by mass of the protein. In addition, the dispersion liquid may contain impurities generated in the process of obtaining the protein.

It is desirable to mix the impermeable substances while suppressing their aggregation. When a protein and a translucent substance are mixed in a dry state, the translucent substance molecules may aggregate and the transparency of the molded product produced from the mixture may be lost. By dispersing in the solution, the agglutinated state is alleviated, and the average particle size can be mixed while maintaining a small state.

The antibacterial agent may be any metal particle as long as it has a biological action such as an antibacterial action, an antifungal action, and an antiviral action. As the metal having an antibacterial action, for example, silver (Ag), cobalt (Co), copper (Cu), nickel (Ni), gold (Au), and palladium (Pd) are known. As the antibacterial agent, for example, Yasuyuki M, Kunihiro K, Kurissery S, etc., Biofouling 2010 Oct; 26 (7): 851-8) can be referred to. When the antibacterial agent is silver particles, it is preferable because it is excellent in versatility and antibacterial property. Furthermore, when the antibacterial agent is silver nanoparticles, it exhibits even better translucency. The average particle size d2 of the silver nanoparticles may be, for example, 1 nm or more, preferably 10 nm or more. The average particle size d2 of the silver nanoparticles may be, for example, 1000 nm or less, preferably 500 nm or less, more preferably 300 nm or less, 150 nm or less, or 50 nm.

Examples of carbon black include hydrophilic carbon black and surface-modified carbon black. Further, carbon black for paint or ink can also be used. The carbon black may be in powder form (carbon black powder). The average particle size d2 of the carbon black powder may be, for example, 1 nm or more, preferably 10 nm or more. The average particle size d2 of the carbon black powder may be, for example, 1000 nm or less, preferably 500 nm or less. In the present specification, the average particle size d2 of the carbon black powder indicates the arithmetic mean diameter obtained by observing with an electron microscope.

The amount of carbon black may be, for example, 0.05 parts by mass or more, preferably 0.5 parts by mass or more, and more preferably 1.0 part by mass or more with respect to 100 parts by mass of the protein. The amount of carbon black blended in the composition may be, for example, 20 parts by mass or less, preferably 10 parts by mass or less, and more preferably 5 parts by mass or less with respect to 100 parts by mass of the protein. When the translucent substance is carbon black, the decrease in strength (particularly, the decrease in bending strength) caused by the deterioration of ultraviolet rays in the heat-pressed molded product is remarkably suppressed.

The salt of polyphosphoric acid and the salt of a polynucleotide are a salt consisting of polyphosphoric acid or a polynucleotide and a cation. A polynucleotide is a polymer having a nucleotide composed of a nucleobase, sugar and phosphoric acid as a monomer unit, and sugar and phosphoric acid are alternately bonded to the main chain thereof. The nucleobase constituting the nucleotide is a group having a heteroaromatic ring structure such as a purine skeleton or a pyrimidine skeleton, and specific examples thereof include adenine, guanine, cytosine, thymine and uracil. The polynucleotides have the same or different nucleobases for each nucleotide unit, and each nucleobase may be any of adenine, guanine, cytosine, thymine and uracil. The cation may be an organic cation such as an onium ion or a metal ion. Examples of onium ions include ammonium ions. Examples of the metal ion include alkali metal ions such as lithium ion, sodium ion and potassium ion, and alkaline earth metal ions such as magnesium ion and calcium ion. Preferred cations are ammonium ions or alkali metal ions. The preferred salt of polyphosphoric acid is ammonium polyphosphate, and the preferred salt of polynucleotide is sodium deoxyribonucleic acid. These contents may be 1 to 50% by mass, 5 to 40% by mass, or 10 to 30% by mass based on the total mass of the composition. When the translucent substance is a salt of polyphosphoric acid or a salt of a polynucleotide, a heat-press molded product having more excellent flame retardancy can be obtained.

The metal oxide is not limited as long as it is a metal oxide that can exist stably. Preferred metal oxides are transition metal oxides. Examples of transition metal oxides include scandium oxide, yttrium oxide, titanium oxide, zirconium oxide, hafnium oxide, vanadium oxide, niobium oxide, tantalum oxide, chromium oxide, molybdenum oxide, tungsten oxide, manganese oxide, technetium oxide, and oxidation. Examples thereof include renium, iron oxide, ruthenium oxide, cobalt oxide, rhodium oxide, iridium oxide, nickel oxide, palladium oxide, platinum oxide, copper oxide and silver oxide. A preferred metal oxide is titanium oxide.

The crystal form of the metal oxide is not particularly limited. A preferred metal oxide is a rutile crystal. When the metal oxide is a rutile-type crystal, it is superior in physical and chemical stability. The metal oxide is preferably surface-treated. For example, surface treatment adds hydrophilicity or water repellency with aluminum hydroxide, stearic acid, or the like. Specific examples of such metal oxides include TTO-51A, TTO-51C, TTO-55A, TTO-55B, TTO-55C, and TTO-55D (trade names, all manufactured by Ishihara Sangyo Co., Ltd.). Be done.

Clay minerals are minerals whose main component is phyllosilicate minerals. In addition, calcite, dolomite, feldspar, quartz, and zeolite are also included in clay minerals. The preferred clay mineral is smectite, and the more preferred clay mineral is montmorillonite or bentonite. Smectite is a swelling clay mineral, and specific examples thereof include montmorillonite, biderite, nontronite, saponite, hectorite, saponite, and stevensite. Bentonite is a clay containing associated minerals such as quartz, cristobalite, zeolite, feldspar, and calcite in addition to the main mineral montmorillonite. The metal oxide or clay mineral preferably has a number average particle size of 30 nm or less.

When a metal oxide or clay mineral is added, this operation causes the inorganic substances to be more evenly dispersed in the protein, so that the bending strength and flexural modulus of the heat-pressed molded product are further improved.

