Patent Application
20220095728
The present invention relates to a fiber for artificial hairs and an artificial hair. The present invention also relates to a method for producing a fiber for artificial hairs that is given a predetermined shape, and a method for producing an artificial hair.
Regarding materials for artificial hairs such as wigs and false hair pieces, human hair or non-natural or synthetic fibers have been conventionally used. As the non-natural fibers for artificial hairs, polyester fibers or acrylic fibers are generally used (for example, Patent Literature 1). Non-natural fibers have an advantage that fibers having higher strength than human hair and having a desired length are stably available in large quantities with uniform product quality. Furthermore, artificial protein fibers for hairs, such as regenerated collagen fibers that are expected to have lower environmental load, have also been proposed (for example, Patent Literature 2).
However, in order to secure a wide variation of style, artificial hairs may be given a shape including a curved part or a linear shape.
Patent Literature 1: JP 2007-297737 A
Patent Literature 2: WO 2016/158702 A
Non-natural fibers feel significantly different to the touch compared to human hair and also have significant differences in view of characteristics. For example, human hair has a characteristic of being elongated by a predetermined length when absorbing water and then restoring the original length when dried, whereas none of the non-natural fibers have such a characteristic. Therefore, when a fiber for artificial hairs formed from a non-natural fiber is used, the occurrence of a feeling of discomfort between the non-natural fiber and human hair cannot be avoided. Therefore, realization of artificial hairs that are not likely to cause a feeling of discomfort between the artificial hairs and human hair is desirable. In addition, from the viewpoint of styleability, artificial hairs may be required to have a predetermined shape such as a curved part (curl part) or a linear shape.
Thus, according to an aspect of the present invention, there is provided a fiber for artificial hairs that is given a predetermined shape, the fiber being stably suppliable while using a non-natural fiber and being capable of suppressing the occurrence of a feeling of strangeness between the fiber and human hair.
In the case of a non-natural fiber for artificial hairs, generally, a shape including a curved part is given to the fiber by winding a synthetic fiber around a core material such as an aluminum pipe and heating the synthetic fiber in that state. In order to satisfactorily give a desired shape and retain the shape, heating at a high temperature of 100° C. or higher may be needed. Heating at a high temperature is likely to cause a change in the feeling of an artificial hair to the touch, a decrease in strength, and a color change. Furthermore, even from the viewpoint of securing safety of the operator, it is desirable that a desired shape can be given to a synthetic fiber for artificial hairs without requiring heating at a high temperature.
Thus, according to another aspect of the present invention, there is provided a method for obtaining a fiber for artificial hairs that is given a predetermined shape, the fiber not requiring heating at a high temperature while using a non-natural fiber.
In the case of conventional artificial protein fibers for hairs, it has been required to crosslink the protein fibers in order to give a predetermined shape and to secure retainability of the given shape. Furthermore, there has also been a need for a pretreatment using a special treatment liquid.
Thus, according to still another aspect of the present invention, there is provided a method for conveniently obtaining a fiber for artificial hairs having excellent retainability of the given shape, while using a protein fiber.
The inventors of the present invention found that fibroin fibers containing modified fibroin expand and contract similarly to human hair concomitantly with wetting and drying, and that fibroin fibers are given a predetermined shape by a simple and easy method. That is, an aspect of the present invention relates to the following.
[1] A fiber for artificial hairs that is given a predetermined shape, the fiber being formed from a synthetic fibroin fiber containing a modified fibroin, and the fiber expanding when placed in a wet state and contracting when dried from a wet state.
[2] An artificial hair including the fiber for artificial hairs according to [1].
As a result of various investigations, the inventors of the present invention found that a fibroin fiber containing a modified fibroin is given a predetermined shape without requiring heating at a high temperature. That is, another aspect of the present invention relates to the following.
[1] A method for producing a fiber for artificial hairs that is given a predetermined shape, the method including retaining a fibroin fiber containing a modified fibroin in a state conforming to a predetermined shape, in which the predetermined shape is a shape including a curved part, a linear part, or both of these.
[2] The method according to [1], in which the fibroin fiber is retained in a state conforming to a shape including a curved part, by being wound around a core material.
[3] The method according to [1] or [2], in which the diameter of the fibroin fiber is more than 30 μm.
[4] The method according to any one of [1] to [3], in which the fibroin fiber that is retained in the state conforming to a predetermined shape is heated.
[5] The method according to [4], in which the fibroin fiber that is retained in the state conforming to a predetermined shape is heated to a temperature below 100° C.
[6] The method according to any one of [1] to [5], in which the modified fibroin includes a modified spider thread fibroin.
[7] The method according to any one of [1] to [6], further including drying the fibroin fiber, in which the fibroin fiber that has been dried is wound around the core material.
[8] A method for producing an artificial hair, the method including obtaining an artificial hair fiber that is given a predetermined shape, by the method according to any one of [1] to [7].
In these methods, the fibroin fiber may be a fiber that expands when placed in a wet state and contracts when dried from a wet state.
As a result of various investigations, the present inventors found that a fibroin fiber containing a modified fibroin is easily given a predetermined shape by heating a fibroin fiber that has been wetted with water while retaining the fibroin fiber in a state conforming to a predetermined shape, without necessarily requiring crosslinking by a chemical reaction and a pretreatment using a treatment liquid, and the given shape is satisfactorily retained. That is, an aspect of the present invention relates to the following.
[1] A method for producing a fiber for artificial hairs that is given a predetermined shape, the method including heating a fibroin fiber that contains a modified fibroin and has been wetted with water, while retaining the fibroin fiber in a state conforming to a predetermined shape, in which the predetermined shape is a shape including a curved part, a linear part, or both of these.
[2] The method according to [1], further including wetting the fibroin fiber with water before retaining the fibroin fiber in the state conforming to a predetermined shape.
[3] The method according to [1] or [2], in which the fibroin fiber that is retained in the state conforming to a predetermined shape is immersed in water.
[4] The method according to [1] or [2], in which the fibroin fiber that is retained in the state conforming to a predetermined shape is exposed to water vapor.
[5] The method according to any one of [1] to [4], in which the fibroin fiber is retained in a state conforming to a predetermined shape including a curved part by being wound around a core material.
[6] The method according to any one of [1] to [5], in which the modified fibroin includes a modified spider thread fibroin.
[7] A method for producing an artificial hair, the method including obtaining a fiber for artificial hairs that is given a predetermined shape by the method according to any one of [1] to [6].
In these methods, too, the fibroin fiber may be a fiber that expands when placed in a wet state and contracts when dried from a wet state.
According to an aspect of the present invention, a fiber for artificial hairs that is given a predetermined shape, the fiber being stably suppliable while using a non-natural fiber and being capable of suppressing the occurrence of a feeling of strangeness between the fiber and human hair, can be provided.
According to another aspect of the present invention, a fiber for artificial hairs that is given a predetermined shape can be obtained, the fiber not requiring heating at a high temperature while using a non-natural fiber that is stably suppliable.
According to still another aspect of the present invention, a fiber for artificial hairs having excellent retainability of a given shape can be conveniently obtained while using a protein fiber that can be supplied stably and has a low environmental load.
Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not intended to be limited to the following embodiments.
(Method for Producing Fiber for Artificial Hairs)
An embodiment of a method for producing a fiber for artificial hairs that is given a predetermined shape includes retaining a fibroin fiber containing a modified fibroin (synthetic fibroin fiber) in a state conforming to a predetermined shape. The predetermined shape may be a curled shape. An embodiment of the method for producing a fiber for artificial hairs that is given a predetermined shape may include heating a fibroin fiber that contains a modified fibroin and has been wetted with water while retaining the fibroin fiber in a state conforming to a predetermined shape. Through these methods, a fiber for artificial hairs that is given a predetermined shape and is formed from a fibroin fiber, can be easily obtained. The predetermined shape as referred to herein can be a shape including a curved part, a linear part, or both of these. A shape including a curved part can also be a curled shape.
The diameter of the core material 75 is not particularly limited; however, the diameter is usually in the range of 1 to 100 mm. The shape of the core material is not limited to a cylindrical shape.
In the method according to the present embodiment, the fibroin fiber 70 that has been dried or wetted by water is wound around the core material 75. Here, the “dried fibroin fiber” means a fibroin fiber that has not been wetted by water. However, the dried fibroin fiber 70 may contain a small amount of unavoidable moisture. Therefore, the method according to the present embodiment may further include drying the fibroin fiber 70 before being wound around the core material 75. The fibroin fiber 70 is dried by, for example, retaining the fibroin fiber 70 in an environment at 10° C. to 100° C. The fibroin fiber 70 after drying is wound around the core material 75 without coming into contact with water or a liquid including water.
The fibroin fiber 70 wound around the core material 75 is retained in an environment at a predetermined temperature. This predetermined temperature may be a temperature that is not associated with heating in a case where the fibroin fiber 70 wound around the core material 75 is in a wet state, and specifically, the predetermined temperature may be, for example, 0° C. or higher, 10° C. or higher, or 20° C. or higher. When the fibroin fiber 70 wound around the core material 75 is in a dried state, the fibroin fiber 70 wound around the core material 75 may be heated or may not be heated. The heating temperature in a case where the fibroin fiber 70 wound around the core material 75 is heated may be higher than 40° C., 45° C. or higher, 50° C. or higher, 60° C. or higher, 70° C. or higher, 80° C. or higher, or 90° C. or higher and may be lower than 100° C. For heating, hot air may be blown toward the fibroin fiber 70. When the temperature at which the fibroin fiber is retained in a state conforming a predetermined shape is high, there is a tendency that the predetermined shape is easily given to the fibroin fiber in a short period of time.
The time for retaining the fibroin fiber 70 wound around the core material 75 is adjusted such that a shape is given to the fibroin fiber 70. Usually, the retention time may be in the range of 10 to 360 minutes and may be in the range of 30 to 90 minutes.
When the fibroin fiber 70 that has been wetted with water is retained in a state conforming to a predetermined shape, even when the fibroin fiber is not crosslinked by a chemical reaction or the like, not only the predetermined shape is easily given, but also the given shape is easily retained. It is thought that by heating the fibroin fiber 70 while retaining the fibroin fiber 70 in the presence of water, or heating the fibroin fiber 70 in a state of having been retained in the presence of water, the quantity of the β sheet structure of the modified fibroin in the fibroin fiber is increased. The present inventors speculate that as this β sheet structure functions as pseudo-crosslinking, movement of the molecular chain of the modified fibroin is limited, and as a result, the given shape is satisfactorily retained.
