CN108456246A - 重组蜘蛛丝蛋白及其制备方法和应用 - Google Patents

重组蜘蛛丝蛋白及其制备方法和应用 Download PDF

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CN108456246A
CN108456246A CN201810093672.XA CN201810093672A CN108456246A CN 108456246 A CN108456246 A CN 108456246A CN 201810093672 A CN201810093672 A CN 201810093672A CN 108456246 A CN108456246 A CN 108456246A
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马永
赵百学
王安良
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Abstract

本发明公开了一种重组蜘蛛丝蛋白，及其编码基因、表达、纯化方法以及所述重组蛛丝蛋白在成丝、成膜等方面的应用。本发明的重组蜘蛛丝蛋白，其由N末端非重复区域(N‑NRT)、核心重复区域(REP)、C末端非重复区域(C‑NRT)组成，其中核心重复区域由1‑3个ADF3Rep和/或1‑6个REPA组成。本发明设计的一系列新型蜘蛛丝蛋白序列，它集高强度、高弹性等蜘蛛丝主要特性于一体。并且，通过基因工程技术将这些序列分别导入到大肠杆菌并建立相应的高密度发酵工艺成功实现了蛛丝蛋白的高效原核表达，发酵产量可达到0.5g/L以上。此外，发明人根据蜘蛛丝蛋白的性质开发了非层析的蛋白纯化手段，纯化后重组蜘蛛丝蛋白纯度符合成丝或成膜的要求。

Description

重组蜘蛛丝蛋白及其制备方法和应用
技术领域
本发明涉及重组蜘蛛丝蛋白，及其编码基因、表达、纯化方法以及所述重组蛛丝蛋白在成丝、成膜等方面的应用。
背景技术
蜘蛛丝既有钢般的坚硬,又有橡胶般的弹性。其突出的性能主要表现在:高强度、高弹性、高断裂功,可以说是迄今为止最强韧的材料,被誉为“生物钢”。随着对蜘蛛丝的研究深入,发现蜘蛛丝还具有生物可降解性、超收缩性、耐高温、耐低温及与生物组织的相容性等特性。由于蜘蛛丝这些独特的物理和生物学特性,它在医学、材料、军事和纺织等方面都有着广泛的应用前景。瑞典科学家Jan Johansson利用无刺激行化学物质制造出的人工蜘蛛丝具有良好的生物相容性，可应用于脊髓修复或帮助干细胞生长来修复损伤的心脏组织等再生医学研究中，亦可用于自身防护用具等纺织工业应用中(Nature ChemicalBiology,DOI:10.1038/nchembio.2269)；
鉴于蜘蛛丝蛋白巨大的潜在应用性,国内外学者加强对蜘蛛丝的研究,希望蜘蛛丝能够像蚕丝那样大规模地应用于实际。由于蜘蛛无法驯养和天然蜘蛛丝产量少等原因,惟有通过基因工程手段才能大量获取蜘蛛丝，满足蜘蛛丝潜在的应用需求。目前研究人员可以借助大肠杆菌，酵母，昆虫细胞，哺乳动物细胞等表达系统进行蜘蛛丝蛋白的生物工程制备。其中，大肠杆菌表达系具有生长快、产量高、生产规模大、成本低、培养条件简单、遗传背景清楚等多重优点，目前被广泛应用于蜘蛛丝蛋白的重组表达研究中。但是，天然蜘蛛丝分子量非常大(>300KDa)，而大肠杆菌表达的外源蛋白大于60KDa时就会出现表达量降低的现象，天然的蜘蛛丝蛋白分子量过大，几乎不可能表达成功。例如：Fahnestock等人就报道了在大肠杆菌中表达了蜘蛛牵引丝蛋白，但发现大于3kb的基因表达效率降低，并有基因删节表达现象。
研究表明，蜘蛛丝蛋白有四种通用的氨基酸模块:(l)GPGXX；(2)GGx；(3)An/(GA)n；(4)间隔区(spacer)。GPGXX(主要是GPGGx和GPGQQ两种形式)是一个五聚肽,主要存在于牵引丝蛋白和鞭毛丝蛋白,形成β成转角结构,一系列的β系转角串联折叠在一起,类似弹簧结构。而这个“弹簧”结构与蜘蛛丝蛋白的弹性相关，蛛丝蛋白弹性的大小与该模块的含量正相关。GGX形成310-螺旋结构,即每三个氨基酸组成一个螺旋。An/(GA)n形成β成片层结构,结构片层之间的分子作用决定了蜘蛛丝蛋白的强度,也是导致蜘蛛丝不溶于水的原因之一。间隔区是一个保守区域,用来间隔富含甘氨酸的区域。总的来说：(l)蜘蛛丝的化学本质是蛋白质；(2)蜘蛛丝蛋白氨基酸序列高度重复性；(3)蜘蛛丝蛋白中含量较多的氨基酸主要为丙氨酸、甘氨酸和丝氨酸；(4)富含聚丙氨酸,形成β成片层刚性结构；(5)脯氨酸形成的β氨转角结构为蜘蛛丝提供良好的弹性。
研究人员可以依据上述蜘蛛丝蛋白特性设计重组的蜘蛛丝蛋白，重组蜘蛛丝蛋白一级结构含有上述决定蜘蛛丝蛋白力学性能的模块，并且可以根据不同材料的研发用途进行个性化的序列设计，这样通过人工的序列设计不仅控制了蛛丝蛋白的力学性能而且可以控制蛋白分子量以便实现蛋白在大肠杆菌表达系统中的高效表达。
发明内容
基于目前重组蜘蛛丝蛋白的结构与功能研究和现已公布的蜘蛛丝蛋白序列，本申请发明人设计了衍生自天然蜘蛛丝的蛋白序列，同时将编码重组蜘蛛丝蛋白序列的基因导入到宿主细胞中进行蛋白表达，并建立高效的重组蜘蛛丝蛋白表达和制备工艺。在此基础上研究了重组蜘蛛丝蛋白序列在静电纺丝和湿法纺丝中的应用。
本发明第一目的在于提供重组蜘蛛丝蛋白，其由N末端非重复区域(N-NRT)、核心重复区域(REP)、C末端非重复区域(C-NRT)组成，其中核心重复区域由1-3个ADF3Rep和/或1-6个REPA组成，单个ADF3Rep氨基酸序列如SEQ ID NO：1所示，单个REPA氨基酸序列如SEQID NO：2所示。
ADF3Rep和REPA可以以自身串联体或者两者的杂交串联体作为核心重复区。以自身串联体作为核心重复区时，优选含有1-3个ADF3Rep作为核心重复区或者含有1-6个REPA作为核心重复区。
以两者杂交串联体作为核心重复区时，优选1个ADF3Rep与1-6个REPA的串联体作为核心重复区。更优选地将ADF3Rep定位在整个融合体的靠近N端位置，而REPA的串联体则在靠近的C端位置。
上述N末端非重复区域(N-NRT)，氨基酸序列如SEQ ID NO：3所示，C末端非重复区域(C-NRT)，氨基酸序列如SEQ ID NO：4所示。
本发明第二目的在于提供编码上述单个ADF3Rep的核苷酸序列，如SEQ ID NO：5所示。
提供编码上述单个REPA的核苷酸序列，如SEQ ID NO：6所示。
本发明所涉及的部分序列如表1所示：
表1
所述核苷酸序列均经过针对大肠杆菌表达系统进行密码子优化，从而以提高在大肠杆菌中的表达量。所述核苷酸序列可由PCR的方法制备，也可由基因合成的方法制备。另外为了便于后续的基因操作，编码ADF3Rep及REPA的核苷酸的5’端和3’端分别引入了同尾酶酶切位点BamHI和BglII。
本发明的第三目的在于提供包含上述核苷酸序列的表达载体，该载体只要与大肠杆菌表达系统相匹配即可，优选具有拷贝数高、表达效率高的载体，如pET21b、pET28a、pBV220等。
本发明的第四目的在于提供包含上述表达载体的大肠杆菌菌株，优选BL21(DE3)、BL21(DE3)plys、Rosetta(DE3)等。
本发明的第五目的在于提供重组蜘蛛丝蛋白高密度发酵方法，具体包括：发酵种子活化、发酵一级种子液制备、发酵二级种子制备、高密度发酵，所述高密度发酵具体包括如下步骤：
将制备好的二级种子液按照5-15％的接种量接入到灭菌后的发酵罐中；
设定发酵温度为37℃、pH为6.8-7.2之间、DO设定在30-40％之间；
发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升的时候表明分批培养基中葡萄糖耗尽，开始进行补料培养(补料培养基的流加速度维持在8-12g/L/h)；
待菌体生长至0D600至L/-55之间，降低发酵温度至25-30℃，待温度稳定后向发酵罐中加入终浓度为0.5-1.0mM的IPTG进行诱导表达；诱导表达6-10h结束培养。
所述分批发酵培养基成分包括：一水合柠檬酸0.5-3g/L，磷酸二氢钾8-15g/L，磷酸氢二铵3-7g/L，葡萄糖10-20g/L，七水合硫酸镁2-4g/L。在发酵接种之前还要向分批发酵培养基中加入1/1000(V/V)的微量元素母液用于维持菌体正常生长和代谢。
所述补料培养基主要成分包括：甘油1024g/L，七水合硫酸镁8-12g/L，酵母粉50-100g/L，在补料开始之前还要在补料培养基中加入1/1000(V/V)的上述微量元素母液用于维持菌体正常生长和代谢。
所述微量元素母液成分包括：FeSO4.7H2O 10g/L、ZnSO4.7H2O 2.25g/L、CuSO4.5H2O15g/L、MnSO4.5H2O 5g/L、CaCl2.7H2O 1g/L、CoCl.6H2O 1g/L、Na2MoO4.2H2O 1.125g/L、H3BO30.0625g/L、HCl 41.75mL、Biotin 0.5g/L。
所述发酵种子活化方法具体为：将构建好的重组蜘蛛丝蛋白大肠杆菌菌株冻存管使用三区划线的方法接种到LB固体培养基中，37℃过夜培养进行活化。
所述发酵一级种子液制备方法具体为：从固体培养基上挑取形状饱满、大小适中的单菌落接种到LB液体培养基中，37℃，220rpm摇床培养8-10h，此为一级种子液。
所述发酵二级种子制备方法具体为：将一级种子液按照1％的接种量转接到新鲜的LB液体培养基中，37℃，220rpm摇床培养至OD600≈3-5之间，此为二级种子液制备。
上述任何用于菌体培养的器皿和培养基在使用前均应进行过滤或者湿热灭菌，所述任何培养基在灭菌结束冷却后使用前均应加入终浓度为50任何/ml的卡那霉素以保证纯种培养。
本发明的第六目的在于提供重组蜘蛛丝蛋白纯化方法，包括如下步骤：
1、取一定量的高密度发酵菌体使用bufferA(20mM Tris、100mM氯化钠、1mM EDTA、1.5M脲，pH8.0)重悬至100g/L，将上述菌体悬浮液使用高压均质机进行菌体破碎。
2、将上述菌体裂解液使用低温离心机10000g离心30min，丢弃上清，收集蜘蛛丝蛋白包涵体。将包涵体使用bufferB(20mM Tris、100mM氯化钠、1-3M脲、0.1-0.3％十二烷基肌氨酸钠，pH8.0)重悬至20g/L(湿重计)，室温搅拌2h，再次离心收集沉淀，重复一次。
3、离心收集第2步中包涵体，使用bufferC(20mM Tris、100mM氯化钠、1-3M脲、0.5-2％TritonX-100，pH8.0)，重悬至20g/L(湿重计)，室温搅拌2h，再次离心收集沉淀，重复一次。
4、离心收集第3步中包涵体，使用含有1-10mM氯化锂的DMSO，重悬至20g/L(湿重计)，40-80℃搅拌2h，再次离心收集沉淀，重复一次。
5、将第4步中包涵体沉淀使用单蒸水重悬后离心丢弃上清以去除残留的DMSO，然后将包涵体使用bufferD(20mM Tris、8-10M脲、10mM DTT，pH8.0)重悬至20g/L(湿重计)，室温搅拌溶解5h以上，彻底离心后去除沉淀得到重组蜘蛛丝蛋白包涵体变性液。
6、取一定量的蜘蛛丝蛋白变性液加入终浓度为3-8％硫酸铵，混匀后，加入等体积的叔丁醇，静置1h，4000g离心5min，此时液体分为两相，上相为叔丁醇有机溶剂，下相含有目的蛋白的变性液，两相界面为蛋白沉淀。
7、收集第6步的下相，加入3M硫酸铵，室温搅拌30min，离心收集蛋白沉淀，将沉淀使用去离子水重悬，然后离心收集蛋白沉淀，反复操作数次直至离心后的上清电导不高于0.1mS/cm。最终离心收集得到的蛋白可用冷冻干燥、热风干燥、喷雾干燥等方法进行干燥处理即可得到纯化后的蜘蛛丝蛋白。
所述重组蜘蛛丝蛋白纺丝液所用溶剂可以选择任何可以将其溶解的溶液体系，作为例子可以选择六氟异丙醇、六氟丙酮、甲酸、溴化锂、氯化锂、二甲基亚砜、二甲酰胺、水等作为溶剂或者制备成一定比例的混合溶剂。所述纺丝液制备，若用于静电纺丝，那么所使用的溶剂系统优选使用六氟异丙醇、六氟丙酮、甲酸，等低沸点、易挥发性溶剂或者复合溶剂，作为复合溶剂可选六氟异丙醇/甲酸＝100/1-1/100。若用于湿法纺丝，所述溶剂系统可以选用二甲基亚砜、二甲酰胺等极性非质子溶剂。所述纺丝液中含有蜘蛛丝蛋白的含量优选3-30％,更优选5-20％，最优选8-15％。为了赋予所制材料特殊的性能，所述纺丝液除了重组蜘蛛丝蛋白以外还可以加入其他聚合物以开发复合材料，如PEO、聚乳酸等。聚合物的添加量与重组蜘蛛丝蛋白的添加量应控制在1/5-1/200之间。
采用静电纺丝时，具体方法为：将上述纺丝液加入静电纺丝进样系统，控制电场强度为13-20KV，进样速度为0.5-2mL/h，接收距离为：11-15cm。
采用湿法纺丝时，具体方法是：
1、将湿法纺丝纺丝液加入注射器中，使用定速注射泵进行挤出，挤出速度为0.5-2ml/h；