The solution in the first step may contain a translucent additive component (for example, a plasticizer, a colorant, a filler, a synthetic resin, etc.) in addition to the translucent substance.

(Second step)
The second step is a step of removing the solvent from the dispersion obtained in the first step to obtain a protein composition.

Methods for removing the solvent include a method of evaporating the solvent and a method of adding a poor solvent to the solvent to precipitate a composition (protein composition) containing a protein and a translucent substance.

When the mixed composition is precipitated, it is advisable to remove the solvent by centrifugation after forming the precipitate and wash the composition containing the protein and the translucent substance with water. In this case, the solubilizer can also be removed by centrifugation or washing with water.

The water content (moisture content) of the protein composition may be 5 to 15%, preferably 5 to 9%, based on the total mass of the protein composition at the start of the third step. ..

The water content can be measured by a known method such as a near infrared method, an electric resistance method, an electric capacity method, a dry gravimetric method, or a chemical measurement method including a Karl Fischer method. Specifically, after the protein composition is sufficiently stirred and homogenized, a part thereof may be taken out and the water content of the part may be measured. To adjust the moisture content, the protein composition may be dried or humidified under predetermined conditions.

By heat-press molding the protein mixture obtained in the first step and the second step, a molding material that does not lose its translucency can be obtained.

(Third step)
The third step is a step of heating and pressurizing the obtained protein composition. For example, in the third step, the protein composition may be introduced into a mold and molded by heating and pressurizing. After the third step, a heat-press molded body (material for molding) is obtained.

The heat-press molded body can be molded using, for example, a pressure molding machine. FIG. 1 is a schematic cross-sectional view of a pressure molding machine that can be used to manufacture a heat-press molded body. The pressure molding machine 10 shown in FIG. 1 includes a mold 2 having a through hole formed and capable of heating, and an upper pin 4 and a lower pin 6 capable of moving up and down in the through hole of the mold 2. Is. In the pressure molding machine 10, the protein composition is introduced into the gap formed by inserting the upper pin 4 or the lower pin 6 into the through hole of the mold 2, and the upper pin 4 is heated while heating the mold 2. By compressing the protein composition with the lower pin 6 and the lower pin 6, a heat-press molded product can be obtained.

FIG. 2 shows a process diagram for obtaining a heat-pressurized molded product, in which (a) is before the introduction of the composition, (b) is immediately after the introduction of the composition, and (c) is the heating and pressurization of the composition. It is a schematic cross-sectional view of the pressure molding machine in the state of being As shown in FIG. 2A, the composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the composition 8a before heating and pressurizing is formed through the through hole. Heat and pressurize inside. The upper pin 4 is lowered until a predetermined pressing force is reached, and heating and pressurization are continued until the composition reaches a predetermined temperature in the state shown in FIG. 2C, and the composition after heating and pressurizing. Obtain 8b. Then, the temperature of the mold 2 is lowered by using a cooler (for example, a spot cooler), and when the molding composition 8b reaches a predetermined temperature, the upper pin 4 or the lower pin 6 is removed from the mold 2 and the contents are removed. Take out things. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lower pin 4 may be lowered and the lower pin 6 may be raised. The removed contents are dried to obtain a molding composition. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lower pin 4 may be lowered and the lower pin 6 may be raised.

The heating is preferably performed at a mold temperature of 80 to 300 ° C., more preferably 100 to 180 ° C., still more preferably 100 to 130 ° C. Pressurization is preferably performed at 5 kN or higher, more preferably 10 kN or higher, and even more preferably 20 kN or higher. Further, after reaching a predetermined heating and pressurizing condition, the time (heat retention condition) for continuing the treatment under that condition is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, still more preferably 5 to 30 minutes.

By changing the shape of the mold used, a heat-press molded body (material for molding) having a desired shape can be obtained. For example, a mold having a special shape may be used to form a shogi or chess piece, a dice, or the like into an uneven shape.

Prior to the second step, a step of adding water to the protein composition may be performed. The amount of water added in the step is preferably 10 to 40 wt%, more preferably 20 to 30 wt% with respect to the mass of the protein.

In that case, as shown in FIG. 2A, the water-containing composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2, but water is added before or after the introduction. Stir on top. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the water-containing composition 8a before heating and pressurizing is penetrated. Heat and pressurize in the hole.

If necessary, the obtained heat-press molded product may be dried. In that case, the drying is preferably allowed to stand in a vacuum and / or in an environment of 100 ° C. for 30 minutes to 3 days. If water is added prior to the second step, the drying is preferably allowed to stand in vacuum and / or in an environment of 100 ° C. for 1 minute to 10 days.

〔protein〕
The protein used in this embodiment will be described below. The protein is a structural protein or a recombinant polypeptide derived from it. Structural proteins are proteins that have a role in constructing biological structures, and are different from functional proteins such as enzymes, hormones, and antibodies. Specific examples thereof include fibroin (for example, spider silk fibroin, silk fibroin, etc.), collagen, resilin, elastin and keratin, and proteins derived from these. The protein may be of natural origin or may be an artificially produced artificial protein. In addition, the amino acid sequence of the artificial protein may be the same as or different from the amino acid sequence of the naturally occurring protein.

The protein is preferably fibroin. The protein is more preferably spider silk fibroin.

Examples of naturally occurring arachnid fibroin include arachnid fibroin produced by spiders such as large spider tube bookmark thread protein, weft protein, and vial-shaped gland protein. Since the large spit tube bookmark thread has a repeating region consisting of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of the spider silk has a characteristic that it does not have a crystalline region but has a repeating region consisting of an amorphous region. The weft thread has a lower stress than the large discharge tube bookmark thread, but has high elasticity.

