JPWO2018221498A1 - Method for producing protein fiber - Google Patents
Method for producing protein fiber Download PDFInfo
- Publication number
- JPWO2018221498A1 JPWO2018221498A1 JP2019521226A JP2019521226A JPWO2018221498A1 JP WO2018221498 A1 JPWO2018221498 A1 JP WO2018221498A1 JP 2019521226 A JP2019521226 A JP 2019521226A JP 2019521226 A JP2019521226 A JP 2019521226A JP WO2018221498 A1 JPWO2018221498 A1 JP WO2018221498A1
- Authority
- JP
- Japan
- Prior art keywords
- protein
- coagulation bath
- solvent
- dissolution
- bath solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 186
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- 239000002904 solvent Substances 0.000 claims abstract description 98
- 238000009987 spinning Methods 0.000 claims abstract description 65
- 230000015271 coagulation Effects 0.000 claims description 124
- 238000005345 coagulation Methods 0.000 claims description 124
- 238000004090 dissolution Methods 0.000 claims description 75
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 57
- 108010022355 Fibroins Proteins 0.000 claims description 52
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- 229910017053 inorganic salt Inorganic materials 0.000 claims description 32
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- YNQRWDDCTKWYJP-UHFFFAOYSA-L strontium;dithiocyanate Chemical compound [Sr+2].[S-]C#N.[S-]C#N YNQRWDDCTKWYJP-UHFFFAOYSA-L 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- PUGUQINMNYINPK-UHFFFAOYSA-N tert-butyl 4-(2-chloroacetyl)piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCN(C(=O)CCl)CC1 PUGUQINMNYINPK-UHFFFAOYSA-N 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000006097 ultraviolet radiation absorber Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
- 229940102001 zinc bromide Drugs 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- RXBXBWBHKPGHIB-UHFFFAOYSA-L zinc;diperchlorate Chemical compound [Zn+2].[O-]Cl(=O)(=O)=O.[O-]Cl(=O)(=O)=O RXBXBWBHKPGHIB-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
- D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Textile Engineering (AREA)
- Artificial Filaments (AREA)
- Peptides Or Proteins (AREA)
- Spinning Methods And Devices For Manufacturing Artificial Fibers (AREA)
Abstract
本発明は、タンパク質と、第１の溶解溶媒と、を含有する紡糸原液を、凝固浴液に導入して、タンパク質を凝固させる工程を含み、凝固浴液が、第２の溶解溶媒を含有する、タンパク質繊維の製造方法に関する。The present invention includes a step of introducing a spinning stock solution containing a protein and a first dissolving solvent into a coagulating bath solution to coagulate the protein, and the coagulating bath solution contains a second dissolving solvent. , A method for producing a protein fiber.
Description
本発明は、タンパク質繊維の製造方法に関する。 The present invention relates to a method for producing a protein fiber.
従来から、人造繊維の製造方法として、ノズルから吐出させた紡糸原液を凝固浴液中で凝固させて繊維を形成する湿式紡糸法及び乾湿式紡糸法が知られている。湿式紡糸法及び乾湿式紡糸法は、タンパク質を主成分として含むタンパク質繊維を製造する際にも利用されている（例えば、特許文献１参照）。 2. Description of the Related Art Conventionally, as a method for producing artificial fibers, a wet spinning method and a dry-wet spinning method in which a spinning solution discharged from a nozzle is coagulated in a coagulation bath to form fibers are known. The wet spinning method and the dry-wet spinning method are also used when producing protein fibers containing protein as a main component (for example, see Patent Document 1).
ところで、そのような湿式紡糸法、乾湿式紡糸法等によって人造繊維を得る際には、凝固浴液中で繊維が凝固する際に、繊維表面等にボイドが生ずることも知られている（例えば、特許文献２参照）。 By the way, it is also known that when artificial fibers are obtained by such a wet spinning method, a dry-wet spinning method, or the like, voids are generated on the fiber surface or the like when the fibers are coagulated in a coagulation bath solution (for example, And Patent Document 2).
このため、例えば、アクリル繊維、アクリロニトリル繊維等の化学繊維を湿式紡糸法によって得る際には、繊維表面等のボイド発生を抑制するために、紡糸原液中の水分含量を減少させたり（例えば、特許文献３参照）、溶剤の水溶液からなる凝固浴液の溶剤濃度をコントロールしたり（例えば、特許文献４参照）する対策が講じられている。 For this reason, for example, when obtaining a chemical fiber such as an acrylic fiber or an acrylonitrile fiber by a wet spinning method, in order to suppress the generation of voids on the fiber surface or the like, the water content in the spinning solution is reduced (for example, see Patent Measures have been taken to control the solvent concentration of a coagulation bath solution composed of an aqueous solution of a solvent (see, for example, Patent Document 4).
しかしながら、湿式紡糸法、乾湿式紡糸法等により、タンパク質繊維を得る際には、タンパク質繊維のボイド発生を抑制するための対策が何等講じられていないのが現状である。 However, at present, no measures have been taken to suppress the generation of voids in protein fibers when obtaining protein fibers by wet spinning, dry-wet spinning, or the like.
そこで、本発明は、ボイドの発生が充分に抑制されたタンパク質繊維の製造方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for producing a protein fiber in which the generation of voids is sufficiently suppressed.
本発明者らは、上記課題を解決するため、タンパク質繊維でのボイドの発生原因を検討した。そして、ボイドの発生原因が、タンパク質を溶解溶媒に溶解させた紡糸原液を凝固浴液に導入する際に、紡糸原液から溶解溶媒が急激に離脱することにあると推定した。このような推定に基づいて、鋭意検討を重ねた結果、凝固浴液が溶解溶媒を含有することにより、凝固浴液中での紡糸原液の脱溶媒をゆっくりと時間を掛けて行うことが可能であることを見出し、係る知見に基づき本発明を完成するに至った。 The present inventors have studied the causes of voids in protein fibers in order to solve the above problems. Then, it was presumed that the cause of the voids was that when the spinning solution in which the protein was dissolved in the dissolving solvent was introduced into the coagulation bath solution, the dissolving solvent was rapidly removed from the spinning solution. Based on such an estimation, as a result of intensive studies, the coagulation bath solution contains a dissolving solvent, so that it is possible to slowly take time to remove the solvent of the spinning stock solution in the coagulation bath solution. The inventors have found that the present invention has been completed based on such findings.
すなわち、本発明は、タンパク質と、第１の溶解溶媒と、を含有する紡糸原液を、凝固浴液に導入して、タンパク質を凝固させる工程を含み、凝固浴液が、第２の溶解溶媒を含有する、タンパク質繊維の製造方法を提供する。 That is, the present invention includes a step of introducing a spinning stock solution containing a protein and a first dissolution solvent into a coagulation bath solution to coagulate the protein, wherein the coagulation bath solution contains the second dissolution solvent. Provided is a method for producing a protein fiber.
本発明の製造方法によれば、凝固浴液が、溶解溶媒（第２の溶解溶媒）を含有していることから、得られるタンパク質繊維のボイドの発生を充分に抑制することができる。 According to the production method of the present invention, since the coagulation bath solution contains a dissolving solvent (second dissolving solvent), it is possible to sufficiently suppress the generation of voids in the obtained protein fiber.
本発明の製造方法において、第１の溶解溶媒は、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサフルオロイソプロノール、ヘキサフルオロアセトン、ギ酸及びこれらに溶解促進剤を添加したもの、並びに水に溶解促進剤を添加したものからなる群より選択される少なくとも１種であってよく、第２の溶解溶媒は、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサフルオロイソプロノール、ヘキサフルオロアセトン、ギ酸及びこれらに溶解促進剤を添加したもの、並びに水に溶解促進剤を添加したものからなる群より選択される少なくとも１種であってよい。 In the production method of the present invention, the first dissolving solvent is dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid, a dissolving promoter added thereto, or a dissolution promoting agent in water. And at least one solvent selected from the group consisting of those added with an agent, and the second dissolving solvent is dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and the like. It may be at least one selected from the group consisting of a dissolution promoter added and a water added dissolution promoter.
凝固浴液における、第２の溶解溶媒の含有量は、凝固浴液全量に対して、１０〜６０質量％であることが好ましい。この場合、本発明の効果がより一層顕著に奏されることとなる。 The content of the second dissolution solvent in the coagulation bath solution is preferably 10 to 60% by mass based on the total amount of the coagulation bath solution. In this case, the effects of the present invention are more remarkably exhibited.
凝固浴液の温度は、４０℃以下であってもよく、０℃以上であってもよい。 The temperature of the coagulation bath may be 40 ° C. or lower, or 0 ° C. or higher.
タンパク質は、構造タンパク質であってもよい。構造タンパク質は、フィブロインであってもよい。フィブロインは、クモ糸フィブロインであってもよい。 The protein may be a structural protein. The structural protein may be fibroin. The fibroin may be spider silk fibroin.
凝固浴液は、メタノールと、第２の溶解溶媒と、を含有し、かつ第２の溶解溶媒が、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群より選択される少なくとも１種であってよい。 The coagulation bath solution contains methanol and a second dissolution solvent, and the second dissolution solvent is selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution promoter to these. It may be at least one selected.
凝固浴液は、無機塩を含んでいてもよい。この場合、ボイドの発生が充分に抑制されたタンパク質繊維をより一層容易に製造することができる。 The coagulation bath solution may contain an inorganic salt. In this case, a protein fiber in which the generation of voids is sufficiently suppressed can be produced more easily.
凝固浴液が無機塩を含む場合、凝固浴液中における無機塩の含有量は、凝固浴液の全量基準で０質量％を超え且つ３０質量％以下であってもよい。 When the coagulation bath solution contains an inorganic salt, the content of the inorganic salt in the coagulation bath solution may be more than 0% by mass and 30% by mass or less based on the total amount of the coagulation bath solution.
紡糸原液が無機塩を含み、凝固浴液中における無機塩の含有量が、紡糸原液中の無機塩の含有量よりも低くてもよい。この場合、より温和な条件下でタンパク質が凝固するようになり、それによって、タンパク質繊維のボイドの発生をより有利に抑制することが可能となる。 The spinning dope may contain an inorganic salt, and the content of the inorganic salt in the coagulation bath may be lower than the content of the inorganic salt in the spinning dope. In this case, the protein coagulates under milder conditions, thereby making it possible to more advantageously suppress the generation of voids in the protein fiber.
本発明によれば、ボイドの発生が充分に抑制されたタンパク質繊維の製造方法を提供することができる。 According to the present invention, it is possible to provide a method for producing a protein fiber in which generation of voids is sufficiently suppressed.