Before the fibroin fiber 70 is wound around the core material 75, the fibroin fiber 70 may be wetted with water in advance. When the fibroin fiber 70 is wetted with water in advance, there is a tendency that the fibroin fiber 70 that is retained in a state conforming to a predetermined shape is not likely to swell as a result of absorbing moisture. When swelling of the fibroin fiber 70 is suppressed, a predetermined shape can be given more easily to the fibroin fiber 70. For example, the fibroin fiber 70 may be brought into contact with water or an aqueous solution by immersing the fibroin fiber 70 in water or the aqueous solution or impregnating a fiber bundle including a plurality of single yarns of the fibroin fiber 70 with water or the aqueous solution. It is not necessarily essential to bring the fibroin fiber 70 into contact with pure water, and the fibroin fiber 70 may be brought into contact with an aqueous solution including additive components or a very small amount of unavoidable impurities. The concentration of components other than water in the aqueous solution to be brought into contact with the fibroin fiber 70 may be 1% by mass or less, 0.5% by mass or less, or less than 0.05% by mass, based on the total mass of the aqueous solution.
The temperature of the water or aqueous solution to be brought into contact with the fibroin fiber 70 is not particularly limited but may be, for example, 10° C. to 80° C. The time for immersion or impregnation is not particularly limited but may be, for example, 5 to 60 minutes. The fibroin fiber 70 after immersion is wound around the core material 75 while maintaining a state of being wetted with water. When the surface of the fibroin fiber 70 may get wet with water, and excessive moisture may be removed.
The fibroin fiber 70 that has been wetted with water and wound around the core material 75 may be heated at a predetermined heating temperature. The heating temperature may be 40° C. or higher, 45° C. or higher, or 50° C. or higher and may be 150° C. or lower, 140° C. or lower, 130° C. or lower, 120° C. or lower, 110° C. or lower, or lower than 100° C. For heating, hot air may be blown toward the fibroin fiber 70. When the heating temperature is high, retainability of the given shape tends to be further enhanced.
The fibroin fiber 70 wound around the core material 75 may be heated by leaving in a wet state to stand in a heating atmosphere, immersing in water that has been heated to a predetermined temperature or higher, or contacting with water vapor. The fibroin fiber 70 may be heated to a temperature exceeding 100° C. by contacting with water vapor. When the fibroin fiber 70 wound around the core material 75 is immersed in heated water or exposed to water vapor, since the fibroin fiber 70 is wetted with water at that time point, the fibroin fiber 70 to be wound around the core material 75 may not be wetted with water in advance.
The time for heating while retaining the fibroin fiber 70 wound around the core material 75 is adjusted such that a shape is given to the fibroin fiber 70. The retention time may be 5 minutes or more or 10 minutes or more and may be 240 minutes or less, 180 minutes or less, or 120 minutes or less.
While the fibroin fiber 70 wound around the core material 75 is retained in that state, it is necessary for the fibroin fiber 70 to retain a state of being wetted with water after being subjected to a heating step in a wet state, and the fibroin fiber 70 may be finally in a dry state. The fibroin fiber 70 wound around the core material 75, or the fibroin fiber 70 detached from the core material 75 may be dried by any means such as heating.
The fibroin fiber 70 to which a predetermined shape or a curled shape has been given can be used as a fiber for artificial hairs constituting an artificial hair. The fiber for artificial hairs formed from a fibroin fiber can be stably supplied and can suppress the occurrence of a feeling of strangeness between the fiber and human hair. An artificial hair can be used as, for example, hair for wigs, hair for hair pieces, hair for hair increase that is directly attached to the head, or hair for extension.
(Fibroin Fiber)
The fibroin fiber 70 can be a fiber that contains a modified fibroin as a main component and is formed by spinning a modified fibroin. The fibroin fiber 70 to be wound around the core material 75 may be a fiber that is not in direct contact with liquid water after spinning.
The fibroin fiber 70 may be a single yarn or may be a fiber bundle composed of a plurality of single yarns. The diameter (single yarn diameter) of the fibroin fiber 70 may be, for example, 10 to 100 μm. From the viewpoint of the ease of shaping and maintaining the given shape, the single yarn diameter of the fibroin fiber 70 may be more than 30 μm and may be 35 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, or 80 μm or more. From a similar point of view, the single yarn diameter of the fibroin fiber 70 may be 90 μm or less, 85 μm or less, less than 80 μm, or 70 μm or less.
The term “modified fibroin” as used in the present specification refers to an artificially produced fibroin (synthetic fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally derived fibroin or may be a fibroin whose domain sequence is the same as the amino acid sequence of naturally derived fibroin.
The modified fibroin may be 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. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either or both of the N-terminus side and the C-terminus side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this but are typically regions that do not have repetitions of amino acid motifs characteristic of fibroin and include about 100 residues of amino acids.
The “modified fibroin” may be a fibroin that utilizes the amino acid sequence of naturally derived fibroin as it is, may be a fibroin whose amino acid sequence has been modified based on the amino acid sequence of the naturally derived fibroin (for example, a fibroin whose amino acid sequence has been modified by modifying a cloned gene sequence of naturally derived fibroin), or may be a fibroin that has been artificially designed and synthesized independently of naturally derived fibroin (for example, a fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding a designed amino acid sequence).
In the present specification, the term “domain sequence” is an amino acid sequence that produces a crystalline region (typically, corresponding to an (A)n motif of an amino acid sequence) and an amorphous region (typically, corresponding to REP of an amino acid sequence) characteristic of 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 represents an amino acid sequence mainly including alanine residues, and the number of amino acid residues is 2 to 27. 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 proportion of the number of alanine residues with respect to the total number of amino acid residues in the (A)n motif may be 40% or more, and the proportion 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%. A plurality of (A)n motifs present in the domain sequence may be such that at least seven of the motifs are composed only of alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also 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. A plurality of (A)n motifs present in the domain sequence may be amino acid sequences that are identical to each other or may be different amino acid sequences. A plurality of REPs present in the domain sequence may be amino acid sequences that are identical to each other or may be different amino acid sequences.
The modified fibroin according to the present embodiment can be obtained by, for example, subjecting a cloned gene sequence of naturally derived fibroin to modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one amino acid residue or a plurality of amino acid residues. Substitution, deletion, insertion, and/or addition of amino acid residues can be performed by methods well known to those ordinarily skilled in the art, such as a site-directed mutagenesis method. Specifically, the modification may be performed according to a method described in literatures such as Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).
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 spiders.
Examples of the fibroins produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama; and hornet silk proteins secreted by larvae of Vespa simillima xanthoptera.
More specific examples of the fibroins produced by insects include, for example, silkworm fibroin L chain (GenBank Accession Nos. M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).
Examples of the fibroins produced by spiders include spider silk proteins produced by spiders belonging to the genus Araneus, such as Araneus ventricosus, Araneus diadematus, Araneus pinguis, Araneus pentagrammicus, and Araneus nojimai; spiders belonging to the genus Neoscona, such as Neoscona scylla, Neoscona nautica, Neoscona adianta, and Neoscona scylloides; spiders belonging to the genus Pronus, such as Pronous minutes; spiders belonging to the genus Cyrtarachne, such as Cyrtarachne bufo and Cyrtarachne inaequalis; spiders belonging to the genus Gasteracantha, such as Gasteracantha kuhli and Gasteracantha mammosa; spiders belonging to the genus Ordgarius, such as Ordgarius hobsoni and Ordgarius sexspinosus; spiders belonging to the genus Argiope, such as Argiope amoena, Argiope minuta, and Argiope bruennich; spiders belonging to the genus Arachnura, such as Arachnura logio; spiders belonging to the genus Acusilas, such as Acusilas coccineus; spiders belonging to the genus Cytophora, such as Cyrtophora moluccensis, Cyrtophora exanthematica, and Cyrtophora unicolor; spiders belonging to the genus Poltys, such as Poltys illepidus; spiders belonging to the genus Cyclosa, such as Cyclosa octotuberculata, Cyclosa sedeculata, Cyclosa vallate, and Cyclosa atrata; and spiders belonging to the genus Chorizopes, such as Chorizopes nipponicus; and spider silk proteins produced by spiders belonging to the genus Tetragnatha, such as Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa, and Tetragnatha squamata; spiders belonging to the genus Leucauge, such as Leucauge magnifica, Leucauge blanda, and Leucauge subblanda; spiders belonging to the genus Nephila, such as Nephila clavate and Nephila pilipes; spiders belonging to the genus Menosira, such as Menosira ornate; spiders belonging to the genus Dyschiriognatha, such as Dyschiriognatha tenera; spiders belonging to the genus Latrodectus, such as Latrodectus mactans, Latrodectus hasseltii, Latrodectus geometricus, and Latrodectus tredecimguttatus; and spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include traction yarn proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
More specific examples of the spider silk proteins produced by spiders include, for example, 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 Number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [derived from 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)), and minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession Number ABR37278.1 (amino acid sequence).
As a more specific example of the naturally derived fibroin, fibroin whose sequence information is registered with NCBI GenBank may be further mentioned. For example, sequences of naturally derived fibroins can be confirmed by extracting sequences for which the term spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION among the sequences including INV as DIVISION in the sequence information registered with the NCBI GenBank, and sequences in which a particular character string of product is described from CDS, or a particular character string is described from SOURCE to TISSUE TYPE.
The modified fibroin according to the present embodiment may be modified silk fibroin (a fibroin obtained by modifying the amino acid sequence of silk protein produced by silkworms), or may be modified spider thread fibroin (a fibroin obtained by modifying the amino acid sequence of spider silk protein produced by spiders). The modified fibroin may be a modified spider thread fibroin.
Specific examples of the modified fibroin include a modified fibroin derived from a large spinneret dragline protein produced at the major ampullate gland of a spider (first modified fibroin), a modified fibroin having a domain sequence in which the content of glycine residues has been reduced (second modified fibroin), a modified fibroin having a domain sequence in which the content of the (A)n motif has been reduced (third modified fibroin), a modified fibroin in which the content of glycine residues and the content of the (A)n motif have been reduced (fourth modified fibroin), a modified fibroin having a domain sequence that includes a region in which the hydropathy index is locally large (fifth modified fibroin), and a modified fibroin having a domain sequence in which the content of glutamine residues has been reduced (sixth modified fibroin).
Examples of the first modified fibroin include 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 of the (A)n motif may be 3 to 20, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. In the first modified fibroin, the number of amino acid residues constituting an REP in Formula 1 may be 10 to 200 residues, 10 to 150 residues, 20 to 100 residues, or 20 to 75 residues. In the first modified fibroin, the total number of residues of glycine residues, serine residues, and alanine residues included in the amino acid sequence represented by Formula 1: [(A)n motif-REP]m may be 40% or more, 60% or more, or 70% or more, with respect to the total number of amino acid residues.