The large spider canal bookmark thread protein is produced in the large bottle-shaped gland of a spider and has a characteristic of being excellent in toughness. Examples of the large spider thread protein include large bottle-shaped gland spiders MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from the European garden spider (Araneus diadematus). ADF3 is one of the two major bookmark thread proteins of the European garden spider. The spider silk protein derived from ADF3 is relatively easy to synthesize, and has excellent properties in terms of strength and elongation and toughness.

The weft protein is produced in the flagelliform gland of the spider. Examples of the weft protein include flagellar silk protein derived from Nephila clavipes in the United States.

Further examples of spider silk fibroins produced by spiders include spiders belonging to the genus Araneus, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus (Neoscona), spiders belonging to the genus Koonigumomodoki (Pronus genus) such as Koonigumomodoki, spiders belonging to the genus Torinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi Spiders belonging to the genus Spider (Gasteracantha genus) such as Chibusatoge spider, spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogeisekigumo, spiders belonging to the genus Ordgarius, spiders belonging to the genus Ordgarius, spiders belonging to the genus Ar Spiders belonging to the genus Arachunura such as spiders and spiders belonging to the spider, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Acusilas, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora Spider silk produced by spiders belonging to the genus Poltys, spiders belonging to the genus Cyclosa such as spiders, spiders, spiders, spiders, spiders, and spiders belonging to the genus Cholizopes. Proteins, spiders belonging to the genus Tetragnatha, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila such as Joro spiders, spiders belonging to the genus Azumi spiders such as Kinyou spiders (genus Menosira), spiders belonging to the genus Dyschiriognata and spiders belonging to the genus Dyschiriognatha, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Latrodictus, such as the spider, and spiders belonging to the genus Euprostenops, which belong to the family Tetragnathidae. Examples include spider silk proteins produced by mo.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence No. AAC47011 (amino acid sequence), U47856 (base sequence)) ), U37520 (base sequence)), major amplifier spidroin 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein (derived from radinic protein) Numbers AAL32472 (amino acid sequence), AF441245 (base sequence)), major amplifier protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major protein (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC1458.91 (amino acid sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank access) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

The fibroin may be modified fibroin. The modified fibroin is, for example, one in which the amino acid sequence of naturally-derived fibroin is modified based on the amino acid sequence of naturally-derived fibroin (for example, the amino acid sequence is modified by modifying the gene sequence of cloned naturally-derived fibroin). It may be artificially designed and synthesized regardless of naturally occurring fibroin (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). ..

The modified fibroin is, for example, modifying the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. Can be obtained at. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).

The modified fibroin is, for example, a fibroin containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} There may be. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to the (A) n} motif of an amino acid sequence) and an amorphous region (typically to the REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to.), And is represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Further, (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). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of spider, and modified fibroin with a reduced content of glycine residues. (Second modified fibroin), (A) _{reduced content of n} motifs Modified fibroin (third modified fibroin), content of glycine residues, and (A) reduced content of _{n motifs} It has a modified fibroin (fourth modified fibroin), a modified fibroin having a domain sequence containing a region having a locally high hydrophobicity index (fifth modified fibroin), and a domain sequence having a reduced content of glutamine residues. Modified fibroin (sixth modified fibroin) can be mentioned.

The modified fibroin (first modified fibroin) derived from the large spider canal bookmark thread protein produced in the large bottle-shaped gland of the spider is a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. Examples include proteins containing. In the first modified fibroin, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and even more preferably an integer of 10 to 20 in Equation 1. An integer of ~ 16 is even more preferred, an integer of 8-16 is particularly preferred, and an integer of 10-16 is most preferred. m represents an integer of 2 to 300 and may be an integer of 10 to 300. In the first modified fibroin, the number of amino acid residues constituting REP in the formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first modified fibroin, the total number of residues of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _{m is the amino acid residue.} It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 45 can be mentioned.

The first modified fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3. Alternatively, it may be a protein which is an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C-terminal of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first modified fibroin, 90% or more sequence identity with the amino acid sequence shown in (1-i) SEQ ID NO: 4 or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 Examples thereof include modified fibroin containing an amino acid sequence having. The sequence identity is preferably 95% or more.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of the start codon, the His10 tag and the HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.

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

Modified fibroin with a reduced content of glycine residues (second modified fibroin) has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring spider fibroin. Have. The second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in the REP being replaced with another amino acid residue as compared with the naturally occurring spider silk fibroin. Can be done.

The second modified fibroin has a domain sequence of GGX and GPGXX in REP as compared with naturally occurring spider fibroin (where G is a glycine residue, P is a proline residue, and X is an amino acid other than glycine. Residues are shown.) In at least one motif sequence selected from), those having an amino acid sequence corresponding to at least one or a plurality of glycine residues in the motif sequence being replaced with another amino acid residue. It may be.

In the second modified fibroin, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the total motif sequence.

The second modified fibroin contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the domain sequence from the (A) _n motif located closest to the C-terminal side from the above domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The _{number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. 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.

The method of calculating z / w will be described in more detail. First, in the spider silk fibroin (modified fibroin) containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , the domain from the (A) n} motif located closest to the C-terminal side from the domain sequence. The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence except the sequence up to the C-terminal of the 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. Further, for example, when X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 2, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 ( _{excluding the (A) n} motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is modified, for example, from the cloned naturally occurring spider silk fibroin gene sequence by substituting at least part of the nucleotide sequence encoding the glycine residue to encode another amino acid residue. Can be obtained by 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 replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above aspects from the amino acid sequence of naturally occurring spider fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of the glycine residue in the REP with another amino acid residue from the amino acid sequence of the naturally occurring spider fibroin, one or more amino acid residues were further substituted. The amino acid sequence corresponding to the deletion, insertion and / or addition may be modified.

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

As a more specific example of the second modified fibroin, (2-i) the amino acid sequence shown by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or (2-ii) SEQ ID NO: 6, sequence. Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in No. 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10, which corresponds to naturally occurring spider silk fibroin. _{In the amino acid sequence shown by SEQ ID NO: 7, every two (A) n} motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above 4 times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. Further, the value of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.

