CN111073864A - 提高苹果酸产量的新突变蛋白 - Google Patents

提高苹果酸产量的新突变蛋白 Download PDF

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CN111073864A
CN111073864A CN201811223531.1A CN201811223531A CN111073864A CN 111073864 A CN111073864 A CN 111073864A CN 201811223531 A CN201811223531 A CN 201811223531A CN 111073864 A CN111073864 A CN 111073864A
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田朝光
赵军旗
李金根
张路
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

本发明提供了一类提高苹果酸产量的新突变蛋白。具体地，本发明提供了一类新的丙酮酸羧化酶突变蛋白和苹果酸转运蛋白突变蛋白或其组合，以及其制备方法以及在提高苹果酸产量中的应用。

Description

提高苹果酸产量的新突变蛋白
技术领域
本发明属于生物技术领域，具体地，本发明涉及苹果酸合成途径中的丙酮酸羧化酶以及苹果酸转运蛋白的新的突变蛋白及其应用。
背景技术
苹果酸(2-羟基-1，4-丁二酸)是一种重要的平台化合物，被广泛应用于药物、化妆品、食品及饮料行业。以往的合成主要是采用化学的方法，然而该过程产生的主要是D、L-苹果酸的混合物，很难分离。
苹果酸合成目前报道的主要有5条途径，分别是TCA循环，还原型TCA(或者反向TCA)rTCA,环形乙醛酸循环和非环形乙醛酸循环，及依赖于苹果酸酶反向催化丙酮酸直接合成苹果酸。其中以rTCA循环和苹果酸酶途径最为有效，其葡萄糖对苹果酸的理论转换率为2，然而苹果酸酶主要催化苹果酸分解为丙酮酸，因此，目前最常用的方法是rTCA途径来合成苹果酸。
从葡萄糖出发经过糖酵解转化为丙酮酸，丙酮酸经过丙酮酸羧化酶转变为草酰乙酸，草酰乙酸经过苹果脱氢酶转化为苹果酸，苹果酸经过转运蛋白运出细胞。该过程中每一步的转化效率，副反应分流，催化每一步反应的辅酶和还原力及细胞能量状态都会对苹果酸合成造成影响。
苹果酸合成中有三个关键的指标，分别是产量，生产能力和转化率。为了构建高效的苹果酸生产菌株，人们在多个物种中构建了苹果酸的合成途径，如酿酒酵母(Saccharomyces cerevisiae)、毕赤酵母(Pichia pastoris)、大肠杆菌(Escherichiacoli)、曲霉属(Aspergillus spp.)、光滑球拟酵母(Torulopsis glabrata)、稗草黑粉病菌(Ustilago trichophora)、枯草芽孢杆菌(Bacillus subtilis)和嗜热子囊菌(Thermobifida fusca)等(Dai et al.,Current advance in biological production ofmalic acid using wild type and metabolic engineered strains.BioresourTechnol.2018,258:345-353.Liu et al.,Biological production of L-malate:recentadvances and future prospects.World J Microbiol Biotechnol.2017,34(1):6.)。然而这些工作主要是在不同物种中尝试构建苹果酸合成途径及进行部分优化，如选用不同强度的启动子表达苹果酸合成中的关键酶来提升苹果酸合成能力，然而这需要一个多轮组合过程且过量表达合成关键酶也会对细胞造成额外的负担。
因此，本领域迫切需要开发一种能够直接高效获得纯的苹果酸的方法。
发明内容
本发明提供了苹果酸合成途径中的丙酮酸羧化酶以及苹果酸转运蛋白的新的突变蛋白及其应用。
本发明第一个方面，提供了一种丙酮酸羧化酶突变蛋白，所述丙酮酸羧化酶突变蛋白基于SEQ ID NO:1(野生型)所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
A762D、A762P、A762V或A762T；和/或
P826E、P826Q、P826Y、P826T、P826D或P826I；