The first modified fibroin may be a polypeptide which contains a unit of an amino acid sequence represented by Formula 1: [(A)n motif-REP]m and in which the C-terminal sequence is an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 3, or an amino acid sequence having an identity of 90% or higher with an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 3.
The amino acid sequence set forth in SEQ ID NO: 1 is identical to an amino acid sequence including 50 residues of amino acids at the C-terminus of the amino acid sequence of ADF3 (GI: 1263287, NCBI), the amino acid sequence set forth in SEQ ID NO: 2 is identical to an amino acid sequence obtained by eliminating 20 residues from the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence set forth in SEQ ID NO: 3 is identical to an amino acid sequence obtained by eliminating 29 residues from the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1.
As a more specific example of the first modified fibroin, a modified fibroin containing: (1-i) an amino acid sequence set forth in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 4, can be mentioned. The sequence identity may be 95% or higher.
The amino acid sequence set forth in SEQ ID NO: 4 is an amino acid sequence obtained by mutating the amino acid sequence of ADF3, in which an amino acid sequence (SEQ ID NO: 5) including a start codon, a His10 tag, and a HRV3C protease (Human rhinovirus 3C protease) recognition site has been added to the N-terminus, such that the 1st to 13th repeating regions are increased to approximately double, and the translation is terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence set forth in SEQ ID NO: 4 is identical to the amino acid sequence set forth in SEQ ID NO: 3.
The modified fibroin of (1-i) may include the amino acid sequence set forth in SEQ ID NO: 4.
Regarding the second modified fibroin, its domain sequence has an amino acid sequence in which the content of glycine residues has been reduced compared to naturally derived fibroin. It can be said that the second modified fibroin is a modified fibroin having an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of glycine residues in an REP has been substituted by other amino acid residue(s), as compared to naturally derived fibroin.
Regarding the second modified fibroin, its domain sequence may have an amino acid sequence corresponding to an amino acid sequence in which one glycine residue in at least one or a plurality of the motif sequences is substituted with another amino acid residue, in at least one motif sequence selected from GGX and GPGXX (provided that G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in an REP, as compared to naturally derived fibroin.
In the second modified fibroin, the proportion of the above-mentioned motif sequence in which a glycine residue has been substituted with another amino acid residue, may be 10% or more with respect to the entire motif sequence.
The second modified fibroin may be a modified fibrin having an amino acid sequence which contains a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in which when the total number of amino acid residues in an amino acid sequence including XGX (provided that X represents an amino acid residue other than glycine) included in all REP's in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the above-described domain sequence, from the above-described domain sequence, is denoted by z; and the total number of amino acid residues in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the above-described domain sequence, from the above-described domain sequence, is denoted by w, z/w is 30% or more, 40% or more, 50% or more, or 50.9% or more. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif may be 83% or more, 86% or more, 90% or more, 95% or more, or 100%. The number of alanine residues with respect to the total number of amino acid residues being 100% means that the (A)n motif is composed only of alanine residues.
The second modified fibroin may be such that the content proportion of the amino acid sequence including XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content proportion of the amino acid sequence including GGX in the domain sequence may be 30% or less, 20% or less, 10% or less, 6% or less, 4% or less, or 2% or less. The content proportion of the amino acid sequence including GGX in the domain sequence can be calculated by a method similar to a method for calculating the content proportion (z/w) of the amino acid sequence including XGX as described below.
The method for calculating z/w will be described in more detail. First, for a fibroin (a modified fibroin or a naturally derived fibroin) containing a domain sequence represented by Formula 1: [(A)n motif-REP]m, an amino acid sequence including XGX is extracted from all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus 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, in a case where 50 amino acid sequences including XGX have been extracted (there is no overlap), z is 50×3=150. For example, in a case where there is an X included in two XGX sequences (central X) as in the case of an amino acid sequence including XGXGX, calculation is performed by deducting the overlapping portion (in the case of XGXGX, the overlapping portion is five amino acid residues). w represents the total number of amino acid residues included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence, from the domain sequence. For example, in the case of the domain sequence illustrated in
Here, z/w in the naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered with the NCBI GenBank were checked by the method mentioned as an example, and 663 kinds of fibroins (among these, there were 415 kinds of spider-derived fibroins) were extracted. z/w was calculated by the above-mentioned calculation method from the amino acid sequences of naturally derived fibroins, which contain a domain sequence represented by Formula 1: [(A)n motif-REP]m and in which the content proportion of the amino acid sequence including GGX in the fibroin is 6% or less, among all the extracted fibroins. As a result, it was confirmed that z/w in the naturally derived fibroins is less than 50.9% in all cases, and the maximum is 50.86%.
With regard to the second modified fibroin, z/w may be 50.9% or more, 56.1% or more, 58.7% or more, 70% or more, or 80% or more. The upper limit of z/w is not particularly limited; however, for example, z/w may be 95% or less.
The second modified fibroin can be obtained by, for example, performing modification such that at least some of nucleotide sequences encoding a glycine residue are substituted in a cloned gene sequence of naturally derived fibroin so as to encode other amino acid residues. In this case, one glycine residue in a GGX motif or a GPGXX motif may be selected as the glycine residue to be modified, and substitution may be performed such that z/w is 50.9% or more. Furthermore, the second modified fibroin can also be obtained by, for example, designing an amino acid sequence that satisfies the above-described aspects from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In all cases, in addition to the modification corresponding to a substitution of a glycine residue in an REP from the amino acid sequence of naturally derived fibroin with another amino acid residue, modification of the amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may also be carried out.
The above-described other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue; however, the other amino acid residue may be 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, and 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, and a glutamic acid (E) residue. The other amino acid residue may be selected from a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, and a glutamine (Q) residue, or may be a glutamine (Q) residue.
As a more specific example of the second modified fibroin, a modified fibroin containing: (2-i) an amino acid sequence set forth 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 a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, can be mentioned.
The modified fibroin of (2-i) will be described. The amino acid sequence set forth in SEQ ID NO: 6 is an amino acid sequence obtained by substituting GQX for all GGX's in the REPs of an amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313), which corresponds to naturally derived fibroin. The amino acid sequence set forth in SEQ ID NO: 7 is an amino acid sequence obtained by deleting every two (A)n motifs from the amino acid sequence set forth in SEQ ID NO: 6 from the N-terminus side toward the C-terminus side, and further inserting one [(A)n motif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 8 is an amino acid sequence obtained by inserting two alanine residues into the C-terminus side of each (A)n motif of the amino acid sequence set forth in SEQ ID NO: 7, substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminus side so that the molecular weight of the product becomes almost the same as the molecular weight of SEQ ID NO: 7. The amino acid sequence set forth in SEQ ID NO: 9 is an amino acid sequence obtained by adding a predetermined hinge sequence and a His tag sequence to the C-terminus of a sequence in which a region of twenty domain sequences (provided that several amino acid residues on the C-terminus side of this region have been substituted) present in the amino acid sequence set forth in SEQ ID NO: 7 is repeated four times.
The value of z/w for the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to naturally derived fibroin) is 46.8%. The values of z/w for the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid sequence set forth in SEQ ID NO: 9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the values of x/y for the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 at a jagged ratio (will be described below) of 1:1.8 to 11.3 are 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.
The modified fibroin of (2-i) may include an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
The modified fibroin of (2-ii) is a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing the domain sequence represented by Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (2-ii) may have a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and when the total number of amino acid residues of an amino acid sequence including XGX (provided that X represents an amino acid residue other than glycine) included in the REP is denoted by z, and the total number of amino acid residues in the REP in the domain sequence is denoted by w, z/w may be 50.9% or more.
The second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, and visualization of the modified fibroin.
Regarding the tag sequence, for example, an affinity tag utilizing specific affinity (binding property, affinity) with another molecule may be mentioned. A specific example of the affinity tag may be a histidine tag (His tag). A His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and since the His tag has a property of specifically binding to a metal ion such as nickel, the His tag can be utilized for isolation of a modified fibroin by chelating metal chromatography. Specific examples of the tag sequence include an amino acid sequence set forth in SEQ ID NO: 11 (an amino acid sequence containing a His-tag sequence and a hinge sequence).
Furthermore, a tag sequence such as glutathione-S-transferase (GST) that specifically binds to glutathione, or a maltose-binding protein (MBP) that specifically binds to maltose, can also be utilized.
Furthermore, an “epitope tag” that utilizes an antigen-antibody reaction can also be utilized. By adding a peptide exhibiting antigenicity (epitope) as a tag sequence, an antibody against this epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. The modified fibroin can easily be purified with high specificity by utilizing an epitope tag.
A tag sequence that has been made cleavable with a particular protease can also be used. By treating a protein adsorbed through the tag sequence with a protease, a modified fibroin from which the tag sequence has been detached can be collected.
As a more specific example of a modified fibroin containing a tag sequence, a modified fibroin containing: (2-iii) an amino acid sequence set forth 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 a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, can be mentioned.
The amino acid sequences set forth in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are amino acid sequences obtained by adding an amino acid sequence set forth in SEQ ID NO: 11 (containing a His tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
The modified fibroin of (2-iii) may include the amino acid sequence set forth 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 a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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 the domain sequence represented by Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (2-iv) may also be a modified fibroin which has a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and in which when the total number of amino acid residues in the amino acid sequence including XGX (provided that X represents an amino acid residue other than glycine) included in the REP is denoted by z, and the total number of amino acid residues in the REP in the domain sequence is denoted by w, z/w is 50.9% or more.
The second modified fibroin may contain a secretory signal for releasing a protein produced in the recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set according to the type of the host.
Regarding the third modified fibroin, its domain sequence has an amino acid sequence in which the content of the (A)n motif has been reduced as compared to naturally derived fibroin. The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of the (A)n motifs have been deleted, as compared to naturally derived fibroin.
The third modified fibroin may have an amino acid sequence corresponding to an amino acid sequence obtained by deleting 10% to 40% of the (A)n motifs from naturally derived fibroin.
Regarding the third modified fibroin, its domain sequence may have an amino acid sequence corresponding to an amino acid sequence in which at least one (A)n motif in every one to three (A)n motifs has been deleted from the N-terminal side to the C-terminal side, as compared to naturally derived fibroin.
Regarding the third modified fibroin, its domain sequence may have an amino acid sequence corresponding to an amino acid sequence in which at least a deletion of two consecutive (A)n motifs and a deletion of one (A)n motif are repeated in this order from the N-terminus side to the C-terminus side, as compared to naturally derived fibroin.
Regarding the third modified fibroin, its domain sequence may have an amino acid sequence corresponding to an amino acid sequence in which at least every two (A)n motifs have been deleted from the N-terminus side toward the C-terminus side.
