PRODUCTION METHOD OF PROTEIN FIBER, PRODUCTION METHOD OF PROTEIN FIBER FABRIC, AND PRODUCTION METHOD OF PROTEIN FIBER SHRINKPROOF TREATMENT

Abstract

The purpose of the present invention is to provide a method whereby a protein fiber, in which shrinkage is more certainly suppressed upon first contact with water after manufactured and the occurrence of curling is also suppressed, can be manufactured. The method according to the present invention for manufacturing a protein fiber comprises: an earlier shrinking step for shrinking a material protein fiber, which has been stretched but not wound up, in a relaxed state; a winding step for winding up the material protein fiber, which has been treated in the earlier shrinking step, to thereby give a wound matter; and a later shrinking step for shrinking the material protein fiber, which has been treated in the winding step, in a relaxed state.

Description

[Technical field]

[0001] The present invention relates to a method for producing protein fibers, a method for producing a protein fiber fabric, and a method for shrink-proofing protein fibers. [Background technology]

[0002] Traditionally, protein fibers such as silk and wool have been widely used as materials for clothing and bedding, taking advantage of their characteristics such as excellent touch and heat retention. Recently, spider silk, which has excellent strength and high elasticity and high toughness, has attracted attention, and research has been actively conducted to commercialize artificial spider silk with the characteristics of spider silk. The artificial spider silk is not only used for clothing, but also for various materials such as industrial materials and medical materials that require high strength and high toughness. Unlike synthetic fibers, all of these protein fibers are biodegradable and require little energy for production and processing, so demand is expected to increase in response to the recent increase in environmental conservation awareness.

[0003] Some protein fibers shrink when they come into contact with moisture (e.g., when immersed in water or hot water or exposed to a high humidity environment). If such unexpected changes in length due to shrinkage of protein fibers occur, particularly when the fibers come into contact with moisture for the first time after production, there is concern that various problems may occur as a result. For example, if an unexpected change in length occurs when a protein fiber is exposed to a high humidity environment during storage immediately after production, or when a treatment using moisture such as washing and wet heat setting is performed for the first time after production, not only may the workability and quality be reduced, but post-processing may even be adversely affected.

[0004] Under such circumstances, various methods for shrink-proofing protein fibers have been disclosed. For example, Patent Document 1 discloses a method for producing protein fibers, which includes a stretching step of contacting protein raw material fibers containing protein with liquid or steam to shrink or stretch the protein raw material fibers, and a drying step of drying the protein raw material fibers that have been subjected to the stretching step while adjusting them to a desired length. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 164020 Summary of the Invention [Problem to be solved by the invention]

[0006] In general in-line (continuous) spinning, tension in the fiber axis direction is inevitably applied to the protein fiber in order to move the protein fiber from the upstream side to the downstream side. According to the studies of the present inventors, it was found that when shrink-proofing is performed during in-line spinning, residual stress occurs in the produced protein fiber due to tension in the fiber axis direction (e.g., tension during winding) that is inevitably generated after the shrink-proofing, and that this residual stress causes shrinkage when the protein fiber is brought into contact with moisture for the first time after production, and that the shrinkage is particularly remarkable when the protein fiber is brought into contact with high-temperature moisture.

[0007] In order to more reliably suppress shrinkage when the fiber comes into contact with water for the first time after production, it is possible to perform a shrink-proofing treatment on the in-line spun protein fiber while minimizing tension in the fiber axis direction so as to minimize the generation of residual stress. However, in this case, shrinkage occurs when the protein fiber comes into contact with water. This causes a problem in that the applications for which the obtained protein fiber can be used are limited.

[0008] Therefore, the present invention aims to provide a method for producing protein fibers that are more reliably prevented from shrinking when they come into contact with water for the first time after production, and that also suppress the occurrence of crimping.The present invention also aims to provide a method for producing a protein fiber fabric that is more reliably prevented from shrinking when they come into contact with water for the first time after production, and that also suppresses the occurrence of crimping.The present invention also aims to provide a method for shrink-proofing protein fibers that is more reliably prevented from shrinking when they come into contact with water for the first time after production, and that also suppresses the occurrence of crimping. [Means for solving the problem]

[0009] The present invention relates to, for example, the following inventions. [1] A method for producing protein fibers, comprising: a pre-shrinkage step of shrinking a protein fibril in a relaxed state after stretching and before being wound up; a winding step of winding the protein fibril that has undergone the pre-shrinkage step to obtain a wound product; and a post-shrinkage step of shrinking the protein fibril that has undergone the winding step in a relaxed state. [2] The manufacturing method according to [1], wherein the post-shrinkage step is a wet shrinkage step in which the protein fibrils that have been subjected to the winding step are brought into contact with liquid or steam in a relaxed state to shrink them, or a dry shrinkage step in which the protein fibrils that have been subjected to the winding step are heated in a relaxed state to shrink them. [3] The manufacturing method according to [2], wherein the wet shrinking step is a step of immersing the protein fibrils that have been subjected to the winding step in a relaxed state in a liquid to cause them to shrink. [4] The manufacturing method described in [2] or [3], wherein the liquid or vapor has polarity. [5] The manufacturing method described in [4], wherein the liquid is water and the vapor is water vapor. [6] The method according to any one of [1] to [5], wherein the protein fibrils contain structural proteins. [7] The method of producing a fiber according to [6], wherein the structural protein is a modified fibroin. [8] The manufacturing method described in [7], wherein the modified fibroin is modified spider silk fibroin. [9] A method for producing a protein fiber fabric, comprising a step of producing fabric using the protein fibers obtained by the manufacturing method described in any one of [1] to [8].

[10] The method for shrink-proofing a protein fiber includes a pre-shrinkage step of shrinking the protein fiber in a relaxed state after stretching and before being wound up, a winding step of winding the protein fiber that has been through the pre-shrinkage step to obtain a wound product, and a post-shrinkage step of shrinking the protein fiber that has been through the winding step in a relaxed state.

[11] The shrink-proof processing method according to

[10] , wherein the post-shrinkage step is a wet shrinkage step in which the protein fibers that have been subjected to the winding step are brought into contact with liquid or steam in a relaxed state to cause them to shrink, or a dry shrinkage step in which the protein fibers that have been subjected to the winding step are heated in a relaxed state to cause them to shrink. Effect of the Invention

[0010] According to the present invention, it is possible to provide a method for producing protein fibers in which shrinkage upon contact with water for the first time after production is more reliably suppressed and the occurrence of crimping is also suppressed. According to the present invention, it is also possible to provide a method for producing a protein fiber fabric in which shrinkage upon contact with water for the first time after production is more reliably suppressed and the occurrence of crimping is also suppressed. According to the present invention, it is also possible to provide a shrink-proof processing method for protein fibers in which shrinkage upon contact with water for the first time after production is more reliably suppressed and the occurrence of crimping is also suppressed. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram illustrating an apparatus for producing protein fibrils according to one embodiment. [Diagram 2]FIG. 1 is a schematic diagram illustrating an apparatus for producing protein fibrils according to one embodiment. [Diagram 3] 3 is a diagram showing a speed adjusting means and a temperature adjusting means that can be provided in the high-temperature heating furnace in FIG. 2. [Figure 4] FIG. 2 is a diagram illustrating a schematic diagram of a protein fiber production apparatus (post-shrinkage device) according to one embodiment. [Diagram 5] FIG. 2 is a diagram illustrating a schematic diagram of a protein fiber production apparatus (post-shrinkage device) according to one embodiment. [Figure 6] 1A is a schematic diagram of an apparatus for producing a protein fiber (post-shrinkage apparatus) according to one embodiment of the present invention; FIG. 1B is a schematic diagram of an apparatus for obtaining a skein from a wound material of protein fibrils; FIG. 1C is a schematic diagram of a wet-type shrinkage apparatus; and FIG. 1C is a schematic diagram of a dry-type shrinkage apparatus. [Figure 7] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Figure 8] FIG. 1 shows the distribution of z / w (%) values ​​of naturally derived fibroin. [Figure 9] FIG. 1 shows the distribution of x / y (%) values ​​of naturally derived fibroin. [Figure 10] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. [Figure 11] FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings as needed, but the present invention is not limited to the following embodiments. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and duplicated explanations will be omitted as appropriate.

[0013] [Method of producing protein fiber] The method for producing a protein fiber according to this embodiment includes a pre-shrinkage step of shrinking the protein fibril in a relaxed state after stretching and before winding, a winding step of winding the protein fibril that has undergone the pre-shrinkage step to obtain a wound product, and a post-shrinkage step of shrinking the protein fibril that has undergone the winding step in a relaxed state.

[0014] The method for producing a protein fiber according to the present embodiment includes a post-shrinkage step, which more reliably suppresses shrinkage of the resulting protein fiber when it comes into contact with water for the first time after production. This effect is particularly noticeable when the temperature of the water in contact is high. Furthermore, the method for producing a protein fiber according to the present embodiment includes a pre-shrinkage step in addition to a post-shrinkage step, which sufficiently suppresses the occurrence of crimping of the resulting protein fiber when it comes into contact with water.

[0015] As used herein, "contracting protein fibrils in a relaxed state" includes not only the case where the protein fibrils are naturally contracted in a relaxed state without any tension applied thereto, but also the case where the protein fibrils are contracted under tension to such an extent that the amount of contraction of the protein fibrils is substantially the same as the amount of contraction when the protein fibrils are naturally contracted in a relaxed state without any tension applied thereto. Note that, as used herein, "substantially the same" refers to the same amount or value, or an amount or value that is close thereto, and an amount or value that is close thereto refers to an amount that can produce an effect similar to that obtained when the amount or value is the same.

