CN108368271A - 制造蛋白质溶液的方法 - Google Patents
制造蛋白质溶液的方法 Download PDFInfo
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- CN108368271A CN108368271A CN201680070357.4A CN201680070357A CN108368271A CN 108368271 A CN108368271 A CN 108368271A CN 201680070357 A CN201680070357 A CN 201680070357A CN 108368271 A CN108368271 A CN 108368271A
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Abstract
一种制造蛋白质溶液的方法，其包括下述步骤：对含有蛋白质和分散有该蛋白质的极性溶剂的分散液施加压力，得到含有蛋白质和溶解有蛋白质的极性溶剂的蛋白质溶液。
Description
技术领域
本发明涉及制造蛋白质溶液的方法。另外，本发明涉及使用蛋白质溶液制造成形体的方法。
背景技术
由于环境保护意识的提高，正在进行石油来源材料的替代物质的研究，作为其候选，可以列举在强度等方面优良的结构蛋白质。例如，提出了由结构蛋白质构成的流延膜、纤维等成形体。
但是，结构蛋白质通常为疏水性的高分子，在水等极性溶剂中的溶解性不足。也考虑过通过从结构蛋白质的稀溶液中加热馏去溶剂而得到浓缩的蛋白质溶液，但该操作会产生蛋白质的变性、凝胶化、分解等，可能导致成品率的降低。
作为用于解决上述问题的方法，在专利文献1中提出了一种蛋白质水溶液的浓缩方法，其特征在于，使蛋白质水溶液隔着透析膜与聚醚类高分子或多元醇类高分子水溶液接触。
现有技术文献
专利文献
专利文献1：日本特开平10-036676号公报
发明内容
发明所要解决的问题
但是，在专利文献1中记载的浓缩方法的情况下，在制备蛋白质溶液后，需要另行设置透析工序等独立的工序，制造工序变得繁杂。希望以更简易的方法得到高浓度的蛋白质溶液。
因此，本发明的主要目的在于提供不需要进行透析等、能够容易地得到高浓度的蛋白质溶液的方法。
用于解决问题的方法
本发明人为了解决上述问题而进行了深入研究，结果发现，通过在压力下进行溶液的制备，能够抑制蛋白质的变性等，并且能够以更温和的条件得到蛋白质溶液，基于该发现，完成了本发明。
本发明的一个方面提供一种制造蛋白质溶液的方法，其包括下述步骤：对含有蛋白质和分散有该蛋白质的极性溶剂的分散液施加压力，得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液。
根据该方法，即使不经过透析等工序，也能够容易地得到高浓度的蛋白质溶液。例如能够得到一种蛋白质溶液，其为含有蛛丝丝心蛋白和溶解有该蛛丝丝心蛋白的极性溶剂的蛋白质溶液，其中，以该蛋白质溶液的质量为基准，溶解在极性溶剂中的蛛丝丝心蛋白的浓度为15～70质量％。
本发明的另一个方面提供一种制造成形体的方法，其包括下述步骤：利用上述方法得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液；和从蛋白质溶液中除去极性溶剂，得到含有蛋白质的成形体。
根据该方法，能够使用高浓度的蛋白质溶液容易地制造蛋白质的成形体。
本发明的又一个方面提供一种由成形体再生蛋白质的方法，其包括下述步骤：对与含有蛋白质的成形体接触的极性溶剂施加压力，得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液；和从蛋白质溶液中回收蛋白质。
发明效果
根据本发明的一个方面，可以提供不需要进行透析等、能够容易地得到高浓度的结构蛋白质溶液的方法。
附图说明
图1是示出由实施例4中得到的蛋白质溶液的SDS-PAGE得到的分析结果的照片。
图2是示出由实施例5中得到的蛋白质溶液的SDS-PAGE得到的分析结果的照片。
图3是示出由实施例14～19中得到的蛋白质溶液的SDS-PAGE得到的分析结果的照片。
具体实施方式
以下，对本发明的若干实施方式详细地进行说明。但是，本发明并不限定于以下的实施方式。
制造蛋白质溶液的方法的一个实施方式包括下述步骤：制备含有蛋白质和分散有该蛋白质的极性溶剂的分散液；和对分散液施加压力，得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液。
通过对含有蛋白质的分散液施加压力，能够提高蛋白质在溶剂中的溶解性。其结果，即使不经过透析等工序，也能够制备高浓度的蛋白质溶液。本发明人推测，通过对含有蛋白质的溶液施加压力，溶剂浸渗到溶解性差的蛋白质的由β折叠或α螺旋等构成的结晶部分或利用氢键以高密度凝集的部分中，这些部分发生松解，结果蛋白质变得易于溶解，因此得到了如上所述的效果。
含有蛋白质的分散液可以通过将粉末状或其他任意形态的蛋白质与极性溶剂混合而制备。分散液中含有的蛋白质未必需要全部分散在极性溶剂中，也可以是蛋白质的一部分溶解在极性溶剂中。
用于制备分散液的极性溶剂例如可以包含选自由水、醇、二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)和六氟丙酮(HFA)组成的组中的一种以上的溶剂。从得到更高浓度的溶液的观点出发，极性溶剂可以为单独的二甲基亚砜或者二甲基亚砜与水的混合溶剂。从降低对环境的不良影响的观点出发，极性溶剂可以为水。从以更温和的条件得到蛋白质溶液的观点出发，极性溶剂可以包含醇。例如，极性溶剂可以为水与醇的混合溶剂、二甲基亚砜与醇的混合溶剂、或者水、醇和二甲基亚砜的混合溶剂。醇相对于极性溶剂(或混合溶剂)的总量的比例可以为5～100质量％或10～50质量％。使用包含醇的极性溶剂时，具有能够在更低的压力下使蛋白质溶解于极性溶剂的倾向。
本说明书中，“醇”是指由可以具有取代基的脂肪族基团和键合在该脂肪族基团上的羟基构成的化合物。脂肪族基团例如可以被氟原子等卤素原子取代，也可以无取代。作为具有被氟原子取代的脂肪族基团的氟代醇的示例，有六氟异丙醇(HFIP)。
具有低沸点的醇在能够使醇溶液的制备及其浓缩、成形体的形成等的条件温和的方面特别有利。从该观点出发，醇的沸点例如在1个大气压下可以为99℃以下、50℃以上。醇的沸点在1个大气压下可以为60℃以上。另外，通常具有下述倾向：具有一个羟基的醇具备比具有两个以上羟基的醇低的沸点。
醇的碳原子数(脂肪族基团的碳原子数)没有特别限制，可以为1～10。从以特别温和的条件得到蛋白质溶液的观点出发，醇的碳原子数可以为2～8或2～5。极性溶剂中含有的醇例如可以为选自由甲醇、乙醇、丙醇、丁醇、戊醇、己醇、庚醇、辛醇、壬醇、癸醇和这些醇的异构体组成的组中的一种以上的碳原子数为1～10的醇，也可以为选自由乙醇、丙醇、丁醇和这些醇的异构体组成的组中的一种以上的碳原子数为2～5的醇，也可以为选自由乙醇、1-丙醇和2-丙醇组成的组中的一种以上的醇。
蛋白质可以为结构蛋白质。结构蛋白质是指在生物体内形成或保持结构、形态等的蛋白质。结构蛋白质包括疏水性蛋白质和具有在极性溶剂中容易发生自凝集的倾向的多肽。这些结构蛋白质通常在极性溶剂中的溶解性低，因此，为了制造这些结构蛋白质的溶液，本实施方式的方法特别有用。
结构蛋白质可以包含选自由丝心蛋白和角蛋白组成的组中的一种以上。丝心蛋白例如可以为选自由蚕丝丝心蛋白、蛛丝丝心蛋白和蜂丝丝心蛋白组成的组中的一种以上。结构蛋白质也可以为蚕丝丝心蛋白、蛛丝丝心蛋白或它们的组合。在组合使用蚕丝丝心蛋白和蛛丝丝心蛋白的情况下，蚕丝丝心蛋白的比例相对于蛛丝丝心蛋白100质量份例如可以为40质量份以下、30质量份以下或10质量份以下。
蚕丝是由作为家蚕(Bombyx mori)的幼虫的蚕所形成的茧得到的纤维。通常，一根茧丝由两根蚕丝丝心蛋白和从外侧包覆它们的胶质(丝胶蛋白)构成。蚕丝丝心蛋白由多个原纤维构成。蚕丝丝心蛋白被四层丝胶蛋白所包覆。实际使用中，通过精炼将外侧的丝胶蛋白溶解去除，所得到的蚕丝长丝被用于衣料用途。一般的蚕丝具有1.33的比重、平均3.3分特克斯(decitex)的纤度和约1300m～约1500m的纤维长度。蚕丝丝心蛋白以野生蚕或家蚕的茧、或者旧的或废弃的丝绸料作为原料而得到。
蚕丝丝心蛋白可以为除去丝胶蛋白的蚕丝丝心蛋白、未除去丝胶蛋白的蚕丝丝心蛋白或它们的组合。除去丝胶蛋白的蚕丝丝心蛋白是对将包覆蚕丝丝心蛋白的丝胶蛋白和其他脂肪成分等除去并纯化后的蚕丝丝心蛋白进行冷冻干燥而得到的粉末。未除去丝胶蛋白的蚕丝丝心蛋白是未将丝胶蛋白等除去的未纯化的丝心蛋白。
蛛丝丝心蛋白可以含有选自由天然蛛丝蛋白和来源于天然蛛丝蛋白的多肽组成的组中的蛛丝多肽。
作为天然蛛丝蛋白，可以列举例如大吐丝管牵引丝蛋白、横丝蛋白和小壶状腺蛋白。大吐丝管牵引丝具有由结晶区域和无定形区域构成的重复区域，因此推测其兼具高的应力和伸缩性。蛛丝的横丝的一大特征是，具有不具有结晶区域而由无定形区域构成的重复区域。另一方面，与大吐丝管牵引丝相比，横丝的应力差，但具有高伸缩性。认为这是因为横丝的大部分由无定形区域构成。
大吐丝管牵引丝蛋白由蜘蛛的大壶状腺产生，具有强韧性优良的特征。作为大吐丝管牵引丝蛋白，可以列举例如来源于金纺蜘蛛(Nephila clavipes)的大壶状腺丝蛋白MaSp1和MaSp2、以及来源于十字园蛛(Araneus diadematus)的ADF3和ADF4。ADF3是十字园蛛的两种主要牵引丝蛋白之一。来源于天然蛛丝蛋白的多肽可以为来源于这些牵引丝蛋白的多肽。来源于ADF3的多肽比较容易合成，并且在强伸度和强韧性方面具有优良的特性。
横丝蛋白由蜘蛛的鞭状腺(flagelliform gland)产生。作为横丝蛋白，可以列举例如来源于金纺蜘蛛(Nephila clavipes)的鞭毛状丝蛋白(flagelliform silkprotein)。
来源于天然蛛丝蛋白的多肽可以为重组蛛丝蛋白。作为重组蛛丝蛋白，可以列举天然型蛛丝蛋白的突变体、类似体或衍生物等。这样的多肽的适当的一例为大吐丝管牵引丝蛋白的重组蛛丝蛋白(也称为“来源于大吐丝管牵引丝蛋白的多肽”)。
来源于大吐丝管牵引丝蛋白的多肽可以包含2个以上、5个以上或10个以上的式1：REP1-REP2(1)所表示的氨基酸序列单元(也称为基序)。式1：REP1-REP2(1)所示的氨基酸序列单元的数目的上限没有特别限定，例如可以为300个以下或200个以下。或者，来源于大吐丝管牵引丝蛋白的多肽可以是包含式1：REP1-REP2(1)所示的氨基酸序列单元、且C末端序列为序列号1～3中任一个序列所示的氨基酸序列或与序列号1～3中任一个序列所示的氨基酸序列具有90％以上的同源性的氨基酸序列的多肽。来源于大吐丝管牵引丝蛋白的多肽中，式1：REP1-REP2(1)所示的氨基酸序列单元可以相同，也可以不同。
式1中，丙氨酸残基数相对于REP1基序中的全部氨基酸残基数的比例通常为83％以上，可以为86％以上、90％以上或95％以上。REP1也可以是丙氨酸残基数的比率为100％的聚丙氨酸。REP1中连续排列的丙氨酸(Ala)可以为2个残基以上、3个残基以上、4个残基以上或5个残基以上。REP1中，连续排列的丙氨酸可以为20个残基以下、16个残基以下、12个残基以下或10个残基以下。REP1基序除了包含丙氨酸(Ala)以外，还可以包含选自丝氨酸(Ser)、甘氨酸(Gly)和谷氨酰胺(Gln)等中的其他氨基酸残基。
式1中，REP2为由10～200个残基的氨基酸构成的氨基酸序列，氨基酸序列中含有的甘氨酸(Gly)、丝氨酸(Ser)、谷氨酰胺(Gln)和丙氨酸(Ala)的合计残基数相对于氨基酸残基数整体可以为40％以上、60％以上或70％以上。
大吐丝管牵引丝中，REP1相当于在纤维内形成结晶β折叠的结晶区域，REP2相当于在纤维内更有柔软性、大部分缺乏有序结构的无定形区域。[REP1-REP2]相当于由结晶区域和无定形区域构成的重复区域(重复序列)，是牵引丝蛋白的特征性序列。