且所述的丙酮酸羧化酶突变蛋白具有将丙酮酸羧化合成苹果酸前体草酰乙酸的活性。
在另一优选例中，所述丙酮酸羧化酶突变蛋白还具有选自以下一个或多个位点的核心氨基酸突变：
P824V、P824A、P824I、P824Y、P824L、P824M、P824N、P824R、或P824T；和/或
P1031E、1031S、P1031N或P1031G。
在另一优选例中，所述丙酮酸羧化酶突变蛋白还具有选自以下一个或多个位点的核心氨基酸突变：
G837D、或G837H；
A254K、A254E、A254R、或A254F；
H51N、或H51G；和
F453E、或F453P。
在另一优选例中，所述丙酮酸羧化酶突变蛋白来源于野生型丙酮酸羧化酶。
在另一优选例中，所述野生型丙酮酸羧化酶来源于谷氨酸棒杆菌(Corynebacterium glutamicum)
在另一优选例中，所述丙酮酸羧化酶突变蛋白还包括其活性片段、变异形式或衍生蛋白，所述丙酮酸羧化酶突变蛋白活性片段、变异形式或衍生蛋白与所述丙酮酸羧化酶突变蛋白具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性，且具有将丙酮酸羧化合成苹果酸前体草酰乙酸的活性。
在另一优选例中，所述丙酮酸羧化酶突变蛋白衍生蛋白除一个或多个所述核心氨基酸突变以外，其余序列与SEQ ID NO:1所示的序列相同或基本相同。
在另一优选例中，所述丙酮酸羧化酶突变蛋白的序列与SEQ ID NO:1(野生型)相比，具有以下位点的核心氨基酸突变：
A762D和P826E；
A762D和P824V；
P824V和P826E
P824V和P1031E；或
A762D、P826E和P1031E。
在另一优选例中，所述丙酮酸羧化酶突变蛋白如SEQ ID NO:2-20、175所示。
本发明第二方面，提供了一种苹果酸转运蛋白突变蛋白，所述苹果酸转运蛋白突变蛋白基于SEQ ID NO:22所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
I80K、I80W、或I80C；和
R272A、或R272F；
且所述的苹果酸转运蛋白突变蛋白具有将苹果酸从细胞内转运至细胞的活性。
在另一优选例中，所述丙酮酸羧化酶突变蛋白还具有选自以下一个或多个位点的核心氨基酸突变：
E8Q、E8Y、E8R、或E8P；和/或
V142I、V142S或V142C。
在另一优选例中，所述丙酮酸羧化酶突变蛋白还具有选自下组一个或多个位点的核心氨基酸突变：
F97V、或F97R；
F101Y、或F101W；
Y115E；
S123K、S123H、S123R、或S123D；
I301W、I301F、I301H、I301K、或I301Y；
L140N、L140H、或L140K；
P163I、P163V、P163F、P163L、P163Q、或P163Y；
A191R、或A191K；
F201Q、F201G、F201N或F201K；
A214V，A214G或A214D；
E222S、或E222H；
H227F、或H227W；和/或
M251E、或M251G。
在另一优选例中，所述苹果酸转运蛋白突变蛋白的序列与SEQ ID NO:22(野生型)相比，具有以下位点的核心氨基酸突变：
I80K和R272A；或
I80K、E8Q和R272A。
在另一优选例中，所述苹果酸转运蛋白突变蛋白序列如SEQ ID NO:23-42、176所示。
在另一优选例中，所述苹果酸转运蛋白突变蛋白还包括其活性片段、变异形式或衍生蛋白，所述苹果酸转运蛋白突变蛋白活性片段、变异形式或衍生蛋白与所述苹果酸转运蛋白突变蛋白具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性，且将苹果酸从细胞内转运至细胞外的活性。
本发明第三方面，提供了一种分离的多核苷酸或其组合，所述多核苷酸或其组合编码本发明第一方面所述的丙酮酸羧化酶突变蛋白和/或本发明第二方面所述的苹果酸转运蛋白突变蛋白。
在另一优选例中，所述分离的多核苷酸分别编码本发明第一方面所述的丙酮酸羧化酶突变蛋白或本发明第二方面所述的苹果酸转运蛋白突变蛋白。
在另一优选例中，所述分离的多核苷酸组合同时含有编码本发明第一方面所述的丙酮酸羧化酶突变蛋白和本发明第二方面所述的苹果酸转运蛋白突变蛋白的多核苷酸。
本发明第四方面，提供了一种载体，所述载体含有本发明第三方面所述的分离的多核苷酸。
在另一优选例中，所述表达载体含有编码本发明第一方面所述丙酮酸羧化酶突变蛋白和本发明第二方面所述苹果酸转运蛋白突变蛋白。