[0016] [Pre-shrinkage process] The pre-shrinkage step is a step of shrinking the protein fibrils in a relaxed state after stretching and before winding. The pre-shrinkage step may be performed before the winding step, and may be performed, for example, on protein fibrils that have been spun using a dope (spinning stock solution) by a known spinning method (dry spinning, wet spinning, dry-wet spinning, melt spinning, etc.) and then stretched by a known stretching method (wet heat stretching, dry heat stretching, etc.).

[0017] Methods for shrinking protein fibrils in a relaxed state include, for example, a method including a wet shrinkage step in which protein fibrils in a relaxed state are brought into contact with liquid or steam to shrink them (wet shrinkage method), and a method including a dry shrinkage step in which protein fibrils in a relaxed state are heated to shrink them (dry shrinkage method).

[0018] In the wet shrinkage method, protein fibrils are brought into contact with liquid or steam, and the protein fibrils shrink without the application of external force. This is thought to occur when the liquid or steam penetrates between or into the fibers of protein fibrils that have residual stress due to stretching, thereby relieving the residual stress.

[0019] The type of liquid or steam used in the wet shrinkage method is not particularly limited as long as it can naturally shrink the protein fibrils by contacting with the protein fibrils. Examples of the liquid or steam include liquid or steam having polarity. Examples of the liquid having polarity include water, tetrahydrofuran, acetonitrile, acetone, and methanol, and among these, water is preferably used. This is because water is not only inexpensive and easy to handle, but also can shrink the protein fibrils more quickly and reliably by contacting with the protein fibrils. Examples of the steam having polarity include the steam of the liquid having polarity described above, and among these, water vapor is preferably used. This is because water vapor can shrink the protein fibrils more quickly and reliably by contacting with the protein fibrils, like water.

[0020] In the wet shrinkage method, it is preferable to bring the liquid into contact with the protein fibrils in a heated state. This can advantageously further shorten the shrinkage time of the protein fibrils. From the same viewpoint, it is more preferable that the liquid used in the wet shrinkage method is hot water (heated water) rather than water.

[0021] When the wet shrinkage method is carried out using a heated liquid, the temperature of the liquid may be lower than the temperature at which the protein contained in the protein fibrils decomposes or the protein fibrils are thermally damaged. However, in consideration of the handleability of the liquid and the workability of the wet shrinkage method, the upper limit of the liquid temperature is preferably lower than the boiling point. From the viewpoint of obtaining a sufficient effect of shortening the shrinkage time, the lower limit of the liquid temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. When water is used as the liquid, the upper limit of the water temperature is preferably 90°C or lower, and more preferably 80°C or lower. The lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. During the contact of the liquid with the protein fibrils, the temperature of the liquid may be constant, or may be varied to a predetermined temperature.

[0022] In the wet shrinkage method, the method of bringing the liquid or steam into contact with the protein fibrils is not particularly limited. Examples of such methods include a method of immersing the protein fibrils in a liquid, a method of spraying a liquid at room temperature or in a state of heated steam or the like onto the protein fibrils, and a method of exposing the protein fibrils to a high humidity environment filled with steam. Among these methods, the method of immersing the protein fibrils in a liquid is preferred because it can effectively shorten the shrinkage time and can simplify the shrinkage processing equipment.

[0023] The time for contacting the protein fibrils with the liquid is not particularly limited, and may be, for example, 1 minute or more. The time may be 10 minutes or more, 20 minutes or more, or 30 minutes or more. There is no particular upper limit to the time, but from the viewpoint of shortening the time of the production process and eliminating the risk of decomposition (hydrolysis, etc.) of the protein fibrils, the time may be, for example, 120 minutes or less, 90 minutes or less, or 60 minutes or less.

[0024] The wet shrinkage method may further include a step of drying the protein fibrils (drying step) following the wet shrinkage step.

[0025] The drying method in the drying step is not particularly limited, and may be, for example, natural drying or forced drying using drying equipment. The drying temperature is not particularly limited as long as it is lower than the temperature at which the protein is thermally damaged, but is generally within the range of 20 to 150°C, preferably within the range of 40 to 120°C, and more preferably within the range of 60 to 100°C. Within such a temperature range, the protein fibrils can be dried more quickly and efficiently without causing thermal damage to the protein. The drying time is appropriately selected depending on the drying temperature, etc., and, for example, a time is adopted that can eliminate the influence of overdrying of the protein fibrils on the quality and physical properties of the woven fabric.

[0026] The dry shrinkage method includes a dry shrinkage step of heating and shrinking the protein fibrils in a relaxed state. The dry shrinkage step may include a step of heating the protein fibrils (heating step) and a step of shrinking the heated protein fibrils in a relaxed state (relaxation and shrinkage step).

[0027] The heating temperature of the protein fibrils is preferably equal to or higher than the softening temperature of the protein used in the protein fibrils. The softening temperature of the protein in this specification is the temperature at which the protein fibrils start to shrink due to stress relaxation. In the heat relaxation shrinkage at or higher than the softening temperature of the protein, the protein fibrils shrink to a degree that cannot be obtained by simply removing water from the protein fibrils. The heating temperature is preferably 80°C or higher, more preferably 180°C to 280°C, even more preferably 200°C to 240°C, and even more preferably 220°C to 240°C. From the viewpoint of the elongation of the protein fibrils after the heat treatment, the heating time is preferably 60 seconds or less, more preferably 30 seconds or less, and even more preferably 5 seconds or less. It is considered that the length of the heating time does not have a significant effect on the stress of the protein fibrils.

[0028] [Winding process] The winding step is a step of winding the protein fibrils that have been subjected to the pre-shrinking step to obtain a wound product. The winding step may be performed immediately after the pre-shrinking step, or may be performed after performing another step (e.g., a drying step for drying the protein fibrils) after the pre-shrinking step.

[0029] Fig. 1 is a schematic diagram showing an example of a protein fibril production apparatus (spinning apparatus). The spinning apparatus 100 shown in Fig. 1 includes, in this order from the upstream side, an extrusion apparatus 1, a coagulation bath 20, a washing bath 21, a water bath 46 for carrying out a pre-shrinkage step, and a drying apparatus 4. Stretching is carried out in the coagulation bath 20 and / or the washing bath 21.

[0030] The extrusion device 1 has a storage tank 7, in which a dope (spinning stock solution) 6 is stored. A coagulation liquid 11 (e.g., methanol) is stored in a coagulation bath 20. The dope 6 is extruded from a nozzle 9 provided with an air gap 19 between the nozzle 9 and the coagulation liquid 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded dope 6 is supplied into the coagulation liquid 11 through the air gap 19. In the coagulation liquid 11, the solvent is removed from the dope 6, and the protein is coagulated. The coagulated protein is guided to a washing bath 21, washed with a washing liquid 12 in the washing bath 21, and then sent to a water bath 46 by a first nip roller 13 and a second nip roller 14 installed in the washing bath 21. At this time, for example, if the rotation speed of the second nip roller 14 is set faster than the rotation speed of the first nip roller 13, the protein fibrils are stretched at a ratio corresponding to the rotation speed ratio. After leaving the cleaning bath 21, the protein fibrils stretched in the cleaning solution 12 are shrunk in a relaxed state when passing through a water bath 46 (pre-shrinkage step), then dried when passing through a drying device 4, and then wound up by a winder (winding step). In this way, the protein fibrils are finally obtained as a wound product 5 wound up by the winder. Reference numerals 18a to 18g denote thread guides.

[0031] Relay rollers 50 and 52 that relay the protein fibrils are disposed on the upstream and downstream sides of the water bath 46. The water bath 46 has a heater 54, and liquid (hot water) 47 heated by the heater 54 is contained in the water bath 46. A press roller 56 is also disposed in the water bath 46 in a state in which it is immersed in the liquid (hot water) 47. As a result, the protein fibrils sent from the upstream side are wrapped around the press roller 56 and are immersed in the liquid (hot water) 47 while being pressed down, and travel toward the downstream side in the water bath 46. The immersion time of the protein fibrils in the hot water 47 is appropriately controlled according to the traveling speed of the protein fibrils.

[0032] In the water bath 46, the delivery speed of the protein fibrils by the second nip roller 14 and the winding speed of the protein fibrils by the winder are controlled, so that the protein fibrils in a relaxed state naturally shrink when they come into contact with the liquid (hot water) 47. For example, when using protein fibrils that shrink about 20% in the fiber axial direction when immersed in the liquid (hot water) 47 in a relaxed state, the protein fibrils can be naturally shrunk in a relaxed state in the liquid (hot water) 47 by setting the winding speed and delivery speed so that the latter is less than 80% of the former.

[0033] The coagulation liquid 11 may be any solution capable of removing the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and 2-propanol, and acetone. The coagulation liquid 11 may contain water as appropriate. The temperature of the coagulation liquid 11 is preferably 0 to 30° C. The distance over which the coagulated protein passes through the coagulation liquid 11 (effectively, the distance from the thread guide 18a to the thread guide 18b) may be a length that allows efficient removal of the solvent, and is, for example, 200 to 500 mm. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes. In addition, drawing (pre-drawing) may be performed in the coagulation liquid 11.

[0034] In addition, the stretching performed when obtaining protein fibrils may be, for example, the above-mentioned pre-stretching in the coagulation bath 20 and stretching in the cleaning bath 21, as well as wet heat stretching and dry heat stretching.

[0035] The wet heat drawing can be carried out in hot water, in a solution of hot water with an organic solvent added, or under steam heating. The temperature may be, for example, 50 to 90° C., and preferably 75 to 85° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

[0036] The hot drawing can be carried out using an electric tubular furnace, a hot plate, etc. The temperature may be, for example, 140° C. to 270° C., and preferably 160° C. to 230° C. In the hot drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.