序列号1所示的氨基酸序列与由ADF3的氨基酸序列(NCBI登录号：AAC47010、GI：1263287)的C末端的50个残基的氨基酸构成的氨基酸序列相同。序列号2所示的氨基酸序列与从序列号1所示的氨基酸序列的C末端去除20个残基后的氨基酸序列相同。序列号3所示的氨基酸序列与从序列号1所示的氨基酸序列的C末端去除29个残基后的氨基酸序列相同。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽例如可以为由序列号7所示的氨基酸序列构成的多肽。由序列号7所示的氨基酸序列构成的多肽是在N末端附加有由起始密码子、His10标签和HRV3C蛋白酶(人鼻病毒(Human rhinovirus)3C蛋白酶)识别位点构成的氨基酸序列(序列号4)的ADF3的氨基酸序列(NCBI登录号：AAC47010、GI：1263287)中以在第543位氨基酸残基处终止翻译的方式发生突变而得到的多肽。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽可以为由在序列号7所示的氨基酸序列中置换、缺失、插入和/或添加一个或多个氨基酸而得到的氨基酸序列构成、具有由结晶区域和无定形区域构成的重复区域的蛋白质。本说明书中，“一个或多个”是指例如选自1～40个、1～35个、1～30个、1～25个、1～20个、1～15个、1～10个、以及一个或几个中的范围。本说明书中，“一个或几个”是指1～9个、1～8个、1～7个、1～6个、1～5个、1～4个、1～3个、1～2个或1个。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽可以为具有序列号8所示的氨基酸序列的ADF4来源的重组蛋白。序列号8所示的氨基酸序列是在由NCBI数据库获得的ADF4的部分氨基酸序列(NCBI登录号：AAC47011、GI：1263289)的N末端附加有由起始密码子、His10标签和HRV3C蛋白酶(人鼻病毒3C蛋白酶)识别位点构成的氨基酸序列(序列号4)的氨基酸序列。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽可以为由在序列号8所示的氨基酸序列中置换、缺失、插入和/或添加一个或多个氨基酸而得到的氨基酸序列构成、具有由结晶区域和无定形区域构成的重复区域的多肽。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽也可以是具有序列号9所示的氨基酸序列的MaSp2来源的重组蛋白。序列号9所示的氨基酸序列是在由NCBI数据库获得的MaSp2的部分序列(NCBI登录号：AAT75313、GI：50363147)的N末端附加有由起始密码子、His10标签和HRV3C蛋白酶(人鼻病毒3C蛋白酶)识别位点构成的氨基酸序列(序列号4)的氨基酸序列。
包含2个以上的式1：REP1-REP2(1)所示的氨基酸序列单元的多肽可以是由在序列号9所示的氨基酸序列中置换、缺失、插入和/或添加一个或多个氨基酸而得到的氨基酸序列构成、具有由结晶区域和无定形区域构成的重复区域的多肽。
来源于横丝蛋白的多肽可以包含10个以上、20个以上或30个以上的式2：REP3(2)所示的氨基酸序列单元。式2：REP3(2)所示的氨基酸序列单元的数目的上限没有特别限定，例如可以为300个以下或200个以下。
式2中，REP3是指由Gly-Pro-Gly-Gly-X构成的氨基酸序列，X是指选自由丙氨酸(Ala)、丝氨酸(Ser)、酪氨酸(Tyr)和缬氨酸(Val)组成的组中的一种氨基酸。
包含10个以上的式2：REP3(2)所示的氨基酸序列单元的多肽例如可以为具有序列号10所示的氨基酸序列的鞭毛状丝蛋白来源的重组蛋白。序列号10所示的氨基酸序列是将由NCBI数据库获得的金纺蜘蛛的鞭毛状丝蛋白的部分序列(NCBI登录号：AAF36090、GI：7106224)的与重复部分和基序相当的N末端起第1220位残基至第1659位残基为止的氨基酸序列(记作PR1序列)与由NCBI数据库获得的金纺蜘蛛的鞭毛状丝蛋白的部分序列(NCBI登录号：AAC38847、GI：2833649)的C末端起第816位残基至第907位残基为止的C末端氨基酸序列结合，并在结合后的序列的N末端附加由起始密码子、His10标签和HRV3C蛋白酶识别位点构成的氨基酸序列(序列号4)而得到的氨基酸序列。
包含10个以上的式2：REP3(2)所示的氨基酸序列单元的多肽可以是由在序列号10所示的氨基酸序列中置换、缺失、插入和/或添加一个或多个氨基酸而得到的氨基酸序列构成、具有由无定形区域构成的重复区域的多肽。
从以大肠杆菌等微生物为宿主进行重组蛋白生产的情况下的生产率的观点出发，蛋白质或多肽的分子量可以为500kDa以下、300kDa以下、200kDa以下或100kDa以下，可以为10kDa以上。
蜂丝丝心蛋白是蜂的幼虫所产生的蛋白质，可以含有选自由天然蜂丝丝心蛋白和来源于天然蜂丝丝心蛋白的多肽组成的组中的多肽。
多肽例如可以使用利用含有编码多肽的基因的表达载体进行了转化的宿主来制造。
编码多肽的基因的制造方法没有特别限制。例如，在天然型蛛丝蛋白的情况下，可以通过利用聚合酶链式反应(PCR)等由蜘蛛来源的细胞扩增出编码蛋白质的基因并进行克隆的方法或者化学合成来制造基因。基因的化学合成方法也没有特别限制，例如可以利用下述方法进行基因的化学合成：基于由NCBI的网络数据库等获得的天然型蛛丝蛋白的氨基酸序列信息，通过PCR等将利用AKTA oligopilot plus10/100(GE Health Japan株式会社)等自动合成的寡核苷酸连接。此时，为了易于进行蛋白质的纯化和确认，可以合成编码在上述氨基酸序列的N末端附加有由起始密码子和His10标签构成的氨基酸序列的氨基酸序列所构成的蛋白质的基因。
作为表达载体，可以使用能够由DNA序列表达蛋白质的质粒、噬菌体、病毒等。作为质粒型表达载体，只要能够在宿主细胞内表达目的基因、且其自身能够进行扩增即可，没有特别限定。例如在使用大肠杆菌Rosetta(DE3)作为宿主的情况下，可以使用pET22b(+)质粒载体、pCold质粒载体等。其中，从蛋白质的生产率的观点出发，可以使用pET22b(+)质粒载体。作为宿主，可以使用例如动物细胞、植物细胞、微生物等。
分散液和由其得到的蛋白质溶液可以包含蚕丝丝心蛋白和蛛丝丝心蛋白等上述结构蛋白质与其他蛋白质的组合。作为其他蛋白质，可以列举例如胶原蛋白、大豆蛋白、酪蛋白、角蛋白和乳清蛋白。通过将其他蛋白质与结构蛋白质组合使用，能够制备来源于蛋白质的物性。相对于结构蛋白质100质量份，其他蛋白质的比例例如可以为40质量份以下、30质量份以下或10质量份以下。
以分散液的质量为基准，分散液中的蛋白质的浓度可以为1质量％以上、15质量％以上、30质量％以上、40质量％以上或50质量％以上。从蛋白质溶液的制造效率的观点出发，以分散液或蛋白质溶液的质量为基准，蛋白质的浓度可以为70质量％以下、65质量％以下或60质量％以下。
分散液可以进一步含有一种或两种以上的无机盐。通过在分散液中添加无机盐，由加压产生的溶解性提高效果能够变得更为显著。无机盐可以列举例如由以下所示的路易斯酸和路易斯碱构成的无机盐。路易斯碱例如可以为含氧酸根离子(硝酸根离子、高氯酸根离子等)、金属含氧酸根离子(高锰酸根离子等)、卤化物离子、硫氰酸根离子、氰酸根离子等。路易斯酸例如可以为碱金属离子、碱土金属离子等金属离子、铵根离子等多原子离子、络离子等。作为无机盐的具体例，可以列举：氯化锂、溴化锂、碘化锂、硝酸锂、高氯酸锂和硫氰酸锂之类的锂盐；氯化钙、溴化钙、碘化钙、硝酸钙、高氯酸钙和硫氰酸钙之类的钙盐；氯化铁、溴化铁、碘化铁、硝酸铁、高氯酸铁和硫氰酸铁之类的铁盐；以及氯化铝、溴化铝、碘化铝、硝酸铝、高氯酸铝和硫氰酸铝之类的铝盐；氯化钾、溴化钾、碘化钾、硝酸钾、高氯酸钾和硫氰酸钾之类的钾盐；氯化钠、溴化钠、碘化钠、硝酸钠、高氯酸钠和硫氰酸钠之类的钠盐；氯化锌、溴化锌、碘化锌、硝酸锌、高氯酸锌和硫氰酸锌之类的锌盐；氯化镁、溴化镁、碘化镁、硝酸镁、高氯酸镁和硫氰酸镁之类的镁盐；氯化钡、溴化钡、碘化钡、硝酸钡、高氯酸钡和硫氰酸钡之类的钡盐；氯化锶、溴化锶、碘化锶、硝酸锶、高氯酸锶和硫氰酸锶之类的锶盐等。以蛋白质的总量为基准，无机盐的浓度可以为1.0质量％以上、5.0质量％以上、9.0质量％以上、15质量％以上或20.0质量％以上。另外，以蛋白质的总量为基准，无机盐的浓度可以为40质量％以下、35质量％以下或30质量％以下。
分散液和蛋白质溶液可以根据需要含有各种添加剂。作为添加剂，可以列举例如增塑剂、结晶成核剂、抗氧化剂、紫外线吸收剂、着色剂、填料和合成树脂。以蛋白质的总量为基准，添加剂的浓度可以为50质量％以下。
对含有蛋白质和极性溶剂的分散液施加规定的压力，使蛋白质溶解在极性溶剂中，由此生成蛋白质溶液。通过对分散液施加压力，即使在比较低的温度下，也能够使蛋白质溶解在极性溶剂中。因此，能够抑制蛋白质的变性、凝胶化、分解的诱发，并且能够制造高浓度的溶液。此外，也未必需要使用透析等的浓缩工序，因此，还能够提高蛋白质溶液的生产率。
对分散液施加的压力根据蛋白质和极性溶剂的种类、所期望的浓度等，以使蛋白质适当地溶解在极性溶剂中的方式进行调整。对分散液施加的压力高时，容易得到更高浓度的溶液。从该观点出发，对分散液施加的压力可以为0.05MPa以上、0.06MPa以上、0.07MPa以上、0.08MPa以上、0.1MPa以上、1.0MPa以上、5.0MPa以上或10MPa以上。对分散液施加的压力可以为300MPa以下、150MPa以下、50MPa以下或30MPa以下。
对分散液施加压力的方法没有特别限制。例如可以在收容有分散液的耐压容器内封入氮气、氩气等惰性气体或空气，通过加热等对耐压容器内的压力进行调整，由此对分散液施加压力。
可以在将分散液加热的同时对分散液施加压力。加热不限于在施加压力的期间，例如可以将分散液加热至规定温度后对分散液施加压力。从进一步抑制蛋白质的分解的观点出发，加热温度可以为150℃以下、140℃以下、135℃以下或130℃以下。在极性溶剂为水的情况下，从进一步抑制水所介导的蛋白质的分解的观点出发，加热温度优选为140℃以下。从得到更高浓度的溶液的观点出发，加热温度可以为70℃以上、90℃以上或100℃以上。可以将这些上限和下限任意组合。例如，加热温度可以为70℃以上且150℃以下、90℃以上且140℃以下、或100℃以上且130℃以下。加热温度可以为恒定的温度，也可以发生变动。
可以在将分散液搅拌的同时对分散液施加压力。搅拌不限于在施加压力的期间，也可以在施加压力的前后对分散液进行搅拌。搅拌的方法没有特别限制。例如可以利用倾斜桨叶、涡轮桨叶等对分散液进行搅拌。
加压后得到的蛋白质溶液有时会包含加压用的气体。因此，一个实施方式的蛋白质溶液的制造方法可以进一步包括从蛋白质溶液中除去气体的步骤。除去气体的方法没有特别限制，可以列举例如利用离心分离器的方法。通过将蛋白质溶液置于离心分离器中，能够除去较多量地含有气体的层。
通过一个实施方式的方法得到的蛋白质可以包含以高浓度溶解在极性溶剂中的蛋白质。蛋白质溶液中溶解在极性溶剂中的蛋白质的浓度可以与分散液中的浓度相同。具体而言，以蛋白质溶液的质量为基准，溶解在极性溶剂中的蛋白质的浓度可以为1质量％以上、15质量％以上、30质量％以上、40质量％以上或50质量％以上，可以为70质量％以下、65质量％以下或60质量％以下。
以高浓度包含蛋白质的蛋白质溶液例如可以用于以各种方法制造蛋白质的成形体。一个实施方式的制造成形体的方法可以包括从蛋白质溶液中除去极性溶剂而得到含有蛋白质的成形体的步骤。根据该方法，能够削减在制造成形体的过程中除去的溶剂的量，因此能够降低生产成本。特别是以高浓度含有蛛丝丝心蛋白的蛋白质溶液可以用于制造发挥出蛛丝丝心蛋白的特性的具有优良物性的成形体。
例如，可以使用蛋白质溶液作为胶液(ドープ溶液)，制造凝胶、膜、纤维等成形体。膜例如可以通过形成蛋白质溶液(胶液)的膜并从所形成的膜中除去极性溶剂的方法来制造。纤维例如可以通过将蛋白质溶液纺丝并从纺丝后的蛋白质溶液中除去极性溶剂的方法来制造。
也可以利用上述制造蛋白质溶液的方法对形成了成形体的蛋白质进行再生。由成形体再生蛋白质的方法的一个实施方式可以包括下述步骤：对与含有蛋白质的成形体接触的极性溶剂施加压力，得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液；和从蛋白质溶液中回收蛋白质。
再生蛋白质的方法中使用的极性溶剂和可向其中添加的材料可以与制造蛋白质溶液的上述方法同样地选择。可以将成形体浸渍在极性溶剂中，对极性溶剂施加压力。或者，可以使通过粉碎等进行了微粒化的成形体分散在极性溶剂中而制备分散液，对该分散液施加压力。压力和加热等的条件可以与制造蛋白质溶液的上述方法同样地进行设定。
从蛋白质溶液中回收蛋白质的方法没有特别限定，可以应用从蛋白质溶液中除去极性溶剂的方法、再沉淀等通常的方法。可以从包含自成形体中溶出的蛋白质的蛋白质溶液中回收粉末状等任意形态的蛋白质，也可以利用上述方法直接制造成形体。
实施例
以下，列举实施例进一步详细地对本发明进行说明。但是，本发明并不限于这些实施例。
<基因合成>
(1)ADF3Kai的基因的合成