The third modified fibroin may also have an amino acid sequence which contains a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in which when the numbers of amino acid residues in the REP in two adjacent [(A)n motif-REP] units are sequentially compared from the N-terminus side toward the C-terminus side, and in a case where the number of amino acid residues in the REP having a smaller number of amino acid residues is designated as 1, the maximum value of the sum value obtained by adding the numbers of amino acid residues in two adjacent [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is designated as x, while the total number of amino acid residues in the domain sequence is designated as y, x/y is 20% or more, 30% or more, 40% or more, or 50% or more. The number of alanine residues with respect to the total number of amino acid residues in the (A)n motif may be 83% or more, 86% or more, 90% or more, 95% or more, or 100%. The number of alanine residues with respect to the total number of amino acid residues being 100% means that the (A)n motif is composed only of alanine residues.
A calculation method for x/y will be described in more detail with reference to
Two adjacent [(A)n motif-REP] units are sequentially selected from the N-terminus side to the C-terminus side so as to have no overlaps. At this time, there may be an unselected [(A)n motif-REP] unit.
Next, for each of the patterns, the numbers of amino acid residues of the respective REPs in the selected two adjacent [(A)n motif-REP] units are compared with each other. The comparison is carried out, when the unit having a smaller number of amino acid residues is designated as 1, by determining the ratio of the number of amino acid residues on the other unit. For example, in the case of making a comparison between the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is designated as 1, the ratio of the number of amino acid residues of the second REP is 100/50=2. Similarly, in the case of making a comparison between the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is designated as 1, the ratio of the number of amino acid residues of the fifth REP is 30/20=1.5.
In
In each pattern, all the numbers of amino acid residues in two adjacent [(A)n motif-REP] units shown by the solid lines are summed up (the numbers of amino acid residues in the REPs as well as in the (A)n motifs are added). The sum values obtained by summing up are compared, and the sum value of the pattern whose sum value is the maximum (maximum value of the sum value) is denoted by x. In the example shown in
Then, x/y (%) can be calculated by dividing x by the total number of amino acid residues y of the domain sequence.
In the third modified fibroin, x/y may be 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. The upper limit of x/y is not particularly limited, and for example, x/y may be 100% or less. The jagged ratio may be 1:1.9 to 11.3, and x/y may be 89.6% or more. The jagged ratio may be 1:1.8 to 3.4, and x/y may be 77.1% or more. The jagged ratio may be 1:1.9 to 8.4, and x/y may be 75.9% or more. The jagged ratio may be 1:1.9 to 4.1, and x/y may be 64.2% or more.
When the third modified fibroin is a modified fibroin in which at least seven of a plurality of the (A)n motifs present in the domain sequence are composed only of alanine residues, x/y may be 46.4% or more, 50% or more, 55% or more, 60% or more, 70% or more, or 80% or more. The upper limit of x/y is not particularly limited and may be 100% or less.
Here, x/y in the naturally derived fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered with the NCBI GenBank were checked by the method mentioned as an example, and 663 kinds of fibroins (among these, there were 415 kinds of spider-derived fibroins) were extracted. x/y was calculated by the above-mentioned calculation method from the amino acid sequences of naturally derived fibroins composed of a domain sequence represented by Formula 1: [(A)n motif-REP]m among all the extracted fibroins. As a result, x/y in the naturally derived fibroins was less than 64.2% in all cases, and the maximum was 64.14%.
The third modified fibroin can be obtained by, for example, deleting one or a plurality of sequences encoding the (A)n motif from a cloned gene sequence for the naturally derived fibroin such that x/y is 64.2% or more. In addition, the third modified fibroin can also be obtained by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of (A)n motifs have been deleted from the amino acid sequence of naturally derived fibroin such that x/y is 64.2% or more, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In all cases, in addition to the modification corresponding to a deletion of the (A)n motif from the amino acid sequence of naturally derived fibroin, modification of the amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may also be carried out.
As a more specific example of the third modified fibroin, a modified fibroin containing: (3-i) an amino acid sequence set forth 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 a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, can be mentioned.
The modified fibroin of (3-i) will be described. The amino acid sequence set forth in SEQ ID NO: 17 is an amino acid sequence obtained by deleting every two (A)n motifs from the amino acid sequence set forth in SEQ ID NO: 10 (Met-PRT313), which corresponds to naturally derived fibroin, from the N-terminus side toward the C-terminus side, and inserting one [(A)n motif-REP] before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described in connection with the second modified fibroin.
The value of x/y for the amino acid sequence set forth in SEQ ID NO: 10 (corresponding to naturally derived fibroin) at a jagged ratio of 1:1.8 to 11.3 is 15.0%. The values of x/y for the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ ID NO: 7 are both 93.4%. The value of x/y for the amino acid sequence set forth in SEQ ID NO: 8 is 92.7%. The value of x/y for the amino acid sequence set forth in SEQ ID NO: 9 is 89.8%. The values of z/w for the amino acid sequences set forth 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.
The modified fibroin of (3-i) may include an amino acid sequence set forth 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 a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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 the domain sequence represented by Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (3-ii) may have a sequence identity of 90% or higher with an amino acid sequence set forth 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 in the REPs in two adjacent [(A)n motif-REP] units are sequentially compared from the N-terminus side toward the C-terminus side, and the number of amino acid residues in the REP having a smaller number of amino acid residues is designated as 1, and when the maximum value of the sum value obtained by adding the numbers of amino acid residues in two adjacent [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 (jagged ratio is 1:1.8 to 11.3) is designated as x, while the total number of amino acid residues in the domain sequence is designated as y, x/y is 64.2% or more.
The third modified fibroin may be such that either or both of the N-terminus and the C-terminus contain the above-mentioned tag sequence.
As a more specific example of a modified fibroin containing a tag sequence, a modified fibroin containing: (3-iii) an amino acid sequence set forth 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 a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, can be mentioned.
The amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are amino acid sequences obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
The modified fibroin of (3-iii) may be a modified fibroin including an amino acid sequence set forth 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 a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth 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 the domain sequence represented by Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (3-iv) may have a sequence identity of 90% or higher with an amino acid sequence set forth 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 in the REPs of two adjacent [(A)n motif-REP] units are sequentially compared from the N-terminus side toward the C-terminus side, and the number of amino acid residues in an REP having a smaller number of amino acid residues is designated as 1, and when the maximum value of the sum value obtained by adding the numbers of amino acid residues in two adjacent [(A)n motif-REP] units where the ratio of the number of amino acid residues in the other REP is 1.8 to 11.3 is designated as x, and the total number of amino acid residues in the domain sequence is designated as y, x/y is 64.2% or more.
The third modified fibroin may contain a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set according to the type of the host.
Regarding the fourth modified fibroin, its domain sequence has an amino acid sequence in which the content of glycine residues has been reduced, and the content of the (A)n motifs has also been reduced, as compared to naturally derived fibroin. It can be said that the domain sequence of the fourth modified fibroin has an amino acid sequence corresponding to an amino acid sequence in which at least one or a plurality of (A)n motifs have been deleted, and at least one or a plurality of glycine residues in the REP have also been substituted with other amino acid residue(s), as compared to naturally derived fibroin. That is, the fourth modified fibroin is a modified fibroin having the features of the second modified fibroin and third modified fibroin in combination. Specific embodiments and the like are as described in connection with the second modified fibroin and the third modified fibroin.
As a more specific example of the fourth modified fibroin, a modified fibroin containing: (4-i) an amino acid sequence set forth 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) an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth 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, can be mentioned. Specific embodiments of the modified fibroin containing the amino acid sequence set forth 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.
Regarding the fifth modified fibroin, its domain sequence may have an amino acid sequence containing a region in which the hydropathy index is locally high, the amino acid sequence corresponding to an amino acid sequence in which one or a plurality of amino acid residues in the REP have been substituted with an amino acid residue(s) having a high hydropathy index, and/or an amino acid sequence in which one or a plurality of amino acid residues having a high hydropathy index have been inserted into the REP, as compared to naturally derived fibroin.
The region in which the hydropathy index is locally high may be composed of two to four consecutive amino acid residues.
The above-mentioned amino acid residue having a high hydropathy index may be an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A).
The fifth modified fibroin may further have modifications of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues as compared to naturally derived fibroin, in addition to modification corresponding to substitution of one or a plurality of amino acid residues in the REP with an amino acid residue having a large hydropathy index, and/or insertion of one or a plurality of amino acid residues having a large hydropathy index into the REP, as compared to naturally derived fibroin.
The fifth modified fibroin can be obtained by, for example, substituting one or a plurality of hydrophilic amino acid residues (for example, amino acid residues having a negative hydropathy index) in the REP from a cloned gene sequence of naturally derived fibroin with a hydrophobic amino acid residue(s) (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydrophobic amino acid residues into the REP. Furthermore, the fifth modified fibroin can also be obtained by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of hydrophilic amino acid residues in the REP have been substituted with a hydrophobic amino acid residue(s), and/or one or a plurality of hydrophobic amino acid residues have been inserted into the REP, from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In all cases, in addition to the modification of the amino acid sequence corresponding to substitution of one or a plurality of hydrophilic amino acid residues in the REP from the amino acid sequence of naturally derived fibroin substituted with hydrophobic amino acid residue(s), and/or insertion of one or a plurality of hydrophobic amino acid residues into the REP, modification of an amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be further carried out.
The fifth modified fibroin may also have an amino acid sequence which contains a domain sequence represented by Formula 1: [(A)n motif-REP]m, and in which for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence, when the total number of amino acid residues included in a region in which the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more is designated as p, and the total number of amino acid residues included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence is designated as q, p/q is 6.2% or more.
Regarding the hydropathy index of the amino acid residue, 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) is used. Specifically, the hydropathy index (hereinafter, also referred to as “HI”) of each amino acid is indicated in the following Table 1.
A calculation method for p/q will be described in more detail. For the calculation, a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from a domain sequence represented by Formula 1: [(A)n motif-REP]m (hereinafter, referred to as “sequence A”) is used. First, for all the REPs included in the sequence A, the average value of the hydropathy indices of four consecutive amino acid residues is calculated. The average value of the hydropathy index is determined by dividing the total sum of HI of the respective amino acid residues included in the four consecutive amino acid residues by 4 (number of amino acid residues). The average value of the hydropathy index is determined for all of the four consecutive amino acid residues (each of the amino acid residues is used for calculating the average values for 1 to 4 times). Next, a region in which the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more is specified. Even in a case where a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of the hydropathy indices is 2.6 or more”, the amino acid residue is counted as one amino acid residue in the region. Then, the total number of amino acid residues included in the region is p. Also, the total number of amino acid residues included in the sequence A is q.