[0037] The wet heat stretching and the dry heat stretching may be performed alone, or may be performed in multiple stages or in combination. That is, the wet heat stretching and the dry heat stretching may be appropriately combined, for example, the first stage stretching may be performed by wet heat stretching and the second stage stretching by dry heat stretching, or the first stage stretching may be performed by wet heat stretching, the second stage stretching may be performed by wet heat stretching, and the third stage stretching may be performed by dry heat stretching.

[0038] The lower limit of the final draw ratio, relative to the undrawn yarn (or pre-drawn yarn), is preferably any one of more than 1, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more, and the upper limit is preferably 40 or less, 30 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less.

[0039] Fig. 2 is a schematic diagram showing another example of a protein fibril production apparatus (spinning apparatus). Fig. 3 is a diagram showing a speed adjusting means and a temperature adjusting means that can be provided in the high-temperature heating furnace in Fig. 2. The spinning apparatus 200 shown in Fig. 2 is used for a dry shrinkage method, and includes a spinning apparatus (spinning means) 25 that spins protein fibrils, and a high-temperature heating relaxation apparatus 60 that heats the protein fibrils spun by the spinning apparatus 25 at a high temperature in a relaxed state to cause them to shrink. In the spinning apparatus 200, a protein fibril spinning process and a dry shrinkage process that heats the protein fibrils at a high temperature in a relaxed state to cause them to shrink are successively performed.

[0040] The spinning device 25 is, for example, a spinning device for dry-wet spinning, and includes an extrusion device 1, a coagulation device 2, a washing device 3, and a drying device 4, in this order from the upstream side. The extrusion device 1 has a storage tank 7, in which a dope liquid (raw spinning liquid) 6 is stored. The coagulation device 2 has a coagulation bath 20, in which a coagulation liquid 11 (e.g., methanol) is stored. The dope liquid 6 is extruded from a nozzle 9 provided with an air gap 19 between the nozzle 9 and the coagulation liquid 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded dope liquid 6 is supplied into the coagulation liquid 11 through the air gap 19. In the coagulation liquid 11, the solvent is removed from the dope liquid 6, and the protein is coagulated.

[0041] The washing device 3 has a washing bath 21, in which a washing liquid 12 (e.g., water) is stored. The protein coagulated in the coagulation bath 20 is guided to the washing bath 21 and washed with the washing liquid 12. The protein is sent to the drying device 4 by a first nip roller 13 and a second nip roller 14 installed in the washing bath 21. For example, the rotation speed of the second nip roller 14 is set faster than the rotation speed of the first nip roller 13, so that the protein fibrils are stretched at a ratio according to the rotation speed ratio. Note that 18a to 18e are thread guides.

[0042] The stretching carried out in the washing bath 21 when obtaining protein fibrils may be so-called wet heat stretching, which is carried out in warm water or in a solution of warm water to which an organic solvent or the like has been added. The temperature for this wet heat stretching may be, for example, 0 to 90° C., preferably 20 to 70° C., and more preferably 30 to 60° C. The stretching ratio of the unstretched yarn (or pre-stretched yarn) in the wet heat stretching may be, for example, 1 to 10 times, or 2 to 8 times.

[0043] The protein fibrils stretched in the cleaning solution 12 are dried when they leave the cleaning bath 21 and pass through the drying device 4. The drying device 4 has, for example, a dry heat type drying oven 17. A delivery roller 31 and a take-up roller 32 are provided in the drying oven 17. The protein fibrils are allowed to stay in the drying oven 17 for a predetermined stay time by the delivery roller 31 and the take-up roller 32, and are then sent to the high-temperature heating relaxation device 60. In the drying oven 17, for example, the rotation speed of the take-up roller 32 may be set faster than the rotation speed of the delivery roller 31, so that the protein fibrils are stretched at a ratio according to the rotation speed ratio. A heater (not shown) is provided in the drying oven 17. The temperature in the drying oven 17, i.e., the drying temperature of the protein fibrils, is, for example, 80°C. An oiling device 30 may be provided between the cleaning device 3 and the drying device 4.

[0044] The high-temperature heating relaxation device 60 is provided downstream of the spinning device 25 in the running direction of the protein fibrils. The high-temperature heating relaxation device 60 has, for example, a dry-heat type high-temperature heating furnace 63. A feed roller (feed-out means) 61 and a take-up roller (take-up means) 62 are provided inside the high-temperature heating furnace 63. Both the feed roller 61 and the take-up roller 62 are cylindrical, and the protein fibrils are wound around their peripheral surfaces. The protein fibrils are kept in the high-temperature heating furnace 63 for a predetermined residence time by the feed roller 61 and the take-up roller 62, and are then taken up by a winder.

[0045] In the high-temperature heating furnace 63, for example, the rotation speed of the take-up roller 62 is set slower than the rotation speed of the feed roller 61, so that the protein fibrils are relaxed at a rate corresponding to the rotation speed ratio. That is, the feed roller 61 is configured to continuously deliver the protein fibrils at a predetermined delivery speed. The take-up roller 62 is configured to continuously take up the protein fibrils delivered by the feed roller 61 at a take-up speed slower than the delivery speed of the feed roller 61. With the feed roller 61 and take-up roller 62 configured in this way, the protein fibrils are overfed, and a relaxed state of the protein fibrils occurs between the feed roller 61 and the take-up roller 62.

[0046] The high-temperature heating relaxation device 60 will be described in more detail with reference to FIG. 3. A speed controller 66 is connected to the feed roller 61 and the winding roller 62. For example, the speed controller 66 is connected to a drive motor (not shown) provided on each of the feed roller 61 and the winding roller 62. The speed controller 66 is a computer configured with hardware such as a CPU, a ROM, and a RAM, and software such as a program stored in the ROM. The speed controller 66 adjusts the feed speed and / or the winding speed as described above by controlling both or either one of the feed roller 61 and the winding roller 62. That is, the speed controller 66 constitutes a speed adjustment means that adjusts at least one of the feed speed of the feed roller 61 and the winding speed of the winding roller 62. The speed controller 66 can adjust the feed speed and / or the winding speed so that the feed speed by the feed roller 61 is an arbitrary ratio (relaxation ratio) within a range of 1 to 3 times the winding speed by the winding roller 62.

[0047] In the high-temperature heating furnace 63 of the high-temperature heating relaxation device 60, for example, a high-temperature heater 64 is installed. A temperature controller 67 is connected to the high-temperature heater 64. The temperature controller 67 is a computer composed of hardware such as a CPU, a ROM, and a RAM, and software such as a program stored in the ROM. The temperature controller 67 controls the high-temperature heater 64 to adjust the temperature in the high-temperature heating furnace 63. Information on the temperature in the high-temperature heating furnace 63 may be input to the temperature controller 67 from a temperature sensor (not shown) provided in the high-temperature heating furnace 63, and the temperature controller 67 may control the high-temperature heater 64 based on the information. The temperature controller 67 constitutes a temperature adjustment means for adjusting the heating temperature of the protein fibrils in the high-temperature heating furnace 63. The temperature controller 67 controls the high-temperature heater 64 so that the heating temperature in the high-temperature heating furnace 63 is higher than the heating temperature in the drying furnace 17 of the drying device 4. The temperature controller 67 can adjust the heating temperature of the protein fibrils to any temperature within the range of, for example, 80 to 300°C.

[0048] In this embodiment, the speed controller 66 and the temperature controller 67 are shown separately, but the present invention is not limited to this. For example, the speed controller 66 and the temperature controller 67 may be integrated into an integrated controller, or a controller that controls the entire spinning apparatus 200 may be provided with functions corresponding to the speed controller 66 and the temperature controller 67.

[0049] The high-temperature heating and relaxation device 60 comprises a heating means for heating the protein fibrils and a relaxation and contraction means for shrinking the heated protein fibrils in a relaxed state. In other words, the high-temperature heating and relaxation device 60 is a device that serves both as a heating means and a relaxation and contraction means. In the dry shrinkage method using the high-temperature heating and relaxation device 60, a heating step and a relaxation and contraction step are performed simultaneously. As shown in FIG. 2, for example, a winder is provided downstream of the high-temperature heating and relaxation device 60 in the running direction of the protein fibrils. After undergoing a shrinkage treatment in the high-temperature heating and relaxation device 60, the protein fibrils are wound up by the winder to obtain a wound product 5.

[0050] In the relaxation contraction step, the relaxation ratio is preferably more than 1, more preferably 1.4 or more, even more preferably 1.7 or more, and particularly preferably 2 or more. The relaxation ratio is understood as, for example, the ratio of the delivery speed to the winding speed of the protein fibrils.

[0051] [Post-shrinkage process] In the post-shrinkage step, the protein fibrils that have been through the winding step are shrunk in a relaxed state. By carrying out the post-shrinkage step, the shrinkage of the obtained protein fibers when they come into contact with water for the first time after production can be more reliably suppressed. As will be shown in the examples described later, it has been found that when the post-shrinkage step is omitted, shrinkage occurs when the fibers come into contact with water for the first time after production, and the shrinkage is particularly remarkable when the fibers come into contact with water at high temperatures. This is considered to be due to the fact that residual stress is generated in the protein fibrils due to the inevitable tension in the fiber axis direction being applied to the protein fibrils during winding in the winding step. The post-shrinkage step is carried out for the purpose of relaxing all residual stress in the protein fibrils, including the residual stress generated in the winding step.

[0052] In the post-shrinkage step, examples of the method for shrinking the protein fibrils in a relaxed state include a method including a wet shrinkage step in which the protein fibrils are brought into contact with liquid or steam in a relaxed state to shrink them (wet shrinkage method), and a method including a dry shrinkage step in which the protein fibrils are heated in a relaxed state to shrink them (dry shrinkage method). Specific aspects of the wet shrinkage method and the dry shrinkage method are as described in the pre-shrinkage step.