本发明第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的载体，或所述宿主细胞的核酸中整合有本发明第三方面所述的分离的多核苷酸。
在另一优选例中，所述宿主细胞包括来源于以下微生物的细胞：酿酒酵母(Saccharomyces cerevisiae)、毕赤酵母(Pichia pastoris)、摩纳酵母(Saccharomycesmonacensis)、贝酵母(Saccharomyces bayanus)、巴氏酵母(Saccharomycespastorianus)、卡氏酵母(Saccharomyces carlsbergensis)、粟酒裂殖酵母(Saccharomyces pombe)、马克斯克鲁维酵母(Kluyveromyces marxiamus)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(Kluyveromyces fragilis)，树干毕赤酵母(Pichia stipites)、休哈塔假丝酵母(Candida shehatae)、热带假丝酵母(Candidatropicalis)、大肠杆菌(Escherichia coli)、枯草杆菌(Bacillus subtilis)、光滑球拟酵母(Torulopsis glabrata)、米曲霉(Aspergillus oryzae)、米根霉(Rhizopus oryzae)、稗草黑粉病菌(Ustilago trichophora)嗜热子囊菌(Thermobifida fusca)、嗜热毁丝酶(Myceliophthora thermophila)、异梭毁丝霉(Myceliophthora heterothallica)。
在另一优选例中，所述宿主细胞包括酿酒酵母、毕赤酵母或嗜热毁丝霉。
在另一优选例中，所述宿主细胞仅表达丙酮酸羧化酶突变蛋白。
在另一优选例中，所述宿主细胞仅表达苹果酸转运蛋白突变蛋白。
在另一优选例中，所述宿主细胞同时表达丙酮酸羧化酶突变蛋白和苹果酸转运蛋白突变蛋白。
本发明第六方面，提供了一种制备本发明第一方面所述丙酮酸羧化酶突变蛋白和/或本发明第二方面所述苹果酸转运蛋白突变蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述丙酮酸羧化酶突变蛋白和/或本发明第二方面所述苹果酸转运蛋白突变蛋白；和
分离表达产物，从而获得本发明第一方面所述丙酮酸羧化酶突变蛋白和/或本发明第二方面所述苹果酸转运蛋白突变蛋白。
本发明第七方面，提供了一种提高苹果酸产量的方法，包括步骤：
i)将本发明第三方面所述的多核苷酸或其组合导入合适的宿主细胞；
ii)在碳源的存在下，培养所述的宿主细胞，从而提高苹果酸产量。
在另一优选例中，所述碳源包括葡萄糖、蔗糖、半乳糖、木糖、甘油、或甲醇。
在另一优选例中，所述合适的宿主细胞包括来源于以下微生物的细胞：毕赤酵母、酿酒酵母和/或嗜热毁丝霉、异梭毁丝霉。
本发明第八方面，提供了一种突变蛋白组合，所述突变蛋白组合含有本发明第一方面所述的丙酮酸羧化酶突变蛋白，和本发明第二方面所述的苹果酸转运蛋白突变蛋白。
本发明第九方面，提供了本发明第一方面所述丙酮酸羧化酶突变蛋白和/或本发明第二方面所述苹果酸转运蛋白突变蛋白、本发明第三方面所述分离的多核苷酸或其组合、本发明第四方面所述载体、本发明第五方面所述宿主细胞、或本发明第八方面所述突变蛋白组合的应用，用于提高苹果酸产量和/或用于制备苹果酸。
在另一优选例中，所述苹果酸产量的提高包括与生产苹果酸的野生型菌株相比，苹果酸的产量至少提高10％，优选至少提高20％、30％、40％、50％。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了pyc表达质粒图。
图2显示了mdh表达质粒图。
图3显示了mdh和pyc串联表达质粒图。
图4显示了二元羧酸转运蛋白比较表达及定向进化质粒图。
图5显示了Aomae表达质粒图。
图6显示了pyc定向进化改造质粒图。
图7显示了过表达pyc和mdh产苹果酸及不同同转运蛋白过表达对产苹果酸影响。
图8显示了基于水解圈的转运蛋白筛选改造点板初筛。
图9显示了Aomae I80饱和突变对苹果酸产量影响，其中，I80K，I80W，I80C分别提升苹果酸产量14.3％，11.3％，11.5％。
图10显示了Aomae V142饱和突变对苹果酸产量影响，其中，V142I，V142S，V142C分别提升苹果酸产量21.1％，16.2％，14.1％。