2、纺丝液挤出后在凝固浴中进行脱溶剂，形成白色未经拉伸的纤维；纤维在凝固浴中经过的距离为20cm以上。所述凝固浴优选使用甲醇、乙醇、异丙醇或者与水的混合系统；
3、所述纤维经过凝固脱溶剂处理以后，在空气浴(40-80℃)中拉伸1.5-2倍。
本申请发明人根据己报道的蜘蛛丝蛋白的氨基酸序列，结合蜘蛛丝蛋白的模块结构特性和功能的关系，优化设计了一系列新型蜘蛛丝蛋白序列，它集高强度、高弹性等蜘蛛丝主要特性于一体。并且，通过基因工程技术将这些序列分别导入到大肠杆菌并建立相应的高密度发酵工艺成功实现了蛛丝蛋白的高效原核表达，发酵产量可达到0.5g/L以上。此外，在生物工程领域，一般蛋白的精制需要借助层析的手段，如离子交换或者分子筛层析等。但是重组蜘蛛丝蛋白的应用规模很大(Kg级以上)，层析手段用于重组蜘蛛丝蛋白的纯化显然成本过高。鉴于此，发明人根据蜘蛛丝蛋白的性质开发了非层析的蛋白纯化手段，经过纯化后重组蜘蛛丝蛋白纯度(电泳纯)可以达到70％左右，需要明确的是，其残余的杂质对于后续的纺丝等操作无明显影响。
附图说明
图1重组蜘蛛丝蛋白大肠杆菌菌株构建原理
图2-1 BL21(DE3)-REPA1表达鉴定，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-2 BL21(DE3)-REPA2表达鉴定，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-3 BL21(DE3)-REPA4表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-4 BL21(DE3)-REPA6表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-5 BL21(DE3)-REPA8表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-6 BL21(DE3)-ADF3Rep1表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-7 BL21(DE3)-ADF3Rep2表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-8 BL21(DE3)-ADF3Rep3表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图2-9 BL21(DE3)-ADF3Rep1-REPA1、BL21(DE3)-ADF3Rep1-REPA2表达鉴定图，其中M：蛋白分子量标准；lane1-2：ADF3Rep1-REPA1超声裂解物沉淀；lane3-4：ADF3Rep1-REPA2超声裂解物沉淀；
图2-10 BL21(DE3)-ADF3Rep1-REPA4表达鉴定图，其中M：蛋白分子量标准；lane1：超声裂解物上清；lane2：超声裂解物沉淀；
图3-1 BL21(DE3)-ADF3Rep1发酵表达分析图，其中M：蛋白分子量标准；lane1：BL21(DE3)-ADF3Rep1超声裂解物沉淀
图3-2 BL21(DE3)-ADF3Rep2发酵表达分析图，其中M：蛋白分子量标准；lane1：BL21(DE3)-ADF3Rep2超声裂解物沉淀
图3-3 BL21(DE3)-ADF3Rep1-REPA4发酵表达分析图，其中M：蛋白分子量标准；lane1：BL21(DE3)-ADF3Rep1-REPA4超声裂解物沉淀
图4-1 ADF3Rep1-REPA4纯化样品分析图，其中M：蛋白分子量标准；lane1：ADF3Rep1-REPA4纯化样品
图4-2 ADF3Rep2纯化样品分析图，其中M：蛋白分子量标准；lane1：ADF3Rep2纯化样品
图5 ADF3Rep1-REPA4静电纺丝制备的纤维膜
图6 ADF3Rep1-REPA4静电纺丝制备的纤维膜扫描电镜观察
图7 ADF3Rep1-REPA4湿法纺丝凝固浴中形成的纤维
图8 ADF3Rep1-REPA4湿法纺丝拉伸后形成的纤维丝(400倍显微镜观察)
图9 ADF3Rep1-REPA4湿法纺丝制备的纤维丝扫描电镜观察(放大2000倍)
具体实施方式
以下实施例是针对本专利进行的进一步说明，需要说明的是实施例所述内容只是用于解释本专利并不构成对本专利发明内容的限制。
实施例1重组蜘蛛丝蛋白大肠杆菌表达菌株构建
本专利涉及到多个重组蜘蛛丝蛋白菌株的构建。重复模块的拼接(重复模块是指REPA、ADF3Rep)通过同尾酶的分子操作实现，方案流程如图1所示。下面以ADF3Rep与4个REPA串联体形成的杂交串联体(ADF3Rep1-REPA4)为例进行说明，具体如下：
1、参考GenBank中已经公布的十字园蛛大壶腺拖丝蛋白ADF3mRNA序列(GenBank基因登录号为：U47855.1)，从中截取核心重复区域并委托金斯瑞生物科技有限公司对该基因序列针对大肠杆菌表达系统进行密码子优化得到单个ADF3Rep的核苷酸序列，如SEQ IDNO：5所示，为了方便后续的分子操作，在5’端和3’端非别引入了酶切位点BamH I和BglI I。ADF3Rep被克隆至pUC57质粒，形成重组质粒pUC57-ADF3Rep。
2、单个REPA的核苷酸序列如SEQ ID NO：6所示，由金斯瑞生物科技有限公司合成由REPA核苷酸序列，并在5’端和3’端非别引入了酶切位点BamH I和BglI I。记为REPA。REPA被克隆至pUC57质粒，形成重组质粒pUC57REPA。
3、将N-NRT与C-NRT基因序列(如SEQ ID NO：18和如SEQ ID NO：19所示)委托金斯瑞生物科技有限公司对该基因序列针对大肠杆菌表达系统进行密码子优化并由金斯瑞生物科技有限公司合成，其中N-NRT的5’端含有Xba I酶切位点，3’端含有BamH I酶切位点(该酶切位点作为N-NRT重组核苷酸的一部分，不改变N-NRT的氨基酸序列)；C-NRT的5’端含有Bgl II酶切位点(该酶切位点作为N-NRT重组核苷酸的一部分，不改变N-NRT的氨基酸序列)，3’端含有Xho I酶切位点；N-NRT与C-NRT对应的核苷酸以融合的形式合成，即N-NRT-C-NRT(5’→3’)。N-NRT-C-NRT通过酶切、酶连的方式插入pET28aplus载体(将pET28a原始载体去除原有Bgl II改造而成)，记为pET28aplus-N-NRT-C-NRT。
4、pUC57-ADF3Rep使用BamH I和Bgl II酶切获取带有相应粘性末端的ADF3Rep片段。将pET28aplus-N-NRT-C-NRT使用BamH I和Bgl II酶切获取带有相应粘性末端的载体骨架片段。将酶切获取的粘性片段酶连后转化到大肠杆菌BL21(DE3)中形成重组表达菌株。
5、从步骤4中的转化平板中挑取单克隆进行培养，提取质粒后使用BamHI和BglII酶切进行鉴定，根据同尾酶的切割原理分析鉴定获得的阳性克隆，阳性克隆即为BL21(DE3)-ADF3Rep1，对应的阳性质粒为pET28aplus-N-NRT-ADF3Rep1-C-NRT。
6、pET28aplus-N-NRT-ADF3Rep1-C-NRT使用BglII酶切获取带有相应粘性末端的载体骨架片段，pUC57-ADF3Rep使用BamHI和BglII酶切获取带有相应粘性末端的ADF3Rep片段。将2种粘性片段连后转化到大肠杆菌BL21(DE3)中形成重组表达菌株，鉴定后得到BL21(DE3)-ADF3Rep2(含有两个ADF3Rep的串联体)，对应的阳性质粒为pET28aplus-N-NRT-ADF3Rep2-C-NRT。同理，对pET28aplus-N-NRT-ADF3Rep2-C-NRT进行重复操作即可获得含有n个ADF3Rep的串联体重组大肠杆菌菌株，即为BL21(DE3)-ADF3Repn。
7、同理，将pUC57-REPA和pET28aplus-N-NRT-ADF3Rep-C-NRT分别按照步骤4-6中的操作，即可获得BL21(DE3)-REPA1(含有1个REPA)、BL21(DE3)-REPA2(含有2个REPA的串联体)、BL21(DE3)-REPA3(含有3个REPA的串联体)、BL21(DE3)-REPA4(含有4个REPA的串联体)、BL21(DE3)-REPAn(含有n个REPA的串联体)。
8、将pET28aplus-N-NRT-ADF3Rep1-C-NRT质粒使用Bgl II酶切获取带有相应粘性末端的载体骨架片段，将pET28aplus-N-NRT-REPA4-C-NRT使用BamH I和Bgl II酶切获取带有相应粘性末端的REPA4片段。将带有粘性末端的REPA4与带有粘性末端的pET28aplus-N-NRT-ADF3Rep1-C-NRT酶连后转化到大肠杆菌BL21(DE3)中形成重组表达菌株。从转化平板中挑取单克隆进行培养，提取质粒后使用BamH I和Bgl II酶切进行鉴定，根据同尾酶的切割原理分析鉴定获得的阳性克隆，阳性克隆即为BL21(DE3)-ADF3Rep1-REPA4(核心重复区域为1个ADF3Rep加4个REPA串联体)，对应的阳性质粒为pET28aplus-N-NRT-ADF3Rep1-REPA4-C-NRT。
9、将上述构建的重组蜘蛛丝样蛋白菌株接种至新鲜的LB液体培养基(含有50培g/mL的卡那霉素)中，37那，220rpm摇床培养10-12h。
10、将步骤9中的种子液转接到新鲜的LB液体培养基(含有50培g/mL的卡那霉素)中，37那，220rpm摇床培养至OD600为0.6-1.0，加入终浓度为1mM IPTG进行诱导表达，诱导表达4-6h结束培养，收集菌体。
11、将步骤10中的菌体使用PBS重悬至50g/L(湿重计)，使用探针式超声波破碎仪冰浴超声破碎20min，功率：200W，工作3s，间隔4s。离心收集超声裂解上清和沉淀，超声沉淀即为包涵体蛋白。将包涵体蛋白使用10M脲的水溶液彻底变性，至均匀透亮。然后将包涵体变性液和超声裂解上清使用SDS-PAGE分析，分析结果如图2所示。
实施例2重组蜘蛛丝蛋白大肠杆菌菌株高密度发酵
蜘蛛丝蛋白含有大量的重复氨基酸序列，这一特性导致其在大肠杆菌中重组表达时会遇到tRNA池枯竭、基因复制障碍、蛋白翻译截断等问题，最终导致表达量不高。有研究表明，随着人工蛛丝蛋白的分子量增大(大于60KDa)，蛋白的表达难度会极大上升，并且可能出现表达失败的问题。而天然蛛丝蛋白的分子量可以达到200KDa以上，虽然没有直接研究表明蛛丝蛋白的分子量和其力学性能呈正比例关系，但仍需要使人工设计的蛛丝蛋白在分子量上与天然蛛丝蛋白尽可能相近。所以，重组蜘蛛丝蛋白就要在分子量和大肠杆菌表达量上做好平衡，分子量过大则表达量降低或者表达失败，分子量过小则对天然序列的仿生程度低。
本部分阐述了发明人关于重组蜘蛛丝蛋白的高密度发酵工艺，以BL21(DE3)-ADF3Rep1-REPA4的发酵工艺进行说明(ADF3Rep1-REPA4分子量在实施例1中的蛛丝蛋白构建体中分子量较大，达到100KDa以上，对于工艺产量的评价具有一定代表性)。
1、将构建好的重组重组蜘蛛丝蛋白大肠杆菌菌株冻存管使用三区划线的方法接种到LB固体培养基中，37培过夜培养进行活化；
2、从固体培养基上挑取形状饱满、大小适中的单菌落接种到LB液体培养基中，37培，220rpm摇床培养8-10h，此为一级种子液。
3、将一级种子液按照1％的接种量转接到新鲜的LB液体培养基中，37培，220rpm摇床培养至OD600≈3-5之间，此为二级种子液制备。
4、将制备好的二级种子液接种到发酵培养基中开始补料-分批高密度发酵培养，该高密度发酵工艺可以实现重组蜘蛛丝蛋白的大量表达。所述分批发酵培养基成分包括：一水合柠檬酸1.7g/L，磷酸二氢钾12g/L，磷酸氢二铵4g/L，葡萄糖20g/L，七水合硫酸镁2g/L，在发酵接种之前还要向分批发酵培养基中加入1/1000(V/V)的微量元素母液用于维持菌体正常生长和代谢，所述微量元素母液成分包括：FeSO4.7H2O 10g/L、ZnSO4.7H2O 2.25g/L、CuSO4.5H2O 15g/L、MnSO4.5H2O 5g/L、CaCl2.7H2O 1g/L、CoCl.6H2O 1g/L、Na2MoO4.2H2O1.125g/L、H3BO3 0.0625g/L、HCl 41.75ml、Biotin 0.5g/L。所述补料培养基主要成分包括：甘油1024g/L，七水合硫酸镁12g/L，在补料开始之前还要补料培养基中加入1/1000(V/V)的上述微量元素母液用于维持菌体正常生长和代谢。所述的高密度发酵方法步骤包括：将制备好的二级种子液按照5-15％的接种量接入到灭菌后的发酵罐中→设定发酵温度为37发、pH为7.0之间、DO设定在30-40％之间(通过转速/空气/高纯氧的关联进行控制)→发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升的时候表明分批培养基中葡萄糖耗尽，开始进行补料培养(补料培养基的流加速度维持在12g/L/h(补料培养基质量/发酵液初始体积/时间))→待菌体生长至0D600≈00-55之间，降低发酵温度至30℃，待温度稳定后向发酵罐中加入终浓度为0.5-1.0mM的IPTG进行诱导表达→诱导表达6-10h结束培养。发酵结束后，按照实施例1中的方法进行表达分析，分析结果如图3所示。