For example, in a case where the “four consecutive amino acid residues whose average value of the hydropathy indices is 2.6 or more” are extracted at 20 sites (no overlap), in a region where the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more, twenty sets of four consecutive amino acid residues (no overlap) are included, and p is such that 20×4=80. For example, in a case where two sets of the “four consecutive amino acid residues whose average value of the hydropathy indices is 2.6 or more” overlap by only one amino acid residue, in a region where the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more, the number of amino acid residues included is 7 (p=2×4 −1=7. “−1” is the deduction for overlap). For example, in the case of the domain sequence shown in
With regard to the fifth modified fibroin, p/q may be 6.2% or more, 7% or more, 10% or more, 20% or more, or 30% or more. The upper limit of p/q is not particularly limited but may be, for example, 45% or less.
The fifth modified fibroin can be obtained by, for example, modifying a cloned amino acid sequence of naturally derived fibroin into an amino acid sequence containing a region in which the hydropathy index is locally high, by substituting one or a plurality of hydrophilic amino acid residues (for example, amino acid residues having a negative hydropathy index) in the REP with hydrophobic amino acid residues (for example, amino acid residues having a positive hydropathy index), and/or inserting one or a plurality of hydrophobic amino acid residues into the REP, so as to satisfy the above-described conditions for p/q. Furthermore, the fifth modified fibroin can also be obtained by, for example, designing an amino acid sequence that satisfies the above-described conditions for p/q from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In all cases, in addition to the modification corresponding to a substitution of one or a plurality of amino acid residues in the REP with an amino acid residue(s) having a high hydropathy index, and/or an insertion of one or a plurality of amino acid residues having a high hydropathy index into the REP, modification corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues may be further carried out, as compared to naturally derived fibroin.
The amino acid residue with a high hydropathy index is not particularly limited but may be an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). The amino acid residue having a high hydropathy index may also be an amino acid residue selected from valine (V), leucine (L), and isoleucine (I).
As a more specific example of the fifth modified fibroin, a modified fibroin containing: (5-i) an amino acid sequence set forth 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 a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, can be mentioned.
The modified fibroin of (5-i) will be described. The amino acid sequence set forth in SEQ ID NO: 19 is an amino acid sequence obtained by inserting an amino acid sequence each including three amino acid residues (VLI) at two sites in every other REP in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410), except for the domain sequence at the end on the C-terminus side, further substituting some of glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminus side. The amino acid sequence set forth in SEQ ID NO: 20 is an amino acid sequence obtained by inserting an amino acid sequence each including three amino acid residues (VLI) at one site in every other REP in the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525). The amino acid sequence set forth in SEQ ID NO: 21 is an amino acid sequence obtained by inserting an amino acid sequence each including three amino acid residues (VLI) at two sites in every other REP in the amino acid sequence set forth in SEQ ID NO: 8.
The modified fibroin of (5-i) may include an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
The modified fibroin of (5-ii) contains an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein including a domain sequence set forth in Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (5-ii) may have a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence, when the total number of amino acid residues included in a region in which the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more is designated as p, and the total number of amino acid residues included in a sequence obtained by excluding the sequence from the (A)n motif located at the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence is designated as q, p/q is 6.2% or more.
The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.
As a more specific example of the modified fibroin containing a tag sequence, a modified fibroin containing: (5-iii) an amino acid sequence set forth 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 a sequence identity of 90% or higher with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, can be mentioned.
The amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are amino acid sequences obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (containing a His tag sequence and a hinge sequence) to the N-termini of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
The modified fibroin of (5-iii) may include an amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The modified fibroin of (5-iv) is a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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 the domain sequence represented by Formula 1: [(A)n motif-REP]m. The above-described sequence identity may be 95% or higher.
The modified fibroin of (5-iv) may have a sequence identity of 90% or higher with an amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence, when the total number of amino acid residues included in a region in which the average value of the hydropathy indices of four consecutive amino acid residues is 2.6 or more is designated as p, and the total number of amino acid residues included in a sequence obtained by excluding the sequence from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence is designated as q, p/q is 6.2% or more.
The fifth modified fibroin may contain a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set according to the type of the host.
The sixth modified fibroin has an amino acid sequence in which the content of glutamine residues has been reduced, as compared to naturally derived fibroin.
Regarding the sixth modified fibroin, at least one motif selected from a GGX motif and a GPGXX motif may be included in the amino acid sequence of REP.
In a case where the sixth modified fibroin contains a GPGXX motif in the REP, the percentage content of the GPGXX motif is usually 1% or more and may be 5% or more or 10% or more. The upper limit of the percentage content of the GPGXX motif is not particularly limited and may be 50% or less or 30% or less.
In the present specification, the “percentage content of the GPGXX motif” is a value calculated by the following method.
With regard to 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, for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence, when the number at which the total number of the numbers of GPGXX motifs included in the region becomes three times (that is, corresponding to the total number of G and P in the GPGXX motif) is designated as s, and the total number of amino acid residues of all the REPs obtained by excluding the sequence from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence and further excluding the (A)n motif is designated as t, the percentage content of the GPGXX motif is calculated as s/t.
With regard to the calculation of the percentage content of the GPGXX motif, “a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence” is the target of the calculation because in the “sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence” (sequence corresponding to an REP), a sequence having low correlation with a sequence characteristic of fibroin may be included, and when m is small (that is, when the domain sequence is short), since the sequence affects the results of calculation of the percentage content of the GPGXX motif, this influence needs to be eliminated. Incidentally, in a case where a “GPGXX motif” is located at the C-terminus of REP, even when “XX” is, for example, “AA”, the motif is dealt with as “GPGXX motif”.
The percentage content of glutamine residue in the sixth modified fibroin may be 9% or less, 7% or less, 4% or less, or 0%.
According to the present specification, the “percentage content of glutamine residue” is a value calculated by the following method.
With regard to 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, for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in
Regarding the sixth modified fibroin, its domain sequence may have an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of glutamine residues in the REP have been deleted or substituted with other amino acid residue(s), as compared to naturally derived fibroin.
The “other amino acid residue(s)” may be an amino acid residue other than a glutamine residue but may also be an amino acid residue with a higher hydropathy index than that of a glutamine residue. The hydropathy indices of the amino acid residues are as shown in Table 1.
As shown in Table 1, examples of the amino acid residue with a higher hydropathy index than that of a glutamine residue 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, the amino acid residue may be an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A), or may be an amino acid residue selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F).
The degree of hydrophobicity of REP in the sixth modified fibroin may be −0.8 or higher, −0.7 or higher, 0 or higher, 0.3 or higher, or 0.4 or higher. The upper limit of the degree of hydrophobicity of REP is not particularly limited, and the degree of hydrophobicity of REP may be 1.0 or lower or 0.7 or lower.
According to the present specification, the “degree of hydrophobicity of REP” is a value calculated by the following method.
With regard to 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, for all the REPs included in a sequence obtained by excluding the sequence ranging from the (A)n motif located on the furthermost C-terminus side to the C-terminus of the domain sequence from the domain sequence (sequence corresponding to “region A” in
Regarding the sixth modified fibroin, its domain sequence may have further modification of the amino acid sequence corresponding to substitution, deletion, insertion, and/or addition of one or a plurality of amino acid residues, in addition to the modification of deletion of one or a plurality of glutamine residues in the REP and/or substitution of one or a plurality of glutamine residues in the REP with other amino acid residue(s), as compared to naturally derived fibroin.
The sixth modified fibroin can be obtained from, for example, a cloned gene sequence of naturally derived fibroin by deleting one or a plurality of glutamine residues in the REP and/or substituting one or a plurality of glutamine residues in the REP with other amino acid residue(s). Furthermore, the sixth modified fibroin can also be obtained by, for example, designing an amino acid sequence corresponding to an amino acid sequence in which one or a plurality of glutamine residues in the REP have been deleted and/or one or a plurality of glutamine residues in the REP have been substituted with other amino acid residue(s), from the amino acid sequence of naturally derived fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
As a more specific example of the sixth modified fibroin, (6-i) a modified fibroin containing an amino acid sequence set forth 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) a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with the amino acid sequence set forth 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, can be mentioned.
The modified fibroin of (6-i) will be described. The amino acid sequence set forth in SEQ ID NO: 25 is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) with VL. The amino acid sequence set forth in SEQ ID NO: 26 is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with TS and substituting the remaining Qs with As. The amino acid sequence set forth in SEQ ID NO: 27 is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with VL and substituting the remaining Qs with Is. The amino acid sequence set forth in SEQ ID NO: 28 is obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with VI and substituting the remaining Qs with Ls. The amino acid sequence set forth in SEQ ID NO: 29 is obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with VF and substituting the remaining Qs with Is.
The amino acid sequence set forth in SEQ ID NO: 30 is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence set forth in SEQ ID NO: 31 is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 8 with VL and substituting the remaining Qs with Is.
The amino acid sequence set forth in SEQ ID NO: 32 is an amino acid sequence obtained by substituting every QQ with VF and substituting the remaining Qs with Is, in a sequence in which a region of twenty domain sequences present in the amino acid sequence set forth in SEQ ID NO: 7 (Met-PRT410) is repeated twice.
The amino acid sequence set forth in SEQ ID NO: 41 (Met-PRT917) is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with LI and substituting the remaining Qs with Vs. The amino acid sequence set forth in SEQ ID NO: 42 (Met-PRT1028) is an amino acid sequence obtained by substituting every QQ in the amino acid sequence set forth in SEQ ID NO: 7 with IF and substituting the remaining Qs with Ts.
In the amino acid sequences set forth 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, the percentage content of glutamine residue is 9% or less in all cases (Table 2).
The modified fibroin of (6-i) may be a modified fibroin including an amino acid sequence set forth 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 a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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 above-described sequence identity may be 95% or higher.
The percentage content of glutamine residue in the modified fibroin of (6-ii) may be 9% or less. Furthermore, the percentage content of GPGXX motif in the modified fibroin of (6-ii) may be 10% or more.
The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, and visualization of the modified fibroin.
As a more specific example of the modified fibroin having a tag sequence, (6-iii) a modified fibroin containing an amino acid sequence set forth 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) a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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, can be mentioned.
The amino acid sequences set forth 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 amino acid sequences obtained by adding the amino acid sequence set forth in SEQ ID NO: 11 (containing a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences set forth 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 this is merely addition of a tag sequence to the N-terminus, there is no change in the percentage content of glutamine residue, and in the amino acid sequences set forth 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, the percentage content of glutamine residue is 9% or less in all cases (Table 3).
The modified fibroin of (6-iii) may be a modified fibroin including the amino acid sequence set forth 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 a modified fibroin containing an amino acid sequence having a sequence identity of 90% or higher with an amino acid sequence set forth 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 above-described sequence identity may be 95% or higher.