图11显示了Aomae R272饱和突变对苹果酸产量影响，其中，R272A，R272F分别提升苹果酸产量21％，22.1％。
图12显示了Aomae E8饱和突变对苹果酸产量影响，其中，E8Q，E8Y，E8R，E8P分别提升苹果酸产量15.5％，10.3％，9.3％，10.7％。
图13显示了Aomae I 109饱和突变对苹果酸产量影响，其中，I 109M，I109P分别提升苹果酸产量12％，10.2％。
图14显示了Aomae S123饱和突变对苹果酸产量影响，S123，S123H，S123R，S123D分别提升苹果酸产量27.4％，28.2％，23.4％，23.6％。
图15显示了Aomae I301饱和突变对苹果酸产量影响，I301W，I301F，I301H，I301K，I301Y分别提升苹果酸产量25.2％，18.6％，18.3％，17.6％，17.6％。
图16显示了Aomae P163饱和突变对苹果酸产量影响，其中，P163I，P163V，P163F，P163L，P163Q，P163Y分别提升苹果酸产量27.4％，25.2％，20.6％，18.1％，16.5％，15.3％。
图17显示了Aomae F101饱和突变对苹果酸产量影响，其中，F101Y，F101W分别提升苹果酸产量25％，21％。
图18显示了Aomae L140饱和突变对苹果酸产量影响，其中，L140N，L140K，L140H分别提升苹果酸产量22％，10％，20％。
图19显示了Aomae F97饱和突变对苹果酸产量影响，其中，F97V，F97R分别提升苹果酸产量25％，22.7％。
图20显示了Aomae A191饱和突变对苹果酸产量影响，其中，A191R，A191K分别提升苹果酸产量29.2％，22.6％。
图21显示了Aomae Y115饱和突变对苹果酸产量影响，其中，Y115E，Y115L分别提升苹果酸产量28％，16％。
图22显示了Aomae A214饱和突变对苹果酸产量影响，其中，A214V，A214G，A214D分别提升苹果酸产量25％，22.1％，21.3％。
图23显示了Aomae F201饱和突变对苹果酸产量影响，其中，F201Q，F201G，F201K，F201N分别提升苹果酸产量32％，22％，18％，19％。
图24显示了Aomae H227饱和突变对苹果酸产量影响，其中，H227F，H227W分别提升苹果酸产量22％，18％。
图25显示了Aomae E222饱和突变对苹果酸产量影响，其中，E222S，E222G分别提升苹果酸产量22％，22％。
图26显示了Aomae M251饱和突变对苹果酸产量影响，其中，M251E，M251G提升苹果酸产量28％，23％。
图27显示了Aomae组合突变对苹果酸产量影响，其中，I80K/R272A可以提升苹果酸产量30％以上。
图28显示了不同来源pyc对苹果酸产量影响，其中，比较谷氨酸棒杆菌合成苹果酸产量最高达到53.2g/L。
图29显示了基于水解圈对PYC进行点板初筛
图30显示了CgPyc H51饱和突变对苹果酸产量影响，其中，H51N,H51G分别提升苹果酸产量19％，18％。
图31显示了CgPyc F453饱和突变对苹果酸产量影响，其中，F453E，F453P分别提升苹果酸产量17％，17％。
图32显示了CgPyc A254饱和突变对苹果酸产量影响，其中，A254K，A254E，A254R，A254F分别提升苹果酸产量19％，17％，16％，15％。
图33显示了CgPyc G837饱和突变对苹果酸产量影响，其中，G837D，G837H分别提升苹果酸产量24％，17％。
图34显示了CgPyc P824饱和突变对苹果酸产量影响，其中，P824V，P824A，P824I，P824Y，P824L，P824M，P824N，P824R，P824T分别提升苹果酸产量29.4％，28.2％，25.9％，22.4％，23.5％，23.5％，23.6％，23.2％，20％。
图35显示了CgPyc A762饱和突变对苹果酸产量影响，其中，A762D，A762P，A762V，A762T分别提升苹果酸产量33.5％，30.6％，29.6％，22.4％。
图36显示了CgPyc P826饱和突变对苹果酸产量影响，其中，P826E，P826Q，P826Y，P826T，P826D，P826I分别提升苹果酸产量28.3％，20.9％，24.1％，22.9％，20.9％，21.6％。
图37显示了CgPyc P1031饱和突变对苹果酸产量影响，其中，P1031E，P1031N，P1031S，P1031G分别提升苹果酸产量34.2％，30.8％，128.2％，122.2％。