[0053] Fig. 4 is a schematic diagram showing an example of a protein fiber production apparatus (post-shrinkage apparatus). The post-shrinkage apparatus 300 shown in Fig. 4 is used in a wet shrinkage method, and includes a feed roller 42 that sends out protein fibrils, a winder 44 that winds up the protein fibers, a water bath 46 that performs the post-shrinkage process, and a dryer 48 that performs the drying process.

[0054] More specifically, the feed roller 42 can be fitted with a wound material of protein fibrils 36, and can continuously and automatically feed the protein fibrils 36 from the wound material of protein fibrils 36 by the rotation of an electric motor (not shown). The winder 44 can continuously and automatically wind up the protein fibers 38, which are produced through a post-shrinkage process and a drying process after being fed from the feed roller 42, by the rotation of an electric motor (not shown). Here, the feed speed of the protein fibrils 36 by the feed roller 42 and the winding speed of the protein fibers 38 by the winder 44 can be controlled independently of each other.

[0055] The water bath 46 and the dryer 48 are arranged side by side, respectively, on the upstream side and the downstream side in the feeding direction of the protein fibrils 36, between the feed roller 42 and the winder 44. Note that the post-shrinkage device 300 shown in FIG. 4 has relay rollers 50 and 52 that relay the protein fibrils 36 traveling from the feed roller 42 toward the winder 44.

[0056] The water bath 46 has a heater 54, and liquid (hot water) 47 heated by the heater 54 is contained in the water bath 46. A press roller 56 is also placed in the water bath 46 in a state in which it is immersed in the liquid (hot water) 47. As a result, the protein fibrils 36 sent out from the feed roller 42 are wrapped around the press roller 56 in the water bath 46 and run toward the winder 44 while being immersed in the liquid (hot water) 47 in a pressed state. The immersion time of the protein fibrils 36 in the liquid (hot water) 47 is appropriately controlled according to the running speed of the protein fibrils 36.

[0057] The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 can wrap the protein fibrils 36 that have left the water bath 46 and are traveling toward the winder 44. In this way, the protein fibrils 36 immersed in the liquid (hot water) 47 in the water bath 46 are heated and dried by the pair of hot rollers 58 in the dryer 48, and then are further sent toward the winder 44.

[0058] When the target protein fiber 38 is produced using the post-shrinking device 300 having such a structure, first, a wound product 5 of protein fibrils 36 spun using, for example, the spinning device 100 shown in FIG. 1 is attached to the feed roller 42. Next, the protein fibrils 36 are continuously sent out from the feed roller 42 and immersed in a liquid (hot water) 47 in a water bath 46. At this time, by controlling the ratio of the sending speed of the feed roller 42 and the winding speed of the winder 44, the protein fibrils naturally shrink in a relaxed state due to contact with the liquid (hot water) 47. For example, when a protein fibril that shrinks by about 20% in the fiber axis direction when immersed in the liquid (hot water) 47 in a relaxed state is used, the protein fibrils can be naturally shrunk in a relaxed state in the liquid (hot water) 47 by setting the winding speed and sending speed to a speed that is 80% or less faster than the former.

[0059] Next, the protein fibrils 36 shrunk in the liquid (hot water) 47 in the water bath 46 are heated by a pair of hot rollers 58 of a dryer 48. This dries the shrunken protein fibrils 36 to form protein fibers 38. The obtained protein fibers 38 are then wound by a winder 44 to obtain a wound product of the protein fibers 38. Note that instead of the pair of hot rollers 58, the protein fibrils 36 may be dried using drying equipment consisting of only a heat source, such as a known dry heat plate.

[0060] Fig. 5 is a schematic diagram showing another example of a protein fiber production apparatus (post-shrinkage apparatus). The post-shrinkage apparatus 400 shown in Fig. 5 is used in a dry shrinkage method, and includes a feed roller 42 that sends out protein fibrils, a winder 44 that winds up the protein fibers, and a high-temperature heating relaxation apparatus 60 that performs the post-shrinkage process.

[0061] The feed roller 42 is adapted to be able to receive a wound product of the protein fibrils 36. The high-temperature heating relaxation device 60 has a configuration as described in FIG. 3. That is, the high-temperature heating relaxation device 60 has, for example, a dry-heat type high-temperature heating furnace 63. A feed roller (feeding means) 61 and a take-up roller (take-up means) 62 are provided in the high-temperature heating furnace 63. The feed roller 61 and the take-up roller 62 are both cylindrical, and the protein fibrils are wound around their peripheral surfaces. The protein fibrils stay in the high-temperature heating furnace 63 for a predetermined stay time by the feed roller 61 and the take-up roller 62, and are contracted in a relaxed state, and are then taken up by the winder 44.

[0062] Figure 6 is a schematic diagram of yet another example of a protein fiber production apparatus (post-shrinkage apparatus). Figure 6(a) is a schematic diagram of an apparatus for obtaining a skein from a wound material of protein fibrils. Figure 6(b) is a schematic diagram of a wet-type shrinkage apparatus. Figure 6(c) is a schematic diagram of a dry-type shrinkage apparatus.

[0063] The post-shrinking device shown in Fig. 6 has a feed roller 42 on which a wound material of protein fibrils 36 can be attached. First, for example, a wound material 5 of protein fibrils 36 spun using the spinning device 100 shown in Fig. 1 is attached to the feed roller 42. Next, the protein fibrils 36 are sent out from the feed roller 42 and wound around a support 71 to obtain a skein-shaped protein fibril 70 (Fig. 6(a)). The skein-shaped protein fibrils 70 are not under tension and are in a relaxed state.

[0064] Next, the skein-like protein fibrils 70 are shrunk in a relaxed state by a wet shrinkage method or a dry shrinkage method. Figure 6(b) is a schematic diagram of an apparatus used in the wet shrinkage method, and Figure 6(c) is a schematic diagram of an apparatus used in the dry shrinkage method.

[0065] The apparatus shown in FIG. 6(b) has a water bath 46, a heater 54 capable of heating the liquid in the water bath 46, and a liquid (e.g., water or hot water) 47 contained in the water bath 46. In the apparatus shown in FIG. 6(b), skein-shaped protein fibrils 70 are immersed in the heated liquid (e.g., hot water) 47. This allows the protein fibrils to shrink in a relaxed state. The preferred aspects of the liquid (type, temperature, etc.) and the preferred aspects of the immersion time are as explained in the pre-shrinkage step (wet shrinkage method).

[0066] The device shown in Fig. 6(c) has a high-temperature heater 64. In the device shown in Fig. 6(c), a skein-shaped protein fibril 70 is held above the high-temperature heater 64. This heats the protein fibril and causes it to shrink in a relaxed state. The temperature of the high-temperature heater 64 is preferably set so that the temperature of the protein fibril (heating temperature) is equal to or higher than the softening temperature of the protein used in the protein fibril. The preferred aspects of the heating temperature and heating time are as described in the pre-shrinkage step (dry shrinkage method).

[0067] [Protein fibrils and protein fibers] The protein fiber produced according to the production method of the present invention, or the protein fibril which is an intermediate product, contains a protein as a main component. There is no particular limitation on the protein, and any protein can be used. Examples of the protein include natural proteins and recombinant proteins (artificial proteins). Examples of the recombinant protein include any protein that can be produced on an industrial scale, such as proteins that can be used for industrial purposes, proteins that can be used for medical purposes, and structural proteins. Specific examples of proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccination, vaccines, antigen-binding proteins, immunostimulating proteins, allergens, full-length antibodies, or antibody fragments or derivatives. Specific examples of structural proteins include spider silk, silkworm silk, keratin, collagen, elastin, and resilin, as well as proteins derived therefrom. The protein used is preferably modified fibroin, and more preferably modified spider silk fibroin.

[0068] (Modified fibroin) The modified fibroin according to this embodiment is a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif. The modified fibroin may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and the C-terminal sequence are typically, but are not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin, and consist of about 100 amino acid residues.

[0069] As used herein, "modified fibroin" refers to artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be a fibroin whose amino acid sequence is identical to that of naturally occurring fibroin. As used herein, "naturally occurring fibroin" is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif.

[0070] A "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as is, or one that has had its amino acid sequence modified based on the amino acid sequence of naturally occurring fibroin (for example, one that has had its amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has been artificially designed and synthesized independent of naturally occurring fibroin (for example, one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence).

[0071] As used herein, the term "domain sequence" refers to an amino acid sequence that produces a crystalline region (typically corresponding to the (A)n motif in the amino acid sequence) and an amorphous region (typically corresponding to REP in the amino acid sequence) specific to fibroin, and means an amino acid sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif. Here, the (A)n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. The number of amino acid residues in the (A)n motif may be an integer of 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. Furthermore, the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A)n motifs present in the domain sequence may be composed of only alanine residues. REP represents an amino acid sequence composed 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 (A)n motifs present in multiple locations may be the same amino acid sequence as each other or different amino acid sequences. The REPs present in multiple locations may be the same amino acid sequence as each other or different amino acid sequences.

[0072] The modified fibroin according to the present embodiment can be obtained, for example, by modifying the amino acid sequence of the cloned gene sequence of naturally occurring fibroin by, for example, substituting, deleting, inserting, and / or adding one or more amino acid residues. Substitution, deletion, insertion, and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as site-specific mutagenesis. Specifically, it can be performed according to the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0073] Naturally derived fibroin is a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif, and specific examples include fibroins produced by insects or arachnids.

[0074] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as hornet silk proteins excreted by the larvae of the Japanese hornet (Vespa simillima xanthoptera).