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

The modified fibroin of (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 12 (amino acid sequence including His tag sequence and hinge sequence).

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

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

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the second modified fibroin comprising the tag sequence, (2-iii) SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or the amino acid sequence set forth in SEQ ID NO: 15 or (2-iv). SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 can be mentioned as a modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 15, or SEQ ID NO: 15.

The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15, are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. And the amino acid sequence shown in SEQ ID NO: 12 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence shown in SEQ ID NO: 9.

The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

The second modified 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.

(A) The modified fibroin (third modified fibroin) having a reduced content of _{n motifs has a reduced domain sequence of (A) n} motifs as compared with naturally occurring spider silk fibroin. Has an amino acid sequence. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of _{at least one or more (A) n motifs as compared with the naturally occurring spider silk fibroin.}

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

The third modified fibroin has a domain sequence of at least 1 to 3 (A) from the N-terminal side to the C-terminal side, one (A) for each _{n motif, as compared with the naturally occurring spider silk fibroin.} It may have an amino acid sequence corresponding to the deletion of the _n-motif.

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

The third modified fibroin may have an amino acid sequence corresponding to the deletion _{of the (A) n} motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , and two adjacent [(A) n} motifs from the N-terminal side to the C-terminal side. -REP] When the number of amino acid residues in the REP of the unit is sequentially compared and the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 1. When x is the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _{n motif-REP] units, which is 11.3, and y is the total number of amino acid residues in the domain sequence.} In addition, 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 with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The method of calculating x / y will be described in more detail with reference to FIG. FIG. 3 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from spider silk fibroin. From the N-terminal side (left side), the domain sequence consists of (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 an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, _{there may be a [(A) n} motif-REP] unit that is not selected. In FIG. 3, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. 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.} The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with a smaller number of amino acid residues is set to 1, the fifth REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

_{In FIG. 3, a set of [(A) n} motif-REP] units in which the ratio of the number of amino acid residues to the other is 1.8 to 11.3 when the one having the smaller number of amino acid residues is 1. Shown by solid line. Hereinafter, such a ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of _{(A) n motif.} is there.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 3, the total value of pattern 1 is the maximum.

Next, 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 fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which _{at least 7 of the (A) n} motifs present in the domain sequence are composed only of alanine residues, the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

The third modified fibroin, for example, lacks one or more of the sequences encoding the _{(A) n} motif so that x / y is 64.2% or more from the cloned naturally occurring spider silk fibroin gene sequence. It can be obtained by losing it. Further, for example, from the amino acid sequence of naturally-derived spider silk fibroin, an amino acid sequence corresponding to the deletion _{of one or more (A) n motifs so that x / y is 64.2% or more is designed.} It can also be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. _{In each case, in addition to the modification corresponding to the deletion of (A) n} motif from the amino acid sequence of naturally occurring spider fibroin, one or more amino acid residues were further substituted, deleted, inserted and /. Alternatively, the amino acid sequence corresponding to the addition may be modified.

As a more specific example of the third modified fibroin, (3-i) the amino acid sequence shown by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18, sequence. Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in No. 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-i) will be described. The amino acid sequence shown by SEQ ID NO: 18 lacks the _{(A) n} motif every other two from the N-terminal side to the C-terminal side from the amino acid sequence shown by SEQ ID NO: 10, which corresponds to the naturally occurring spider silk fibroin. _{It was lost, and one [(A) n} motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 7 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 18. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). This is a sequence in which the His tag is added to the C-terminal of the sequence obtained by repeating the above 4 times.

The value of x / y in the jagged ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 18 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.3%. The values of z / w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When the maximum value of the total value obtained by adding the radix is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

As a more specific example of the third modified fibroin containing the tag sequence, the amino acid sequence set forth in (3-iii) SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 or the (3-iv) sequence. Examples thereof include modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15, are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. And the amino acid sequence shown in SEQ ID NO: 12 (including His tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence shown in SEQ ID NO: 9.

The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third modified 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.

Modified fibroin (fourth modified fibroin) with reduced glycine residue content and (A) _n motif content has a domain sequence of (A) compared to naturally occurring spider fibroin. _In addition to having a reduced content of n motifs, it has an amino acid sequence with a reduced content of glycine residues. The domain sequence of the fourth modified fibroin lacked at least one or more (A) _n motifs as compared to naturally occurring spider fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to the group being replaced with another amino acid residue. That is, the fourth modified fibroin includes the above-mentioned modified fibroin having a reduced content of glycine residue (second modified fibroin) and (A) modified fibroin having a reduced content of _{n motif (third).} It is a modified fibroin that also has the characteristics of modified fibroin). Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

As a more specific example of the fourth modified fibroin, (4-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 9. Specific embodiments of the modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are as described above.

A modified fibroin (fifth modified fibroin) having a domain sequence containing a region having a locally high hydrophobicity index has one or more amino acids in the REP whose domain sequence is compared with that of naturally occurring spider fibroin. Local hydrophobicity index, corresponding to the fact that the residue was replaced with an amino acid residue with a high hydrophobicity index and / or one or more amino acid residues with a large hydrophobicity index were inserted in the REP. It may have an amino acid sequence containing a large region of.

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 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). It is more preferably a residue.

In the fifth modified fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a high hydrophobicity index as compared with naturally occurring spider fibroin, and / or 1 in REP. Or, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, further, one or more amino acid residues are substituted, deleted, or inserted as compared with the naturally occurring spider fibroin. And / or there may be a modification of the amino acid sequence corresponding to the addition.