以下、本発明を実施するための形態について詳細に説明する。但し、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
本実施形態に係るタンパク質繊維の製造方法は、タンパク質と第１の溶解溶媒（ドープ溶媒）とを含有する紡糸原液を、凝固浴液に導入して、タンパク質を凝固させる工程を含み、凝固浴液が、第２の溶解溶媒を含有する。凝固浴液が、溶解溶媒を含有していることにより、得られるタンパク質繊維におけるボイドの発生を充分に抑制することができる。 The method for producing a protein fiber according to this embodiment includes a step of introducing a spinning stock solution containing a protein and a first dissolution solvent (dope solvent) into a coagulation bath solution to coagulate the protein. Contains a second dissolution solvent. When the coagulation bath solution contains the dissolving solvent, the generation of voids in the obtained protein fiber can be sufficiently suppressed.
（タンパク質）
本実施形態の製造方法に従って製造されるタンパク質繊維は、タンパク質を主成分として含む。タンパク質は、特に限定されるものではなく、遺伝子組換え技術により微生物等で製造したものであってもよく、合成により製造されたものであってもよく、また天然由来のタンパク質を精製したものであってもよい。(protein)
The protein fiber produced according to the production method of the present embodiment contains protein as a main component. The protein is not particularly limited, and may be one produced by a microorganism or the like by genetic recombination technology, one produced by synthesis, or one obtained by purifying a naturally-derived protein. There may be.
上記タンパク質は、例えば、構造タンパク質、又は当該構造タンパク質に由来する人造構造タンパク質であってもよい。構造タンパク質とは、生体内で構造、形態等を形成又は保持するタンパク質を意味する。構造タンパク質としては、例えば、フィブロイン、ケラチン、コラーゲン、エラスチン、レシリン等を挙げることができる。構造タンパク質は、好ましくは、フィブロインである。 The protein may be, for example, a structural protein or an artificial structural protein derived from the structural protein. A structural protein refers to a protein that forms or retains a structure, form, and the like in a living body. Examples of the structural protein include fibroin, keratin, collagen, elastin, resilin and the like. The structural protein is preferably fibroin.
フィブロインは、例えば、絹フィブロイン、クモ糸フィブロイン、及びホーネットシルクフィブロインからなる群より選択される１種以上であってよい。特に、構造タンパク質は、絹フィブロイン、クモ糸フィブロイン又はこれらの組み合わせであってもよい。絹フィブロインとクモ糸フィブロインとを併用する場合、絹フィブロインの割合は、例えば、クモ糸フィブロイン１００質量部に対して、４０質量部以下、３０質量部以下、又は１０質量部以下であってよい。 The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. In particular, the structural protein may be silk fibroin, spider silk fibroin or a combination thereof. When silk fibroin and spider silk fibroin are used in combination, the proportion of silk fibroin may be, for example, 40 parts by weight or less, 30 parts by weight or less, or 10 parts by weight or less based on 100 parts by weight of spider silk fibroin.
絹フィブロインとしては、セリシン除去絹フィブロイン、セリシン未除去絹フィブロイン、又はこれらの組み合わせであってもよい。セリシン除去絹フィブロインは、絹フィブロインを覆うセリシン、及びその他の脂肪分などを除去して精製したものである。このようにして精製した絹フィブロインは、好ましくは、凍結乾燥粉末として用いられる。セリシン未除去絹フィブロインは、セリシンなどが除去されていない未精製の絹フィブロインである。 The silk fibroin may be a silk fibroin without sericin, a silk fibroin without sericin, or a combination thereof. Sericin-removed silk fibroin is purified by removing sericin covering silk fibroin and other fats. The silk fibroin purified in this manner is preferably used as a lyophilized powder. Silk fibroin without sericin is unpurified silk fibroin from which sericin and the like have not been removed.
クモ糸フィブロインは、天然クモ糸タンパク質、及び天然クモ糸タンパク質に由来するポリペプチド（人造クモ糸タンパク質）からなる群より選ばれるクモ糸ポリペプチドを含有していてもよい。 The spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of natural spider silk proteins and polypeptides derived from the natural spider silk proteins (artificial spider silk proteins).
天然クモ糸タンパク質としては、例えば、大吐糸管しおり糸タンパク質、横糸タンパク質、及び小瓶状腺タンパク質が挙げられる。大吐糸管しおり糸は、結晶領域と非晶領域（無定形領域とも言う。）からなる繰り返し領域を持つため、高い応力と伸縮性を併せ持つ。クモ糸の横糸は、結晶領域を持たず、非晶領域からなる繰り返し領域を持つという特徴を有する。横糸は、大吐糸管しおり糸に比べると応力は劣るが、高い伸縮性を持つ。 Natural spider silk proteins include, for example, large spinal canal thread proteins, weft proteins, and small bottle gland proteins. Since the large spinneret thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of spider silk has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft has a lower stress than the large spinneret and has a high elasticity.
大吐糸管しおり糸タンパク質は、クモの大瓶状腺で産生され、強靭性に優れるという特徴を有する。大吐糸管しおり糸タンパク質としては、例えば、アメリカジョロウグモ（Ｎｅｐｈｉｌａ ｃｌａｖｉｐｅｓ）に由来する大瓶状腺スピドロインＭａＳｐ１及びＭａＳｐ２、並びに二ワオニグモ（Ａｒａｎｅｕｓ ｄｉａｄｅｍａｔｕｓ）に由来するＡＤＦ３及びＡＤＦ４が挙げられる。ＡＤＦ３は、ニワオニグモの２つの主要なしおり糸タンパク質の一つである。天然クモ糸タンパク質に由来するポリペプチドは、これらのしおり糸タンパク質に由来するポリペプチドであってもよい。ＡＤＦ３に由来するポリペプチドは、比較的合成し易く、また、強伸度及びタフネスの点で優れた特性を有する。 The large spinal cord marker thread protein is produced in the large ampullate gland of a spider and has the feature of being excellent in toughness. Examples of the large spinal cord marker thread proteins include the large ampullate spidroins MaSp1 and MaSp2 derived from the American spider, Nephila clavipes, and ADF3 and ADF4 derived from the Araneus diadematus. ADF3 is one of the two major bookmarker thread proteins of the Japanese spider. Polypeptides derived from natural spider silk proteins may be polypeptides derived from these bookmarked silk proteins. ADF3-derived polypeptides are relatively easy to synthesize and have excellent properties in terms of strength and elongation and toughness.
横糸タンパク質は、クモの鞭毛状腺（ｆｌａｇｅｌｌｉｆｏｒｍ ｇｌａｎｄ）で産生される。横糸タンパク質としては、例えばアメリカジョロウグモ（Ｎｅｐｈｉｌａ ｃｌａｖｉｐｅｓ）に由来する鞭毛状絹タンパク質（ｆｌａｇｅｌｌｉｆｏｒｍ ｓｉｌｋ ｐｒｏｔｅｉｎ）が挙げられる。 Weft protein is produced in the flagellar gland of spiders. Examples of the weft protein include a flagellaform silk protein derived from the American spider spider (Nephila clavipes).
天然クモ糸タンパク質に由来するポリペプチドは、組換えクモ糸タンパク質であってよい。組換えクモ糸タンパク質としては、天然型クモ糸タンパク質の変異体、類似体又は誘導体等が挙げられる。このようなポリペプチドの好適な一例は、大吐糸管しおり糸タンパク質の組換えクモ糸タンパク質（「大吐糸管しおり糸タンパク質に由来するポリペプチド」ともいう。）である。 A polypeptide derived from a natural spider silk protein may be a recombinant spider silk protein. Examples of the recombinant spider silk protein include a mutant, analog or derivative of a natural spider silk protein. A preferred example of such a polypeptide is a recombinant spider silk protein of a large spinal canal thread protein (also referred to as a "polypeptide derived from a large spinal canal thread protein").
フィブロイン様タンパク質である大吐糸管しおり糸由来のタンパク質及びカイコシルク由来のタンパク質としては、例えば、式１：［（Ａ）ｎモチーフ−ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ−ＲＥＰ］ｍ−（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質が挙げられる。ここで、（Ａ）ｎモチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は２〜２７である。（Ａ）ｎモチーフのアミノ酸残基数は、２〜２０、４〜２７、４〜２０、８〜２０、１０〜２０、４〜１６、８〜１６、又は１０〜１６の整数であってよい。また、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は４０％以上であればよく、６０％以上、７０％以上、８０％以上、８３％以上、８５％以上、８６％以上、９０％以上、９５％以上、又は１００％（アラニン残基のみで構成されることを意味する。）であってもよい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されてもよい。ＲＥＰは２〜２００アミノ酸残基から構成されるアミノ酸配列を示す。ＲＥＰは、１０〜２００アミノ酸残基から構成されるアミノ酸配列であってもよい。ｍは２〜３００の整数を示し、１０〜３００の整数であってもよい。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。大吐糸管しおり糸由来のタンパク質の具体例としては、配列番号１及び配列番号８で示されるアミノ酸配列を含むタンパク質を挙げることができる。Examples of the protein derived from the major spinal cord and the silkworm silk that are fibroin-like proteins include, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif- REP] m - (a) a protein comprising a domain sequence represented by n motifs, and the like. Here, the (A) n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues of the 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. . (A) The ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences. As a specific example of the protein derived from the large spinal cord marker thread, a protein containing the amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 8 can be mentioned.
横糸タンパク質に由来するタンパク質としては、例えば、式３：［ＲＥＰ２］ｏで表されるドメイン配列を含むタンパク質（ここで、式３中、ＲＥＰ２はＧｌｙ−Ｐｒｏ−Ｇｌｙ−Ｇｌｙ−Ｘから構成されるアミノ酸配列を示し、Ｘはアラニン（Ａｌａ）、セリン（Ｓｅｒ）、チロシン（Ｔｙｒ）及びバリン（Ｖａｌ）からなる群から選ばれる一つのアミノ酸を示す。ｏは８〜３００の整数を示す。）を挙げることができる。具体的には配列番号２で示されるアミノ酸配列を含むタンパク質を挙げることができる。配列番号２で示されるアミノ酸配列は、ＮＣＢＩデータベースから入手したアメリカジョロウグモの鞭毛状絹タンパク質の部分的な配列（ＮＣＢＩアクセッション番号：ＡＡＦ３６０９０、ＧＩ：７１０６２２４）のリピート部分及びモチーフに該当するＮ末端から１２２０残基目から１６５９残基目までのアミノ酸配列（ＰＲ１配列と記す。）と、ＮＣＢＩデータベースから入手したアメリカジョロウグモの鞭毛状絹タンパク質の部分配列（ＮＣＢＩアクセッション番号：ＡＡＣ３８８４７、ＧＩ：２８３３６４９）のＣ末端から８１６残基目から９０７残基目までのＣ末端アミノ酸配列を結合し、結合した配列のＮ末端に配列番号７で示されるアミノ酸配列（タグ配列及びヒンジ配列）が付加されたものである。As a protein derived from the weft protein, for example, a protein containing a domain sequence represented by Formula 3: [REP2] o (wherein, in Formula 3, REP2 is composed of Gly-Pro-Gly-Gly-X X represents an amino acid sequence, and X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr), and valine (Val), and o represents an integer of 8 to 300. Can be mentioned. Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence represented by SEQ ID NO: 2 is obtained from the N-terminus corresponding to the repeat portion and the motif of the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAF36090, GI: 7106224). The amino acid sequence from residues 1220 to 1659 (referred to as PR1 sequence) and the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649) A C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus is linked, and the amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence) is added to the N-terminus of the linked sequence. is there.