[0075] A more specific example of fibroin produced by an insect is, for example, silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0076] Examples of fibroin produced by spiders include spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as the Japanese orb spider, the Japanese garden orb spider, the red orb spider, the green orb spider, and the like; spiders belonging to the genus Neoscona, such as the Japanese orb spider, the Japanese house orb spider, and the Japanese orb spider; spiders belonging to the genus Pronus, such as the Japanese orb spider; spiders belonging to the genus Cyrtarachne, such as the Japanese orb spider and the Japanese orb spider; spiders belonging to the genus Togegumo and the Japanese orb spider; Spiders of the genus Gasteracantha, spiders of the genus Ordgarius such as the Orbweaver Spider and the Orbweaver Spider, spiders of the genus Argiope such as the Orbweaver Spider, the Orbweaver Spider and the Orbweaver Spider, spiders of the genus Arachnura such as the Orbweaver Spider, spiders of the genus Acusilas such as the Breeding Spider, the Orbweaver Spider and the Orbweaver Spider, spiders of the genus Cytophorus such as the Orbweaver Spider, the Orbweaver Spider and the Orbweaver Spider, spider silk proteins produced by spiders belonging to the genus Poltys such as the Poltys spider, spiders belonging to the genus Cyclosa such as the Japanese bush spider, the four-headed bush spider, the round bush spider and the black bush spider, and spiders belonging to the genus Chorizopes such as the Japanese bush spider, as well as spiders belonging to the genus Tetragnatha such as the long-legged spider, the long-legged spider, the Japanese bush spider and the scaled long-legged spider, spiders belonging to the genus Orbweaver spider, Spiders of the genus Leucauge, such as the medium-sized orb-weaver spider and the small orb-weaver spider; spiders of the genus Nephila, such as the orb-weaver spider and the giant orb-weaver spider; spiders of the genus Menosira, such as the golden spider; spiders of the genus Dyschiriognatha, such as the long-legged spider; spiders of the genus Latrodectus, such as the black widow spider, the redback spider, the gray widow spider, and the three-spotted widow spider;and spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag, etc.

[0077] More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenBank accession numbers CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

[0078] More specific examples of naturally derived fibroins include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequence information registered in NCBI GenBank, sequences that include INV as DIVISION can be confirmed by extracting sequences with spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" as keywords in DEFINITION, sequences with a specific product character string in CDS, and sequences with a specific character string in TISSUE TYPE in SOURCE.

[0079] The modified fibroin in this embodiment may be modified silk fibroin (a silk protein produced by silkworms with a modified amino acid sequence) or modified spider silk fibroin (a spider silk protein produced by spiders with a modified amino acid sequence).

[0080] Specific examples of modified fibroins include a modified fibroin derived from the major ampullate gland protein produced in spiders (first modified fibroin), a modified fibroin having a domain sequence with a reduced content of glycine residues (second modified fibroin), a modified fibroin having a domain sequence with a reduced content of (A)n motifs (third modified fibroin), a modified fibroin having a reduced content of glycine residues and a reduced content of (A)n motifs (fourth modified fibroin), a modified fibroin having a domain sequence including a region with a locally high hydrophobicity index (fifth modified fibroin), and a modified fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified fibroin).

[0081] The first modified fibroin includes a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. In the first modified fibroin, the number of amino acid residues in the (A)n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, even more preferably an integer of 10 to 20, even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. In the first modified fibroin, the number of amino acid residues constituting REP in formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, even more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by formula 1: [(A)n motif-REP]m is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more, of the total number of amino acid residues.

[0082] The first modified fibroin may be a polypeptide comprising an amino acid sequence unit represented by formula 1: [(A)n motif-REP]m and having a C-terminal sequence which is an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 or an amino acid sequence having 90% or more homology to an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.

[0083] The amino acid sequence shown in SEQ ID NO:1 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminus of the amino acid sequence of ADF3 (GI:1263287, NCBI), the amino acid sequence shown in SEQ ID NO:2 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:3 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0084] A more specific example of the first modified fibroin is (1-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The sequence identity is preferably 95% or more.

[0085] The amino acid sequence shown in SEQ ID NO:4 is an amino acid sequence of ADF3 with an amino acid sequence (SEQ ID NO:5) consisting of an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus, in which the repeat region at positions 1 to 13 is increased approximately twice and mutated so that translation terminates at amino acid residue 1154. The amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO:4 is identical to the amino acid sequence shown in SEQ ID NO:3.

[0086] The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO:4.

[0087] The second modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of glycine residues compared to naturally-derived fibroin. It can be said that the second modified fibroin has an amino acid sequence in which at least one or more glycine residues in REP have been replaced with another amino acid residue compared to naturally-derived fibroin.

[0088] The second modified fibroin may have an amino acid sequence in which, compared to a naturally occurring fibroin, at least one glycine residue in at least one motif sequence selected from GGX and GPGXX (wherein G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP is replaced with another amino acid residue in at least one or more of the motif sequences.

[0089] In the second modified fibroin, the proportion of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more relative to the total motif sequence.

[0090] The second modified fibroin may have an amino acid sequence having a domain sequence represented by formula 1: [(A)n motif-REP]m, in which z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, and w is the total number of amino acid residues in the sequence excluding the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence from the domain sequence, and z / w is 30% or more, 40% or more, 50% or more, or 50.9% or more. The number of alanine residues relative to the total number of amino acid residues in the (A)n motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

[0091] The second modified fibroin is preferably one in which one glycine residue of the GGX motif is replaced with another amino acid residue to increase the content of the amino acid sequence consisting of XGX. The second modified fibroin preferably has a content of the amino acid sequence consisting of GGX in the domain sequence of 30% or less, more preferably 20% or less, even more preferably 10% or less, even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the calculation method for the content (z / w) of the amino acid sequence consisting of XGX below.

[0092] The calculation method of z / w will be explained in more detail. First, in a fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by formula 1: [(A)n motif-REP]m, an amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no overlap), z is 50×3=150. In addition, for example, in the case of an amino acid sequence consisting of XGXGX, when there is an X (a central X) contained in two XGX, the overlap is deducted from the calculation (in the case of XGXGX, it is 5 amino acid residues). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in Figure 7, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A)n motif located at the most C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

[0093] Here, z / w in naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types are spider-derived fibroins) were extracted. From all the extracted fibroins, z / w was calculated by the above-mentioned calculation method from the amino acid sequence of naturally derived fibroin that contains a domain sequence represented by formula 1: [(A)n motif-REP]m and has an amino acid sequence content of GGX in the fibroin of 6% or less. The results are shown in FIG. 8. The horizontal axis of FIG. 8 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 8, z / w in all naturally derived fibroins is less than 50.9% (the highest is 50.86%).

[0094] In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, even more preferably 70% or more, and even more preferably 80% or more. There is no particular upper limit to z / w, but it may be, for example, 95% or less.

[0095] The second modified fibroin can be obtained, for example, by modifying the gene sequence of a cloned naturally-derived fibroin by substituting at least a part of the base sequence encoding a glycine residue to encode another amino acid residue. In this case, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z / w is 50.9% or more. Alternatively, for example, an amino acid sequence satisfying the above-mentioned aspects may be designed from the amino acid sequence of a naturally-derived fibroin, and a nucleic acid encoding the designed amino acid sequence may be chemically synthesized to obtain the second modified fibroin. In either case, in addition to the modification equivalent to substituting the glycine residue in REP with another amino acid residue from the amino acid sequence of a naturally-derived fibroin, the amino acid sequence may be modified by substituting, deleting, inserting and / or adding one or more amino acid residues.

[0096] The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, or a tryptophan (W) residue, or a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, or a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, or a glutamine (Q) residue, and even more preferably a glutamine (Q) residue.

[0097] More specific examples of the second modified fibroin include (2-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0098] The modified fibroin (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally derived fibroin with GQX. The amino acid sequence shown in SEQ ID NO: 7 is obtained by deleting every third (A)n motif from the N-terminus side to the C-terminus side of the amino acid sequence shown in SEQ ID NO: 6, and further inserting one [(A)n motif-REP] before the C-terminus sequence. The amino acid sequence shown in SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminus side of each (A)n motif of the amino acid sequence shown in SEQ ID NO: 7, further replacing some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminus side so that the molecular weight is approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO: 7 is repeated four times (however, several amino acid residues on the C-terminal side of the region have been replaced), to which a specific hinge sequence and His tag sequence have been added at the C-terminus.

[0099] The z / w value in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 46.8%. The z / w values ​​in the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the x / y values ​​in the jagged ratio (described later) of 1:1.8 to 11.3 in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.

[0100] The modified fibroin (2-i) may consist of the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0101] The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0102] The modified fibroin (2-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and has a z / w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

[0103] The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows for isolation, immobilization, detection, visualization, etc. of the modified fibroin.

[0104] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). The His tag is a short peptide consisting of about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by metal chelating chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 11 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0105] Furthermore, 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.

[0106] Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, the modified fibroin can be easily purified with high specificity.

[0107] Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with the protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

[0108] More specific examples of modified fibroins containing a tag sequence include (2-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0109] The amino acid sequences shown in SEQ ID NO:16 (PRT313), SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0110] The modified fibroin (2-iii) may consist of the amino acid sequence shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0111] The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0112] The modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and preferably has a z / w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

[0113] The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0114] The third modified fibroin has an amino acid sequence in which the content of (A)n motifs is reduced compared to naturally-occurring fibroin, and the domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to the deletion of at least one or more (A)n motifs compared to naturally-occurring fibroin.

[0115] The third modified fibroin may have an amino acid sequence corresponding to a naturally occurring fibroin in which 10 to 40% of the (A)n motif has been deleted.

[0116] The third modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of at least one (A)n motif out of every one to three (A)n motifs from the N-terminus to the C-terminus, compared to naturally occurring fibroin.

[0117] The third modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of at least two consecutive (A)n motifs from the N-terminus to the C-terminus, and one (A)n motif repeated in this order, compared to naturally occurring fibroin.