The fifth modified fibroin is hydrophobic, for example, from the gene sequence of cloned naturally occurring spider silk fibroin to one or more hydrophilic amino acid residues in REP (eg, amino acid residues having a negative hydrophobicity index). It can be obtained by substituting an amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in REP were replaced 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 insertion of a residue and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic in the REP. In addition to the modification corresponding to the insertion of the sex amino acid residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , from the (A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the above domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more is defined as p. _{When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydropathic index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs contained in the sequence A. The average value of the hydrophobicity index is obtained by dividing the total 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 consecutive 4 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 hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 3, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

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

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, hydrophobic) in the REP so that the amino acid sequence of the cloned naturally occurring spider silk fibroin satisfies the above p / q condition. Replacing an amino acid residue with a negative sex index) with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues in the REP. Can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can be obtained by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally-derived spider silk fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring spider fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is further performed. May be good.

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

As a specific example of the fifth modified fibroin, (5-i) the amino acid sequence shown by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 21 can be mentioned.

The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 22 is an amino _{acid sequence in which alanine residues in the (A) n} motif of naturally occurring spider fibroin are contiguous and deleted so that the number of contiguous alanine residues is five. Is. The amino acid sequence shown by SEQ ID NO: 19 is the amino acid sequence shown by SEQ ID NO: 22, in which two amino acid sequences (VLI) consisting of 3 amino acid residues are inserted every other REP, and the amino acid sequence is shown by SEQ ID NO: 22. A part of the amino acid on the C-terminal side is deleted so that the molecular weight of the amino acid sequence is almost the same as that of the amino acid sequence. In the amino acid sequence shown in SEQ ID NO: 23, two alanine residues are inserted on the C-terminal side of _{each (A) n} motif with respect to the amino acid sequence shown in SEQ ID NO: 22, and a part of glutamine (Q) remains. The group was replaced with a serine (S) residue, and some amino acids on the C-terminal side were deleted so as to have substantially the same molecular weight as the amino acid sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence shown in SEQ ID NO: 23. The amino acid sequence shown by SEQ ID NO: 21 is the amino acid sequence shown by SEQ ID NO: 23, in which two amino acid sequences (VLI) consisting of 3 amino acid residues are inserted every other REP.

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

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most on the C-terminal side (A) _n. Amino acids 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 sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

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

As a more specific example of the fifth modified fibroin comprising a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24, sequence. Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by No. 25 or SEQ ID NO: 26.

The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 are the amino acid sequences shown in SEQ ID NO: 12 (His tag) at the N-terminal of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. The modified fibroin of (5-iv) is also a protein containing the domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most on the C-terminal side (A) _n. Amino acids 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 sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth modified 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.

Modified fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified fibroin) has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring spider silk fibroin. ..

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

When the sixth modified fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif is usually 1% or more, may be 5% or more, and is 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.

In the present specification, 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) Most C-terminal in spider silk fibroin containing a domain sequence represented by _{n motif. In all REPs included in the sequence excluding the sequence from the (A) n} motif located on the side to the C-terminal of the domain sequence from the domain sequence, the total number of GPGXX motifs contained in the region is tripled (3). That is, (corresponding to the total number of G and P in the GPGXX motif) is s, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding the motif.

In the calculation of the GPGXX motif content, "the _{sequence obtained by excluding the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is targeted at "the most C-terminal side". _{The sequence from (A) n} motif to the C-terminal of the domain sequence located in (A) (sequence corresponding to REP) may include a sequence having a low correlation with the sequence characteristic of spider silk fibroin, and m When is small (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing the domain sequence of spider silk fibroin. The calculation method of the GPGXX motif content rate will be specifically described with reference to FIG. First, in the domain sequence of spider silk fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “positioned on the most C-terminal side”. (A) Since the _{sequence from the n} motif to the C end of the domain sequence is excluded from the domain sequence ”(the sequence shown by“ region A ”in FIG. 5), s is calculated. The number of GPGXX motifs is 7, and s is 7 × 3 = 21. _{Similarly, all REPs are "sequences obtained by removing the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 5). Since it is contained in (.), The total number t of amino acid residues in all REPs excluding the _{(A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150.} Next, s / t (%) can be calculated by dividing s by t, which is 21/150 = 14.0% in the case of fibroin in FIG.

The sixth modified fibroin has a glutamine residue content of preferably 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%. ..

In the present specification, the "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) Most C-terminal in spider fibroin containing a domain sequence represented by _{n motif.} In all REPs included in the sequence (the sequence corresponding to "region A" in FIG. 5) excluding the sequence from _{the (A) n} motif located on the side to the C-terminal of the domain sequence from the domain sequence, the region is included. _Let u be the total number of glutamine residues contained, and remove the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence, and further (A) of all REPs excluding the _{n motif.} The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted or replaced with one or more glutamine residues in the REP as compared to naturally occurring spider fibroin. It may have a corresponding amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

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

The sixth modified fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, 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) Most C-terminal in spider fibroin containing a domain sequence represented by _{n motif. In all REPs included in the sequence obtained by excluding the sequence from the (A) n} motif located on the side to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 5), the region _Let v be the sum of the hydrophobicity indexes of each amino acid residue, and the sequence from the (A) n motif located most on the C-terminal side to the C-terminal of the domain sequence was removed from the domain sequence, and (A) _{n motif was further removed.} The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues in all REPs. In the calculation of the hydrophobicity of REP, the _{reason for targeting "the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring spider fibroin, and / or one or more glutamines in REP. In addition to the modification corresponding to the substitution of the residue with another amino acid residue, there is a modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. May be good.

The sixth modified fibroin, for example, deletes one or more glutamine residues in REP from the cloned naturally occurring spider silk fibroin gene sequence and / or one or more glutamine residues in REP. Can be obtained by substituting with another amino acid residue. Also, for example, one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally occurring spider fibroin, and / or one or more glutamine residues in REP have been replaced with 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 a more specific example of the sixth modified fibroin, (6-i) 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. A modified fibroin containing the indicated amino acid sequence, or (6-ii) 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. Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence.

The modified fibroin of (6-i) will be described.

Amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) is a fibroin naturally occurring Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415) based on the nucleotide sequence and amino acid sequence of, _{(A) n} Amino acids have been modified to improve productivity, such as increasing the number of consecutive alanine residues to 5 in the amino acid sequence in which the alanine residues are continuous in the motif. On the other hand, since Met-PRT410 did not modify the glutamine residue (Q), the glutamine residue content was about the same as the glutamine residue content of naturally occurring fibroin.

The amino acid sequence (M_PRT888) shown in SEQ ID NO: 27 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with VL.

In the amino acid sequence (M_PRT965) shown in SEQ ID NO: 28, all QQs in Met-PRT410 (SEQ ID NO: 7) are replaced with TS, and the remaining Qs are replaced with A.

The amino acid sequence (M_PRT889) shown in SEQ ID NO: 29 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and substituting the remaining Qs with I.

In the amino acid sequence (M_PRT916) shown in SEQ ID NO: 30, all QQs in Met-PRT410 (SEQ ID NO: 7) are replaced with VI, and the remaining Qs are replaced with L.

The amino acid sequence (M_PRT918) shown in SEQ ID NO: 31 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with VF and substituting the remaining Qs with I.

_{The amino acid sequence (M_PRT525) shown in SEQ ID NO: 34 has two alanine residues inserted in a region (A 5} ) where alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 7), and the molecular weight of Met-PRT410 Two domain sequences on the C-terminal side were deleted, and 13 glutamine residues (Q) were replaced with serine residues (S) or proline residues (P) so as to be substantially the same as.

The amino acid sequence (M_PRT699) shown in SEQ ID NO: 32 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 34) with VL.

The amino acid sequence (M_PRT698) shown in SEQ ID NO: 33 is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 34) with VL and substituting the remaining Qs with I.

The amino acid sequence shown by SEQ ID NO: 43 (Met-PRT966) contains all the QQs in the amino acid sequence shown by SEQ ID NO: 9 (the amino acid sequence before the amino acid sequence shown by SEQ ID NO: 42 is added to the C-terminal) VF. And the remaining Q is replaced with I.

The amino acid sequences shown in 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 and SEQ ID NO: 43 all have a glutamine residue content of 9% or less. Yes (Table 2).

The modified fibroin of (6-i) comprises the amino acid sequence 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. There may be.

The modified fibroin of (6-ii) is 90% or more of the amino acid sequence 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. It contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-ii) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

As a more specific example of the sixth modified fibroin containing the tagged sequence, (6-iii) 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: 41 or Modified fibroin containing the amino acid sequence set forth in SEQ ID NO: 44, or (6-iv) 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: 41 or SEQ ID NO: A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 44 can be mentioned.

The amino acid sequences shown by 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: 41 and SEQ ID NO: 44 are SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively. , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 added to the N-terminal of the amino acid sequence shown by SEQ ID NO: 12 (including His tag sequence and hinge sequence). It is a thing. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and 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: 41. And the amino acid sequences shown in SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin (6-iii) comprises the amino acid sequence set forth in 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: 41 or SEQ ID NO: 44. There may be.

The modified fibroin of (6-iv) is 90% or more of the amino acid sequence represented by 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: 41 or SEQ ID NO: 44. It contains an amino acid sequence having the sequence identity of. The modified fibroin of (6-iv) is also a domain 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 sequence. The sequence identity is preferably 95% or more.

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

The sixth modified 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 fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the above-mentioned characteristics.

The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. Hydrophobic modified fibroin is the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then the sum divided by the total number of amino acid residues (mean HI) is 0 or more. Is a modified fibroin. The hydrophobicity index is as shown in Table 1. Further, the hydrophilic modified fibroin is a modified fibroin having the above-mentioned average HI of less than 0.

Examples of the hydrophobic modified fibroin include the above-mentioned sixth modified fibroin. More specific examples of the hydrophobically modified fibroin include the amino acid sequences set forth in 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. Examples thereof include modified fibroins containing the amino acid sequences set forth in 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.

Examples of the hydrophilic modified fibroin include the above-mentioned first modified fibroin, second modified fibroin, third modified fibroin, fourth modified fibroin, and fifth modified fibroin. More specific examples of the hydrophilic spider silk protein include the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 9. 11, amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15, 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: Examples thereof include the amino acid sequence shown by 15, SEQ ID NO: 19, SEQ ID NO: 20 or modified fibroin containing the amino acid sequence shown by SEQ ID NO: 21.

The above-mentioned modified fibroin can be used alone or in combination of two or more.

The modified fibroin expresses the nucleic acid, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding modified fibroin, or a method of chemically synthesizing the nucleic acid. The chemical synthesis method of nucleic acid is not particularly limited. For example, based on the amino acid sequence information of spider silk protein obtained from NCBI's web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.) The gene can be chemically synthesized by a method of linking oligonucleotides automatically synthesized by the above method by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal may be synthesized.

The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the desired modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector containing a promoter at a position capable of autonomous replication in a host cell, integration into a host chromosome, and transcription of a nucleic acid encoding modified fibroin is preferably used.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

When a prokaryote such as a bacterium is used as a host, the expression vector is a vector that can replicate autonomously in the prokaryote and at the same time contains a promoter, a ribosome binding sequence, a nucleic acid encoding a modified fibroin, and a transcription termination sequence. It is preferable to have. It may contain genes that control promoters.

Examples of prokaryotes include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Philum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, as a vector into which a nucleic acid encoding modified fibroin is introduced, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples thereof include pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

When a eukaryote is used as a host, examples of the vector into which the nucleic acid encoding the modified fibroin is introduced include YEp13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like can be mentioned.

As a method for expressing nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

The modified fibroin can be produced, for example, by culturing a transformed host in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing the transformed host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and the host can be efficiently cultured. Either a natural medium or a synthetic medium may be used as long as it can be used.