コラーゲン由来のタンパク質として、例えば、式４：［ＲＥＰ３］ｐで表されるドメイン配列を含むタンパク質（ここで、式４中、ｐは５〜３００の整数を示す。ＲＥＰ３は、Ｇｌｙ−Ｘ−Ｙから構成されるアミノ酸配列を示し、Ｘ及びＹはＧｌｙ以外の任意のアミノ酸残基を示す。複数存在するＲＥＰ３は、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。）を挙げることができる。具体的には、配列番号３で示されるアミノ酸配列を含むタンパク質を挙げることができる。配列番号３で示されるアミノ酸配列は、ＮＣＢＩデータベースから入手したヒトのコラーゲンタイプ４の部分的な配列（ＮＣＢＩのＧｅｎＢａｎｋのアクセッション番号：ＣＡＡ５６３３５．１、ＧＩ：３７０２４５２）のリピート部分及びモチーフに該当する３０１残基目から５４０残基目までのアミノ酸配列のＮ末端に配列番号７で示されるアミノ酸配列（タグ配列及びヒンジ配列）が付加されたものである。As a collagen-derived protein, for example, a protein containing a domain sequence represented by Formula 4: [REP3] p (where p represents an integer of 5 to 300. In Formula 4, REP3 is Gly-XY. Wherein X and Y each represent an amino acid residue other than Gly. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) . Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 3 can be mentioned. The amino acid sequence represented by SEQ ID NO: 3 corresponds to a repeat portion and a motif of a partial sequence of human collagen type 4 obtained from the NCBI database (Accession number of GenBank of NCBI: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.
レシリン由来のタンパク質として、例えば、式５：［ＲＥＰ４］ｑで表されるドメイン配列を含むタンパク質（ここで、式５中、ｑは４〜３００の整数を示す。ＲＥＰ４はＳｅｒ−Ｊ−Ｊ−Ｔｙｒ−Ｇｌｙ−Ｕ−Ｐｒｏから構成されるアミノ酸配列を示す。Ｊは任意のアミノ酸残基を示し、特にＡｓｐ、Ｓｅｒ及びＴｈｒからなる群から選ばれるアミノ酸残基であることが好ましい。Ｕは任意のアミノ酸残基を示し、特にＰｒｏ、Ａｌａ、Ｔｈｒ及びＳｅｒからなる群から選ばれるアミノ酸残基であることが好ましい。複数存在するＲＥＰ４は、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。）を挙げることができる。具体的には、配列番号４で示されるアミノ酸配列を含むタンパク質を挙げることができる。配列番号４で示されるアミノ酸配列は、レシリン（ＮＣＢＩのＧｅｎＢａｎｋのアクセッション番号ＮＰ ６１１１５７、Ｇｌ：２４６５４２４３）のアミノ酸配列において、８７残基目のＴｈｒをＳｅｒに置換し、かつ９５残基目のＡｓｎをＡｓｐに置換した配列の１９残基目から３２１残基目までのアミノ酸配列のＮ末端に配列番号７で示されるアミノ酸配列（タグ配列及びヒンジ配列）が付加されたものである。As a protein derived from resilin, for example, a protein containing a domain sequence represented by Formula 5: [REP4] q (where, in Formula 5, q represents an integer of 4 to 300. REP4 is Ser-JJ- 1 shows an amino acid sequence composed of Tyr-Gly-U-Pro, wherein J represents any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. And preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser .. A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ). Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 4 can be mentioned. In the amino acid sequence of SEQ ID NO: 4, in the amino acid sequence of resilin (NCBI GenBank Accession No. NP 611157, Gl: 246654243), Thr at the 87th residue is replaced with Ser, and Asn at the 95th residue is replaced with Ser. Is an amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence) added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence obtained by substituting Asp with Asp.
エラスチン由来のタンパク質として、例えば、ＮＣＢＩのＧｅｎＢａｎｋのアクセッション番号ＡＡＣ９８３９５（ヒト）、Ｉ４７０７６（ヒツジ）、ＮＰ７８６９６６（ウシ）等のアミノ酸配列を有するタンパク質を挙げることができる。具体的には、配列番号５で示されるアミノ酸配列を含むタンパク質を挙げることができる。配列番号５で示されるアミノ酸配列は、ＮＣＢＩのＧｅｎＢａｎｋのアクセッション番号ＡＡＣ９８３９５のアミノ酸配列の１２１残基目から３９０残基目までのアミノ酸配列のＮ末端に配列番号７で示されるアミノ酸配列（タグ配列及びヒンジ配列）が付加されたものである。 Examples of elastin-derived proteins include proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine) and the like. Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 5. The amino acid sequence represented by SEQ ID NO: 5 corresponds to the amino acid sequence represented by SEQ ID NO: 7 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of GenBank Accession No. AAC98395 of NCBI. And a hinge sequence).
ケラチン由来のタンパク質として、例えば、カプラ・ヒルクス（Ｃａｐｒａ ｈｉｒｃｕｓ）のタイプＩケラチン等を挙げることができる。具体的には、配列番号６で示されるアミノ酸配列（ＮＣＢＩのＧｅｎＢａｎｋのアクセッション番号ＡＣＹ３０４６６のアミノ酸配列）を含むタンパク質を挙げることができる。 Examples of keratin-derived proteins include, for example, Capra hircus type I keratin and the like. Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 6 (the amino acid sequence of GenBank Accession No. ACY30466 of NCBI).
上述した構造タンパク質及び当該構造タンパク質に由来するタンパク質は、１種を単独で、又は２種以上を組み合わせて用いることができる。 One of the above-mentioned structural proteins and proteins derived from the structural proteins can be used alone or in combination of two or more.
タンパク質繊維に主成分として含まれるタンパク質は、例えば、当該タンパク質をコードする核酸配列と、当該核酸配列に作動可能に連結された１又は複数の調節配列とを有する発現ベクターで形質転換された宿主により、当該核酸を発現させることにより生産することができる。 A protein contained as a main component in a protein fiber can be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. , By expressing the nucleic acid.
タンパク質繊維に主成分として含まれるタンパク質をコードする核酸の製造方法は、特に制限されない。例えば、天然の構造タンパク質をコードする遺伝子を利用して、ポリメラーゼ連鎖反応（ＰＣＲ）などで増幅しクローニングする方法、又は、化学的に合成する方法によって、当該核酸を製造することができる。核酸の化学的な合成方法も特に制限されず、例えば、ＮＣＢＩのウェブデータベースなどより入手した構造タンパク質のアミノ酸配列情報をもとに、ＡＫＴＡ ｏｌｉｇｏｐｉｌｏｔ ｐｌｕｓ １０／１００（ＧＥヘルスケア・ジャパン株式会社）などで自動合成したオリゴヌクレオチドをＰＣＲなどで連結する方法によって遺伝子を化学的に合成することができる。この際に、タンパク質の精製及び／又は確認を容易にするため、上記のアミノ酸配列のＮ末端に開始コドン及びＨｉｓ１０タグからなるアミノ酸配列を付加したアミノ酸配列からなるタンパク質をコードする核酸を合成してもよい。 The method for producing a nucleic acid encoding a protein contained as a main component in a protein fiber is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by polymerase chain reaction (PCR) or the like using a gene encoding a natural structural protein, or a method of chemically synthesizing. The method for chemically synthesizing nucleic acids is not particularly limited. For example, AKTA oligopilot plus 10/100 (GE Healthcare Japan) based on amino acid sequence information of structural proteins obtained from the NCBI web database or the like. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized by the method such as PCR. At this time, in order to facilitate purification and / or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence obtained by adding an initiation codon and an amino acid sequence consisting of a His10 tag to the N-terminal of the above amino acid sequence was synthesized. Is also good.
調節配列は、宿主における組換えタンパク質の発現を制御する配列（例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等）であり、宿主の種類に応じて適宜選択することができる。プロモーターとして、宿主細胞中で機能し、目的とするタンパク質を発現誘導可能な誘導性プロモーターを用いてもよい。誘導性プロモーターは、誘導物質（発現誘導剤）の存在、リプレッサー分子の非存在、又は温度、浸透圧若しくはｐＨ値の上昇若しくは低下等の物理的要因により、転写を制御できるプロモーターである。 The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and can induce the expression of a target protein may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等、宿主の種類に応じて適宜選択することができる。発現ベクターとしては、宿主細胞において自立複製が可能、又は宿主の染色体中への組込みが可能で、目的とするタンパク質をコードする核酸を転写できる位置にプロモーターを含有しているものが好適に用いられる。 The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a protein of interest can be transcribed are suitably used. .
宿主として、原核生物、並びに酵母、糸状真菌、昆虫細胞、動物細胞及び植物細胞等の真核生物のいずれも好適に用いることができる。 As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
原核生物の宿主の好ましい例として、エシェリヒア属、ブレビバチルス属、セラチア属、バチルス属、ミクロバクテリウム属、ブレビバクテリウム属、コリネバクテリウム属及びシュードモナス属等に属する細菌を挙げることができる。エシェリヒア属に属する微生物として、例えば、エシェリヒア・コリ等を挙げることができる。ブレビバチルス属に属する微生物として、例えば、ブレビバチルス・アグリ等を挙げることができる。セラチア属に属する微生物として、例えば、セラチア・リクエファシエンス等を挙げることができる。バチルス属に属する微生物として、例えば、バチルス・サチラス等を挙げることができる。ミクロバクテリウム属に属する微生物として、例えば、ミクロバクテリウム・アンモニアフィラム等を挙げることができる。ブレビバクテリウム属に属する微生物として、例えば、ブレビバクテリウム・ディバリカタム等を挙げることができる。コリネバクテリウム属に属する微生物として、例えば、コリネバクテリウム・アンモニアゲネス等を挙げることができる。シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に属する微生物として、例えば、シュードモナス・プチダ等を挙げることができる。 Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like. Examples of microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis. Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
原核生物を宿主とする場合、目的タンパク質をコードする核酸を導入するベクターとしては、例えば、ｐＢＴｒｐ２（ベーリンガーマンハイム社製）、ｐＧＥＸ（Ｐｈａｒｍａｃｉａ社製）、ｐＵＣ１８、ｐＢｌｕｅｓｃｒｉｐｔＩＩ、ｐＳｕｐｅｘ、ｐＥＴ２２ｂ、ｐＣｏｌｄ、ｐＵＢ１１０、ｐＮＣＯ２（特開２００２−２３８５６９号公報）等を挙げることができる。 When a prokaryote is used as a host, examples of a vector for introducing a nucleic acid encoding a target protein include pBTrp2 (Boehringer Mannheim), pGEX (Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
真核生物の宿主としては、例えば、酵母及び糸状真菌（カビ等）を挙げることができる。酵母としては、例えば、サッカロマイセス属、ピキア属、シゾサッカロマイセス属等に属する酵母を挙げることができる。糸状真菌としては、例えば、アスペルギルス属、ペニシリウム属、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属等に属する糸状真菌を挙げることができる。 Eukaryotic hosts include, for example, yeasts and filamentous fungi (such as mold). Examples of the yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
真核生物を宿主とする場合、目的タンパク質をコードする核酸を導入するベクターとしては、例えば、ＹＥＰ１３（ＡＴＣＣ３７１１５）、ＹＥｐ２４（ＡＴＣＣ３７０５１）等を挙げることができる。上記宿主細胞への発現ベクターの導入方法としては、上記宿主細胞へＤＮＡを導入する方法であればいずれも用いることができる。例えば、カルシウムイオンを用いる方法〔Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ，６９，２１１０（１９７２）〕、エレクトロポレーション法、スフェロプラスト法、プロトプラスト法、酢酸リチウム法、コンピテント法等を挙げることができる。 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a target protein is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, and competent methods.