图38显示了CgPyc组合突变对苹果酸产量影响，其中，A762D/P826E，A762D/P824V，P824V/P826E，P824V/P1031E，A762D/P826E/P1031E分别提升苹果酸产量41.4％，38.6％，36.7％，37.1％，42.3％。
图39显示了酿酒酵母表达PYC、AoMae和MDH产苹果酸，其中，三个基因共表达菌株可以产22g/L苹果酸。
图40过表达改造后苹果酸转运蛋白菌株的苹果酸产量，其中，过表达突变AomaeI80K/R272A，苹果酸产量提升9.5％。
图41过表达改造后丙酮酸羧化酶菌株的苹果酸产量，其中，过表达突变CgPYCA762D/P826E/P1031E，苹果酸产量提升16％。
具体实施方式
本发明人通过广泛而深入的研究，首次意外地发现，针对对苹果酸合成的催化机器及转运蛋白进行改造，即对丙酮酸羧化酶和苹果酸转运蛋白特定位点的核心氨基酸进行突变后，微生物菌株生产苹果酸的能力具有显著提高，从而能够使更多的丙酮酸前体流向目标产物苹果酸，从而有效地提高苹果酸产量。具体地，本发明人首先在毕赤酵母中构建了苹果酸合成偶联的高通量筛选方案，对不同来源的丙酮酸羧化酶、苹果酸转运蛋白进行了比较，找到要改造的目标蛋白，对两个目标蛋白分别构建随机突变文库，多次筛选后获得苹果酸产量提升的丙酮酸羧化酶和苹果酸转运蛋白突变体，然后进行单氨基酸饱和突变和组合突变，获得了催化活性提高的丙酮酸羧化酶突变体和苹果酸转运能力提升的苹果酸转运蛋白突变体，并构建了一系列高效合成苹果酸的工程菌株。在此基础上，完成了本发明。
苹果酸及其合成
苹果酸(2-羟基-1，4-丁二酸)是一种重要的平台化合物，被广泛应用于药物、化妆品、食品及饮料行业。
苹果酸合成目前报道的主要有5条途径，分别是TCA循环，还原型TCA(或者反向TCA)rTCA,环形乙醛酸循环和非环形乙醛酸循环，及依赖于苹果酸酶反向催化丙酮酸直接合成苹果酸。其中以rTCA循环和苹果酸酶途径最为有效，其葡萄糖对苹果酸的理论转换率为2，然而苹果酸酶主要催化苹果酸分解为丙酮酸，因此，目前最常用的方法是rTCA途径来合成苹果酸。
从葡萄糖出发经过糖酵解转化为丙酮酸，丙酮酸经过丙酮酸羧化酶转变为草酰乙酸，草酰乙酸经过苹果脱氢酶转化为苹果酸，苹果酸经过转运蛋白运出细胞。该过程中每一步的转化效率，副反应分流，催化每一步反应的辅酶和还原力及细胞能量状态都会对苹果酸合成造成影响。
以往的合成主要是采用化学的方法，然而该过程产生的主要是D、L-苹果酸的混合物，很难分离。而本发明采用微生物的方法则可以合成高纯度的L-苹果酸。
丙酮酸羧化酶
丙酮酸羧化酶(Pyruvate carboxylase，PYC)是生物体内参与中心代谢途径的关键酶之一，通过羧化丙酮酸形成草酰乙酸参与到TCA循环中，同时这步反应也是糖异生途径的第一步反应。大多数丙酮酸羧化酶都由四个相同的亚基组成，单个亚基的分子量为120-130kDa，其发挥功能的四聚体分子量约520kDa。于丙酮酸羧化酶需要四聚化才能发挥功能，该四聚化后的蛋白有520kDa之多，难以分泌到细胞外，虽然丙酮酸羧化酶可以通过测定酶活的方法进行筛选，然而其只能驻留胞内，需要破碎细胞才能进行酶活测定，若对一个突变库中所有突变体进行破壁处理很难实施，造成了该蛋白改造起来难度很大。目前针对这类形成复合体的蛋白改造也只有极少数基于晶体结构基础上针对特定位点的定点突变，所涉及到的突变体非常之少，而对没有晶体结构的CgPYC则没有进行过蛋白质工程改造。
本发明的丙酮酸羧化酶来源于谷氨酸棒杆菌(Corynebacterium glutamicum)其野生型(命名为CgPYC)序列号为WP_011013816.1，序列如SEQ ID NO:1所示，其编码核酸如SEQ ID NO:21所示。是首次被用于苹果酸合成的菌株中。通过构建随机突变，定点突变和组合突变，获得多个可以显著提升毕赤酵母苹果酸合成30％以上的单点突变和2个提升苹果酸40％以上的双突变和三突变，同时，突变蛋白在嗜热毁丝霉中也可提高苹果酸16％。
苹果酸转运蛋白
苹果酸转运蛋白在苹果酸合成过程中发挥着重要作用，本发明用于苹果酸生产的转运蛋白主要有来源于米曲霉Aspergillus oryzae(AO090023000318(C4T318))，野生型(命名为AoMae)的蛋白序列如SEQ ID NO:22所示，其编码核酸如SEQ ID NO:43所示；来源于Schizosaccharomyces pombe的(NM_001020205.2)和来源于Aspergillus carbonarius的DCT(AQW79505.1)，其中关键的AoMae蛋白理论分子量42.2kDa。