The carbon source may be any assimilated by the host, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolyzate, and organic acids such as acetic acid and propionic acid. , And alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used.

As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

Modified fibroin produced by a transformed host can be isolated and purified by methods commonly used for protein isolation and purification. For example, when the modified fibroin is expressed in a lysed state in the cells, after the culture is completed, the host cells are collected by centrifugation, turbid in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or manton gaulin. Crush the host cells with a homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting-out method using sulfuric acid, a desalting method, or an organic solvent Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

As the above chromatography, column chromatography using phenyl-toyopearl (Tosoh), DEAE-toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cell, crushing it, and centrifuging it. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.

When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

[Recombinant cells expressing proteins]
The method for producing modified fibroin will be described in detail below. The modified fibroin of interest comprises, for example, a gene transformed by a host transformed with an expression vector having a gene sequence encoding a structural protein and one or more regulatory sequences operably linked to the gene sequence. It can be produced by expressing it.

The method for producing the gene encoding the desired modified fibroin is not particularly limited. For example, a gene encoding a naturally occurring structural protein can be utilized to produce the gene by amplification and cloning by a polymerase chain reaction (PCR) or the like, or by chemical synthesis. The chemical synthesis method of the gene is also not particularly limited. For example, based on the amino acid sequence information of the structural protein obtained from the NCBI web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized in (1) by PCR or the like. At this time, in order to facilitate purification and confirmation of the protein, a gene encoding a polypeptide having an amino acid sequence consisting of a start codon and a His10 tag added to the N-terminal of the above amino acid sequence may be synthesized.

The regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the protein of interest may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. The expression vector preferably contains a promoter at a position capable of autonomous replication in the host cell, integration into the host chromosome, and transcription of the gene encoding the recombinant polypeptide of interest. Used for.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Preferred examples of prokaryotes include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.

Examples of the vector into which the gene encoding the desired modified fibroin is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002). -238569A) and the like can be mentioned.

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.).

Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

Examples of the vector include YEp13 (ATCC37115) and YEp24 (ATCC37051).

As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like can be mentioned.

As a method for expressing a gene by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

The desired modified fibroin is produced, for example, by culturing a host transformed with the expression vector according to the present invention in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. be able to. The method for culturing the host according to the present invention in a culture medium can be carried out according to the method usually used for culturing the host.

When the host according to the present invention is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium of the host according to the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host. However, either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the host.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates such as molasses, starch and starch hydrolysate containing them, acetic acid, propionic acid and the like. Organic acids and alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used.

As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The modified fibroin according to the present invention can be isolated and purified by a method usually used for protein isolation and purification. For example, when the modified fibroin is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher, a French press, or a manton. Crush the host cells with a gaulin homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting out method using ammonium sulfate or the like, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cell, crushing it, and centrifuging it. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.

When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

[Antibacterial]
When the translucent substance is an antibacterial agent (for example, an antibacterial metal compound), antibacterial properties can be imparted to the heat-pressed molded product. Antibacterial properties can be evaluated using, for example, the method described in ISO 22196. The antibacterial activity value means a value indicating the difference in logarithmic number of viable bacteria after inoculation and culture of bacteria between the antibacterial processed product and the non-antibacterial processed product, and can be calculated by the following formula. If the viable cell count is less than 0.40, the logarithmic average value is calculated as 0.40.

R = (Ut-Uo)-(At-Uo) = At-Uo
In the formula, R indicates the antibacterial activity value, Uo indicates the average logarithmic value of the viable cell count immediately after inoculation of the unprocessed test piece, and Ut indicates the logarithmic value of the viable cell count 24 hours after the unprocessed test piece. At shows the mean value of the logarithmic value of the viable cell count after 24 hours of the antibacterial processed test piece.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.

(Preparation of artificial spider silk powder)
(1) Preparation of structural protein expression strain The base sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes are obtained from the GenBank web database and then produced. Amino acids shown in SEQ ID NO: 39 are substituted, inserted and deleted for the purpose of improving sex, and the amino acid sequence shown in SEQ ID NO: 12 (tag sequence and hinge sequence) is added to the N-terminal. A recombinant fibroin having a sequence (hereinafter, also referred to as “PRT918”) was designed.

Next, the gene encoding PRT918 was outsourced for synthesis. As a result, a gene having an NdeI site immediately upstream of the 5'end and an EcoRI site immediately downstream of the 3'end was obtained. The gene was cloned into a cloning vector (pUC118), treated with restriction enzymes NdeI and EcoRI, and recombined into a protein expression vector pET-22b (+).

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT918 obtained above. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours, and then the same culture solution was added to _{100 mL of the seed culture medium shown in Table 4 so that the OD 600 was 0.005.} The temperature of the culture solution was kept at 30 ° C., and the cells were further cultured in a flask for about 15 hours until _{the OD 600 reached 5, to obtain a seed culture solution.}

The obtained seed culture solution was added to a jar fermenter to which 500 mL of the production medium shown in Table 5 was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., controlled to be constant at pH 6.9, and the culture was carried out so that the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. As the antifoaming agent, ADEKANOLG-295S (manufactured by ADEKA Corporation) was used.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / L, Yeast Extract 120 g / L) was added at a rate of 1 mL / min. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, a 1 M aqueous solution of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the target protein size depending on the addition of IPTG.

(3) Purification of Structural Protein The cells collected 2 hours after the addition of IPTG 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 phenylmethylsulfonyl fluoride (PMSF), and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl buffer, pH 7.0) to a concentration of 100 mg / mL. Stirred with a stirrer at 60 ° C. for 30 minutes 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 agglutinated protein obtained after dialysis was recovered by centrifugation, water was removed by a lyophilizer, and the lyophilized powder was recovered.