从NCBI网络数据库获取作为十字园蛛的两种主要牵引丝蛋白之一的ADF3的部分氨基酸序列(NCBI登录号：AAC47010、GI：1263287)，委托GenScript公司合成编码在该序列的N末端附加有由起始密码子、His10标签和HRV3C蛋白酶(人鼻病毒3C蛋白酶)识别位点构成(序列号4)的氨基酸序列(序列号5)的基因。其结果，获得导入有由序列号6所示的碱基序列构成的ADF3Kai的基因的pUC57载体(具有紧挨在基因的5’末端上游的NdeI位点和紧挨在5’末端下游的Xba I位点)。然后，用Nde I和EcoR I对该基因进行限制酶处理，重组至pET22b(+)表达载体中。
(2)ADF3Kai-noNR的基因的合成
以上述中得到的导入有ADF3Kai的基因的pET22b(+)载体作为模板，通过使用PrimeStar诱变基础试剂盒(宝生物株式会社制造)的定点突变导入，使ADF3Kai的氨基酸序列(序列号5)中的第543位的氨基酸残基缬氨酸(Val)所对应的密码子GTG突变为终止密码子TAA，在pET22b(+)上构建序列号11所示的ADF3Kai-noNR的基因。关于突变的导入的准确性，通过使用3130xl基因分析仪(Applied Biosystems)的测序反应来确认。需要说明的是，ADF3Kai-noNR的氨基酸序列如序列号7所示。
<蛋白质的表达>
将上述中得到的包含ADF3Kai-noNR的基因序列的pET22b(+)表达载体转化至大肠杆菌Rosetta(DE3)中。将所得到的单菌落在含有氨苄青霉素的2mL的LB培养基中培养15小时后，将该培养液1.4mL添加至含有氨苄青霉素的140mL的LB培养基中，在37℃、200rpm的条件下培养至培养液的OD600达到3.5为止。接着，将OD600为3.5的培养液与50％葡萄糖140mL一同添加至含有氨苄青霉素的7L的2×YT培养基中，进一步培养至OD600达到4.0为止。然后，向所得到的OD600为4.0的培养液中添加异丙醇-β-硫代吡喃半乳糖苷(IPTG)，以使终浓度达到0.5mM，诱导蛋白质表达。在IPTG添加后经过2小时的时刻，将培养液离心分离，回收菌体。将由IPTG添加前和IPTG添加后的培养液制备的蛋白质溶液在聚丙烯酰胺凝胶中进行电泳，结果依赖于IPTG添加地观察到目标大小的条带，确认到目标蛋白质进行了表达。将表达ADF3Kai-noNR的蛋白质的大肠杆菌在冷冻室(-20℃)中保存。
<蛛丝多肽的制备例>
(I)在离心管(50mL)中添加表达ADF3Kai-noNR的蛋白质的大肠杆菌的菌体约4.5g和缓冲液AI(20mMTris-HCl、pH7.4)30mL，利用混合器(GE公司制造的“SI-0286”、级别10)使菌体分散后，利用离心分离机(TOMY SEIKO制造的“MX-305”)进行离心分离(10000rpm、10分钟、室温)，弃去上清。
(II)在通过离心分离得到的沉淀物(菌体)中添加缓冲液AI 30mL和0.1M的PMSF(用异丙醇溶解)0.3mL，利用上述GE公司制造的混合器(级别10)分散3分钟。然后，使用超声波破碎机(SONIC&MATERIALSINC制造的“VCX500”)将菌体破碎，进行离心分离(10000rpm、10分钟、室温)。
(III)在通过离心分离得到的沉淀物中加入缓冲液AI 30mL，利用混合器(IKA公司制造的“T18基础型ULTRA-TURRAX”、级别2)分散3分钟后，利用上述TOMY SEIKO制造的离心分离机进行离心分离(10000rpm、10分钟、室温)，除去上清。
(IV)在弃去上清后的离心管中加入7.5M的尿素缓冲液I(7.5M尿素、10mM磷酸二氢钠、20mM NaCl、1mM Tris-HCl、pH7.0)，利用上述SMT公司制造的超声波破碎机(级别7)使沉淀充分分散。然后，利用上述Tietech公司制造的振荡器(200rpm、60℃)溶解120分钟。将溶解后的蛋白质溶液利用上述TOMY SEIKO制造的离心分离机进行离心分离(11000×g、10分钟、室温)，将上清使用透析管(三光纯药株式会社、纤维素管36/32)在水中进行透析。通过离心分离回收透析后得到的白色凝集蛋白质，利用冷冻干燥机除去水分，回收冷冻干燥粉末。所得到的冷冻干燥粉末中的目标蛋白质ADF3Kai-noNR的纯化度通过使用Totallab(nonlineardynamics ltd.)对粉末的聚丙烯酰胺凝胶电泳(CBB染色)的结果进行图像分析来确认。其结果，ADF3Kai-noNR的纯化度为约85％。
(实施例1～7、比较例1～3)
<蛛丝多肽的溶解试验>
将上述“蛛丝多肽的制备例”中得到的蛛丝多肽(蛛丝丝心蛋白)的冷冻干燥粉末(分子量：60kDa)与水或二甲基亚砜(DMSO)以表1所示的比例(质量份)进行混合，制备在水中分散有蛛丝多肽的分散液。如表1所示，在一部分分散液中添加氯化锂。将所得到的分散液装入到耐压容器中，将耐压容器密闭后进行加热，以使容器内温度达到90℃。进一步向耐压容器内导入氮气，调整至容器内的压力达到1.0～10MPa的规定压力，对分散液施加压力。在经过2小时的时刻终止加压，将耐压容器冷却至25℃。然后，目视观察多肽的溶解的状态，按照下述基准对溶解性进行评价。将结果示于表1。
A：多肽全部溶解。
B：稍微观察到多肽的溶解残留。
C：多肽未溶解。
<基于SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)的分析>
对于确认到蛛丝多肽的溶解的溶液，基于使用十二烷基硫酸钠(SDS)的SDS-PAGE进行分析。利用下述条件对电泳的结果进行图像分析，由此对溶解的多肽的分解状态进行确认。将结果示于表1。表1中，“A”表示溶液中的大部分结构蛋白质未分解，“C”表示溶液中的大部分结构蛋白质分解。
[SDS-PAGE分析条件]
电源装置：PowerPactm Universal(BIO-RAD制造)
图像分析系统：Gel Doctm EZ Imager(BIO-RAD制造)
电泳用凝胶：Mini-PROTEAN(R)TGXTM Gels(BIO-RAD制造)
分子量标志物：XL-Ladder(APRO SCOEMCE制造)
固定液：NovexTM InVisionTM His-Tag In-Gel染色试剂盒(Thermo FisherScientific制造)
染色剂：OrioleTM荧光凝胶染色剂(BIO-RAD制造)
图1和图2分别为实施例4和实施例5中得到的蛋白质溶液的基于SDS-PAGE的分析结果。图中的“R”表示利用将蛛丝多肽的冷冻干燥粉末浸渍到水中后进行干燥而准备的样品进行的参照试验。由显色强度的比较求出的蛛丝多肽的残留程度在实施例4的情况下为约83％、在实施例5的情况下为约100％。
(实施例8～13、比较例4)
<蚕丝丝心蛋白的溶解试验>
将家蚕(Bombyx mori)来源的蚕丝丝心蛋白(fibroin heavy chain Fib-H[Bombyx mori]、登录号AAF76983；以下也简称为“蚕丝丝心蛋白”；理论分子量：391.5kDa)和水以表2所示的比例(质量份)进行混合，制备在水中分散有蚕丝丝心蛋白的分散液。如表2所示，在一部分分散液中添加氯化锂。将所得到的分散液装入到耐压容器中，将耐压容器密闭后进行加热，以使容器内温度达到90℃。进一步向耐压容器内导入氮气，调整至容器内的压力达到1.0～10MPa的规定压力，对分散液施加压力。在经过48小时的时刻终止加压，将耐压容器冷却至25℃。然后，目视观察蚕丝丝心蛋白的溶解状态，按照与蛛丝丝心蛋白同样的基准对溶解性进行评价。将结果示于表2。蚕丝丝心蛋白的分子量大，因此难以利用SDS-PAGE进行分析。
[表1]
[表2]
如表1和表2所示，确认了：通过对分散有结构蛋白质的分散液施加压力，可得到以高浓度溶解有结构蛋白质的蛋白质溶液。
(实施例14～19)
<蛛丝多肽的溶解试验2>
(1)编码改造丝心蛋白的核酸的合成和表达载体的构建
基于作为天然来源的蛛丝丝心蛋白的金纺蜘蛛(Nephila clavipes)(GenBank登录号：P46804.1、GI：1174415)的碱基序列和氨基酸序列，设计出具有分别由序列号12和13所示的氨基酸序列的改造丝心蛋白。
序列号12所示的氨基酸序列为如下序列：由上述天然来源的丝心蛋白起始，使其(A)n基序中的丙氨酸残基连续而成的氨基酸序列发生缺失以使丙氨酸残基连续的数目变为5个，从N末端侧向C末端侧每隔2个使(A)n基序((A)5)缺失，在C末端序列的近前侧插入1个[(A)n基序-REP]，将REP中的全部GGX置换为GQX。序列号13所示的氨基酸序列是在该序列号12所示的氨基酸序列的N末端附加有序列号14所示的氨基酸序列(标签序列和铰链序列)的氨基酸序列。
合成编码具有在序列号12所示的氨基酸序列的N末端附加有His标签序列和铰链序列(序列号14)的序列号13所示的氨基酸序列的蛋白质的核酸。该核酸中，在5’末端附加有NdeI位点，在终止密码子下游附加有EcoRI位点。将该核酸克隆至克隆载体(pUC118)中。然后，利用NdeI和EcoRI对该核酸进行限制酶处理而切出后，重组到蛋白质表达载体pET-22b(+)中得到表达载体。
(2)蛋白质的表达
利用包含编码具有序列号13所示的氨基酸序列的蛋白质的核酸的pET22b(+)表达载体对大肠杆菌BLR(DE3)进行转化。将该转化大肠杆菌在含有氨苄青霉素的2mL LB培养基中培养15小时。将该培养液添加至含有氨苄青霉素的100mL种子培养用培养基(表3)中以使OD600达到0.005，接种转化大肠杆菌。将培养液温度保持于30℃，进行烧瓶培养直至OD600达到5(约15小时)，得到种子培养液。
[表3]
表3.种子培养用培养基(培养开始时每1L)
添加氨苄青霉素以使终浓度达到100mg/L，制成种子培养用培养基。
将该种子培养液添加至添加有500ml生产培养基(表4)的发酵罐中以使OD600达到0.05，接种转化大肠杆菌。将培养液温度保持于37℃，在pH6.9下控制恒定而进行培养。另外，将培养液中的溶解氧浓度维持为溶解氧饱和浓度的20％。
[表4]
表4.生产培养基(培养开始时每1L)
在生产培养基中的葡萄糖完全被消耗后，立即以1ml/分钟的速度添加补料液(葡萄糖455g/1L、酵母提取物120g/1L)。将培养液温度保持于37℃，在pH6.9下控制恒定而进行培养。另外，将培养液中的溶解氧浓度维持为溶解氧饱和浓度的20％，进行20小时培养。然后，向培养液中添加1M的异丙醇-β-硫代吡喃半乳糖苷(IPTG)以使终浓度达到1mM，诱导表达目标蛋白质。在IPTG添加后经过20小时的时刻，将培养液离心分离，回收菌体。使用由IPTG添加前和IPTG添加后的培养液制备的菌体进行SDS-PAGE，根据依赖于IPTG添加的目标蛋白质大小的条带的出现，确认到目标蛋白质的表达。
(3)蛋白质的纯化
将添加IPTG后2小时后回收的菌体用20mM Tris-HCl缓冲液(pH7.4)进行清洗。使清洗后的菌体悬浮在包含约1mM的PMSF的20mMTris-HCl缓冲液(pH7.4)中，利用高压均质器(GEA Niro Soavi公司)将细胞破碎。将破碎的细胞离心分离，得到沉淀物。利用20mMTris-HCl缓冲液(pH7.4)清洗所得到的沉淀物，直至达到高纯度为止。将清洗后的沉淀物以达到100mg/mL的浓度的方式悬浮在8M胍缓冲液(8M胍盐酸盐、10mM磷酸二氢钠、20mMNaCl、1mMTris-HCl、pH7.0)中，在60℃下用搅拌器搅拌30分钟，使其溶解。溶解后，使用透析管(三光纯药株式会社制造的纤维素管36/32)在水中进行透析。通过离心分离回收透析后得到的白色凝集蛋白质，利用冷冻干燥机除去水分，回收冷冻干燥粉末。
所得到的冷冻干燥粉末中的目标蛋白质的纯化度通过使用Totallab(nonlineardynamics ltd.)对粉末的聚丙烯酰胺凝胶电泳的结果进行图像分析来确认。其结果，蛋白质的纯化度为约85％。
(4)溶解试验
将上述中得到的蛛丝多肽(蛛丝丝心蛋白)的冷冻干燥粉末(分子量：50kDa)与水、乙醇、丙醇、丁醇或クリンソルブP-7(含有乙醇85.5±1.0％、异丙醇小于5.0％、正丙醇9.6±0.5％和水分0.2％以下的混合溶剂、“クリンソルブ”为日本醇贩卖株式会社的注册商标)以表5所示的比例(质量份)进行混合，制备在溶液中分散有蛛丝多肽的分散液。将所得到的分散液装入到耐压容器中，密闭后间歇地进行加热以使容器内温度保持于90～100℃的规定温度。通过将容器内温度提高至90～100℃，使分散液中的溶剂蒸发，容器内的压力上升从而对分散液施加0.08～0.1MPa的压力(计算值)。开始加热后25分钟将容器内温度保持于90～100℃，然后目视观察多肽的溶解状态，按照下述基准对溶解性进行评价。将结果示于表5。
A：多肽全部溶解。