発現ベクターで形質転換された宿主による核酸の発現方法としては、直接発現のほか、モレキュラー・クローニング第２版に記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができる。 As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
タンパク質は、例えば、発現ベクターで形質転換された宿主を培養培地中で培養し、培養培地中に当該タンパク質を生成蓄積させ、該培養培地から採取することにより製造することができる。宿主を培養培地中で培養する方法は、宿主の培養に通常用いられる方法に従って行うことができる。 The protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in the culture medium can be performed according to a method usually used for culturing the host.
宿主が、大腸菌等の原核生物又は酵母等の真核生物である場合、培養培地として、宿主が資化し得る炭素源、窒素源及び無機塩類等を含有し、宿主の培養を効率的に行える培地であれば天然培地、合成培地のいずれを用いてもよい。 When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
炭素源としては、上記形質転換微生物が資化し得るものであればよく、例えば、グルコース、フラクトース、スクロース、及びこれらを含有する糖蜜、デンプン及びデンプン加水分解物等の炭水化物、酢酸及びプロピオン酸等の有機酸、並びにエタノール及びプロパノール等のアルコール類を用いることができる。窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物を用いることができる。無機塩類としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムを用いることができる。 The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used. As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
大腸菌等の原核生物又は酵母等の真核生物の培養は、例えば、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことができる。培養温度は、例えば、１５〜４０℃である。培養時間は、通常１６時間〜７日間である。培養中の培養培地のｐＨは３．０〜９．０に保持することが好ましい。培養培地のｐＨの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。 Cultivation of a prokaryote such as Escherichia coli or a eukaryote such as yeast can be performed under aerobic conditions such as, for example, shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is generally 16 hours to 7 days. The pH of the culture medium during the culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
また、培養中、必要に応じて、アンピシリン及びテトラサイクリン等の抗生物質を培養培地に添加してもよい。プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはイソプロピル−β−Ｄ−チオガラクトピラノシド等を、ｔｒｐプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。 During the culturing, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. When culturing a microorganism transformed with an expression vector using the trp promoter, indole acryl is used. An acid or the like may be added to the medium.
発現させたタンパク質の単離、精製は通常用いられている方法で行うことができる。例えば、当該タンパク質が、細胞内に溶解状態で発現した場合には、培養終了後、宿主細胞を遠心分離により回収し、水系緩衝液に懸濁した後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー及びダイノミル等により宿主細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、タンパク質の単離精製に通常用いられている方法、すなわち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）−セファロース、ＤＩＡＩＯＮ ＨＰＡ−７５（三菱化成社製）等のレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ−Ｓｅｐｈａｒｏｓｅ ＦＦ（Ｐｈａｒｍａｃｉａ社製）等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の方法を単独又は組み合わせて使用し、精製標品を得ることができる。 The expressed protein can be isolated and purified by a commonly used method. For example, when the protein is expressed in a dissolved state in the cells, after culturing, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, and Manton Gaulin. The host cells are crushed with a homogenizer and a dynomill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia). Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. And other methods alone or in combination. Goods can be obtained.
また、タンパク質が細胞内に不溶体を形成して発現した場合は、同様に宿主細胞を回収後、破砕し、遠心分離を行うことにより、沈殿画分としてタンパク質の不溶体を回収する。回収したタンパク質の不溶体はタンパク質変性剤で可溶化することができる。該操作の後、上記と同様の単離精製法によりタンパク質の精製標品を得ることができる。当該タンパク質が細胞外に分泌された場合には、培養上清から当該タンパク質を回収することができる。すなわち、培養物を遠心分離等の手法により処理することにより培養上清を取得し、その培養上清から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 When the protein is expressed by forming an insoluble form in the cell, the host cell is similarly recovered, crushed, and centrifuged to collect the protein insoluble form as a precipitate fraction. The insoluble form of the recovered protein can be solubilized with a protein denaturant. After this operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
（タンパク質繊維の製造方法）
タンパク質繊維は、上述したタンパク質を紡糸したものであり、上述したタンパク質を主成分として含む。本実施形態に係るタンパク質繊維の製造方法は、タンパク質と第１の溶解溶媒とを含有する紡糸原液を、凝固浴液に導入して、タンパク質を凝固させる工程を含むものであり、凝固浴液が、第２の溶解溶媒を含有することに特徴を有する。本実施形態に係るタンパク質繊維の製造方法は、湿式紡糸、乾湿式紡糸等の公知の紡糸方法に準じて実施することができる。(Producing method of protein fiber)
The protein fiber is obtained by spinning the above-described protein, and contains the above-described protein as a main component. The method for producing a protein fiber according to the present embodiment includes a step of introducing a spinning solution containing a protein and a first dissolution solvent into a coagulation bath solution to coagulate the protein. , A second dissolving solvent. The method for producing a protein fiber according to the present embodiment can be performed according to a known spinning method such as wet spinning and dry-wet spinning.
紡糸原液は、上述のタンパク質及び第１の溶解溶媒を含有する。紡糸原液は、上述のタンパク質を溶解溶媒に溶解させたものであってよい。ここで、溶解溶媒とは、タンパク質を溶解する成分（溶媒）を意味する。溶解溶媒は、後述する溶解促進剤と組み合わせて用いることにより、タンパク質を溶解する成分も含む。溶解溶媒は、溶解促進剤を含んでいてもよい。第１の溶解溶媒は、紡糸原液中のタンパク質を溶解する成分であり、ドープ溶媒ともいう。 The spinning dope contains the protein described above and a first dissolution solvent. The spinning dope may be one in which the above-mentioned protein is dissolved in a dissolving solvent. Here, the dissolving solvent means a component (solvent) that dissolves the protein. The dissolution solvent also includes a component that dissolves a protein when used in combination with a dissolution promoter described below. The dissolution solvent may contain a dissolution promoter. The first dissolving solvent is a component that dissolves the protein in the spinning dope and is also called a dope solvent.
溶解溶媒（第１の溶解溶媒）は、例えば、ジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）、ヘキサフルオロイソプロノール（ＨＦＩＰ）、ヘキサフルオロアセトン（ＨＦＡ）、ギ酸等の有機溶媒であってよい。また、第１の溶解溶媒は、上記の有機溶媒に溶解促進剤を添加したもの、又は水に溶解促進剤を添加したものであってよい。第１の溶解溶媒は、１種単独又は２種以上を組み合わせて用いたものであってよい。 The dissolution solvent (first dissolution solvent) is, for example, an organic solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), hexafluoroisopronol (HFIP), hexafluoroacetone (HFA), and formic acid. May be. Further, the first dissolving solvent may be one obtained by adding a dissolution promoter to the above-mentioned organic solvent, or one obtained by adding a dissolution promoter to water. The first dissolving solvent may be used alone or in combination of two or more.
溶解溶媒(第１の溶解溶媒)は、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサフルオロイソプロノール、ヘキサフルオロアセトン、ギ酸及びこれらに溶解促進剤を添加したもの、並びに水に溶解促進剤を添加したものからなる群より選択される少なくとも１種であってもよく、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群から選択される少なくとも１種であってよい。第１の溶解溶媒が、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群から選択される少なくとも１種である場合、これらの溶媒が高沸点であるため、高温条件でのタンパク質の溶解が可能となる。これにより、タンパク質を第１の溶解溶媒に溶解させて紡糸原液を得る際の調製作業がより一層効率化する。さらに、第１の溶解溶媒がジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群から選択される少なくとも１種である場合、当該溶媒の安全性が高いことから、生産作業性が向上し、かつ、当該溶媒自体のコストが安いことから、タンパク質繊維の製造コストが低減する。 The dissolving solvent (first dissolving solvent) is dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid, a dissolution promoter added to these, and a dissolution promoter added to water. At least one selected from the group consisting of dimethylsulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution promoter thereto; Good. When the first dissolving solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a dissolution accelerator to these, since these solvents have a high boiling point, The protein can be dissolved under high temperature conditions. Thereby, the preparation operation when dissolving the protein in the first dissolving solvent to obtain the spinning stock solution is further improved in efficiency. Further, when the first dissolution solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a dissolution promoter to them, the safety of the solvent is high. Since the productivity is improved and the cost of the solvent itself is low, the production cost of the protein fiber is reduced.
第１の溶解溶媒は、溶解促進剤を添加したもの（含有するもの）であってよい。この場合、紡糸原液の調製が容易になる。溶解促進剤は、タンパク質及び溶解溶媒の種類等に応じて、適宜選択することができる。 The first dissolving solvent may be one containing (containing) a dissolution promoter. In this case, the preparation of the spinning stock solution becomes easy. The dissolution promoter can be appropriately selected according to the type of the protein and the dissolution solvent.