上述任何用于菌体培养的器皿和培养基在使用前均应进行过滤或者湿热灭菌，所述任何培养基在灭菌结束冷却后使用前均应加入终浓度为50任何/mL的卡那霉素以保证纯种培养。重组蜘蛛丝蛋白含有大量的氨基酸重复序列，实施例1获得的部分菌株采用该高密度发酵工艺产量可达到0.5g/L，实现了重组蜘蛛丝蛋白的高效生产，为重组蜘蛛丝的大规模应用奠定了基础。
实施例3重组蜘蛛丝蛋白纯化制备
此部分内容，阐述了蜘蛛丝蛋白的非层析的蛋白纯化手段，经过纯化后重组蜘蛛丝蛋白纯度(电泳纯)可以达到70％左右，需要明确的是，其残余的杂质对于后续的纺丝等操作无明显影响。使用实施例2制备的发酵菌体经过相同的纯化工艺进行操作可以达到相似的纯化效果，具体纯化操作如下：
1、取一定量的高密度发酵菌体使用bufferA(20mM Tris 100mM氯化钠1mM EDTA1.5M脲，pH8.0)重悬至100g/L，将上述菌体悬浮液使用高压均质机进行菌体破碎。
2、将上述菌体裂解液使用低温离心机10000g离心30min，丢弃上清，收集蜘蛛丝蛋白包涵体。将包涵体使用bufferB(20mM Tris 100mM氯化钠2M脲0.1％十二烷基肌氨酸钠，pH8.0)重悬至20g/L(湿重计)，室温搅拌2h，再次离心收集沉淀，重复一次。
3、离心收集第2步中包涵体，使用bufferC(20mM Tris 100mM氯化钠2M脲1％TritonX-100，pH8.0)，重悬至20g/L(湿重计)，室温搅拌2h，再次离心收集沉淀，重复一次。
4、离心收集第3步中包涵体，使用含有1-10mM氯化锂的DMSO，重悬至20g/L(湿重计)，40-80℃搅拌2h，再次离心收集沉淀，重复一次。
5、将第4步中包涵体沉淀使用单蒸水重悬后离心丢弃上清以去除残留的DMSO，然后将包涵体使用bufferD(20mM Tris 8M脲10mM DTT，pH8.0)重悬至20g/L(湿重计)，室温搅拌溶解5h以上，彻底离心后去除沉淀得到重组蜘蛛丝蛋白包涵体变性液。
6、取一定量的蜘蛛丝蛋白变性液加入终浓度为5％硫酸铵，混匀后，加入等体积的叔丁醇，静置1h，4000g离心5min，此时液体分为两相，上相为叔丁醇有机溶剂，下相含有目的蛋白的变性液，两相界面为蛋白沉淀。
7、收集第6步的下相，加入3M硫酸铵，室温搅拌30min，离心收集蛋白沉淀，将沉淀使用去离子水重悬，然后离心收集蛋白沉淀，反复操作数次直至离心后的上清电导不高于0.1mS/cm。取部分沉淀蛋白使用实施例1中的方法进行SDS-PAGE分析，分析结果如图4所示，电泳检测结果使用灰度分析得到电泳纯约为70％。最终离心收集得到的蛋白可用冷冻干燥、热风干燥、喷雾干燥等方法进行干燥处理即可得到纯化后的蜘蛛丝蛋白，干燥后的蛛丝蛋白称重后，根据纯度和发酵体积核算重组蜘蛛丝蛋白ADF3Rep1-REPA4的发酵产量为0.53g/L。
申请人采用实施例2、实施例3的制备发酵、纯化工艺分别对分别对部分构建的重组蜘蛛丝蛋白大肠杆菌菌株进行发酵和纯化制备，通过结果核算，不同重组蜘蛛丝蛋白菌株蛋白表达量如表2所示。
表2
实施例4重组蜘蛛丝蛋白静电纺丝纺丝液制备和静电纺丝
1、取干燥后的重组蜘蛛丝蛋白样品使用HFIP进行溶解，投料量为10％(wt％)，室温搅拌溶解48h以上，至溶液均匀透亮。
2、取上述纺丝液2mL加入到注射器中，选择注射针为27G针头，设定注射速率为1mL/h，设定电压为16KV，设定接收距离为15cm，接受基板为锡箔纸。
3、将上述静电纺丝制备的纤维膜从锡箔纸上剥离，可以轻易地实现纤维膜的剥离获得较为完成的膜，如图5所示。所获得的膜可以进行进一步的处理。
4、将上述静电纺丝制备的纤维膜，使用扫描电镜进行检测分析，分析结果如图6所示。
由此可见，采用本专利的蜘蛛丝蛋白可满足静电纺丝对蛋白的要求，电镜显示，获得的蛋白膜均匀完整、结构整齐。
实施例5重组蜘蛛丝蛋白湿法纺丝纺丝液制备和湿法纺丝
1、取干燥后的蛋白样品使用DMSO(含有1-2M溴化锂)进行溶解，投料量为15％(wt％)，60℃搅拌溶解12h，然后快速升温至120℃继续搅拌2h，至溶液均匀透亮。
2、取上述纺丝液2mL加入到注射器中，选择注射针为27G针头，设定注射速率为0.2mL/h，将纺丝液注入凝固浴(100％乙醇)，观察纤维形成情况，如图7所示。
3、将上述形成的纤维取出在空气浴中(60的)进一步拉伸2倍。显微镜下观察所成丝纤维，如图8所示，然后将纤维丝在拉力下拉断，在扫描电镜下观察拉断面，如图9所示。
由此可见，采用本专利的蜘蛛丝蛋白可满足湿法纺丝对蛋白的要求，电镜显示，获得的蛋白丝结构均一，具有一定的韧性和强度。
序列表
<110> 常州京森生物医药研究所有限公司
<120> 重组蜘蛛丝蛋白及其制备方法和应用
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Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
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Met His His His His His His His His His His Ser Ser Gly Ser Ser
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1025 1030 1035 1040
Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
1045 1050 1055
Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser Val Gly Gly Tyr Gly
1060 1065 1070
Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala Val Ala Ser Arg Leu
1075 1080 1085
Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu
1090 1095 1100
Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu Ser Asn Thr Ile Ser
1105 1110 1115 1120
Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys
1125 1130 1135
Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser
1140 1145 1150
Ile Leu
<210> 9
<211> 1677
<212> PRT
<213> Araneus diadematus
<400> 9
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 Ser Arg Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln
545 550 555 560
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly
565 570 575
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly
580 585 590
Gln Gln Gly Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly
595 600 605
Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly
610 615 620
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
625 630 635 640
Tyr Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly
645 650 655
Pro Gly Ser Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln
660 665 670
Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro
675 680 685
Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly
690 695 700
Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr
705 710 715 720
Gly Pro Gly Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln
725 730 735
Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly
740 745 750
Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
755 760 765
Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
770 775 780
Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly
785 790 795 800
Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala
805 810 815
Ser Ala Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln
820 825 830
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro
835 840 845
Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser
850 855 860
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
865 870 875 880
Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
885 890 895
Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
900 905 910
Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
915 920 925
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
930 935 940
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly
945 950 955 960
Pro Gly Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly
965 970 975
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
980 985 990
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
995 1000 1005
Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
1010 1015 1020
Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
1025 1030 1035 1040
Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
1045 1050 1055
Tyr Gly Pro Gly Ala Ala Ser Ala Ala Val Ser Val Ser Arg Ala Arg