B：稍微观察到多肽的溶解残留。
C：多肽未溶解。
(5)基于SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)的分析
对于确认到蛛丝多肽溶解的溶液，基于与实施例1～7同样的条件的SDS-PAGE进行分析，对溶解的多肽的分解状态进行确认。图3为实施例14～19中得到的蛋白质溶液的基于SDS-PAGE的分析结果。将结果示于表5。表5中，“A”表示溶液中的大部分结构蛋白质未分解。在任一实施例中，均在抑制蛋白质的分解的同时得到蛋白质溶液。
[表5]
如表5所示，确认了：通过对分散有结构蛋白质的分散液施加压力，可得到以高浓度溶解有结构蛋白质的蛋白质溶液。
序列表
<110> 丝芭博株式会社（Spiber Inc.）
<120> 制造蛋白质溶液的方法（METHOD OF PRODUCING PROTEIN SOLUTION）
<130> 2015PS008WO1
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> PRT
<213> Araneus diadematus
<400> 1
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala
1 5 10 15
Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly
20 25 30
Ala Ser Ala Gln Tyr Thr Gln Met Val Gly Gln Ser Val Ala Gln Ala
35 40 45
Leu Ala
50
<210> 2
<211> 30
<212> PRT
<213> Araneus diadematus
<400> 2
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala
1 5 10 15
Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn
20 25 30
<210> 3
<211> 21
<212> PRT
<213> Araneus diadematus
<400> 3
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala
1 5 10 15
Leu Val Ser Ile Leu
20
<210> 4
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> His tag and start codon
<400> 4
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro
20
<210> 5
<211> 660
<212> PRT
<213> Artificial Sequence
<220>
<223> recombinant spider silk protein ADF3Kai
<400> 5
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln
20 25 30
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
35 40 45
Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr
50 55 60
Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln
65 70 75 80
Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
85 90 95
Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro
100 105 110
Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala
115 120 125
Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln
130 135 140
Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala
145 150 155 160
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly
165 170 175
Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala
180 185 190
Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln
195 200 205
Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
210 215 220
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly
225 230 235 240
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
245 250 255
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
260 265 270
Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
275 280 285
Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly
290 295 300
Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln
305 310 315 320
Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly
325 330 335
Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
340 345 350
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
355 360 365
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
370 375 380
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
385 390 395 400
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
405 410 415
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly
420 425 430
Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly
435 440 445
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
450 455 460
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
465 470 475 480
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly
485 490 495
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
500 505 510
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
515 520 525
Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser
530 535 540
Val Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala
545 550 555 560
Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser
565 570 575
Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu
580 585 590
Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro
595 600 605
Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val
610 615 620
Ser Ala Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn
625 630 635 640
Tyr Gly Ala Ser Ala Gln Tyr Thr Gln Met Val Gly Gln Ser Val Ala
645 650 655
Gln Ala Leu Ala
660
<210> 6
<211> 1983
<212> DNA
<213> Artificial Sequence
<220>
<223> recombinant DNA of spider silk protein gene ADF3Kai
<400> 6
atgcatcacc atcatcatca tcaccaccac cattcctcgg gctcatcctt ggaagtgtta 60
tttcaaggac cagcacgagc cggttcggga caacaagggc ctggccagca gggcccaggt 120
caacaagggc caggacagca gggtccttat gggcccggcg caagcgcagc agctgcggcc 180
gctggtggct atggtcctgg ctccggtcaa cagggccctt cgcaacaagg tcccgggcag 240
caaggtcctg gtggccaggg tccctacggg ccgggggcga gtgcggcagc agccgctgca 300
ggcggttatg gtccaggaag cggacagcaa ggtccgggag gtcaaggtcc gtatggccca 360
ggctctagcg cggctgccgc tgccgcgggt ggcaacggac cagggagcgg acaacagggc 420
gcgggacaac agggtccagg acagcaaggc ccaggggcgt cggcggctgc agcggcggcc 480
ggaggctatg gacccggctc aggacaacag ggaccgggtc aacaaggacc cggtggccaa 540
ggcccctatg gcccgggcgc cagcgcggcc gcagccgccg cgggcgggta cggccccggt 600
agcggccagg gaccaggtca gcaggggcca ggaggtcagg gcccatacgg tccgggcgca 660
tccgcggcgg cggcagcggc aggtggctac ggtcccggaa gcggccaaca ggggccaggg 720
caacaaggac caggacaaca aggtcctggg ggccaaggac cgtatggacc aggagcatca 780
gctgcagccg cggcagctgg cggttacggt ccaggctacg gccagcaggg tccgggtcag 840
cagggaccgg gaggccaggg gccttatggc cctggcgctt ccgcagccag tgccgcttct 900
ggaggatacg ggccgggaag cggtcagcaa ggccctggcc aacaaggacc tggaggccaa 960
gggccctacg gcccaggagc ctcggcagcc gcagctgccg caggtgggta tgggccaggt 1020