溶解促進剤は、例えば、以下に示すルイス酸とルイス塩基とからなる無機塩であってよい。ルイス塩基としては、例えば、オキソ酸イオン(硝酸イオン、過塩素酸イオン等)、金属オキソ酸イオン(過マンガン酸イオン等)、ハロゲン化物イオン、チオシアン酸イオン、シアン酸イオン等が挙げられる。ルイス酸としては、例えば、アルカリ金属イオン、アルカリ土類金属イオン等の金属イオン、アンモニウムイオン等の多原子イオン、錯イオン等が挙げられる。溶解溶媒が有機溶媒である場合、無機塩としては、例えば、塩化リチウム、臭化リチウム、ヨウ化リチウム、硝酸リチウム、過塩素酸リチウム、及びチオシアン酸リチウム等のリチウム塩、塩化カルシウム、臭化カルシウム、ヨウ化カルシウム、硝酸カルシウム、過塩素酸カルシウム、及びチオシアン酸カルシウム等のカルシウム塩、塩化鉄、臭化鉄、ヨウ化鉄、硝酸鉄、過塩素酸鉄、及びチオシアン酸鉄等の鉄塩、並びに、塩化アルミニウム、臭化アルミニウム、ヨウ化アルミニウム、硝酸アルミニウム、過塩素酸アルミニウム、及びチオシアン酸アルミニウム等のアルミニウム塩、塩化カリウム、臭化カリウム、ヨウ化カリウム、硝酸カリウム、過塩素酸カリウム、及びチオシアン酸カリウム等のカリウム塩、塩化ナトリウム、臭化ナトリウム、ヨウ化ナトリウム、硝酸ナトリウム、過塩素酸ナトリウム、及びチオシアン酸ナトリウム等のナトリウム塩、塩化亜鉛、臭化亜鉛、ヨウ化亜鉛、硝酸亜鉛、過塩素酸亜鉛、及びチオシアン酸亜鉛等の亜鉛塩、塩化マグネシウム、臭化マグネシウム、ヨウ化マグネシウム、硝酸マグネシウム、過塩素酸マグネシウム、及びチオシアン酸マグネシウム等のマグネシウム塩、塩化バリウム、臭化バリウム、ヨウ化バリウム、硝酸バリウム、過塩素酸バリウム、及びチオシアン酸バリウム等のバリウム塩、塩化ストロンチウム、臭化ストロンチウム、ヨウ化ストロンチウム、硝酸ストロンチウム、過塩素酸ストロンチウム、及びチオシアン酸ストロンチウム等のストロンチウム塩などが挙げられる。これらの無機塩は、溶解溶媒に対するタンパク質の溶解促進剤として用いられる。紡糸原液が溶解促進剤（上記の無機塩）を含有することにより、タンパク質が紡糸原液中に高い濃度で溶解可能となる。これにより、タンパク質繊維の生産効率がより一層向上し、かつタンパク質繊維の高品質化と応力等の物性の向上等が期待される。無機塩は、塩化リチウム及び塩化カルシウムからなる群より選択される少なくとも１種であってよい。 The dissolution promoter may be, for example, an inorganic salt composed of the following Lewis acid and Lewis base. Examples of the Lewis base include oxo acid ions (nitrate ions, perchlorate ions, etc.), metal oxo acid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. When the dissolution solvent is an organic solvent, examples of the inorganic salt include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, and calcium bromide. Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate; iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; Aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate; potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and thiocyanate Potassium salts such as potassium acid, sodium chloride, nato bromide Sodium salts such as sodium, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate; zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate and zinc thiocyanate Magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate, barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and thiocyanate And barium salts such as barium acid, and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate. These inorganic salts are used as a promoter for dissolving a protein in a dissolution solvent. When the spinning dope contains a dissolution promoter (the above-mentioned inorganic salt), the protein can be dissolved in the spinning dope at a high concentration. As a result, it is expected that the production efficiency of the protein fiber is further improved, and that the quality of the protein fiber and the physical properties such as stress are improved. The inorganic salt may be at least one selected from the group consisting of lithium chloride and calcium chloride.
溶解溶媒が水である（又は水を含有している）場合、紡糸原液は、溶解促進剤として、例えば、尿素、グアニジン、又はドデシル硫酸ナトリウム（ＳＤＳ）を含んでいてよい。 When the dissolving solvent is water (or contains water), the spinning dope may contain, for example, urea, guanidine, or sodium dodecyl sulfate (SDS) as a dissolution promoter.
上記の溶解促進剤は、１種単独、又は２種以上を組み合わせて用いたものであってよい。 The above-mentioned dissolution promoters may be used alone or in combination of two or more.
溶解促進剤の含有量は、紡糸原液全量に対して、０．１質量％以上、１質量％以上、４質量％以上、７質量％以上、１０質量％以上、又は１５質量％以上であってよく、２０質量％以下、１６質量％以下、１２質量％以下、又は９質量％以下であってよい。 The content of the dissolution accelerator is 0.1% by mass or more, 1% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more based on the total amount of the spinning dope. It may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
紡糸原液（タンパク質溶液）は、必要に応じて、各種の添加剤を更に含有していてよい。添加剤としては、例えば、可塑剤、レベリング剤、架橋剤、結晶核剤、酸化防止剤、紫外線吸収剤、着色剤、フィラー、合成樹脂が挙げられる。添加剤の含有量は、紡糸原液中のタンパク質全量１００質量部に対して、５０質量部以下であってよい。 The spinning dope (protein solution) may further contain various additives as necessary. Examples of the additives include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin. The content of the additive may be 50 parts by mass or less based on 100 parts by mass of the total amount of the protein in the spinning dope.
図１は、タンパク質繊維を製造するための紡糸装置の一例を概略的に示す説明図である。図１に示す紡糸装置１０は、乾湿式紡糸用の紡糸装置の一例であり、押出し装置１と、凝固浴槽２０と、洗浄浴槽（延伸浴槽）２１と、乾燥装置４とを上流側から順に有している。 FIG. 1 is an explanatory view schematically showing an example of a spinning apparatus for producing protein fibers. The spinning device 10 shown in FIG. 1 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1, a coagulation bath 20, a washing bath (drawing bath) 21, and a drying device 4 in this order from the upstream side. doing.
押出し装置１は貯槽７を有しており、ここに紡糸原液（ドープ液）６が貯留される。紡糸原液６は、上述したタンパク質を溶解溶媒（ドープ溶媒）に溶解させて得られる。凝固浴槽２０に凝固浴液１１が貯留される。紡糸原液６は、貯槽７の下端部に取り付けられたギアポンプ８により、凝固浴液１１との間にエアギャップ１９を開けて設けられたノズル９から押し出される。押し出された紡糸原液６は、エアギャップ１９を経て、凝固浴槽２０の凝固浴液１１内に供給（導入）される。凝固浴液１１内で紡糸原液から溶媒が除去されてタンパク質が凝固する。凝固したタンパク質は、洗浄浴槽２１に導かれ、洗浄浴槽２１内の洗浄液１２により洗浄された後、洗浄浴槽２１内に設置された第一ニップローラ１３と第二ニップローラ１４により、乾燥装置４へと送られる。このとき、例えば、第二ニップローラ１４の回転速度を第一ニップローラ１３の回転速度よりも速く設定すると、回転速度比に応じた倍率で延伸されたタンパク質繊維３６が得られる。洗浄液１２中で延伸されたタンパク質繊維は、洗浄浴槽２１内を離脱してから、乾燥装置４内を通過する際に乾燥され、その後、ワインダーにて巻き取られる。このようにして、タンパク質繊維が、紡糸装置１０により、最終的にワインダーに巻き取られた巻回物５として得られる。なお、１８ａ〜１８ｇは糸ガイドである。 The extruder 1 has a storage tank 7 in which a stock spinning solution (dope solution) 6 is stored. The spinning dope 6 is obtained by dissolving the above-described protein in a dissolving solvent (dope solvent). The coagulation bath liquid 11 is stored in the coagulation bath 20. The stock spinning solution 6 is pushed out of a nozzle 9 provided with an air gap 19 opened between the stock solution 7 and a coagulation bath solution 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded spinning solution 6 is supplied (introduced) into the coagulation bath solution 11 of the coagulation bath tank 20 via the air gap 19. In the coagulation bath solution 11, the solvent is removed from the spinning dope to coagulate the protein. The coagulated protein is guided to the washing bath 21 and washed by the washing liquid 12 in the washing bath 21, and then sent to the drying device 4 by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21. Can be At this time, for example, if the rotation speed of the second nip roller 14 is set higher than the rotation speed of the first nip roller 13, the protein fibers 36 drawn at a magnification corresponding to the rotation speed ratio are obtained. The protein fiber stretched in the washing liquid 12 is separated from the inside of the washing bath 21, dried when passing through the drying device 4, and then wound up by a winder. In this way, the protein fiber is finally obtained by the spinning device 10 as the wound material 5 wound on a winder. In addition, 18a-18g is a thread guide.
凝固浴液１１は、第２の溶解溶媒を含有する。ここで、第２の溶解溶媒とは、凝固浴液１１に含まれる溶解溶媒であり、紡糸原液６中のタンパク質を溶解し得る溶媒ということもできる。第２の溶解溶媒として、第１の溶解溶媒で例示したものと同じものを例示できる。第２の溶解溶媒は、第１の溶解溶媒（ドープ溶媒）と、同種の溶媒であってもよく、異種の溶媒であってもよい。紡糸原液６を凝固浴液１１に導入する際、紡糸原液６とともに、紡糸原液６中の第１の溶解溶媒が凝固浴液１１に導入されるが、本実施形態における凝固浴液１１は、紡糸原液６を導入する前に、予め、第２の溶解溶媒を含んでいる。すなわち、本実施形態において、凝固浴液１１は、紡糸原液６を凝固浴液１１に導入する前に、第２の溶解溶媒を添加されてなるものである。凝固浴液１１が第２の溶解溶媒を含有することにより、得られるタンパク質繊維のボイドの発生が充分に抑制される。 The coagulation bath solution 11 contains a second dissolution solvent. Here, the second dissolving solvent is a dissolving solvent contained in the coagulation bath solution 11, and may be a solvent capable of dissolving proteins in the spinning dope 6. As the second dissolving solvent, the same solvent as exemplified for the first dissolving solvent can be exemplified. The second dissolving solvent may be the same kind of solvent as the first dissolving solvent (dope solvent) or may be a different kind of solvent. When the spinning solution 6 is introduced into the coagulation bath solution 11, the first dissolution solvent in the spinning solution 6 is introduced into the coagulation bath solution 11 together with the spinning solution 6. Before introducing the undiluted solution 6, it contains a second dissolution solvent in advance. That is, in the present embodiment, the coagulation bath solution 11 is obtained by adding the second dissolving solvent before introducing the spinning stock solution 6 into the coagulation bath solution 11. When the coagulation bath solution 11 contains the second dissolution solvent, the generation of voids in the obtained protein fiber is sufficiently suppressed.
第２の溶解溶媒は、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサフルオロイソプロノール、ヘキサフルオロアセトン、ギ酸及びこれらに溶解促進剤を添加したもの、並びに水に溶解促進剤を添加したものからなる群より選択される少なくとも１種であってよく、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群より選択される少なくとも１種であることが好ましい。 The second dissolving solvent is composed of dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and those obtained by adding a dissolution promoter thereto, and water obtained by adding a dissolution promoter to water. It may be at least one selected from the group, and is preferably at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution promoter to these.
凝固浴液１１は、第２の溶解溶媒を含み、かつ、脱溶媒できる溶液であればよく、例えば、メタノール、エタノール、２−プロパノール等の炭素数１〜５の低級アルコール、アセトン等を含有していてよい。凝固浴液１１は、適宜水を含んでいてもよい。凝固浴液１１は、メタノールと、第２の溶解溶媒とを含有していてよい。この場合、第２の溶解溶媒は、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド及びこれらに溶解促進剤を添加したものからなる群より選択される少なくとも１種であることが好ましい。 The coagulation bath solution 11 may be any solution that contains the second dissolution solvent and can remove the solvent, and for example, contains a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone. May be. The coagulation bath liquid 11 may appropriately contain water. The coagulation bath liquid 11 may contain methanol and a second dissolution solvent. In this case, the second dissolution solvent is preferably at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution promoter to these.