In the modified fibroin of (6-iv), the percentage content of glutamine residue may be 9% or less. Furthermore, in the modified fibroin of (6-iv), the percentage content of GPGXX motif may be 10% or more.
The sixth modified fibroin may be a modified fibroin containing a secretory signal for releasing a protein produced in a recombinant protein production system to the outside of a host. The sequence of the secretory signal can be appropriately set according to the type of the host.
The modified fibroin may also be a modified fibroin having at least two or more features in combination, among the features possessed by the modified fibroin of the first embodiment, the modified fibroin of the second embodiment, the modified fibroin of the third embodiment, the modified fibroin of the fourth embodiment, the modified fibroin of the fifth embodiment, and the modified fibroin of the sixth embodiment.
The modified fibroin may be a hydrophobic modified fibroin, from the viewpoint that contraction by contact with moisture can be suppressed. According to the present specification, the “hydrophobic modified fibroin” is a modified fibroin whose value obtained by determining the total sum of the hydropathy indices (HI) of all the amino acid residues constituting the modified fibroin and then dividing the total sum by the total number of amino acid residues (average HI) is 0 or more. The average value of the hydropathy index (average HI) of the modified fibroin may be 0.5 or more, or 0.6 or more. The average value of the hydropathy index (average HI) of the modified fibroin may be 1.2 or less, or 0.9 or less. The hydropathy index is as shown in Table 1. Furthermore, a modified fibroin having an average HI or less than 0 is also referred to as a hydrophobic modified fibroin.
Examples of the hydrophobic modified fibroin include modified fibroins containing an amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43; and an amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 44.
Examples of a hydrophilic modified fibroin include modified fibroins containing an amino acid sequence set forth in SEQ ID NO: 4; an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9; an amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15; an amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9; an amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15; or an amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
Each of the modified fibroins according to the above-described embodiments can all be produced by, for example, expressing a nucleic acid encoding the modified fibroin using a host transformed with an expression vector that has the nucleic acid sequence and one or a plurality of regulatory sequences operatively linked to the nucleic acid sequence.
The method for producing a nucleic acid encoding a modified fibroin is not particularly limited. For example, the nucleic acid can be produced by a method of utilizing a gene encoding naturally derived fibroin to amplify and clone the gene by a polymerase chain reaction (PCR) or the like, and modifying the nucleic acid by a genetic engineering technique, or a method of chemically synthesizing the nucleic acid. The method of chemically synthesizing a nucleic acid is also not particularly limited, and for example, a gene can be chemically synthesized by a method of linking an oligonucleotide that has been automatically synthesized with AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) or the like, by PCR or the like based on the amino acid sequence information of fibroin obtained from the NCBI web database and the like. At this time, in order to facilitate purification and/or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin including an amino acid sequence obtained by adding an amino acid sequence including a start codon and a His10 tag to the N-terminus of the above-described amino acid sequence, may be synthesized.
The regulatory sequence is a sequence that controls the expression of the modified fibroin in a host (for example, a promoter, an enhancer, a ribosome binding site, a transcription termination sequence, and the like) and can be appropriately selected according to the type of the host. As a promoter, an inducible promoter that functions in a host cell and can induce expression of a modified fibroin, may be used. An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.
The type of the expression vector can be appropriately selected according to the type of the host, from a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, an expression vector which is capable of autonomous replication in a host cell or is capable of incorporation into the chromosome of a host, and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed, is suitably used.
As the host, prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can all be used.
Preferred examples of a prokaryote host include bacteria belonging to the genus Escherichia, the genus Brevibacillus, the genus Serratia, the genus Bacillus, the genus Microbacterium, the genus Brevibacterium, the genus Corynebacterium, and the genus Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of the microorganism belonging to the genus Pseudomonas include Pseudomonas putida.
In a case where a prokaryote is used as a host, examples of the vector into which a nucleic acid encoding a modified fibroin is introduced include pBTrp2 (manufactured by Boehringer Mannheim Corporation), pGEX (manufactured by Pharmacia Corporation), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).
Examples of a eukaryotic host include yeast and filamentous fungi (molds and the like). Examples of the yeast include yeasts belonging to the genus Saccharomyces, the genus Pichia, and the genus Schizosaccharomyces. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, and the genus Trichoderma.
In a case where a eukaryote is used as a host, examples of the vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). Regarding a method for introducing an expression vector into the above-described host cell, any method can all be used as long as it is a method for introducing a DNA into the host cell. For example, a method of using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], an electroporation method, a spheroplast method, a protoplast method, a lithium acetate method, and a competent method may be mentioned.
Regarding a method for expressing a nucleic acid by a host transformed with an expression vector, secretory production, fusion protein expression, and the like can be carried out, in addition to direct expression, according to the methods described in Molecular Cloning, 2nd Edition, or the like.
A modified fibroin can be produced by, for example, culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing a host in a culture medium can be carried out according to a method that is conventionally used for culturing a host.
In a case where the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any of a natural medium and a synthetic medium may be used as the culture medium, as long as it is a medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host and is capable of efficiently performing culturing of the host.
As the carbon source, any carbon source that can be assimilated by a transformed microorganism may be used, and for example, carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates containing these; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol, can be used. As the nitrogen source, for example, ammonia, ammonium salts of inorganic acids or organic acids, such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; other nitrogen-containing compounds; and peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean cake, and soybean cake hydrolysate, various fermented microbial cells, and digestion products thereof, can be used. As the inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
Culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be carried out, for example, under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is, for example, 15° C. to 40° C. The culturing time is usually 16 hours to 7 days. The pH of the culture medium during culturing may be maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be performed using an inorganic acid, an organic acid, an alkali solution, urea, calcium carbonate, ammonia, and the like.
Furthermore, during culturing, antibiotic substances such as ampicillin and tetracycline may also be added to the culture medium, as necessary. When culturing a microorganism transformed with an expression vector that uses an inducible promoter as a promoter, if necessary, an inducer may be added to the medium. For example, in the case of culturing a microorganism transformed with an expression vector that uses a lac promoter, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and in the case of culturing a microorganism transformed with an expression vector that uses a trp promoter, indole acrylic acid or the like may be added to the medium.
Isolation and purification of the expressed modified fibroin can be performed by a method that is conventionally used. For example, in a case where the modified fibroin is expressed in a state of being dissolved in cells, after completion of culture, the host cells are collected by centrifugal separation and suspended in a water-based buffer solution, subsequently the host cells are disrupted using an ultrasonic disruptor, a French press, a Manton-Gaulin homogenizer, a Dyno Mill, or the like, and a cell-free extract is obtained. From a supernatant obtained by centrifugally separating this cell-free extract, a purified preparation can be obtained by methods that are conventionally used for the isolation and purification of a modified fibroin, that is, methods such as a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE)-sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), a cation exchange chromatography method using a resin such as S-sepharose FF (manufactured by Pharmacia Corporation), a hydrophobic chromatography method using a resin such as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, and an electrophoresis method such as isoelectric point electrophoresis, singly or in combination.
Furthermore, in a case where a modified fibroin has formed an insoluble matter in the cell and is expressed, host cells are similarly collected and then disrupted, centrifugal separation is carried out, and thereby the insoluble matter of the modified fibroin is collected as a precipitated fraction. The insoluble matter of the modified fibroin thus collected can be solubilized with a protein denaturing agent. After this operation, a purified preparation of the modified fibroin can be obtained by a similar isolation and purification method as described above. In a case where the modified fibroin is secreted outside the cells, the modified fibroin can be collected from the culture supernatant. That is, the culture supernatant is obtained by treating a culture product by a technique such as centrifugal separation, and from the culture supernatant, a purified preparation can be obtained by using a similar isolation and purification method as described above.
A fibroin fiber that is given a predetermined shape or a curled shape may expand when placed in a wet state and contract when dried from a wet state. By giving a predetermined shape to the fibroin fiber that expands when placed in a wet state and contracts when dried from a wet state according to the above-mentioned method, a fiber for artificial hairs that is given a predetermined shape, which expands when placed in a wet state and contracts when dried from a wet state, can be easily produced. The predetermined shape may be a shape including a curved part, a linear part, or both of these.
The recovery rate of the fibroin fiber that expands when placed in a wet state and contracts when dried from a wet state, the recovery rate being defined by the following Formula (1), may be 95% or higher.
Recovery rate=(Length of fibroin fiber when dried from wet state/length of fibroin fiber before being placed in wet state)×100(%) Formula (1):
As the recovery rate defined by Formula (1) is higher, the behavior at the time of wetting and drying is close to that of human hair, and therefore, the occurrence of a feeling of strangeness between artificial hair and human hair can be suppressed. The recovery rate of the fibroin fiber as defined by Formula (1) may be 96% or higher, 97% or higher, 98% or higher, or 99% or higher.
The elongation rate of the fibroin fiber as defined by the following Formula (4) may be 17% or less. The elongation rate defined by Formula (4) is an index of the elongation characteristics obtainable when the fibroin fiber is placed in a wet state.
Elongation rate={(Length of fibroin fiber in wet state/length of fibroin fiber before being placed in wet state)−1}×100(%) Formula (4):
The elongation rate defined by Formula (4) may be, for example, 15% or less, 13% or less, 10% or less, or 5% or less. The elongation rate defined by Formula (4) may be, for example, more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more. The elongation rate of the fibroin fiber according to the present embodiment may be, for example, more than 0% and 17% or less, more than 0% and 15% or less, 2% or more and 15% or less, 5% or more and 15% or less, 5% or more and 13% or less, 5% or more and 10% or less, more than 0% and 10% or less, or more than 0% and 5% or less. From the viewpoint of further reducing the feeling of strangeness between artificial hair and human hair, the elongation rate defined by Formula (4) may be small.
The contraction rate C of the fibroin fiber as defined by the following Formula (5) may be 17% or less. The contraction rate C defined by Formula (5) is an index of the contraction characteristics obtainable when the fibroin fiber is dried from a wet state.
Contraction rate C={1−(length of fibroin fiber when dried from wet state/length of fibroin fiber in wet state)}×100(%). Formula (5):
The contraction rate C defined by Formula (5) may be 15% or less, 13% or less, 10% or less, or 5% or less. The contraction rate C defined by Formula (5) may be more than 0%, 1% or more, 2% or more, 5% or more, 10% or more, or 13% or more. The contraction rate C of the fibroin fiber according to the present embodiment may be, for example, more than 0% and 17% or less, more than 0% and 15% or less, 2% or more and 15% or less, 5% or more and 15% or less, 5% or more and 13% or less, 5% or more and 10% or less, more than 0% and 10% or less, or more than 0% and 5% or less. From the viewpoint of further reducing the feeling of strangeness between artificial hair and human hair, the contraction rate C defined by Formula (5) may be low.