agcgggcaac aagggccggg tcagcaagga ccggggcaac agggacctgg gcagcaagga 1080
cccgggggtc aaggcccgta cggacctggt gcgtctgcag ctgctgctgc ggctggtgga 1140
tatggtccgg gatcggggca gcagggtccc ggtcagcagg gccctggtca gcaagggcca 1200
ggccaacagg gacccggaca acaaggcccg ggtcaacagg gtcctggaca gcaggggccg 1260
ggccaacaag gccctgggca acagggtccg gggggacagg gggcctatgg gcctggcgca 1320
tctgccgccg ctggcgcagc cggtgggtac gggcctgggt caggtcaaca ggggcctggt 1380
caacaaggcc ccgggcaaca gggccccggc cagcaaggtc cagggcagca gggcccggga 1440
cagcaagggc ctggacaaca ggggcccgga cagcagggac cttacgggcc cggtgcgagc 1500
gcagcggccg ccgccgcagg gggatatggc cccggatcgg gccagcaggg accaggccag 1560
caaggacctg gccaacaggg cccggggggt caggggccgt atggtcccgg cgctgcaagt 1620
gctgcagtgt ccgttggagg ttacggccct cagtcttcgt ctgttccggt ggcgtccgca 1680
gttgcgagta gactgtcttc acctgctgct tcatcgcgag tatcgagcgc tgtttcgtct 1740
cttgtctcgt cgggtcccac gaaacatgcc gccctttcaa atacgatttc atctgtagtg 1800
tcccaagtta gtgcaagtaa cccggggtta tccggatgcg acgttctcgt tcaggcactc 1860
ctagaagtag tatccgcgtt ggtgagcatc ttaggcagct cctcgatagg tcaaataaac 1920
tatggtgctt cagcccagta tacacagatg gtgggacaga gcgtcgcgca ggcattggct 1980
taa 1983
<210> 7
<211> 542
<212> PRT
<213> Artificial Sequence
<220>
<223> recombinant DNA of spider silk protein gene ADF3Kai
<400> 7
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Ala Arg Ala Gly Ser Gly Gln Gln
20 25 30
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
35 40 45
Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr
50 55 60
Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln
65 70 75 80
Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
85 90 95
Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro
100 105 110
Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala
115 120 125
Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln
130 135 140
Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala
145 150 155 160
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly
165 170 175
Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala
180 185 190
Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln
195 200 205
Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
210 215 220
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly
225 230 235 240
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
245 250 255
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
260 265 270
Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
275 280 285
Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly
290 295 300
Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln
305 310 315 320
Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly
325 330 335
Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
340 345 350
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
355 360 365
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
370 375 380
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
385 390 395 400
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
405 410 415
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly
420 425 430
Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly
435 440 445
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
450 455 460
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
465 470 475 480
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly
485 490 495
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
500 505 510
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
515 520 525
Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ser Ala Ala
530 535 540
<210> 8
<211> 434
<212> PRT
<213> Artificial
<220>
<223> Recombinang spider silk protein ADF4Kai
<400> 8
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Ala Gly Ser Ser Ala Ala Ala Ala
20 25 30
Ala Ala Ala Ser Gly Ser Gly Gly Tyr Gly Pro Glu Asn Gln Gly Pro
35 40 45
Ser Gly Pro Val Ala Tyr Gly Pro Gly Gly Pro Val Ser Ser Ala Ala
50 55 60
Ala Ala Ala Ala Ala Gly Ser Gly Pro Gly Gly Tyr Gly Pro Glu Asn
65 70 75 80
Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Ser
85 90 95
Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly
100 105 110
Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly Ser Gly Gly Tyr Gly
115 120 125
Pro Gly Ser Gln Gly Ala Ser Gly Pro Gly Gly Pro Gly Ala Ser Ala
130 135 140
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly
145 150 155 160
Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Ala Tyr Gly Pro Gly Gly
165 170 175
Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly
180 185 190
Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Val Tyr Gly
195 200 205
Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser
210 215 220
Gly Pro Gly Gly Tyr Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Gly
225 230 235 240
Gly Tyr Gly Pro Gly Gly Ser Gly Ser Ser Ala Ala Ala Ala Ala Ala
245 250 255
Ala Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser
260 265 270
Gly Pro Gly Gly Ser Gly Gly Tyr Gly Pro Gly Ser Gln Gly Gly Ser
275 280 285
Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro
290 295 300
Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Tyr Gln
305 310 315 320
Gly Pro Ser Gly Pro Gly Ala Tyr Gly Pro Ser Pro Ser Ala Ser Ala
325 330 335
Ser Val Ala Ala Ser Val Tyr Leu Arg Leu Gln Pro Arg Leu Glu Val
340 345 350
Ser Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Asn Gly Ala
355 360 365
Ala Val Ser Gly Ala Leu Asn Ser Leu Val Ser Gln Ile Ser Ala Ser
370 375 380
Asn Pro Gly Leu Ser Gly Cys Asp Ala Leu Val Gln Ala Leu Leu Glu