凝固浴液１１における、第２の溶解溶媒の含有量は、凝固浴液１１全量に対して、１０〜６０質量％であってもよい。また、第２の溶解溶媒の含有量は、凝固浴液１１全量に対して、１５質量％以上、２０質量％以上、２５質量％以上、又は３０質量％以上であってもよく、５０質量％以下、４０質量％以下、又は３０質量％以下であってもよい。第２の溶解溶媒の含有量が、上述の範囲内である場合、タンパク質繊維におけるボイドの発生がより一層顕著に抑制される。凝固浴液１１における第２の溶解溶媒の含有量が、凝固浴液１１全量に対して、１５質量％以上である場合、最大延伸倍率がより一層高くなり、２０質量％以上である場合、応力がより一層大きくなる。なお、本明細書における応力とは、タンパク質繊維が繊維軸方向の引張外力により破断するまでの最大荷重を、タンパク質繊維の９０００ｍあたりの重量で除した値（単位：ｇ／Ｄ）を意味する。 The content of the second dissolving solvent in the coagulation bath liquid 11 may be 10 to 60% by mass based on the total amount of the coagulation bath liquid 11. Further, the content of the second dissolving solvent may be 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, Hereinafter, it may be 40% by mass or less, or 30% by mass or less. When the content of the second dissolution solvent is within the above range, the generation of voids in the protein fiber is more remarkably suppressed. When the content of the second dissolved solvent in the coagulation bath liquid 11 is 15% by mass or more based on the total amount of the coagulation bath solution 11, the maximum stretching ratio is further increased. Becomes even larger. In addition, the stress in this specification means the value (unit: g / D) obtained by dividing the maximum load until the protein fiber is broken by the tensile external force in the fiber axis direction by the weight per 9000 m of the protein fiber.
凝固浴液１１は、溶解促進剤を含んでいてよい。凝固浴液１１における溶解促進剤として、例えば、上述の無機塩を用いてもよい。また、凝固浴液１１は、溶解促進剤を含有する第２の溶解溶媒を用いることにより、溶解促進剤を含んでいてよい。凝固浴液１１は、無機塩を含んでいてよく、塩化リチウム及び塩化カルシウムからなる群より選択される少なくとも１種を含んでいてよい。凝固浴液１１における溶解促進剤は、１種単独で、又は２種以上を組み合わせて用いたものであってもよい。 The coagulation bath liquid 11 may contain a dissolution promoter. As the dissolution promoter in the coagulation bath liquid 11, for example, the above-mentioned inorganic salts may be used. The coagulation bath liquid 11 may contain a dissolution promoter by using a second dissolution solvent containing the dissolution promoter. The coagulation bath solution 11 may contain an inorganic salt, and may contain at least one selected from the group consisting of lithium chloride and calcium chloride. The dissolution promoter in the coagulation bath liquid 11 may be used alone or in combination of two or more.
紡糸原液が溶解促進剤として無機塩を含有する場合、凝固浴液１１は、紡糸原液が含有する無機塩と同種の無機塩を含んでいてよく、また異種の無機塩を含んでいてよい。紡糸原液が溶解促進剤として無機塩を含有しない場合であっても、凝固浴液１１は、無機塩を含有していてもよい。 When the spinning dope contains an inorganic salt as a dissolution promoter, the coagulation bath solution 11 may contain the same kind of inorganic salt as the inorganic salt contained in the spinning dope, or may contain a different kind of inorganic salt. Even when the spinning dope does not contain an inorganic salt as a dissolution promoter, the coagulation bath solution 11 may contain an inorganic salt.
凝固浴液１１が無機塩を含有する場合、凝固浴液１１中でのタンパク質の凝固をより温和な条件で行うことができる。これにより、得られるタンパク質繊維のボイドの発生がより一層抑制される。また、得られるタンパク質繊維の応力及び最大延伸倍率がより一層高くなる。凝固浴液１１中の無機塩の含有量を調整することにより、凝固浴液１１中でのタンパク質の凝固速度等の調整が容易になり、ボイドの発生が抑制されたタンパク質繊維の製造がより一層容易になる。 When the coagulation bath solution 11 contains an inorganic salt, coagulation of the protein in the coagulation bath solution 11 can be performed under milder conditions. Thereby, the generation of voids in the obtained protein fiber is further suppressed. Further, the stress and the maximum draw ratio of the obtained protein fiber are further increased. By adjusting the content of the inorganic salt in the coagulation bath solution 11, the coagulation rate of the protein in the coagulation bath solution 11 can be easily adjusted, and the production of the protein fiber in which the generation of voids is suppressed is further improved. It will be easier.
凝固浴液１１が無機塩を含有する場合、凝固浴液１１における無機塩の含有量は、特に限定されないが、凝固浴液１１全量基準で、０質量％超え３０質量％以下、５質量％以上２５質量％以下、又は１０質量％以上２０質量％未満であってよい。紡糸原液６が無機塩を含有する場合、凝固浴液１１における無機塩の含有量は、紡糸原液６中の無機塩の含有量より低いことが好ましい。この場合、紡糸原液６を凝固浴液１１に導入する際に、凝固浴液１１中でタンパク質の凝固が阻害されることなく、より温和な条件下でタンパク質の凝固が可能となる。これにより、タンパク質繊維のボイドの発生がより有利に抑制される。 When the coagulation bath liquid 11 contains an inorganic salt, the content of the inorganic salt in the coagulation bath liquid 11 is not particularly limited, but is more than 0% by mass and 30% by mass or less and 5% by mass or more based on the total amount of the coagulation bath solution 11. It may be 25% by mass or less, or 10% by mass or more and less than 20% by mass. When the spinning dope 6 contains an inorganic salt, the content of the inorganic salt in the coagulation bath solution 11 is preferably lower than the content of the inorganic salt in the spinning dope 6. In this case, when the spinning solution 6 is introduced into the coagulation bath solution 11, the coagulation of the protein can be performed under milder conditions without inhibiting the coagulation of the protein in the coagulation bath solution 11. Thereby, the generation of voids in the protein fiber is more advantageously suppressed.
凝固浴液１１の温度は、特に限定されないが、４０℃以下、３０℃以下、２５℃以下、２０℃以下、１０℃以下、又は５℃以下であってよい。凝固浴液１１の温度は、特に限定されないが、−３０℃以上、−２０℃以上、又は−１０℃以上であってよく、作業性、冷却コスト等の観点から、０℃以上であることが好ましい。凝固浴液１１の温度が上記範囲内であれば、ボイドの発生が充分に抑制されたタンパク質繊維の製造がより一層容易になる。凝固浴液１１の温度を上記範囲に調整することにより、タンパク質繊維のボイドの発生が充分に抑制され、得られるタンパク質繊維の応力がより増大する。なお、凝固浴液の温度は、例えば、熱交換器を内部に備える凝固浴槽２０と、冷却循環装置と、を有する紡糸装置１０を用いることにより調整することができる。例えば、凝固浴槽内に設置した熱交換器に冷却循環装置で所定の温度まで冷却した媒体を流すことにより、凝固浴液と熱交換器間での熱交換により温度を上記範囲内に調整することができる。この場合、媒体として凝固浴液１１に用いる溶媒（例えば、メタノール）を循環することでより効率的な冷却が可能となる。 The temperature of the coagulation bath liquid 11 is not particularly limited, but may be 40 ° C or lower, 30 ° C or lower, 25 ° C or lower, 20 ° C or lower, 10 ° C or lower, or 5 ° C or lower. The temperature of the coagulation bath liquid 11 is not particularly limited, but may be −30 ° C. or higher, −20 ° C. or higher, or −10 ° C. or higher, and may be 0 ° C. or higher from the viewpoint of workability, cooling cost, and the like. preferable. When the temperature of the coagulation bath solution 11 is within the above range, the production of protein fibers in which generation of voids is sufficiently suppressed is further facilitated. By adjusting the temperature of the coagulation bath solution 11 to the above range, the generation of voids in the protein fibers is sufficiently suppressed, and the stress of the obtained protein fibers is further increased. In addition, the temperature of the coagulation bath liquid can be adjusted, for example, by using the spinning device 10 including the coagulation bath 20 having a heat exchanger therein and a cooling circulation device. For example, by flowing a medium cooled to a predetermined temperature by a cooling circulation device through a heat exchanger installed in a coagulation bath, the temperature is adjusted to the above range by heat exchange between the coagulation bath liquid and the heat exchanger. Can be. In this case, by circulating a solvent (for example, methanol) used for the coagulation bath liquid 11 as a medium, more efficient cooling becomes possible.
凝固浴液１１が貯留される凝固浴槽２０は複数設けられていてもよい。この場合、ノズル９から押し出された紡糸原液６が直接供給（導入）される、凝固浴槽の凝固浴液（第１凝固浴液）が、第２の溶解溶媒を含有していればよい。すなわち、凝固浴液１１が貯留される凝固浴槽２０は複数設けられている場合、第１凝固浴液以外の凝固浴液（他の凝固浴液）は、第２の溶解溶媒を含有していなくてもよい。他の凝固浴液の温度は、４０℃以下、２０℃以下、１０℃以下、又は５℃以下であってもよく、０℃以上、又は４０℃超であってもよい。 A plurality of coagulation bath tanks 20 in which the coagulation bath liquid 11 is stored may be provided. In this case, the coagulation bath liquid (first coagulation bath liquid) in the coagulation bath to which the spinning dope 6 extruded from the nozzle 9 is directly supplied (introduced) may contain the second dissolving solvent. That is, when a plurality of coagulation baths 20 for storing the coagulation bath 11 are provided, the coagulation baths other than the first coagulation bath (other coagulation baths) do not contain the second dissolved solvent. You may. The temperature of the other coagulation bath liquid may be 40 ° C. or lower, 20 ° C. or lower, 10 ° C. or lower, or 5 ° C. or lower, and may be 0 ° C. or higher, or higher than 40 ° C.
凝固したタンパク質が凝固浴液１１中を通過する距離（実質的には、糸ガイド１８ａから糸ガイド１８ｂまでの距離）は、脱溶媒が効率的に行えるよく、ノズル９からの紡糸原液の押出速度（吐出速度）等に応じて決定されるものであってよい。凝固したタンパク質（又は紡糸原液）の凝固浴液１１中での滞留時間は、凝固したタンパク質が凝固浴液１１中を通過する距離、ノズル９からの紡糸原液６の押出速度等に応じて決定されるものであってよく、例えば、０．０１〜３分であってよく、０．０５〜０．１５分であることが好ましい。また、凝固浴液１１中で延伸（前延伸）をしてもよい。 The distance at which the coagulated protein passes through the coagulation bath solution 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) is such that the solvent can be efficiently removed and the extrusion speed of the spinning solution from the nozzle 9 is good. (Discharge speed) or the like may be determined. The residence time of the coagulated protein (or spinning solution) in the coagulation bath solution 11 is determined according to the distance that the coagulated protein passes through the coagulation bath solution 11, the extrusion speed of the spinning solution 6 from the nozzle 9, and the like. For example, it may be 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation bath liquid 11.