苹果酸转运蛋白Aomae属于膜蛋白，膜蛋白通常的评价标准是对底物转运能力进行测定，需要将蛋白纯化出来，构建于脂质体中，或者构建于一个没有类似功能转运蛋白的多基因敲除菌株中，然后通过同位素标记的底物进行测定；此外，转运又分为外排型和内运型，加之膜蛋白的蛋白一致性一般较低，这些因素都给改造膜蛋白造成了非常大的困难。
本发明经过随机突变，定点突变和组合突变，获得了一系列突变位点可以显著提高苹果酸合成水平，其中I80K/E8Q/R272A和I80K/R272A可提升毕赤酵母苹果酸合成30％以上，在嗜热毁丝霉中也可提升苹果酸10.5％。
突变蛋白及其衍生物
本领域技术人员知晓，如果要对某种酶进行突变，以便获得活性改善的突变体，关键之处在于发现突变后可以改善活性的位点。在本发明中，对氨基酸序列如SEQ ID NO:1和22所示的酶进行特定位点的突变，得到了活性显著提高的相应的突变体。如本文所用，术语“突变蛋白”、“突变体”可互换使用，指的是丙酮酸羧化酶和/或苹果酸转运蛋白的突变蛋白，其与丙酮酸羧化酶突变蛋白或苹果酸转运蛋白突变蛋白的对应性可于上下文的语境中获得。具体地，本发明丙酮酸羧化酶突变蛋白通常指与SEQ ID NO:1和/或22(野生型)所示的序列，并且经改造后具有选自以下一个或多个位点的核心氨基酸突变：
①当依据SEQ ID NO:22所示序列进行突变时，可以具有选自如下一个或多个位点的核心氨基酸突变：
A762D、A762P、A762V或A762T；
P826E、P826Q、P826Y、P826T、P826D或P826I；
P824V、P824A、P824I、P824Y、P824L、P824M、P824N、P824R、或P824T；
P1031E、1031S、P1031N或P1031G；
P824V、P824A、P824I、P824Y、P824L、P824M、P824N、P824R、或P824T；
P1031E、1031S、P1031N或P1031G；
G837D、或G837H；
A254K、A254E、A254R、或A254F；
H51N、或H51G；和/或
F453E、或F453P。
且所述的丙酮酸羧化酶突变蛋白具有将丙酮酸羧化合成苹果酸前体草酰乙酸的活性；
一种优选的丙酮酸羧化酶突变蛋白，基于SEQ ID NO:1所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
A762D和P826E；
A762D和P824V；
P824V和P826E
P824V和P1031E；或
A762D、P826E和P1031E。
一种优选的丙酮酸羧化酶突变蛋白的序列如SEQ ID NO:2-20、175任一序列所示。
②当依据SEQ ID NO:22所示序列进行突变时，可以具有选自如下一个或多个位点的核心氨基酸突变：
I80K、I80W、或I80C；
R272A、或R272F；
E8Q、E8Y、E8R、或E8P；
V142I、V142S或V142C；
F97V、或F97R；
F101Y、或F101W；
Y115E；
S123K、S123H、S123R、或S123D；
I301W、I301F、I301H、I301K、或I301Y；
L140N、L140H、或L140K；
P163I、P163V、P163F、P163L、P163Q、或P163Y；
A191R、或A191K；
F201Q、F201G、F201N或F201K；
E222S、或E222G；
H227F、或H227W；和/或
M251E、或M251G。
且所述的苹果酸转运蛋白突变蛋白具有将苹果酸从细胞内转运至细胞外的活性。
一种优选的苹果酸转运蛋白突变蛋白，基于SEQ ID NO:22所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
I80K和R272A；或
I80K、E8Q和R272A。
一种优选的苹果酸转运蛋白突变蛋白的序列如SEQ ID NO:23-42、176任一序列所示。
鉴于本发明的教导以及现有技术，本领域技术人员应理解，本发明的突变蛋白还应当包括所述突变蛋白的活性片段、经修饰或未修饰的变异形式或其衍生蛋白。