The fibroin fiber may be a fiber having a contraction history of being irreversibly contracted by being brought into contact with water after spinning. In that case, contraction rate A of the fibroin fiber as defined by the following Formula (2) may be 2% or less. For a fibroin fiber having a contraction rate A of 2% or more is such that the behavior at the time of wetting and drying is closer to that of human hair.
Contraction rate A={1−(length of fiber irreversibly contracted by being brought into contact with water after spinning/length of fiber before being brought into contact with water after spinning)}×100(%) Formula (2):
The contraction rate A defined by Formula (2) may be 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more, 15% or more, 20% or more, or 25% or more. The upper limit of the contraction rate A defined by Formula (2) is not particularly limited; however, the contraction rate A may be 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less.
The fibroin fiber may also be a fiber having a contraction history of being irreversibly contracted by being brought into contact with water after spinning and then being further contracted by drying. In this case, contraction rate B of the fibroin fiber as defined by the following Formula (3) may be more than 7%. The fibroin fiber having a contraction rate B defined by Formula (3) of more than 7% is such that the behavior of the fibroin fiber at the time of wetting and drying is closer to that of human hair.
Contraction rate B={1−(length of fiber being irreversibly contracted by being brought into contact with water after spinning and then being further contracted by drying/length of fiber before being brought into contact with water after spinning)}×100(%) Formula (3):
The contraction rate B defined by Formula (3) may be 10% or more, 15% or more, more than 25%, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72% or more. The upper limit of the contraction rate B defined by Formula (3) is not particularly limited; however, the contraction rate B is usually 80% or less.
Concavities or grooves extending in the direction of fiber axis may be formed on the surface of the fibroin fiber. A fibroin fiber having a surface where concavities or grooves are formed shows suppressed glossiness and can realize an outer appearance similar to that of human hair. Regarding a method of providing concavities on the surface of the fibroin fiber, for example, a method of performing spinning by a wet spinning method at the time of spinning a raw material fiber, a method of lowering the rate of desolventization (for example, a method of adding the solvent of a dope solution to a coagulating liquid), and the methods described in WO 2016/201369 A (for example, a method of lengthening the staying time in a coagulation bath (60 seconds or longer), and a method of changing the solvent ratio in the coagulation bath) can be employed.
The thermal contraction rate of the fibroin fiber defined by the following Formula (6) may be 4% or less.
Thermal contraction rate={1−(length of fibroin fiber when heated to 160° C./length of fibroin fiber before heating)}×100(%) Formula (6):
A fiber for artificial hairs formed from a fibroin fiber containing a modified fibroin has a softening point at a temperature that is higher than that of general synthetic fibers and is comparable to that of human hair. Therefore, the fibroin fiber has a small thermal contraction rate (thermal contraction rate defined by Formula (6)) at 160° C. The hot air temperature of hair dryers is set to 120° C. to 140° C., and the suitable temperature for use of hair curling tongs is 170° C. or lower. Since the thermal contraction rate defined by Formula (6) is small, the damage occurring at the time of using a hair dryer or a hair curling tong can be suppressed. The thermal contraction rate defined by Formula (6) may be, for example, 4% or less, 3% or less, or 2.5% or less.
A fibroin fiber containing a modified fibroin can be used as an animal-free (not containing any animal-derived component) material.
A fibroin fiber 70 can be produced according to a conventional spinning method using a dope solution containing a modified fibroin. The dope solution is produced by, for example, adding a modified fibroin to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution promoter as necessary, and dissolving the mixture. An intended fibroin fiber can be obtained by performing spinning using this dope solution according to a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or dry-wet spinning. The spinning method may also be melt spinning.
A dope solution 6 stored in a reservoir 7 is extruded through a spinneret 9 by a gear pump 8. On a laboratory scale, a cylinder may be filled with the dope solution, and the dope solution may be extruded through a nozzle using a syringe pump. Next, the extruded dope solution 6 is fed into a coagulating liquid 11 in a coagulating liquid tank 20 through an air gap 19, the modified fibroin is coagulated by removing the solvent, and a fibrous coagulated body is formed. Next, the fibrous coagulated body is then fed into warm water 12 in a drawing bath 21 and then drawn. The draw ratio is determined by the speed ratio between a feed nip roller 13 and a take-up nip roller 14. Subsequently, the drawn fibrous coagulated body is fed into the drying device 4 and dried in a thread guide 22, and the fibroin fiber 70 is obtained as a wound yarn body 5. 18a to 18g are yarn guides.
The coagulating liquid 11 may be any solvent that can be desolventized, and examples include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. The coagulating liquid 11 may also contain water. The temperature of the coagulating liquid 11 may be 0° C. to 30° C. In a case where a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret 9, the extrusion speed may be 0.2 to 6.0 ml/hour or 1.4 to 4.0 ml/hour per hole. The distance that coagulated protein passes in the coagulating liquid 11 (substantially, the distance from a thread guide 18a to a thread guide 18b) may be a length over which desolventization can be efficiently performed, and for example, the distance is 200 to 500 mm. The take-up speed for the undrawn yarn may be, for example, 1 to 20 m/min or 1 to 3 m/min. The retention time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes or 0.05 to 0.15 minutes. Drawing (pre-drawing) may be performed in the coagulating liquid 11. The coagulating liquid tank 20 may be provided in multiple stages, and drawing may be performed in each stage or in a specific stage, as necessary.
As the drawing of the fibroin fiber 70, for example, in addition to the pre-drawing performed in the coagulating liquid tank 20 and wet heat drawing performed in the drawing bath 21 described above, dry heat drawing can also be adopted.
Wet heat drawing can be performed in hot water, in a solution obtained by adding an organic solvent or the like to hot water, or during steam heating. The temperature may be, for example, 50° C. to 90° C. or 75° C. to 85° C. During wet heat drawing, an undrawn yarn (or pre-drawn yarn) may be drawn to, for example, 1 to 10 times or 2 to 8 times.
Dry heat drawing can be performed using an electric tubular furnace, a dry heat plate, or the like. The temperature for dry heat drawing may be, for example, 140° C. to 270° C. or 160° C. to 230° C. During dry heat drawing, an undrawn yarn (or pre-drawn yarn) may be drawn to, for example, 0.5 to 8 times or 1 to 4 times.
Wet heat drawing and dry heat drawing may be performed each independently, or these may be performed in multiple stages or in combination. Wet heat drawing and dry heat drawing can be performed in appropriate combination, for example, in a manner in which first-stage drawing is performed by wet heat drawing and second-stage drawing is performed by dry heat drawing, or in a manner in which first-stage drawing is performed by wet heat drawing, second-stage drawing is performed by wet heat drawing, and third-stage drawing is performed by dry heat drawing.
The final draw ratio may be more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more, and may be 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less, 12 times or less, 11 times or less, or 10 times or less, all with respect to undrawn yarn (or pre-drawn yarn). When the fibroin fiber is a fiber spun at a draw ratio of 2 times or more, the contraction rate obtainable at the time of bringing the fibroin fiber into contact with water to be in a wet state becomes higher.
The fibroin fiber 70 formed by spinning may be contracted. The fibroin fiber obtainable through a step of contracting can have a property of expanding when placed in a wet state and contracting when dried from a wet state. The step of contracting may include, for example, after spinning, bringing the fibroin fiber 70 before being brought into contact with water to contract irreversibly. After the contact with water, the fibroin fiber 70 may be further contracted by drying.
In the step of contacting between fibroin and water, after spinning, the fibroin fiber 70 before being brought into contact with water is brought into contact with water to place the fibroin fiber 70 in a wet state. A wet state means a state in which at least a portion of the fibroin fiber 70 is in a state of being wetted with water. As a result, the fibroin fiber 70 can be irreversibly contracted without depending on external force.
The temperature of water to be brought into contact with the fibroin fiber 70 may be lower than the boiling point. As a result, handleability, workability in the contracting step, and the like are enhanced. From the viewpoint of sufficiently shortening the contraction time, the temperature of water may be 10° C. or higher, 40° C. or higher, or 70° C. or higher. The temperature of water may also be 90° C. or lower.
The method of bringing water into contact with the fibroin fiber 70 is not particularly limited. Examples of the method include a method of immersing the fibroin fiber 70 in water, a method of spraying water to the fibroin fiber 70 at normal temperature or in the form of heated steam or the like, and a method of exposing the fibroin fiber 70 to a high-humidity environment filled with water vapor. From the viewpoint that shortening of the contraction time can be effectively promoted, and also simplification of processing facilities and the like can be realized, a method of immersing the fibroin fiber 70 in water may be employed.
When the fibroin fiber 70 is brought into contact with water in a relaxed state, the fibroin fiber 70 may not only contract but also be curled to become wavy. In order to prevent the occurrence of such curling, for example, the fibroin fiber 70 may be brought into contact with water in an unrelaxed state, by bringing the fibroin fiber 70 into contact with water while applying tension in the direction of fiber axis, or the like.
The fibroin fiber 70 in a wet state may be further contracted by drying. Drying may be performed by, for example, natural drying or forced drying using a drying facility. As the drying facility, any known drying facilities of contact type or non-contact type can all be used. The drying temperature may be, for example, a temperature lower than the temperature at which the modified fibroin is decomposed or the fibroin fiber 70 is subjected to thermal damage, and for example, the drying temperature may be 20° C. to 150° C. or 50° C. to 100° C. The drying time is appropriately set according to the drying temperature or the like, and for example, a time during which the influence on the product quality and physical properties of the fibroin fiber 70 due to over-drying can be eliminated as far as possible, is employed.
A wound material of the fibroin fiber 70 is mounted on the feed roller 42. The fibroin fiber 70 is fed out continuously and automatically by the rotation of an electric motor or the like. The winder 44 winds the contracted fibroin fiber 70 continuously and automatically by the rotation of an electric motor. The feeding speed of the fibroin fiber 70 by the feed roller 42 and the winding speed of the fibroin fiber 70 by the winder 44 can be controlled independently of each other. The winding speed of the winder 44 may be slower than the feeding speed of the feed roller 42. Accordingly, since the fibroin fiber 70 contracts due to contact with water 47 in a tense state without being unrelaxed between the feed roller 42 and the winder 44, the occurrence of curling can be prevented. Due to the contact with water 47, the fibroin fiber 70 contracts irreversibly.
The water bath 46 and the dryer 48 are disposed in order between the feed roller 42 and the winder 44 from the upstream side in the feed direction of the fibroin fiber 70. The production apparatus 40 has relay rollers 50 and 52 that mediate the fibroin fiber 70.