なお、タンパク質繊維を得る際に洗浄浴槽２１内で実施される延伸は、温水中、温水に有機溶剤等を加えた溶液中等で行う、いわゆる湿熱延伸であってもよい。この湿熱延伸の温度としては、例えば、５０〜９０℃であってよく、７５〜８５℃が好ましい。湿熱延伸では、未延伸糸（又は前延伸糸）を、例えば、１倍〜１０倍延伸することができ、２〜８倍延伸することが好ましい。 The drawing performed in the washing bath 21 when obtaining the protein fiber may be so-called wet heat drawing performed in hot water, a solution obtained by adding an organic solvent or the like to hot water, or the like. The temperature of the wet heat stretching may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, by 1 to 10 times, and preferably by 2 to 8 times.
最終的な延伸倍率は、その下限値が、未延伸糸（又は前延伸糸）に対して、好ましくは、１倍超、２倍以上、３倍以上、４倍以上、５倍以上、６倍以上、７倍以上、８倍以上、９倍以上のうちのいずれかであり、上限値が、好ましくは４０倍以下、３０倍以下、２０倍以下、１５倍以下、１４倍以下、１３倍以下、１２倍以下、１１倍以下、１０倍以下である。 The final draw ratio is preferably such that the lower limit is more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, and 6 times of the undrawn yarn (or pre-drawn yarn). Or more, 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 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, 10 times or less.
以下、本発明について実施例をもとに説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.
１．クモ糸タンパク質（クモ糸フィブロイン：ＰＲＴ４１０）の製造（クモ糸タンパク質をコードする遺伝子の合成、及び発現ベクターの構築）
ネフィラ・クラビペス（Ｎｅｐｈｉｌａ ｃｌａｖｉｐｅｓ）由来のフィブロイン（ＧｅｎＢａｎｋアクセッション番号：Ｐ４６８０４．１、ＧＩ：１１７４４１５）の塩基配列及びアミノ酸配列に基づき、配列番号１で示されるアミノ酸配列を有する改変フィブロイン（以下、「ＰＲＴ４１０」ともいう。）を設計した。1. Production of spider silk protein (spider silk fibroin: PRT410) (synthesis of gene encoding spider silk protein and construction of expression vector)
A modified fibroin having an amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as “PRT410”) based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 1174415) derived from Nephila clavipes. Also called.))
配列番号１で示されるアミノ酸配列は、ネフィラ・クラビペス由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したアミノ酸配列と、そのＮ末端に付加された配列番号７で示されるアミノ酸配列（タグ配列及びヒンジ配列）とを有する。 The amino acid sequence represented by SEQ ID NO: 1 is obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clabipes, and the N-terminal thereof. And the amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence).
設計したＰＲＴ４１０をコードする核酸を合成した。当該核酸には、５’末端にＮｄｅＩサイト及び終止コドン下流にＥｃｏＲＩサイトを付加した。当該核酸をクローニングベクター（ｐＵＣ１１８）にクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ−２２ｂ（＋）に組換えて発現ベクターを得た。 A nucleic acid encoding the designed PRT410 was synthesized. An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
得られたｐＥＴ２２ｂ（＋）発現ベクターによって、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表１）にＯＤ６００が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ６００が５になるまで約１５時間、フラスコ培養を行って、シード培養液を得た。Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 1) so that the OD 600 was 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured for about 15 hours until the OD 600 reached 5, to obtain a seed culture.
当該シード培養液を５００ｍｌの生産培地（表２）を添加したジャーファーメンターにＯＤ６００が０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持した。The seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 2) had been added so that the OD 600 was 0.05. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ Ｅｘｔｒａｃｔ １２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持しながら、２０時間培養を行った。その後、１Ｍのイソプロピル−β−チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、ＰＲＴ４１０を発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ−ＰＡＧＥを行い、ＩＰＴＧ添加に依存したＰＲＴ４１０に相当するサイズのバンドの出現により、ＰＲＴ４１０の発現を確認した。 Immediately after glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, 120 g / 1 L of Yeast Extract) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce PRT410 expression. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using cells prepared from the culture solution before and after the addition of IPTG, and the expression of PRT410 was confirmed by the appearance of a band having a size corresponding to PRT410 dependent on the addition of IPTG.
（クモ糸フィブロインの精製）
ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ Ｔｒｉｓ−ＨＣｌ ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ Ｎｉｒｏ Ｓｏａｖｉ社）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ グアニジン緩衝液（８Ｍ グアニジン塩酸塩、１０ｍＭ リン酸二水素ナトリウム、２０ｍＭ ＮａＣｌ、１ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質（ＰＲＴ４１０）を遠心分離により回収した。回収した凝集タンパク質から凍結乾燥機で水分を除き、ＰＲＴ４１０の凍結乾燥粉末を得た。(Purification of spider silk fibroin)
Two hours after the addition of IPTG, the recovered cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in an 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein (PRT410) obtained after dialysis was collected by centrifugation. Water was removed from the collected aggregated proteins using a freeze dryer to obtain a freeze-dried powder of PRT410.
２．クモ糸フィブロイン繊維の製造
（紡糸原液の調製）
溶解溶媒（第１の溶解溶媒）であるジメチルスルホキシド（ＤＭＳＯ）に、上述のクモ糸フィブロイン（ＰＲＴ４１０）を濃度２４質量％となるよう添加した後、溶解促進剤として塩化リチウム（ＬｉＣｌ）を濃度４．０質量％となるように添加し、その後、シェーカーを使用して３時間振盪し、溶解させた。その後、ゴミと泡を取り除き、紡糸原液（ドープ液）とした。紡糸原液の溶液粘度は９０℃において５０００ｃＰ（センチポアズ）であった。2. Production of spider silk fibroin fiber (preparation of spinning stock solution)
The above spider silk fibroin (PRT410) was added to dimethyl sulfoxide (DMSO) as a dissolution solvent (first dissolution solvent) to a concentration of 24% by mass, and then lithium chloride (LiCl) was added as a dissolution promoter to a concentration of 4%. 0.0% by mass, and then shaken for 3 hours using a shaker to dissolve. Thereafter, dust and bubbles were removed to obtain a spinning solution (dope solution). The solution viscosity of the spinning dope was 5000 cP (centipoise) at 90 ° C.
（紡糸）
得られた紡糸原液を用いて、図１に示す紡糸装置１０に準じた紡糸装置を用いた乾湿式紡糸によって、紡糸及び延伸されたタンパク質繊維（クモ糸フィブロイン繊維）を製造した。用いた紡糸装置は、図１に示す紡糸装置１０において、未延伸糸製造装置２（第１凝固浴槽）及び湿熱延伸装置３（洗浄浴槽）の間に、第２の未延伸糸製造装置（第２凝固浴槽）及び第３の未延伸糸製造装置（第３凝固浴槽）を備えるものである。第１凝固浴液には、メタノール（ＭｅＯＨ）、及びＤＭＳＯ（第２の溶解溶媒）を表３に示す組成（質量比）で含む溶液を用いた。凝固浴液全量に対する、第２の溶解溶媒の含有量を表３に示す。第２凝固浴槽及び第３凝固浴槽の凝固浴液としては、ＭｅＯＨを用いた。また、洗浄浴槽の洗浄液には、水を用いた。その他の乾湿式紡糸の条件は以下のとおりである。
押出しノズル直径：０．２ｍｍ
第１凝固浴液の滞在時間、温度及び延伸倍率：表３参照
延伸倍率：表３参照
乾燥温度：９５℃(spinning)
Using the obtained spinning dope, protein fibers (spider silk fibroin fibers) were produced by dry and wet spinning using a spinning device according to the spinning device 10 shown in FIG. The spinning apparatus used is the same as the spinning apparatus 10 shown in FIG. 1, except that a second undrawn yarn manufacturing apparatus (a first coagulation bath) and a wet heat drawing apparatus 3 (washing bath) are used. 2 coagulation bath) and a third undrawn yarn producing device (third coagulation bath). As the first coagulation bath solution, a solution containing methanol (MeOH) and DMSO (second dissolution solvent) in the composition (mass ratio) shown in Table 3 was used. Table 3 shows the content of the second dissolving solvent with respect to the total amount of the coagulating bath solution. MeOH was used as the coagulation bath liquid in the second and third coagulation baths. In addition, water was used as a cleaning liquid for the cleaning bath. Other conditions of the dry-wet spinning are as follows.
Extrusion nozzle diameter: 0.2mm
Residence time, temperature and stretching ratio of the first coagulation bath solution: See Table 3 Stretching ratio: See Table 3 Drying temperature: 95 ° C
３．ボイドの評価
製造例１−１〜１−５におけるクモ糸フィブロイン繊維のボイドの評価は、光学顕微鏡を用いた表面観測により行った。具体的には、ボイドの評価は、凝固後及び洗浄後の繊維について、光学顕微鏡を用いた観察により、約１０００μｍ２あたりに１μｍ以上のボイドが存在する割合を評価した。製造例１−１〜１−５におけるクモ糸フィブロイン繊維の光学顕微鏡写真を図２に示す。凝固後の繊維は第１凝固浴液での凝固により得られた繊維である。なお、図２の製造例１−１の洗浄後における光学顕微鏡写真中のスケールバーは、５０μｍである。3. Evaluation of voids The voids of spider silk fibroin fibers in Production Examples 1-1 to 1-5 were evaluated by surface observation using an optical microscope. Specifically, the voids were evaluated by observing the fibers after coagulation and after washing with an optical microscope to evaluate the proportion of voids of 1 μm or more per 1000 μm 2 . An optical micrograph of the spider silk fibroin fiber in Production Examples 1-1 to 1-5 is shown in FIG. The fiber after coagulation is a fiber obtained by coagulation in the first coagulation bath liquid. In addition, the scale bar in the optical micrograph after washing of Production Example 1-1 in FIG. 2 is 50 μm.
図２に示すとおり、凝固浴液が溶解溶媒（第２の溶解溶媒）としてＤＭＳＯを含有する場合（製造例１−２〜１−５）では、凝固浴液がＤＭＳＯを含有しない場合（製造例１−１）と比べて、クモ糸フィブロイン繊維のボイドの発生が抑制された。また、凝固浴液がＤＭＳＯを１０質量％以上含有する場合（製造例１−４〜１−５）では、クモ糸フィブロイン繊維のボイドの発生がより顕著に抑制された。 As shown in FIG. 2, when the coagulation bath solution contains DMSO as a dissolution solvent (second dissolution solvent) (Production Examples 1-2 to 1-5), the coagulation bath solution does not contain DMSO (Production Example). As compared with 1-1), generation of voids in spider silk fibroin fibers was suppressed. In the case where the coagulation bath solution contained 10% by mass or more of DMSO (Production Examples 1-4 to 1-5), generation of voids in spider silk fibroin fibers was more remarkably suppressed.