1060 1065 1070
Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
1075 1080 1085
Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
1090 1095 1100
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Ser
1105 1110 1115 1120
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
1125 1130 1135
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
1140 1145 1150
Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser
1155 1160 1165
Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser Gly Gln
1170 1175 1180
Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Ala Ser
1185 1190 1195 1200
Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln
1205 1210 1215
Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly
1220 1225 1230
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly
1235 1240 1245
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro
1250 1255 1260
Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser
1265 1270 1275 1280
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
1285 1290 1295
Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala
1300 1305 1310
Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly Gln Gln Gly
1315 1320 1325
Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ser Ala
1330 1335 1340
Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln
1345 1350 1355 1360
Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
1365 1370 1375
Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro
1380 1385 1390
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
1395 1400 1405
Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala
1410 1415 1420
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly
1425 1430 1435 1440
Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
1445 1450 1455
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
1460 1465 1470
Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala Ser Ala
1475 1480 1485
Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
1490 1495 1500
Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro
1505 1510 1515 1520
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
1525 1530 1535
Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala
1540 1545 1550
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly
1555 1560 1565
Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala
1570 1575 1580
Ala Ser Ala Ala Val Ser Val Gly Gly Tyr Gly Pro Gln Ser Ser Ser
1585 1590 1595 1600
Val Pro Val Ala Ser Ala Val Ala Ser Arg Leu Ser Ser Pro Ala Ala
1605 1610 1615
Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro
1620 1625 1630
Thr Lys His Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln
1635 1640 1645
Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln
1650 1655 1660
Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu
1665 1670 1675
<210> 10
<211> 277
<212> PRT
<213> Araneus diadematus
<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 Pro Gly Gly Tyr Arg Pro Gly
20 25 30
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
65 70 75 80
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
85 90 95
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
100 105 110
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
115 120 125
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
130 135 140
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
145 150 155 160
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
165 170 175
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala Val Ala
195 200 205
Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val
210 215 220
Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu Ser Asn
225 230 235 240
Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu
245 250 255
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala
260 265 270
Leu Val Ser Ile Leu
275
<210> 11
<211> 444
<212> PRT
<213> Araneus diadematus
<400> 11
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 Pro Gly Gly Tyr Arg Pro Gly
20 25 30
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
65 70 75 80
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
85 90 95
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
100 105 110
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
115 120 125
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
130 135 140
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
145 150 155 160
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
165 170 175
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
195 200 205
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro
210 215 220
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
225 230 235 240
Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln
245 250 255
Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
260 265 270
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala
275 280 285
Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
290 295 300
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala
305 310 315 320
Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
325 330 335
Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
340 345 350
Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val
355 360 365
Pro Val Ala Ser Ala Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser
370 375 380
Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr
385 390 395 400
Lys His Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val
405 410 415
Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala
420 425 430
Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu
435 440
<210> 12
<211> 778
<212> PRT
<213> Araneus diadematus
<400> 12
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 Pro Gly Gly Tyr Arg Pro Gly
20 25 30
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
65 70 75 80
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
85 90 95
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
100 105 110
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
115 120 125
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
130 135 140
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
145 150 155 160
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
165 170 175
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
195 200 205
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro
210 215 220
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
225 230 235 240
Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln
245 250 255
Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
260 265 270
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala
275 280 285
Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
290 295 300
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala
305 310 315 320
Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
325 330 335
Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
340 345 350
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln
355 360 365
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
370 375 380
Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
385 390 395 400
Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala
405 410 415
Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln
420 425 430
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
435 440 445
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln
450 455 460
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro
465 470 475 480
Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly
485 490 495
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro
500 505 510
Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
515 520 525
Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
530 535 540
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
545 550 555 560
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala
565 570 575
Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu
580 585 590
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
595 600 605
Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala
610 615 620
Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala
645 650 655
Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
660 665 670
Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala
675 680 685
Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val
690 695 700
Ala Ser Ala Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg
705 710 715 720
Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His
725 730 735
Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala
740 745 750
Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu
755 760 765
Glu Val Val Ser Ala Leu Val Ser Ile Leu
770 775
<210> 13
<211> 1112
<212> PRT
<213> Araneus diadematus
<400> 13
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 Pro Gly Gly Tyr Arg Pro Gly
20 25 30
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
65 70 75 80
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
85 90 95
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
100 105 110
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
115 120 125
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
130 135 140
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
145 150 155 160
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
165 170 175
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
195 200 205
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro
210 215 220
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
225 230 235 240
Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln
245 250 255
Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
260 265 270
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala
275 280 285
Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
290 295 300
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala
305 310 315 320
Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
325 330 335
Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
340 345 350
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln
355 360 365
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
370 375 380
Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
385 390 395 400
Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala
405 410 415
Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln
420 425 430
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
435 440 445
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln
450 455 460
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro
465 470 475 480
Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly
485 490 495
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro
500 505 510
Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
515 520 525
Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
530 535 540
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
545 550 555 560
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala
565 570 575
Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu
580 585 590
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
595 600 605
Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala
610 615 620
Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala
645 650 655
Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
660 665 670
Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala
675 680 685
Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro
690 695 700
Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro
705 710 715 720
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
725 730 735
Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala
740 745 750
Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly
755 760 765
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro
770 775 780
Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly
785 790 795 800
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
805 810 815
Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly
820 825 830
Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly
835 840 845
Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr
850 855 860
Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala
865 870 875 880
Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
885 890 895
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser
900 905 910
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly
915 920 925
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
930 935 940
Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala
945 950 955 960
Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
965 970 975
Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly
980 985 990
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro
995 1000 1005
Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala
1010 1015 1020
Ala Ala Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser
1025 1030 1035 1040
Ala Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser
1045 1050 1055
Ser Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala
1060 1065 1070
Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn
1075 1080 1085
Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val
1090 1095 1100
Val Ser Ala Leu Val Ser Ile Leu
1105 1110
<210> 14
<211> 1446
<212> PRT
<213> Araneus diadematus
<400> 14
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 Pro Gly Gly Tyr Arg Pro Gly
20 25 30
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
35 40 45
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
65 70 75 80
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
85 90 95
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
100 105 110
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
115 120 125
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
130 135 140
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
145 150 155 160
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
165 170 175
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
195 200 205
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro
210 215 220
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
225 230 235 240
Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln
245 250 255
Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
260 265 270
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala
275 280 285
Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
290 295 300
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala
305 310 315 320
Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
325 330 335
Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
340 345 350
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln
355 360 365
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
370 375 380
Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly
385 390 395 400
Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala
405 410 415
Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln
420 425 430
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
435 440 445
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln
450 455 460
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro
465 470 475 480
Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly
485 490 495
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro
500 505 510
Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
515 520 525
Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala
530 535 540
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
545 550 555 560
Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala
565 570 575
Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu
580 585 590
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
595 600 605
Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala
610 615 620
Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala
645 650 655
Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr
660 665 670
Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala
675 680 685
Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro
690 695 700
Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro
705 710 715 720
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
725 730 735
Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala
740 745 750
Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly
755 760 765
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro
770 775 780
Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly
785 790 795 800
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
805 810 815
Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly
820 825 830
Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly
835 840 845
Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr
850 855 860
Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala
865 870 875 880
Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
885 890 895
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser
900 905 910
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly
915 920 925
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
930 935 940
Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala
945 950 955 960
Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
965 970 975
Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly
980 985 990
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro
995 1000 1005
Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala
1010 1015 1020
Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly
1025 1030 1035 1040
Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
1045 1050 1055
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
1060 1065 1070
Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro
1075 1080 1085
Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
1090 1095 1100
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser
1105 1110 1115 1120
Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly
1125 1130 1135
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser
1140 1145 1150
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln
1155 1160 1165
Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly
1170 1175 1180
Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro
1185 1190 1195 1200
Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala
1205 1210 1215
Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
1220 1225 1230
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala
1235 1240 1245
Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly
1250 1255 1260
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
1265 1270 1275 1280
Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly
1285 1290 1295
Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
1300 1305 1310
Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly
1315 1320 1325
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln
1330 1335 1340
Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala
1345 1350 1355 1360
Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala Val
1365 1370 1375
Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala
1380 1385 1390
Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu Ser
1395 1400 1405
Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly
1410 1415 1420
Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser
1425 1430 1435 1440
Ala Leu Val Ser Ile Leu
1445
<210> 15
<211> 800
<212> PRT
<213> Araneus diadematus
<400> 15
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 Ser Arg Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser
545 550 555 560
Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
565 570 575
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln
580 585 590
Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly
595 600 605
Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
610 615 620
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro
645 650 655
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala
660 665 670
Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
675 680 685
Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro
690 695 700
Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gln
705 710 715 720
Ser Ser Ser Val Pro Val Ala Ser Ala Val Ala Ser Arg Leu Ser Ser
725 730 735
Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser
740 745 750
Ser Gly Pro Thr Lys His Ala Ala Leu Ser Asn Thr Ile Ser Ser Val
755 760 765
Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val
770 775 780
Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu
785 790 795 800
<210> 16
<211> 967
<212> PRT
<213> Araneus diadematus
<400> 16
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 Ser Arg Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser
545 550 555 560
Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
565 570 575
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln
580 585 590
Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly
595 600 605
Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
610 615 620
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro
645 650 655
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala
660 665 670
Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
675 680 685
Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro
690 695 700
Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg
705 710 715 720
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala
725 730 735
Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
740 745 750
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala
755 760 765
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr
770 775 780
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
785 790 795 800
Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala
805 810 815
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
820 825 830
Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
835 840 845
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly
850 855 860
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
865 870 875 880
Ala Gly Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala
885 890 895
Val Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser
900 905 910
Ala Val Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu
915 920 925