[0118] The third modified fibroin may have an amino acid sequence in which the domain sequence is such that at least every third (A)n motif is deleted from the N-terminus to the C-terminus.

[0119] The third modified fibroin may have an amino acid sequence that includes a domain sequence represented by formula 1: [(A)n motif-REP]m, and when the number of amino acid residues of the REP of two adjacent [(A)n motif-REP] units is compared from the N-terminus side to the C-terminus side, and the number of amino acid residues of the REP with the smaller number of amino acid residues is set to 1, the maximum value of the sum of the number of amino acid residues of the two adjacent [(A)n motif-REP] units in which the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 is set to x, and the total number of amino acid residues of the domain sequence is set to y, the ratio of x / y may be 20% or more, 30% or more, 40% or more, or 50% or more. The number of alanine residues relative to the total number of amino acid residues in the (A)n motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

[0120] The method of calculating x / y will be explained in more detail with reference to Fig. 7. Fig. 7 shows the domain sequence of modified fibroin excluding the N-terminal sequence and the C-terminal sequence. The domain sequence has the following sequence from the N-terminus (left side): (A)n motif-first REP (50 amino acid residues)-(A)n motif-second REP (100 amino acid residues)-(A)n motif-third REP (10 amino acid residues)-(A)n motif-fourth REP (20 amino acid residues)-(A)n motif-fifth REP (30 amino acid residues)-(A)n motif.

[0121] Two adjacent [(A)n motif-REP] units are selected in sequence from the N-terminus to the C-terminus so that there is no overlap. At this time, there may be an [(A)n motif-REP] unit that is not selected. FIG. 7 shows pattern 1 (comparison of the first REP with the second REP, and the third REP with the fourth REP), pattern 2 (comparison of the first REP with the second REP, and the fourth REP with the fifth REP), pattern 3 (comparison of the second REP with the third REP, and the fourth REP with the fifth REP), and pattern 4 (comparison of the first REP with the second REP). There are other selection methods as well.

[0122] Next, for each pattern, the number of amino acid residues of each REP in the two adjacent selected [(A)n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the other REP to the number of amino acid residues of the one with the fewer amino acid residues set to 1. For example, when comparing the first REP (50 amino acid residues) with the second REP (100 amino acid residues), when the first REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the second REP is 100 / 50=2. Similarly, when comparing the fourth REP (20 amino acid residues) with the fifth REP (30 amino acid residues), when the fourth REP with the fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the fifth REP is 30 / 20=1.5.

[0123] In Fig. 7, a pair of [(A)n motif-REP] units in which the ratio of the number of amino acid residues of the other is 1.8 to 11.3 when the number of amino acid residues of the other is set to 1 is shown by a solid line. In this specification, this ratio is called the jagged ratio. A pair of [(A)n motif-REP] units in which the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 when the number of amino acid residues of the other is set to 1 is shown by a dashed line.

[0124] In each pattern, the total number of amino acid residues in the two adjacent [(A)n motif-REP] units shown by solid lines is added together (not only the number of amino acid residues in REP, but also the number of amino acid residues in the (A)n motif). The total values ​​thus added are then compared, and the total value (maximum of the total values) of the pattern with the maximum total value is designated as x. In the example shown in FIG. 7, the total value of pattern 1 is the maximum.

[0125] Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues, y, in the domain sequence.

[0126] In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, even more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. There is no particular upper limit for x / y, and it may be, for example, 100% or less. When the knurling ratio is 1:1.9 to 11.3, x / y is preferably 89.6% or more, when the knurling ratio is 1:1.8 to 3.4, x / y is preferably 77.1% or more, when the knurling ratio is 1:1.9 to 8.4, x / y is preferably 75.9% or more, and when the knurling ratio is 1:1.9 to 4.1, x / y is preferably 64.2% or more.

[0127] When the third modified fibroin is a modified fibroin in which at least seven of the (A)n motifs present in the domain sequence are composed of only alanine residues, x / y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x / y, and it is sufficient that it is 100% or less.

[0128] Here, x / y in naturally derived fibroin will be explained. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types were spider-derived fibroin) were extracted. From all the extracted fibroins, x / y was calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by formula 1: [(A)n motif-REP]m, using the calculation method described above. The results when the jagged ratio is 1:1.9 to 4.1 are shown in FIG. 9.

[0129] The horizontal axis of Fig. 9 indicates x / y (%), and the vertical axis indicates frequency. As is clear from Fig. 9, x / y in all naturally derived fibroins is less than 64.2% (the highest is 64.14%).

[0130] The third modified fibroin can be obtained, for example, by deleting one or more sequences encoding the (A)n motif from the gene sequence of a cloned naturally-occurring fibroin so that x / y is 64.2% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the deletion of one or more (A)n motifs from the amino acid sequence of a naturally-occurring fibroin so that x / y is 64.2% or more, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the deletion of the (A)n motif from the amino acid sequence of a naturally-occurring fibroin, the amino acid sequence may be modified by further substituting, deleting, inserting and / or adding one or more amino acid residues.

[0131] More specific examples of the third modified fibroin include (3-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0132] The modified fibroin (3-i) will now be described. The amino acid sequence shown in SEQ ID NO: 17 is obtained by deleting every third (A)n motif from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin, and further inserting one [(A)n motif-REP] just before the C-terminus sequence. The amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described in the second modified fibroin.

[0133] The value of x / y in the jagged ratio of 1:1.8-11.3 of the amino acid sequence shown in SEQ ID NO:10 (corresponding to naturally derived fibroin) is 15.0%. The values ​​of x / y in the amino acid sequence shown in SEQ ID NO:17 and the amino acid sequence shown in SEQ ID NO:7 are both 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 89.8%. The values ​​of z / w in the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 are 46.8%, 56.2%, 70.1%, 66.1% and 70.0%, respectively.

[0134] The modified fibroin (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0135] The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0136] The modified fibroin (3-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and when the numbers of amino acid residues of the REP of two adjacent [(A)n motif-REP] units are compared sequentially from the N-terminus to the C-terminus, and the number of amino acid residues of the REP with the fewer number of amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues of the two adjacent [(A)n motif-REP] units in which the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 (jagged ratio of 1:1.8 to 11.3) is defined as x, and the total number of amino acid residues in the domain sequence is y, the ratio x / y is 64.2% or more.

[0137] The third modified fibroin may contain the above-mentioned tag sequence at either or both of the N-terminus and C-terminus.

[0138] More specific examples of modified fibroins containing a tag sequence include (3-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0139] The amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0140] The modified fibroin (3-iii) may consist of the amino acid sequence shown in SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0141] The modified fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0142] The modified fibroin (3-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and when the numbers of amino acid residues of the REP of two adjacent [(A)n motif-REP] units are compared sequentially from the N-terminus to the C-terminus, and the number of amino acid residues of the REP with the fewer number of amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues of two adjacent [(A)n motif-REP] units such that the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is y, the ratio x / y is 64.2% or more.

[0143] The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0144] The fourth modified fibroin has an amino acid sequence in which the content of (A)n motifs is reduced and the content of glycine residues is reduced, compared to naturally-derived fibroin. The domain sequence of the fourth modified fibroin can be said to have an amino acid sequence in which at least one or more (A)n motifs are deleted and at least one or more glycine residues in REP are replaced with other amino acid residues, compared to naturally-derived fibroin. In other words, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects are as described for the second modified fibroin and the third modified fibroin.

[0145] More specific examples of the fourth modified fibroin include (4-i) modified fibroin containing an amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Specific embodiments of modified fibroin containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

[0146] The fifth modified fibroin may have an amino acid sequence whose domain sequence includes a localized region of high hydrophobicity index, corresponding to the replacement of one or more amino acid residues in REP with amino acid residues with a high hydrophobicity index and / or the insertion of one or more amino acid residues with a high hydrophobicity index into REP, compared to naturally occurring fibroin.

[0147] The region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

[0148] The amino acid residue with a high hydrophobicity index is more preferably an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A).

[0149] The fifth modified fibroin may, in addition to modifications corresponding to the replacement of one or more amino acid residues in REP with amino acid residues having a high hydrophobicity index and / or the insertion of one or more amino acid residues having a high hydrophobicity index into REP, have further modifications in the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues compared to the naturally occurring fibroin.

[0150] The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP from the gene sequence of cloned naturally-derived fibroin with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and / or by inserting one or more hydrophobic amino acid residues into REP. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin and / or the insertion of one or more hydrophobic amino acid residues into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-derived fibroin and / or the insertion of one or more hydrophobic amino acid residues into REP, the amino acid sequence may be modified by further substituting, deleting, inserting and / or adding one or more amino acid residues.

[0151] The fifth modified fibroin may have an amino acid sequence comprising a domain sequence represented by formula 1: [(A)n motif-REP]m, and in all REPs contained in the sequence excluding the sequence from the domain sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence, p is the total number of amino acid residues contained in a region having an average hydrophobicity index of 2.6 or more of four consecutive amino acid residues, and q is the total number of amino acid residues contained in the sequence excluding the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence, in which p / q is 6.2% or more.

[0152] } The hydrophobicity index of amino acid residues is determined by a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.

[0153] [Table 1]

[0154] The calculation method of p / q will be explained in more detail. For the calculation, a sequence (hereinafter referred to as "sequence A") is used, which is obtained by removing the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by formula 1: [(A)n motif-REP]m. First, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs included in sequence A. The average value of the hydrophobicity index is calculated by dividing the sum of the HI of each amino acid residue included in the four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is calculated for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of the four consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "four consecutive amino acid residues having an average value of hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. The total number of amino acid residues included in the region is p. The total number of amino acid residues included in sequence A is q.