385 390 395 400
Leu Val Ser Ala Leu Val Ala Ile Leu Ser Ser Ala Ser Ile Gly Gln
405 410 415
Val Asn Val Ser Ser Val Ser Gln Ser Thr Gln Met Ile Ser Gln Ala
420 425 430
Leu Ser
<210> 9
<211> 355
<212> PRT
<213> Artificial
<220>
<223> Recombinant spider silk protein MaSp2_N
<400> 9
Met His His His His His His Ser Ser Gly Ser Ser Leu Glu Val Leu
1 5 10 15
Phe Gln Gly Pro Ala Arg Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln
20 25 30
Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
50 55 60
Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala
65 70 75 80
Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly
85 90 95
Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
100 105 110
Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly
115 120 125
Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly
130 135 140
Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr
145 150 155 160
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly
165 170 175
Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ala
180 185 190
Gly Pro Gly Gly Tyr Gly Pro Ala Gln Gln Gly Pro Ser Gly Pro Gly
195 200 205
Ile Ala Ala Ser Ala Ala Ser Ala Gly Pro Gly Gly Tyr Gly Pro Ala
210 215 220
Gln Gln Gly Pro Ala Gly Tyr Gly Pro Gly Ser Ala Val Ala Ala Ser
225 230 235 240
Ala Gly Ala Gly Ser Ala Gly Tyr Gly Pro Gly Ser Gln Ala Ser Ala
245 250 255
Ala Ala Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala Arg Val Ala Ser
260 265 270
Ala Val Ser Asn Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu
275 280 285
Ser Ser Val Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro
290 295 300
Gly Leu Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile Val
305 310 315 320
Ser Ala Cys Val Thr Ile Leu Ser Ser Ser Ser Ile Gly Gln Val Asn
325 330 335
Tyr Gly Ala Ala Ser Gln Phe Ala Gln Val Val Gly Gln Ser Val Leu
340 345 350
Ser Ala Phe
355
<210> 10
<211> 559
<212> PRT
<213> Artificial
<220>
<223> Recombinant spider silk protein Flag_92_short2
<400> 10
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Ala Gly Gly Ser Gly Pro Gly
20 25 30
Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly
35 40 45
Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ser
50 55 60
Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro
65 70 75 80
Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro Gly Gly
85 90 95
Ala Tyr Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ser Gly Gly Pro
100 105 110
Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly
115 120 125
Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro
130 135 140
Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Gly Pro Tyr
145 150 155 160
Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly
165 170 175
Pro Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly
180 185 190
Gly Pro Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Gly Gly Pro Gly
195 200 205
Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly
210 215 220
Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr
225 230 235 240
Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly
245 250 255
Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro
260 265 270
Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro Gly
275 280 285
Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly
290 295 300
Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser
305 310 315 320
Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly
325 330 335
Pro Gly Gly Phe Gly Pro Gly Gly Phe Gly Pro Gly Gly Ser Gly Pro
340 345 350
Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly
355 360 365
Gly Val Gly Pro Gly Gly Phe Gly Pro Gly Gly Ala Gly Pro Gly Gly
370 375 380
Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala
385 390 395 400
Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly
405 410 415
Pro Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ser
420 425 430
Gly Gly Ala Gly Gly Ser Gly Gly Thr Thr Ile Ile Glu Asp Leu Asp
435 440 445
Ile Thr Ile Asp Gly Ala Asp Gly Pro Ile Thr Ile Ser Glu Glu Leu
450 455 460
Thr Ile Ser Ala Tyr Tyr Pro Ser Ser Arg Val Pro Asp Met Val Asn
465 470 475 480
Gly Ile Met Ser Ala Met Gln Gly Ser Gly Phe Asn Tyr Gln Met Phe
485 490 495
Gly Asn Met Leu Ser Gln Tyr Ser Ser Gly Ser Gly Thr Cys Asn Pro
500 505 510
Asn Asn Val Asn Val Leu Met Asp Ala Leu Leu Ala Ala Leu His Cys
515 520 525
Leu Ser Asn His Gly Ser Ser Ser Phe Ala Pro Ser Pro Thr Pro Ala
530 535 540
Ala Met Ser Ala Tyr Ser Asn Ser Val Gly Arg Met Phe Ala Tyr
545 550 555
<210> 11
<211> 1629
<212> DNA
<213> Artificial Sequence
<220>
<223> recombinant spider silk protein gene ADF3Kai-noNR
<400> 11
atgcatcacc atcatcatca tcaccaccac cattcctcgg gctcatcctt ggaagtgtta 60
tttcaaggac cagcacgagc cggttcggga caacaagggc ctggccagca gggcccaggt 120
caacaagggc caggacagca gggtccttat gggcccggcg caagcgcagc agctgcggcc 180
gctggtggct atggtcctgg ctccggtcaa cagggccctt cgcaacaagg tcccgggcag 240
caaggtcctg gtggccaggg tccctacggg ccgggggcga gtgcggcagc agccgctgca 300
ggcggttatg gtccaggaag cggacagcaa ggtccgggag gtcaaggtcc gtatggccca 360
ggctctagcg cggctgccgc tgccgcgggt ggcaacggac cagggagcgg acaacagggc 420
gcgggacaac agggtccagg acagcaaggc ccaggggcgt cggcggctgc agcggcggcc 480
ggaggctatg gacccggctc aggacaacag ggaccgggtc aacaaggacc cggtggccaa 540
ggcccctatg gcccgggcgc cagcgcggcc gcagccgccg cgggcgggta cggccccggt 600
agcggccagg gaccaggtca gcaggggcca ggaggtcagg gcccatacgg tccgggcgca 660
tccgcggcgg cggcagcggc aggtggctac ggtcccggaa gcggccaaca ggggccaggg 720