Ser Asn Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro
930 935 940
Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val
945 950 955 960
Ser Ala Leu Val Ser Ile Leu
965
<210> 17
<211> 1301
<212> PRT
<213> Araneus diadematus
<400> 17
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 Ser Arg Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser
545 550 555 560
Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
565 570 575
Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln
580 585 590
Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly
595 600 605
Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
610 615 620
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
625 630 635 640
Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro
645 650 655
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala
660 665 670
Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln
675 680 685
Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro
690 695 700
Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg
705 710 715 720
Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala
725 730 735
Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro
740 745 750
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala
755 760 765
Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr
770 775 780
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
785 790 795 800
Pro Gly Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala
805 810 815
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
820 825 830
Pro Ser Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
835 840 845
Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly
850 855 860
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
865 870 875 880
Ala Gly Pro Gly Gly Tyr Arg Pro Gly Gln Gln Gly Pro Ser Gly Pro
885 890 895
Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr
900 905 910
Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
915 920 925
Pro Ser Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
930 935 940
Gln Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
945 950 955 960
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala
965 970 975
Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro Gly Gly
980 985 990
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ser Ala
995 1000 1005
Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
1010 1015 1020
Pro Gly Gly Tyr Ala Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser
1025 1030 1035 1040
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Tyr Arg Pro Gly
1045 1050 1055
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
1060 1065 1070
Ala Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
1075 1080 1085
Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Ala Gly Ser Ala Ala Ala
1090 1095 1100
Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Leu Gly Gly Tyr Gly Pro
1105 1110 1115 1120
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly
1125 1130 1135
Gly Tyr Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro
1140 1145 1150
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Ser
1155 1160 1165
Gly Pro Gly Ser Ala Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly
1170 1175 1180
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln
1185 1190 1195 1200
Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
1205 1210 1215
Gly Tyr Gly Pro Gln Ser Ser Ser Val Pro Val Ala Ser Ala Val Ala
1220 1225 1230
Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val
1235 1240 1245
Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu Ser Asn
1250 1255 1260
Thr Ile Ser Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu
1265 1270 1275 1280
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala
1285 1290 1295
Leu Val Ser Ile Leu
1300
<210> 18
<211> 72
<212> DNA
<213> Araneus diadematus
<400> 18
atgcatcacc atcaccatca ccatcaccat cactcttccg gttcatcgtt agaagttttg 60
tttcaaggcc ct 72
<210> 19
<211> 345
<212> DNA
<213> Araneus diadematus
<400> 19
ggagggtatg gtccccagag tagctctgtc ccagtagctt ccgccgtggc atcacgtctt 60
tcgagtccgg cggctagctc tcgcgtttcc tcagccgtct cgagtctcgt aagctctggc 120
cctactaaac atgcagcgct atccaatacc atttcatcgg tggttagtca agtcagcgct 180
tctaaccccg gactgtccgg gtgtgatgta ttagtgcagg ccttgcttga ggttgtctca 240
gcactcgtat cgatcctagg tagtagctct ataggccaaa ttaattacgg agcgtccgct 300
cagtatacac aaatggtggg gcagtcagtt gcccaagcac tggcg 345

Claims (10)

1.一种重组蜘蛛丝蛋白，其氨基酸序列由N末端非重复区域(N-NRT)、核心重复区域(REP)、C末端非重复区域(C-NRT)组成，其中核心重复区域由1-3个ADF3Rep和/或1-6个REPA组成，单个ADF3Rep氨基酸序列如SEQ ID NO：1所示，单个REPA氨基酸序列如SEQ ID NO：2所示；所述N末端非重复区域(N-NRT)，氨基酸序列如SEQ ID NO：3所示，C末端非重复区域(C-NRT)，氨基酸序列如SEQ ID NO：4所示。
2.根据权利要求1所述的重组蜘蛛丝蛋白，其特征在于：重组蜘蛛丝蛋白的氨基酸序列由1个ADF3Rep与1-6个REPA的串联体作为核心重复区。
3.根据权利要求2所述的重组蜘蛛丝蛋白，其特征在于：ADF3Rep在整个融合体的靠近N端位置，REPA的串联体在靠近的C端位置。
4.根据权利要求1所述的重组蜘蛛丝蛋白，其特征在于：单个ADF3Rep的核苷酸序列，如SEQ ID NO：5所示。
5.根据权利要求1所述的重组蜘蛛丝蛋白，其特征在于：单个REPA的核苷酸序列，如SEQID NO：6所示。
6.一种包含如权利要求4和/或权利要求5所述的核苷酸序列的载体。
7.一种包含如权利要求6所述的载体的大肠杆菌菌株。
8.一种表达重组蜘蛛丝蛋白的方法，其特征在于包括如下步骤：
(1)将如权利要求7所述的大肠杆菌菌落接入发酵罐中，设定发酵温度为37℃、pH为6.8-7.2之间、DO设定在30-40％之间；
(2)发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升时，开始进行补料培养，补料培养基的流加速度维持在8-12g/L/h；
(3)待菌体生长至0D600≈45-55之间，降低发酵温度至25-30℃，待温度稳定后向发酵罐中加入终浓度为0.5-1.0mM的IPTG进行诱导表达；诱导表达6-10h结束培养。
9.一种重组蜘蛛丝蛋白的纯化方法，其特征在于包括如下步骤：
(1)取表达重组蜘蛛丝蛋白的大肠杆菌，经菌体破碎、收集包涵体及包涵体变性；
(2)取上述变性液加入硫酸铵和叔丁醇后，分为水相和有机相两相；
(3)取水相含有目的蛋白的变性液再加入硫酸铵，离心收集蛋白沉淀，将沉淀使用去离子水重悬，然后离心收集蛋白沉淀，反复操作数次直至离心后的上清电导不高于0.1mS/cm，最终离心收集得到的蛋白。
10.如权利要求1至5任一项所述的重组蜘蛛丝蛋白在制作蛋白丝、蛋白膜中的应用。
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