[0155] For example, if 20 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without overlap), the region in which the average hydrophobicity index of the consecutive 4 amino acid residues is 2.6 or more contains 20 consecutive 4 amino acid residues (without overlap), and p is 20×4=80. Also, for example, if two "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" overlap by one amino acid residue, the region in which the average hydrophobicity index of the consecutive 4 amino acid residues is 2.6 or more contains 7 amino acid residues (p=2×4-1=7, where "-1" is the deduction of the overlap). For example, in the case of the domain sequence shown in FIG. 10, there are 7 "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more" without overlap, and p is 7×4=28. For example, in the case of the domain sequence shown in Figure 10, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A)n motif at the end of the C-terminus). Next, p / q (%) can be calculated by dividing p by q. In the case of Figure 10, the result is 28 / 170=16.47%.

[0156] In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, even more preferably 20% or more, and even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

[0157] The fifth modified fibroin can be obtained, for example, by modifying the amino acid sequence of a cloned naturally-derived fibroin to include a region with a high hydrophobic index locally by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobic index) in the REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobic index) and / or inserting one or more hydrophobic amino acid residues into the REP so as to satisfy the above-mentioned p / q condition. Alternatively, for example, the fifth modified fibroin can be obtained by designing an amino acid sequence that satisfies the above-mentioned p / q condition from the amino acid sequence of a naturally-derived fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more amino acid residues in the REP with amino acid residues with a high hydrophobic index and / or the insertion of one or more amino acid residues with a high hydrophobic index into the REP, modifications corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed.

[0158] The amino acid residues having a high hydrophobicity index are not particularly limited, but are preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A), and more preferably valine (V), leucine (L) and isoleucine (I).

[0159] A more specific example of the fifth modified fibroin includes (5-i) a modified fibroin comprising an amino acid sequence shown in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0160] The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at two positions, excluding the domain sequence at the C-terminus, and further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids at the C-terminus, in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). The amino acid sequence shown in SEQ ID NO: 20 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at one position, in the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525). The amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP at two positions, in the amino acid sequence shown in SEQ ID NO: 8.

[0161] The modified fibroin (5-i) may consist of the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

[0162] The modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0163] The modified fibroin (5-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and in all REPs contained in the domain sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence, where the average hydrophobicity index of four consecutive amino acid residues is 2.6 or more, p is the total number of amino acid residues contained in the domain sequence excluding the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence, q is the total number of amino acid residues contained in the domain sequence, where the p / q is 6.2% or more.

[0164] The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus.

[0165] More specific examples of modified fibroins containing a tag sequence include (5-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0166] The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are obtained by adding the amino acid sequence shown in SEQ ID NO: 11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0167] The modified fibroin (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0168] The modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m. The sequence identity is preferably 95% or more.

[0169] The modified fibroin (5-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and in all REPs contained in the domain sequence excluding the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence, where the total number of amino acid residues contained in a region having an average hydrophobicity index of 2.6 or more of four consecutive amino acid residues is p, and the total number of amino acid residues contained in the domain sequence excluding the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence is q, the p / q ratio is 6.2% or more.

[0170] The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of the host.

[0171] The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

[0172] It is preferable that the sixth modified fibroin contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0173] When the sixth modified fibroin contains a GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

[0174] In the present specification, the "GPGXX motif content" is a value calculated by the following method. In a fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif, the GPGXX motif content is calculated as s / t, where s is the total number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence, multiplied by three (i.e., equivalent to the total number of G and P in the GPGXX motif), and t is the total number of amino acid residues of all REPs excluding the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence and further excluding the (A)n motif from the domain sequence.

[0175] In calculating the GPGXX motif content, the target is "the sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because "the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence" (the sequence corresponding to REP) may contain a sequence that has low correlation with the sequence characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), it affects the calculation result of the GPGXX motif content, so this influence is to be eliminated. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

[0176] FIG. 11 is a schematic diagram showing the domain sequence of the modified fibroin. The calculation method of the GPGXX motif content will be specifically described with reference to FIG. 11. First, in the domain sequence of the modified fibroin shown in FIG. 11 (which is the "[(A)n motif-REP]m-(A)n motif" type), all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 11), so the number of GPGXX motifs for calculating s is 7, and s is 7×3=21. Similarly, all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 11), so the total number of amino acid residues t of all REPs obtained by further removing 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, and in the case of the modified fibroin in FIG. 11, this is 21 / 150=14.0%.

[0177] The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%.

[0178] In the present specification, the "glutamine residue content" is a value calculated by the following method. In a fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif, in all REPs contained in a sequence (sequence corresponding to "region A" in FIG. 11) obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence, the total number of glutamine residues contained in the region is u, and the total number of amino acid residues of all REPs removed from the domain sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence and further removed from the (A)n motif is t. In calculating the glutamine residue content, the reason for targeting "the sequence removed from the domain sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence" is the same as that described above.

[0179] The sixth modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of one or more glutamine residues in the REP or the substitution of other amino acid residues, compared to a naturally occurring fibroin.

[0180] The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine residue. The hydrophobicity indexes of amino acid residues are as shown in Table 1.

[0181] As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M) and alanine (A) are more preferred, and amino acid residues selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are even more preferred.

[0182] In the sixth modified fibroin, the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

[0183] In the present specification, the "hydrophobicity of REP" is a value calculated by the following method. In a fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif, in all REPs contained in a sequence (sequence corresponding to "region A" in FIG. 11) obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the total number of amino acid residues of all REPs, excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence, and further excluding the (A)n motif, is t. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminus side to the C-terminus of the domain sequence" is the same as that described above.

[0184] The sixth modified fibroin may have a domain sequence that, compared to a naturally occurring fibroin, is modified by the deletion of one or more glutamine residues in the REP and / or the replacement of one or more glutamine residues in the REP with other amino acid residues, and may also have an amino acid sequence modification that is equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0185] The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of a cloned naturally-occurring fibroin and / or substituting one or more glutamine residues in REP with other amino acid residues. It can also be obtained, for example, by designing an amino acid sequence corresponding to the deletion of one or more glutamine residues in REP from the amino acid sequence of a naturally-occurring fibroin and / or the substitution of one or more glutamine residues in REP with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0186] More specific examples of the sixth modified fibroin include (6-i) modified fibroin having an amino acid sequence shown in SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or SEQ ID NO: 42 (Met-PRT1028), or (6-ii) modified fibroin having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

[0187] The modified fibroin (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO: 26 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with TS, and substituting the remaining Q with A. The amino acid sequence shown in SEQ ID NO: 27 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VL, and substituting the remaining Q with I. The amino acid sequence shown in SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VI, and substituting the remaining Q with L. The amino acid sequence shown in SEQ ID NO: 29 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 7 with VF, and substituting the remaining Q with I.

[0188] The amino acid sequence shown in SEQ ID NO: 30 is the amino acid sequence shown in SEQ ID NO: 8 (Met-PRT525) in which all QQ are replaced with VL. The amino acid sequence shown in SEQ ID NO: 31 is the amino acid sequence shown in SEQ ID NO: 8 in which all QQ are replaced with VL and the remaining Q is replaced with I.

[0189] The amino acid sequence shown in SEQ ID NO:32 is a sequence in which the 20 domain sequence region present in the amino acid sequence shown in SEQ ID NO:7 (Met-PRT410) is repeated twice, with all QQs replaced with VF and the remaining Qs replaced with I.

[0190] The amino acid sequence shown in SEQ ID NO:41 (Met-PRT917) is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with LI and the remaining Q with V. The amino acid sequence shown in SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with IF and the remaining Q with T.

[0191] The amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41 and SEQ ID NO:42 all have a glutamine residue content of 9% or less (Table 2).

[0192] [Table 2]

[0193] The modified fibroin (6-i) may consist of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

[0194] The modified fibroin of (6-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41, or SEQ ID NO: 42. The modified fibroin of (6-ii) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif. The sequence identity is preferably 95% or more.

[0195] The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

[0196] The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the modified fibroin to be isolated, immobilized, detected, visualized, and the like.

[0197] More specific examples of modified fibroins containing a tag sequence include (6-iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or (6-iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44.

[0198] The amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:44 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41 and SEQ ID NO:42, respectively. Since only a tag sequence is added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:44 all have a glutamine residue content of 9% or less (Table 3).

[0199] [Table 3]

[0200] The modified fibroin (6-iii) may consist of the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44.

[0201] The modified fibroin of (6-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 44. The modified fibroin of (6-iv) is also a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif. The sequence identity is preferably 95% or more.

[0202] The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less, and the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

[0203] The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately selected depending on the type of the host.

[0204] The modified fibroin may be a modified fibroin having 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.

[0205] The modified fibroin according to the present embodiment can be produced by a conventional method using a nucleic acid encoding the modified fibroin. The nucleic acid encoding the modified fibroin may be chemically synthesized based on the base sequence information, or may be synthesized using a PCR method or the like. Furthermore, the produced modified fibroin can be isolated and purified by a conventional method.

[0206] [Method of manufacturing protein fiber fabric] The method for producing a protein fiber fabric according to the present embodiment includes a step of producing a fabric using the protein fibers obtained by the method for producing a protein fiber according to the present invention. The method for producing a fabric using the protein fibers is not particularly limited, and any known method can be used.

[0207] According to the method for producing a protein fiber fabric of this embodiment, by using the protein fibers obtained by the production method of the present invention as described above, it becomes possible to easily produce a protein fiber fabric in which shrinkage upon contact with water for the first time after production is more reliably suppressed and the occurrence of crimping is also suppressed.

[0208] The protein fibers used in the production of protein fiber fabrics may be short or long fibers. Moreover, such protein fibers may be used alone or in combination with other fibers. That is, when producing a protein fiber fabric, a single yarn consisting of only the protein fiber obtained by the production method of the present invention, and a composite yarn consisting of a combination of the protein fiber obtained by the production method of the present invention and other fibers may be used as material yarns, either alone or in combination. The other fibers refer to protein fibers obtained by production methods other than the production method of the present invention, fibers not containing protein, and the like. Moreover, the composite yarns include, for example, blended yarns, blended yarns, covering yarns, and the like.