The water bath 46 includes a heater 54, and water 47 heated by the heater 54 is accommodated in the water bath 46. In the water bath 46, a tension roller 56 is installed in a state of being immersed in the water 47. The fibroin fiber 70 fed out from the feed roller 42 travels in the water bath 46 toward the winder 44 side while being immersed in the water 47 in a state of being wound around the tension roller 56.
The dryer 48 has a pair of hot rollers 58. The fibroin fiber 70 that is released from the water bath 46 and travels toward the winder 44 is wound around the pair of hot rollers 58. The fibroin fiber 70 in a wet state is heated by the pair of hot rollers 58 and is dried thereby in the dryer 48. The fibroin fiber 70 is further contracted by this drying. The fibroin fiber 70 after drying is wound around the winder 44.
By controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, the fibroin fiber 70 can be further contracted, and the length of the fibroin fiber 70 can be made unchanged.
The processing device 60 of
The drying device 62 illustrated in
Hereinafter, the present invention will be described more specifically based on Examples and the like. However, the present invention is not intended to be limited to following Examples.
1. Fibroin Fiber
(1) Modified Fibroin
A nucleic acid encoding a modified fibroin (PRT966) set forth in SEQ ID NO: 40 was synthesized. In the synthesized nucleic acid, an NdeI site was added to the 5′-end and an EcoRI site was added to the downstream of a stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Subsequently, the same nucleic acid was cleaved by subjecting the nucleic acid to a restriction enzyme treatment with NdeI and EcoRI. The cleaved nucleic acid was recombined into a protein expression vector pET-22b(+) to obtain an expression vector.
Escherichia coli BLR (DE3) was transformed with the obtained expression vector pET-22b(+). The transformed Escherichia coli was cultured in 2 mL of an LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) such that the OD600 was 0.005. The culture solution temperature was maintained at 30° C., flask culture was carried out for about 15 hours until the OD600 reached 5, and thus a seed culture solution was obtained.
The seed culture solution was added to a jar fermenter, to which 500 mL of a production medium (Table 5) had been added, such that the OD600 reached 0.05. Culturing was carried out by maintaining the culture solution temperature at 37° C. and controlling the pH to be constant at 6.9. Furthermore, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
Immediately after glucose in the production medium had been completely consumed, a feed solution (455 g of glucose/1 L and 120 g of Yeast Extract/1 L) was added at a rate of 1 mL/min. Culturing was carried out by maintaining the culture solution temperature at 37° C. and controlling the pH to be constant at 6.9. Culturing was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM, and expression of the intended modified fibroin was induced. At a time point when 20 hours had passed after addition of IPTG, the culture solution was centrifuged, and bacterial cells were collected. SDS-PAGE was carried out using the bacterial cells prepared from the culture solution before the addition of IPTG and after the addition of IPTG, and the expression of the intended modified fibroin was confirmed by the IPTG addition-dependent appearance of a band equivalent to the size of the intended modified fibroin.
The bacterial cells collected 2 hours after the addition of IPTG were washed with a 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cells after washing were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF, and the bacterial cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi SpA). The disrupted cells were centrifuged, and a precipitate was obtained. The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4) until the obtained precipitate became highly pure. The precipitate after washing was suspended in an 8 M guanidine buffer solution (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to obtain a concentration of 100 mg/mL, and the precipitate was dissolved by stirring the suspension with a stirrer at 60° C. for 30 minutes. After dissolution, dialysis was carried out in water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the collected aggregated protein in a freeze-dryer, and a freeze-dried powder of the intended modified fibroin was obtained.
(2) Production of Fibroin Fiber
Dimethyl sulfoxide (DMSO) in which 4.0% by mass of lithium chloride was used as a solvent, and the freeze-dried powder of the modified fibroin was dissolved in the solvent. Subsequently, insoluble matters and foams were removed, and a modified fibroin solution was obtained. A fibroin fiber was produced by dry-wet spinning using the obtained modified fibroin solution as a dope solution. The draw ratio was 6 times.
Single yarns of a plurality of the fibroin fibers having a length of about 30 cm were bundled to obtain fiber bundles having a fiber fineness of 150 denier. A 0.8-g lead weight was attached to each fiber bundle. The fiber bundles in that state were immersed in water at 40° C. for 10 minutes, and the fibroin fibers were contracted by the immersion. Subsequently, the fiber bundles taken out from water were dried by leaving the fiber bundles to stand at room temperature for 2 hours. As a result, dried fiber bundles of fibroin fibers containing a modified fibroin and having a single yarn diameter of 15 μm, 30 μm, 40 μm, 60 μm, or 80 μm were obtained. The obtained fiber bundles were submitted to the following shaping test for the fibroin fiber.
2. Shaping Test
(Test 1) The fibroin fiber after drying was wound around a cylindrical-shaped metal core material having a diameter of about 25 mm. The fibroin fiber wound around the core material was retained in a constant-temperature and constant-humidity chamber at 20° C., 40° C., 60° C., or 90° C. for 60 minutes. However, when the fibroin fiber having a single yarn diameter of 15 μm or 30 μm was retained at 20° C. or 40° C., the retention time was set to 180 minutes. Under similar conditions, a shaping test for silk fiber was also carried out.
The fibroin fiber removed from the core material was observed, and the extent to which a curled shape was given was determined by the following criteria.
A: A clear curled shape was retained.
B: Although the extent was weak compared to “A”, it was confirmed that the shape was retained to a certain extent.
C: Retaining of the curled shape was not recognized.
As shown in Table 6, it was confirmed that a predetermined shape (here, a curled shape) can be given to a fibroin fiber by retaining a dry state fibroin fiber in a state conforming to the predetermined shape.
(Test 2-1)
Each fibroin fiber after drying was immersed in water at room temperature (about 20° C.) for 20 minutes. Subsequently, the fibroin fiber in a wet state was wound around a cylindrical-shaped metal core material having a diameter of about 25 mm. The fibroin fiber wound around the core material was retained for 1 hour while being heated at 20° C., 40° C., 60° C., or 90° C. in a constant-temperature and constant-humidity chamber.
The fibroin fiber removed from the core material was observed, and the extent to which a curled shape was given was determined according to criteria similar to those of Test 1.
As shown in Table 7, it was confirmed that a predetermined shape (here, a curled shape) can be given to a fibroin fiber by retaining a wet state fibroin fiber in a state conforming to the predetermined shape.
(Test 2-2)
A fibroin fiber having a single yarn diameter of 80 μm was immersed in water at room temperature (about 20° C.) for 20 minutes. Subsequently, the fibroin fiber in a wet state was wound around a cylindrical-shaped metal core material having a diameter of about 5 mm or 25 mm. The fibroin fiber wound around the core material was retained while being heated in a state of being immersed in water at 20° C., 40° C., 60° C., or 90° C. for 1 hour. The fibroin fiber taken out from water was dried by heating for 1 hour in a state of wound around a core material in a high-temperature and constant-humidity chamber at 60° C. The fibroin fiber removed from the core material was observed, and the extent to which a curled shape was given was determined according to criteria similar to those of Test 1. Results are shown in Table 8.
(Test 2-3)
A fibroin fiber having a single yarn diameter of 80 μm was immersed in water at room temperature (about 20° C.) for 20 minutes. Subsequently, the fibroin fiber in a wet state was wound around a cylindrical-shaped metal core material having a diameter of about 25 mm. The fibroin fiber wound around the core material was retained while being heated in a state of being exposed to water vapor at 115° C. or 125° C. for 2 hours in an autoclave. The fibroin fiber taken out from the autoclave was dried by heating for 20 minutes in a state of wound around a core material in a high-temperature and constant-humidity chamber at 70° C. The fibroin fiber removed from the core material was observed, and the result of determining the extent to which a curled shape was given according to criteria similar to those of Test 1 was “A”.
(Test 2-4: Evaluation of Shape Retainability)
A curled shape was given to fibroin fibers having a single yarn diameter of 80 μm, under the conditions of Test 2-1 of wetting the fibroin fibers by water immersion and then retaining the fibers in an atmosphere at 90° C., the conditions of Test 2-2 of retaining the fibroin fibers in hot water at 90° C., or the conditions of Test 2-3 of retaining the fibroin fibers in a state of being exposed to steam at 115° C. In order to evaluate the retainability of shape, the fibroin fibers that had been given a shape were submitted to a water-washing test of immersing the fibroin fibers in water at room temperature (about 20° C.) for 20 minutes and drying the fibroin fibers after immersion in a dryer.
(Test 2-5: Evaluation of Shape Retainability)
A curled shape was given to a fibroin fiber having a single yarn diameter of 80 μm under the conditions of Test 2-3, while the temperature of water vapor and the retention time were changed as shown in Table 9. Each fibroin fiber that was given a curled shape was repeatedly submitted to a washing test similar to Test 2-4. The number of times of the washing test performed until the given shape was substantially lost was recorded.
Water Stretchability
The lengths in a dry state (length of a fibroin fiber before being placed in a wet state) of a fibroin fiber before being given a shape and of fibroins that were given a curled shape, which were obtained in Test Example 1 or Test Example 2-1, were respectively measured. Next, a 0.8-g lead weight was attached to each fibroin fiber, and the fibroin fiber in that state was immersed in water at 40° C. for 10 minutes. Subsequently, the length of each fibroin fiber in water (length of a fibroin fiber when placed in a wet state) was measured. Measurement of the length of each fibroin fiber in water was carried out while having a 0.8-g lead weight attached to each fibroin fiber, so that curls would be removed from each fibroin fiber. Next, each fibroin fiber taken out from water was dried by leaving the fiber at room temperature for 2 hours while having a 0.8-g lead weight attached to the fibroin fiber. Subsequently, the length of each fibroin fiber in water (length of a fibroin fiber when dried from a wet state) was measured. From the obtained measured values, the recovery rate, elongation rate, and contraction rate C of each fibroin fiber were calculated by the following Formula (1), Formula (4), and Formula (5).
Recovery rate=(Length of fibroin fiber when dried from wet state/length of fibroin fiber before being placed in wet state)×100(%). Formula (1):
Elongation rate={(Length of fibroin fiber when placed in wet state/length of fibroin fiber before being placed in wet state)−1}×100(%) Formula (4):
Contraction rate C={1−(length of fibroin fiber when dried from wet state/length of fibroin fiber when placed in wet state)}×100(%). Formula (5):
The fibroin fibers of various Examples that were given a curled shape exhibited an elongation rate of 2% to 17%, a contraction rate C of 2% to 17%, and a recovery rate of 95% to 100%. That is, these fibroin fibers exhibited a characteristic that the fibroin fibers expand when placed in a wet state and contract when dried from a wet state.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-016458 | Jan 2019 | JP | national |
| 2019-016472 | Jan 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/003543 | 1/30/2020 | WO | 00 |