４．応力の評価
製造例１−１〜１−８により得られたタンパク質繊維について応力を評価した。応力は、ＩＮＳＴＲＯＮ３３４２引張試験機により、試験片長１００ｍｍ、引張り速度１００ｍｍ／ｍｉｎで１０回測定し、その平均値を算出した。平均値間の有意差に関しては、２水準間の比較は２−ｓａｐｍｌｅ ｔ検定により行い、３水準以上の比較は分散分析検定により行った。有意差水準（ｐ値）が０．０５未満の場合に有意差有りと判断した。図３は、応力の評価結果を示す箱ひげ図である。なお、本明細書における応力とは、タンパク質繊維が繊維軸方向の引張外力により破断するまでの最大荷重を、タンパク質繊維の９０００ｍあたりの重量で除した値（単位：ｇ／Ｄ）を意味する。図３中、＊は外れ値を示す。4. Evaluation of stress The stress was evaluated for the protein fibers obtained in Production Examples 1-1 to 1-8. The stress was measured 10 times with an INSTRON 3342 tensile tester at a test piece length of 100 mm and a tensile speed of 100 mm / min, and the average value was calculated. Regarding the significant difference between the mean values, the comparison between two levels was performed by a 2-saplet test, and the comparison of three or more levels was performed by an analysis of variance test. When the level of significant difference (p value) was less than 0.05, it was determined that there was a significant difference. FIG. 3 is a box plot showing the evaluation results of stress. In addition, the stress in this specification means the value (unit: g / D) obtained by dividing the maximum load until the protein fiber is broken by the tensile external force in the fiber axis direction by the weight per 9000 m of the protein fiber. In FIG. 3, * indicates an outlier.
ＤＭＳＯの含有量が２０質量％以上である第１凝固浴液を用いて得たクモ糸フィブロイン繊維（製造例１−４〜１−８）は、ＤＭＳＯの含有量が２０質量％未満である第１凝固浴液を用いて得たクモ糸フィブロイン繊維（製造例１−１〜１−３）と比べて、応力が高かった。また、第１凝固浴液におけるＤＭＳＯの含有量が、３０質量％以上である凝固浴液を用いた場合、より一層応力が高くなることが示された。 The spider silk fibroin fiber (Production Examples 1-4 to 1-8) obtained by using the first coagulation bath liquid having a DMSO content of 20% by mass or more has a DMSO content of less than 20% by mass. 1 Stress was higher than that of the spider silk fibroin fiber obtained using the coagulation bath solution (Production Examples 1-1 to 1-3). Moreover, it was shown that when the content of DMSO in the first coagulation bath solution was 30% by mass or more, the coagulation bath solution further increased the stress.
５．最大延伸倍率の評価
最大延伸倍率は、図１に示した紡糸装置１０の２箇所で評価した。評価した箇所は、１箇所目が１８ｂと１８ｃとの間での延伸（固化浴延伸）であり、２箇所目が１４と１８ｅの間での延伸（洗浄浴延伸）である。評価は、２箇所同時でなく、一方を評価する際は他方は等倍に近い延伸倍率に固定して行った。評価方法としては１倍ずつ延伸倍率を上昇させていき、断糸した際の延伸倍率を最大延伸倍率とした。結果を図４に示す。5. Evaluation of Maximum Stretching Ratio The maximum stretching ratio was evaluated at two points of the spinning device 10 shown in FIG. The evaluated places are the first place stretching between 18b and 18c (solidification bath stretching) and the second place stretching between 14 and 18e (wash bath stretching). The evaluation was not performed simultaneously at two places, and when one was evaluated, the other was fixed at a draw ratio close to unity. As an evaluation method, the draw ratio was increased by one time, and the draw ratio when the yarn was broken was taken as the maximum draw ratio. FIG. 4 shows the results.
凝固浴液がＤＭＳＯを１５質量％以上含有する場合、ＤＭＳＯを１５質量％未満の割合で含有する場合と比べ、最大延伸倍率が高くなった。 When the coagulation bath solution contained 15% by mass or more of DMSO, the maximum stretching ratio was higher than that in the case where DMSO was contained at a ratio of less than 15% by mass.
６．凝固浴液に無機塩を添加した際のボイドの発生量評価
凝固浴液における組成ＭｅＯＨ／ＤＭＳＯ（質量比）、第２の溶解溶媒として用いたＤＭＳＯの含有量並びに無機塩の種類及び含有量を表４に示す条件とすること以外は、製造例１−１と同様にして、クモ糸フィブロイン繊維（タンパク質繊維）を製造した（製造例２−１〜２−８）。ボイドの評価は、製造例１−１〜１−５における方法と同様にして行った。製造例２−１〜２−８において、得られたタンパク質繊維のボイドの評価結果をそれぞれ図５〜１２に示す。6. Evaluation of the amount of voids generated when an inorganic salt was added to the coagulation bath solution The composition MeOH / DMSO (mass ratio) in the coagulation bath solution, the content of DMSO used as the second dissolution solvent, and the type and content of the inorganic salt were determined. Except for the conditions shown in Table 4, spider silk fibroin fibers (protein fibers) were produced in the same manner as in Production Example 1-1 (Production Examples 2-1 to 2-8). The void was evaluated in the same manner as in Production Examples 1-1 to 1-5. In Production Examples 2-1 to 2-8, the evaluation results of voids of the obtained protein fibers are shown in FIGS.
図５〜１２に示すとおり、凝固浴液（第１凝固浴液）が溶解溶媒（第２の溶解溶媒）として、ＤＭＳＯを含み、かつ無機塩を含む場合であっても、ボイドの発生が充分に抑制されることが示された。第１凝固浴液の組成ＭｅＯＨ／ＤＭＳＯ（質量比）が９０／１０である場合、凝固浴液における、ＬｉＣｌの含有量を１０質量％以上、又は、塩化カルシウム（ＣａＣｌ２）の含有量を１０質量％とすることで、より顕著にボイドの発生が抑制された（製造例２−２〜２−４と、製造例２−１との対比）。また、第１凝固浴液の組成ＭｅＯＨ／ＤＭＳＯ（質量比）が８０／２０である場合、ＬｉＣｌの含有量を５質量％以上とすることで、より顕著にボイドの発生が抑制された（製造例２−６〜２−８と、製造例２−５との対比）。As shown in FIGS. 5 to 12, even when the coagulation bath solution (first coagulation bath solution) contains DMSO as a dissolution solvent (second dissolution solvent) and also contains an inorganic salt, voids are sufficiently generated. Was shown to be suppressed. When the composition MeOH / DMSO (mass ratio) of the first coagulation bath liquid is 90/10, the content of LiCl in the coagulation bath liquid is 10% by mass or more, or the content of calcium chloride (CaCl 2 ) is 10%. By setting it as mass%, generation | occurrence | production of a void was suppressed more remarkably (Comparative example of Production Examples 2-2 to 2-4 and Production Example 2-1). When the composition MeOH / DMSO (mass ratio) of the first coagulation bath solution was 80/20, the generation of voids was more remarkably suppressed by setting the LiCl content to 5% by mass or more (manufacturing). Examples 2-6 to 2-8 and Production Example 2-5).
１…押出し装置、２…未延伸糸製造装置、３…湿熱延伸装置、４…乾燥装置、６…紡糸原液、１０…紡糸装置、２０…凝固浴槽、２１…洗浄浴槽、３６…タンパク質繊維。 DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 2 ... Undrawn yarn manufacturing apparatus, 3 ... Wet heat drawing apparatus, 4 ... Drying apparatus, 6 ... Spinning solution, 10 ... Spinning apparatus, 20 ... Coagulation bath, 21 ... Washing bath, 36 ... Protein fiber.
Claims (12)
前記凝固浴液が、第２の溶解溶媒を含有する、タンパク質繊維の製造方法。Introducing a spinning dope containing a protein and a first dissolution solvent into a coagulation bath to coagulate the protein,
A method for producing a protein fiber, wherein the coagulation bath solution contains a second dissolution solvent.
前記第２の溶解溶媒が、ジメチルスルホキシド、Ｎ，Ｎ−ジメチルホルムアミド、ヘキサフルオロイソプロノール、ヘキサフルオロアセトン、ギ酸及びこれらに溶解促進剤を添加したもの、並びに水に溶解促進剤を添加したものからなる群より選択される少なくとも１種である、請求項１に記載のタンパク質繊維の製造方法。The first dissolving solvent is selected from dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and those obtained by adding a dissolution promoter thereto, and those obtained by adding a dissolution promoter to water. At least one selected from the group consisting of
The second dissolving solvent is selected from dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and those obtained by adding a dissolution promoter thereto, and those obtained by adding a dissolution promoter to water. The method for producing a protein fiber according to claim 1, wherein the method is at least one selected from the group consisting of:
前記凝固浴液中における前記無機塩の含有量が、前記紡糸原液中の前記無機塩の含有量よりも低い、請求項１０又は１１に記載のタンパク質繊維の製造方法。The spinning dope contains an inorganic salt,
The method for producing a protein fiber according to claim 10 or 11, wherein the content of the inorganic salt in the coagulation bath solution is lower than the content of the inorganic salt in the spinning dope.
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| JPH09268426A (en) * | 1996-03-28 | 1997-10-14 | Kuraray Co Ltd | Production of fiber |
| WO2003060099A2 (en) * | 2002-01-11 | 2003-07-24 | Nexia Biotechnologies, Inc. | Methods and apparatus for spinning spider silk protein |
| WO2013065650A1 (en) * | 2011-11-02 | 2013-05-10 | スパイバー株式会社 | Polypeptide solution, artificial polypeptide fiber production method using same, and polypeptide refining method |
| WO2016149414A1 (en) * | 2015-03-16 | 2016-09-22 | Bolt Threads, Inc. | Improved silk fibers |
| JP2016531845A (en) * | 2013-09-17 | 2016-10-13 | ボルト スレッズ インコーポレイテッド | Methods and compositions for synthesizing improved silk fibers |
| WO2017030197A1 (en) * | 2015-08-20 | 2017-02-23 | 国立研究開発法人理化学研究所 | Method for manufacturing polypeptide composition having fibroin-like structure |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH09268426A (en) * | 1996-03-28 | 1997-10-14 | Kuraray Co Ltd | Production of fiber |
| WO2003060099A2 (en) * | 2002-01-11 | 2003-07-24 | Nexia Biotechnologies, Inc. | Methods and apparatus for spinning spider silk protein |
| WO2013065650A1 (en) * | 2011-11-02 | 2013-05-10 | スパイバー株式会社 | Polypeptide solution, artificial polypeptide fiber production method using same, and polypeptide refining method |
| JP2016531845A (en) * | 2013-09-17 | 2016-10-13 | ボルト スレッズ インコーポレイテッド | Methods and compositions for synthesizing improved silk fibers |
| WO2016149414A1 (en) * | 2015-03-16 | 2016-09-22 | Bolt Threads, Inc. | Improved silk fibers |
| WO2017030197A1 (en) * | 2015-08-20 | 2017-02-23 | 国立研究開発法人理化学研究所 | Method for manufacturing polypeptide composition having fibroin-like structure |
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