[0209] The type of protein fiber fabric produced according to the method for producing a protein fiber fabric according to this embodiment is not particularly limited. The protein fiber fabric may be, for example, a woven fabric or knitted fabric, or a nonwoven fabric. The woven fabric may have a weave such as plain weave, twill weave, satin weave, etc., and may use one or more types of yarn. The knitted fabric may be, for example, a warp knitted fabric such as tricot or raschel, or a weft knitted fabric such as flat knit or circular knit, and may use one or more types of yarn.

[0210] [Shrink-proof processing method for protein fibers] The method for producing a protein fiber of the present invention described above can also be regarded as a shrink-proof processing method for further shrink-proofing a protein fiber produced by a known production method including a drawing step and a shrink-proof processing step. That is, the method for shrink-proof processing of a protein fiber according to this embodiment includes a pre-shrinkage step of shrinking the protein fiber in a relaxed state after drawing and before being wound up, a winding step of winding the protein fiber that has been through the pre-shrinkage step to obtain a wound product, and a post-shrinkage step of shrinking the protein fiber that has been through the winding step in a relaxed state. Specific aspects of the shrink-proof processing method are the same as those described in the method for producing a protein fiber. EXAMPLES

[0211] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0212] [Test Example 1: Production of modified fibroin] (1) Construction of expression vector A modified fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15 was designed. A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0213] (2) Protein expression Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that the OD600 became 0.005. The culture solution temperature was kept at 30°C, and flask culture was continued until the OD600 became 5 (about 15 hours), to obtain a seed culture solution.

[0214] [Table 4]

[0215] The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) so that the OD600 was 0.05. The culture temperature was kept at 37°C, and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

[0216] [Table 5]

[0217] Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455g / 1L, yeast extract 120g / 1L) was added at a rate of 1mL / min. The culture temperature was kept at 37°C and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Then, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1mM to induce the expression of the modified fibroin. After 20 hours had passed after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the modified fibroin of interest was confirmed by the appearance of a band of the desired modified fibroin size depending on the addition of IPTG.

[0218] (3) Protein purification The cells harvested 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 approximately 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it reached a high purity. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and stirred with a stirrer at 60°C for 30 minutes to dissolve. After dissolution, dialysis was performed against water using a dialysis tube (Cellulose tube 36 / 32, Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, the water was removed using a freeze-dryer, and the freeze-dried powder was collected to obtain the modified fibroin (PRT799).

[0219] [Test Example 2: Production and Evaluation of Protein Fiber] (1) Production of protein fibrils Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0 mass% was prepared as a solvent, and freeze-dried powder of modified fibroin (PRT799) was added to the solvent to a concentration of 24 mass%, and dissolved for 3 hours using a shaker. Insoluble matter and bubbles were then removed to obtain a modified fibroin solution (spinning stock solution).

[0220] In the case where the wet shrinkage method was used as the pre-shrinkage step (Example 1 and Comparative Example 2), protein fibrils were produced using a spinning apparatus similar to the spinning apparatus 100 shown in FIG. 1. Specifically, the prepared spinning solution was filtered through a metal filter with a mesh size of 5 μm at 60° C., then left to stand in a 30 mL stainless steel syringe, degassed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 60° C. After coagulation, the solution was washed and immersed in hot water at 90° C. for 10 seconds to perform the pre-shrinkage step. At this time, the protein fibrils had a take-up speed ratio of less than 1 (0.6 times) to the delivery speed, which could be called a relaxed state. After drying at 100° C., the obtained raw yarn was wound (winding step) to obtain protein fibrils (modified fibroin fibrils).

[0221] In the case where the dry shrinkage method was used as the pre-shrinkage step (Example 2 and Comparative Example 3), protein fibrils were produced using a spinning apparatus similar to the spinning apparatus 200 shown in FIG. 2. Specifically, the prepared spinning solution was filtered at 60°C with a metal filter having an opening of 5 μm, then left to stand in a 30 mL stainless steel syringe, degassed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 60°C. After coagulation, the solution was washed and dried, and then heated at 240°C for 2 seconds to perform the pre-shrinkage step. At this time, the protein fibrils had a take-up speed ratio of less than 1 (0.5) to the delivery speed, which could be called a relaxed state. The obtained raw yarn was wound (winding step) to obtain protein fibrils (modified fibroin fibrils).

[0222] When the pre-shrinkage step was not performed (Comparative Example 1), protein fibrils were produced using a conventional wet-dry spinning apparatus (a spinning apparatus 100 shown in FIG. 1 without the water bath 46). Specifically, the prepared spinning solution was filtered through a metal filter with a mesh size of 5 μm at 60° C., then allowed to stand in a 30 mL stainless steel syringe and degassed, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 60° C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain protein fibrils (modified fibroin fibrils).

[0223] When the wet shrinkage method was used as the post-shrinkage step (Example 2 and Comparative Example 1), the post-shrinkage step was carried out using an apparatus similar to that shown in Fig. 6(a) and Fig. 6(b). Specifically, protein fibrils were pulled out from the wound protein fibrils to obtain skein-shaped protein fibrils (see Fig. 6(a)). The protein fibrils were then immersed in 90°C hot water for 15 minutes to carry out the post-shrinkage step. After that, the protein fibers (modified fibroin fibers) were obtained by natural drying.

[0224] When the dry shrinkage method was used as the post-shrinkage step (Example 1), the post-shrinkage step was performed using an apparatus similar to that shown in Figures 6(a) and 6(c). Specifically, protein fibrils were pulled out from the wound protein fibrils to obtain skein-shaped protein fibrils (see Figure 6(a)). The skein-shaped protein fibrils were then brought into contact with a high-temperature heater heated to 240°C and held there for 10 seconds to perform the post-shrinkage step. As a result, protein fibers (modified fibroin fibers) were obtained.

[0225] (2) Evaluation of shrinkage rate of protein fibers The modified fibroin fibers (test pieces) of each Example and Comparative Example cut to a length of 300 mm were immersed in water at 40°C or 90°C for 10 minutes without load. Each test piece was removed from the water and dried at room temperature for 2 hours. The length of the test piece (fiber length after drying) was then measured, and the shrinkage rate was measured. The results are shown in Table 6. The shrinkage rate is a numerical value calculated by the following formula (1). The "fiber length before immersion" was 300 mm. Shrinkage rate = (1 - fiber length after drying / fiber length before immersion) × 100 (1)

[0226] (3) Evaluation of protein fiber crimp The test pieces (after drying) whose shrinkage rates had been measured in (2) were visually inspected for shrinkage. The results are shown in Table 6.

[0227] [Table 6]

[0228] The protein fibers that were subjected to both the pre-shrinkage step and the post-shrinkage step (Examples 1 and 2) had sufficiently reduced shrinkage when they first came into contact with water after production, and did not shrink. The protein fibers that were subjected to only the post-shrinkage step (Comparative Example 1) had reduced shrinkage when they first came into contact with water after production, but did shrink. The protein fibers that were subjected to only the pre-shrinkage step (Comparative Examples 2 and 3) did not shrink, but did shrink significantly when they first came into contact with water after production (particularly when they came into contact with high-temperature water). [Explanation of symbols]

[0229] 1...extrusion device, 4...drying device, 6...dope solution, 20...coagulation bath, 21...washing bath, 36...protein fibrils, 38...protein fibers, 42...feed roller, 44...winder, 46...water bath, 48...dryer, 54...heater, 56...pressure roller, 58...hot roller, 60...high temperature heating relaxation device, 100,200...spinning device, 300,400...post-shrinkage device.

Claims

1. A method for producing protein fibers, comprising: a pre-shrinkage step of shrinking a protein fibril in a relaxed state after stretching and before being wound up; a winding step of winding the protein fibril that has undergone the pre-shrinkage step to obtain a wound product; and a post-shrinkage step of shrinking the protein fibril that has undergone the winding step in a relaxed state.

2. 2. The manufacturing method according to claim 1, wherein the post-shrinking step is a wet shrinking step in which the protein fibrils that have been through the winding step are brought into contact with liquid or steam in a relaxed state to shrink them, or a dry shrinking step in which the protein fibrils that have been through the winding step are heated in a relaxed state to shrink them.

3. The method according to claim 2 , wherein the wet shrinking step is a step of shrinking the protein fibrils that have been subjected to the winding step by immersing them in a relaxed state in a liquid.

4. The method according to claim 2 or 3, wherein the liquid or vapor has polarity.

5. The method of claim 4 , wherein the liquid is water and the vapor is water vapor.

6. The method of any one of claims 1 to 5, wherein the protein fibrils comprise structural proteins.

7. The method of claim 6 , wherein the structural protein is a modified fibroin.

8. The method of claim 7, wherein the modified fibroin is modified spider silk fibroin.

9. A method for producing a protein fiber fabric, comprising a step of producing fabric using the protein fibers obtained by the manufacturing method according to any one of claims 1 to 8.

10. The method for shrink-proofing a protein fiber includes a pre-shrinkage step of shrinking a protein fiber in a relaxed state after stretching and before being wound up, a winding step of winding the protein fiber that has been through the pre-shrinkage step to obtain a wound product, and a post-shrinkage step of shrinking the protein fiber that has been through the winding step in a relaxed state.

11. The shrink-proof processing method according to claim 10, wherein the post-shrinkage step is a wet shrinkage step in which the protein fibers that have been through the winding step are brought into contact with a liquid or steam in a relaxed state to shrink them, or a dry shrinkage step in which the protein fibers that have been through the winding step are heated in a relaxed state to shrink them.

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