具体地，所述丙酮酸羧化酶突变蛋白的活性片段、变异形式或其衍生蛋白包括基于SEQ ID NO:2-20所示序列，具有一个或几个(例如通常为1-30个，较佳地1-10个，更佳地1-6个，再佳地1-3个、最佳地1个)氨基酸残基的缺失、插入和/或取代后，仍然具有将丙酮酸羧化合成苹果酸前体草酰乙酸的活性(cA1)，该活性显著高于SEQ ID NO:1所示野生型丙酮酸羧化酶的相应活性(cA0)，所述显著高于为(cA1-cA0)/cA0≥10％-500％中的任意数值，例如≥15％、20％、25％、30％、40％、50％、或100％。
所述苹果酸转运蛋白突变蛋白的活性片段、变异形式或其衍生蛋白包括基于SEQID NO:23-42所示序列，具有一个或几个(例如通常为1-30个，较佳地1-10个，更佳地1-6个，再佳地1-3个、最佳地1个)氨基酸残基的缺失、插入和/或取代后，仍然具有将苹果酸从细胞内转运至细胞外的活性(aA1)，该活性显著高于SEQ ID NO:22所示野生型苹果酸转运蛋白的相应活性(aA0)，所述显著高于为(cA1-cA0)/cA0≥10％-500％中的任意数值，例如≥15％、20％、25％、30％、40％、50％、或100％。
本领域技术人员可根据，例如下表所示进行保守的氨基酸替换而产生保守性变异的突变体。
初始残基 代表性的取代残基 优选的取代残基
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
如本文所用，蛋白的修饰(通常不改变一级结构)形式包括：体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。这些技术均为本领域技术人员所知。
此外，本发明突变蛋白的衍生蛋白还包括本发明突变蛋白或其活性片段与其它蛋白或标记物形成的融合蛋白或偶联物。
优选地，本发明突变蛋白的活性片段、变异形式或其衍生蛋白分别与SEQ IDNO:1或22具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性。
本发明突变蛋白可以组合形式，从而进一步对苹果酸的产量进行提升。
编码核酸及其组合
在本发明丙酮酸羧化酶突变蛋白或苹果酸转运蛋白突变蛋白的基础上，本发明还提供了编码丙酮酸羧化酶突变蛋白或苹果酸转运蛋白突变蛋白的分离的多核苷酸或其简并的变异体。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与编码本发明实施例中的丙酮酸羧化酶突变蛋白和/或苹果酸转运蛋白突变蛋白的核苷酸序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码本发明权利要求中的丙酮酸羧化酶突变蛋白和/或苹果酸转运蛋白突变蛋白，但与本发明实施例中的丙酮酸羧化酶突变蛋白或苹果酸转运蛋白突变蛋白的编码核苷酸序列有差别的核酸序列。
载体、宿主细胞
编码多核苷酸序列可插入重组表达载体或基因组。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员可采用熟知的方法能用于构建含“丙酮酸羧化酶突变蛋白和/或苹果酸转运蛋白突变蛋白”编码DNA序列和合适的转录/翻译控制信号的表达载体，包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
本文所述的宿主细胞包括包含上述表达载体或基因组上整合了本发明突变蛋白编码序列的宿主细胞。宿主细胞包括来源于子囊菌类宿主，拥有极高的进化关系，且在丙酮酸羧化酶和苹果酸转运蛋白的序列以及功能性上具有很高的相似性，优选的微生物的细胞如下：
酿酒酵母(Saccharomyces cerevisiae)、毕赤酵母(Pichia pastoris)、摩纳酵母(Saccharomyces monacensis)、贝酵母(Saccharomyces bayanus)、巴氏酵母(Saccharomyces pastorianus)、卡氏酵母(Saccharomyces carlsbergensis)、粟酒裂殖酵母(Saccharomyces pombe)、马克斯克鲁维酵母(Kluyveromyces marxiamus)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(Kluyveromyces fragilis)，树干毕赤酵母(Pichia stipites)、休哈塔假丝酵母(Candida shehatae)、热带假丝酵母(Candidatropicalis)、大肠杆菌(Escherichia coli)、枯草杆菌(Bacillus subtilis)、光滑球拟酵母(Torulopsis glabrata)、米曲霉(Aspergillus oryzae)、米根霉(Rhizopus oryzae)、稗草黑粉病菌(Ustilago trichophora)嗜热子囊菌(Thermobifida fusca)、嗜热毁丝酶(Myceliophthora thermophila)、异梭毁丝霉(Myceliophthora heterothallica)。