(Example 1)
1% by weight of silver nanoparticles (Merck, Silver nanopowder, <150 nm particle size) and protein powder (PRT918) were added to a 6 M aqueous guanidine thiocyanate solution, and the mixture was stirred at room temperature for 18 hours. Then, acetone was added and it was allowed to stand for 1 hour. Centrifugation was carried out at 9000 rpm for 10 minutes to remove the solvent, and a composition containing protein and silver nanoparticles was obtained.

The obtained composition (powder) is filled in a stainless steel jig and heated and pressurized at a temperature of 150 ° C. and a pressure of 30 MPa for 5 minutes using a hot press machine. ) Was obtained. The shape of the obtained heat-press molded body was a rectangular parallelepiped of 25 mm × 35 mm × 0.7 mm.

(Comparative Example 1)
Protein powder (PRT918) was filled in a stainless steel jig without adding silver nanoparticles, and heated and pressurized at a temperature of 150 ° C. and a pressure of 30 MPa for 5 minutes using a hot press machine to prepare a heat-pressed molded product. Obtained. The shape of the heat-press molded body was a rectangular parallelepiped of 25 mm × 35 mm × 0.7 mm.

(Comparative Example 2)
0.3% by weight of silver nanoparticles and protein powder (PRT918) were dry-mixed using an attritamill. The obtained composition (powder) was filled in a stainless steel jig and heated and pressurized at a temperature of 150 ° C. and a pressure of 30 MPa for 5 minutes using a hot press machine to obtain a heat-pressurized molded product. The shape of the heat-press molded body was a rectangular parallelepiped of 25 mm × 35 mm × 0.7 mm.

[Evaluation of translucency]
The materials of Examples 1 and Comparative Examples 1 and 2 were irradiated with white light, and the brightness of the white light was measured using a spectrophotometer (manufactured by Konica Minolta, model number: CM-5).

The results are shown in Table 6. The light transmittance of the material of Comparative Example 2 obtained by the dry mixing method was 0.94%, whereas the light transmittance of the material of Example 1 was 45.6%. The light transmittance of the material of Comparative Example 1 containing no silver nanoparticles was 82.2%.

In addition, each material was placed on the graph paper and visually observed to evaluate whether the straight line of the graph paper under the material could be seen through. FIG. 6 is a photograph of each material placed on graph paper taken from above. Comparative Example 1 containing no silver nanoparticles was yellow, and a straight line was clearly visible. On the other hand, Comparative Example 2 was brown, and no straight line was visible. In Example 1, a straight line could be visually recognized even though it contained more silver nanoparticles than in Comparative Example 2.

[Evaluation of antibacterial properties]
The antibacterial properties of Example 1 and Comparative Examples 1 and 2 against Staphylococcus aureus NBRC 12732 and Escherichia coli NBRC 3972 were evaluated according to the method described in ISO22196. The antibacterial activity value was calculated by the following formula. When the viable cell count was less than 0.40, the logarithmic average value was calculated as 0.40.

R = (Ut-Uo)-(At-Uo) = At-Uo
In the formula, R indicates the antibacterial activity value, Uo indicates the average logarithmic value of the viable cell count immediately after inoculation of the unprocessed test piece, and Ut indicates the logarithmic value of the viable cell count 24 hours after the unprocessed test piece. At shows the mean value of the logarithmic value of the viable cell count after 24 hours of the antibacterial processed test piece. As the unprocessed test piece, a polyethylene film not subjected to antibacterial processing was used.

The results for Staphylococcus aureus are shown in Table 7, and the results for Escherichia coli are shown in Table 8. The material of Comparative Example 1 showed almost no antibacterial property, but the material of Example 1 showed antibacterial property. For the material of Example 1, the antibacterial degree against Staphylococcus aureus was 99.99949%, and the antibacterial degree against Escherichia coli was 0.9997%.

2 ... Mold, 4 ... Upper pin, 6 ... Lower pin, 8a ... Composition before heating and pressurizing, 8b ... Composition after heating and pressurizing, 10 ... Pressurized molding machine.

Claims (14)

The first step of dispersing a translucent substance in a solution in which a protein is dissolved in a solvent to obtain a dispersion liquid, and
The second step of removing the solvent from the obtained dispersion to obtain a protein mixture, and
A method for producing a heat-pressed molded article, which comprises a third step of heating and pressurizing the obtained protein mixture. The method for producing a heat-press molded product according to claim 1, wherein the solution in the first step further contains a solubilizer. The solubilizer is selected from the group consisting of dimethyl sulfoxide, 1,1,1,3,3,3-hexafluoro-2-propanol, urea, guanidine hydrochloride, guanidine thiocyanate, sodium iodide and perchlorate. The method for producing a heat-pressurized molded product according to claim 2, which comprises at least one of these. The method for producing a heat-press molded product according to claim 2, wherein the solubilizer is an aqueous solution containing at least one selected from the group consisting of urea, guanidine hydrochloride and guanidine thiocyanate. The method for producing a heat-press molded product according to any one of claims 1 to 4, wherein the protein is fibroin. The method for producing a heat-press molded product according to claim 5, wherein the fibroin is a modified spider silk fibroin. The production of the heat-press molded article according to any one of claims 1 to 6, wherein the translucent substance contains at least one selected from the group consisting of an antibacterial agent, a strengthening agent, a flame retardant and a nucleating agent. Method. The method for producing a heat-press molded product according to any one of claims 1 to 6, wherein the translucent substance contains an antibacterial metal compound. The method for producing a heat-press molded product according to claim 8, wherein the antibacterial metal compound is silver nanoparticles. A heat-press molded product containing a protein and a translucent substance and having a light transmittance of 40% or more. The heat-press molded product according to claim 10, wherein the protein is fibroin. The heat-press molded product according to claim 11, wherein the fibroin is modified spider silk fibroin. A composition comprising a protein, a solvent, a translucent substance, the protein being solvent in the solvent, and the translucent substance dispersed in the solvent. 13. The composition of claim 13, wherein the protein is modified spider silk fibroin.

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