caacaaggac caggacaaca aggtcctggg ggccaaggac cgtatggacc aggagcatca 780
gctgcagccg cggcagctgg cggttacggt ccaggctacg gccagcaggg tccgggtcag 840
cagggaccgg gaggccaggg gccttatggc cctggcgctt ccgcagccag tgccgcttct 900
ggaggatacg ggccgggaag cggtcagcaa ggccctggcc aacaaggacc tggaggccaa 960
gggccctacg gcccaggagc ctcggcagcc gcagctgccg caggtgggta tgggccaggt 1020
agcgggcaac aagggccggg tcagcaagga ccggggcaac agggacctgg gcagcaagga 1080
cccgggggtc aaggcccgta cggacctggt gcgtctgcag ctgctgctgc ggctggtgga 1140
tatggtccgg gatcggggca gcagggtccc ggtcagcagg gccctggtca gcaagggcca 1200
ggccaacagg gacccggaca acaaggcccg ggtcaacagg gtcctggaca gcaggggccg 1260
ggccaacaag gccctgggca acagggtccg gggggacagg gggcctatgg gcctggcgca 1320
tctgccgccg ctggcgcagc cggtgggtac gggcctgggt caggtcaaca ggggcctggt 1380
caacaaggcc ccgggcaaca gggccccggc cagcaaggtc cagggcagca gggcccggga 1440
cagcaagggc ctggacaaca ggggcccgga cagcagggac cttacgggcc cggtgcgagc 1500
gcagcggccg ccgccgcagg gggatatggc cccggatcgg gccagcaggg accaggccag 1560
caaggacctg gccaacaggg cccggggggt caggggccgt atggtcccgg cgctgcaagt 1620
gctgcataa 1629
<210> 12
<211> 590
<212> PRT
<213> Artificial Sequence
<220>
<223> Met_CRY1_L_A5_giza_QQQ (GizaQ)
<400> 12
Met Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
1 5 10 15
Ala Ala Ala Gly Gln Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly
20 25 30
Gln Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
35 40 45
Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro
50 55 60
Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly
65 70 75 80
Ser Gly Gln Gln Gly Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln
85 90 95
Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala
100 105 110
Gly Gln Tyr Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala
115 120 125
Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr
130 135 140
Gly Pro Gly Ala Ser Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly
145 150 155 160
Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro
165 170 175
Gly Gln Tyr Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr
180 185 190
Gly Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly
195 200 205
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala
210 215 220
Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser
225 230 235 240
Ala Ala Ala Ala Ala Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly
245 250 255
Pro Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln
260 265 270
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala
275 280 285
Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
290 295 300
Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly
305 310 315 320
Pro Gly Ser Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser
325 330 335
Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro
340 345 350
Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln
355 360 365
Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly
370 375 380
Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly
385 390 395 400
Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala
405 410 415
Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr
420 425 430
Gly Pro Gly Gln Ser Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro
435 440 445
Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro
450 455 460
Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala
465 470 475 480
Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly
485 490 495
Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser
500 505 510
Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly
515 520 525
Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly
530 535 540
Ser Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser
545 550 555 560
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala
565 570 575
Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Ser
580 585 590
<210> 13
<211> 601
<212> PRT
<213> Artificial Sequence
<220>
<223> CRY1_L_A5_giza_QQQ (GizaQ)
<400> 13
Met His His His His His His Ser Ser Gly Ser Ser Gly Pro Gly Gln
1 5 10 15
Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln
20 25 30
Asn Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Ser Gly Gln Tyr
35 40 45
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ser Ser Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro
65 70 75 80
Ser Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly Gln Gln Gly
85 90 95
Pro Gly Ala Ser Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln
100 105 110
Gln Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser
115 120 125
Gly Pro Gly Gln Gln Gly Pro Tyr Gly Ser Ala Ala Ala Ala Ala Gly
130 135 140
Pro Gly Ser Gly Gln Tyr Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser
145 150 155 160
Gly Pro Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala
165 170 175
Ala Ala Ala Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro
180 185 190
Tyr Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly
195 200 205
Gln Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly
210 215 220
Pro Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro
225 230 235 240
Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala
245 250 255
Gly Gln Tyr Gly Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly
260 265 270
Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln Tyr Gly Pro Gly Gln
275 280 285
Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln
290 295 300
Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr
305 310 315 320
Gly Pro Gly Gln Gln Gly Pro Gly Gln Tyr Gly Pro Gly Ser Ser Gly
325 330 335
Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala
340 345 350
Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Gln
355 360 365
Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln
370 375 380
Gly Pro Tyr Gly Pro Gly Ala Ser Gly Pro Gly Gln Gln Gly Pro Tyr
385 390 395 400
Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly Gln Tyr Gly
405 410 415
Pro Gly Gln Gln Gly Pro Ser Ala Ser Ala Ala Ala Ala Ala Gly Gln
420 425 430
Tyr Gly Ser Gly Pro Gly Gln Tyr Gly Pro Tyr Gly Pro Gly Gln Ser
435 440 445
Gly Pro Gly Ser Gly Gln Gln Gly Gln Gly Pro Tyr Gly Pro Gly Ala
450 455 460
Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Pro Gly Gln Gln Gly Pro
465 470 475 480
Tyr Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala Gly Pro Gly Ser Gly
485 490 495
Gln Tyr Gly Pro Gly Ala Ser Gly Gln Asn Gly Pro Gly Ser Gly Gln
500 505 510
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gln Ser Ala Ala Ala Ala Ala
515 520 525
Gly Gln Tyr Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly
530 535 540
Ala Ser Ala Ala Ala Ala Ala Gly Gln Tyr Gly Ser Gly Pro Gly Gln
545 550 555 560
Gln Gly Pro Tyr Gly Pro Gly Gln Ser Gly Ser Gly Gln Gln Gly Pro
565 570 575
Gly Gln Gln Gly Pro Tyr Ala Ser Ala Ala Ala Ala Ala Gly Pro Gly
580 585 590
Ser Gly Gln Gln Gly Pro Gly Ala Ser
595 600
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> HisTag
<400> 14
Met His His His His His His Ser Ser Gly Ser Ser
1 5 10
Claims (16)
1.一种制造蛋白质溶液的方法，其包括下述步骤：对含有蛋白质和分散有该蛋白质的极性溶剂的分散液施加压力，得到含有所述蛋白质和溶解有该蛋白质的所述极性溶剂的蛋白质溶液。
2.如权利要求1所述的方法，其中，所述压力为0.05MPa以上。
3.如权利要求1或2所述的方法，其中，所述蛋白质为结构蛋白质。
4.如权利要求3所述的方法，其中，所述结构蛋白质为选自由丝心蛋白和角蛋白组成的组中的一种以上的结构蛋白质。
5.如权利要求3所述的方法，其中，所述结构蛋白质为选自由蚕丝丝心蛋白、蛛丝丝心蛋白和蜂丝丝心蛋白组成的组中的一种以上的丝心蛋白。
6.如权利要求1～5中任一项所述的方法，其中，在将所述分散液加热的同时对所述分散液施加所述压力。
7.如权利要求1～6中任一项所述的方法，其中，所述极性溶剂包含选自由水、醇、二甲基亚砜、二甲基甲酰胺和六氟丙酮组成的组中的一种以上的溶剂。
8.一种蛋白质溶液，其为含有蛛丝丝心蛋白和溶解有该蛛丝丝心蛋白的极性溶剂的蛋白质溶液，
以该蛋白质溶液的质量为基准，溶解在所述极性溶剂中的所述蛛丝丝心蛋白的浓度为1～70质量％。
9.一种制造成形体的方法，其包括下述步骤：
利用权利要求1～7中任一项所述的方法得到含有蛋白质和溶解有该蛋白质的极性溶剂的蛋白质溶液；和
从所述蛋白质溶液中除去所述极性溶剂，得到含有所述蛋白质的成形体。
10.一种由成形体再生蛋白质的方法，其包括下述步骤：
对与含有蛋白质的成形体接触的极性溶剂施加压力，得到含有所述蛋白质和溶解有该蛋白质的所述极性溶剂的蛋白质溶液；和
从所述蛋白质溶液中回收所述蛋白质。
11.如权利要求10所述的方法，其中，所述压力为0.05MPa以上。
12.如权利要求10或11所述的方法，其中，所述蛋白质为结构蛋白质。
13.如权利要求12所述的方法，其中，所述结构蛋白质为选自由丝心蛋白和角蛋白组成的组中的一种以上的结构蛋白质。
14.如权利要求12所述的方法，其中，所述结构蛋白质为选自由蚕丝丝心蛋白、蛛丝丝心蛋白和蜂丝丝心蛋白组成的组中的一种以上的丝心蛋白。
15.如权利要求10～14中任一项所述的方法，其中，在将所述极性溶剂加热的同时对所述极性溶剂施加所述压力。
16.如权利要求10～15中任一项所述的方法，其中，所述极性溶剂包含选自由水、醇、二甲基亚砜、二甲基甲酰胺和六氟丙酮组成的组中的一种以上的溶剂。
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| PCT/JP2016/085409 WO2017094722A1 (ja) | 2015-12-01 | 2016-11-29 | タンパク質溶液を製造する方法 |
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| EP (1) | EP3385305A4 (zh) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109778343A (zh) * | 2019-01-14 | 2019-05-21 | 合肥必更赢科技有限公司 | 一种透气抗菌共混蛋白纤维及其织物的制备方法和应用 |
| CN114222751A (zh) * | 2019-08-22 | 2022-03-22 | 保尔特纺织品公司 | 用于改善蜘蛛丝蛋白的提取的方法 |
| CN114269398A (zh) * | 2019-09-16 | 2022-04-01 | 保尔特纺织品公司 | 通过高剪切溶解来分离蜘蛛丝蛋白的方法 |
| CN114502794A (zh) * | 2019-09-30 | 2022-05-13 | 丝芭博株式会社 | 蛋白质成型体的制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7495707B2 (ja) * | 2016-12-27 | 2024-06-05 | Ｓｐｉｂｅｒ株式会社 | タンパク質の回収方法 |
| WO2018235958A1 (ja) * | 2017-06-23 | 2018-12-27 | Ｓｐｉｂｅｒ株式会社 | タンパク質の精製方法、タンパク質溶液の製造方法、及びタンパク質成形体の製造方法 |
| JP7323766B2 (ja) * | 2018-02-28 | 2023-08-09 | セントラル硝子株式会社 | タンパク質溶液の調製方法およびそれを用いた分子量測定方法 |
| JP7284384B2 (ja) * | 2018-03-26 | 2023-05-31 | セントラル硝子株式会社 | シルクフィブロイン溶液の製造方法およびそれから形成される成形体の製造方法 |
| WO2020054873A1 (ja) * | 2018-09-14 | 2020-03-19 | Ｓｐｉｂｅｒ株式会社 | フィブロイン溶液を製造する方法、及びタンパク質成形体を製造する方法 |
| JP7458619B2 (ja) | 2019-01-31 | 2024-04-01 | Ｓｐｉｂｅｒ株式会社 | フィブロイン繊維の製造方法及びフィブロイン溶液 |
| WO2020241769A1 (ja) * | 2019-05-29 | 2020-12-03 | Ｓｐｉｂｅｒ株式会社 | 構造タンパク質微小体及びその製造方法、ナノファイバーの製造方法、並びにタンパク質構造体の製造方法 |
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- 2016-11-29 CN CN201680070357.4A patent/CN108368271A/zh active Pending
- 2016-11-29 US US15/780,477 patent/US20180355120A1/en not_active Abandoned
- 2016-11-29 WO PCT/JP2016/085409 patent/WO2017094722A1/ja active Application Filing
- 2016-11-29 EP EP16870659.6A patent/EP3385305A4/en not_active Withdrawn
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| US20040038333A1 (en) * | 2000-10-31 | 2004-02-26 | Randolph Theodore W. | Methods for protein refolding |
| US20140193466A1 (en) * | 2011-06-07 | 2014-07-10 | Cornell University | Silk compositions and methods of using same |
| WO2013065651A1 (ja) * | 2011-11-02 | 2013-05-10 | スパイバー株式会社 | タンパク質溶液及びこれを用いたタンパク質繊維の製造方法 |
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| CN109778343A (zh) * | 2019-01-14 | 2019-05-21 | 合肥必更赢科技有限公司 | 一种透气抗菌共混蛋白纤维及其织物的制备方法和应用 |
| CN114222751A (zh) * | 2019-08-22 | 2022-03-22 | 保尔特纺织品公司 | 用于改善蜘蛛丝蛋白的提取的方法 |
| CN114269398A (zh) * | 2019-09-16 | 2022-04-01 | 保尔特纺织品公司 | 通过高剪切溶解来分离蜘蛛丝蛋白的方法 |
| CN114269398B (zh) * | 2019-09-16 | 2024-06-25 | 保尔特纺织品公司 | 通过高剪切溶解来分离蜘蛛丝蛋白的方法 |
| CN114502794A (zh) * | 2019-09-30 | 2022-05-13 | 丝芭博株式会社 | 蛋白质成型体的制备方法 |
Also Published As
| Publication number | Publication date |
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| EP3385305A1 (en) | 2018-10-10 |
| US20180355120A1 (en) | 2018-12-13 |
| EP3385305A4 (en) | 2019-11-13 |
| WO2017094722A1 (ja) | 2017-06-08 |
| JPWO2017094722A1 (ja) | 2018-10-11 |
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