可通过本领域常规的重组转化方法本发明突变蛋白，并且所述突变蛋白可以在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、高压匀浆、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、亲和层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。获得本发明所述宿主细胞可分别和/或同时表达丙酮酸羧化酶突变蛋白和苹果酸转运蛋白突变蛋白。
突变蛋白的制备
本发明还提供了制备丙酮酸羧化酶突变蛋白苹果酸转运蛋白突变蛋白的方法，包括在在适合表达的条件下，培养本发明宿主细胞，从而表达丙酮酸羧化酶突变蛋白和/或所述苹果酸转运蛋白突变蛋白；和
分离所述丙酮酸羧化酶突变蛋白和/或所述苹果酸转运蛋白突变蛋白。
获得的突变蛋白还可任选地进行纯化，从而获得更纯的突变蛋白产物。
优选地，所述的适合表达的条件包括本领域常规的技术，而纯化技术包括镍柱纯化，离子交换层析等。
碳源
可用于本发明的碳源没有特殊限制，是微生物(如本发明宿主细胞)生长的一类营养物，是含碳化合物。优选地，可用于本发明的碳源包括葡萄糖、蔗糖、纤维二糖、乳糖、半乳糖、木糖、甘油、或甲醇。
应用
本发明突变蛋白可分别或同时用于提高苹果酸的产量。本发明丙酮酸羧化酶突变蛋白在进行特定位点的单突变后苹果酸合成提高至少10％以上，在优选的菌株中的突变可达30％以上，而双突变和三突变后可提升苹果酸产量40％。而对于苹果酸转运蛋白突变蛋白，在进行特定位点的突变后苹果酸产量至少提高了10％，在在优选的菌株中的突变可达30％以上。而将本发明两个蛋白进行改造组合后，苹果酸的产量则有了协同性的提高
本发明有益效果
本发明通过在毕赤酵母中构建苹果酸合成途径，将苹果酸合成途径中丙酮酸羧化酶和苹果酸转运蛋白合成能力于苹果酸产量，进而是CaCO3水解圈的大小相耦联，首次实现了两个蛋白的定向进化改造，并显著提升了苹果酸合成水平。本发明丙酮酸羧化酶突变蛋白在进行特定位点的单突变后苹果酸合成提高至少10％以上，在优选的菌株中的突变可达30％以上，而双突变和三突变后可提升苹果酸产量40％。而对于苹果酸转运蛋白突变蛋白，在进行特定位点的突变后苹果酸产量至少提高了10％，在在优选的菌株中的突变可达30％以上。而将本发明两个蛋白进行改造组合后，苹果酸的产量则有了协同性的提高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor LaboratoryPress,2001)中所述的条件，或按照制造厂商所建议的条件。
所用引物均由金唯智生物科技有限公司合成，所涉及到要合成的基因也由金唯智负责合成，本发明所涉及的序列如序列表所示。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
本发明中，苹果酸、琥珀酸购自sigma试剂公司。
实施例1毕赤酵母二元羧酸转运蛋白筛选平台构建
一、毕赤酵母过表达丙酮酸羧化酶和苹果酸脱氢酶载体构建：
利用引物AvrII-F(SEQ ID NO:44):(CCACCGCCCGTTACCGTCCGAAGGAAATTTTACTCTGCTGGAG)和AvrII-R(SEQ ID NO:45):(CTCCAGCAGAGTAAAATTTCCTTCGGACGGTAACGGGCGGTGG)，以pGAPzA为模板，进行PCR,PCR反应体系为：5×phusion HF buffer 10μl 10mM dNTPs 1μl，AvrII-F 1μl，AvrII-R 1μl，pGAPzA 1μl，Phusion DNA polymerase 0.5μl，水35.5μl。PCR反应条件为：先98℃30s；然后98℃10s，60℃30s，72℃3min,30个循环；最后72℃10min，4℃10min。PCR反应结束后，将产物进行过纯化柱纯化，以1μg质粒加入1μl限制性内切酶DpnI消化去除模板，然后85℃热失活5min，取5μl产物转化大肠杆菌感受态细胞DH5α，涂布于25μg/ml Zeocin的低盐LB培养基平板上筛选阳性克隆，进行测序，测序结果表明pGAPzA上的AvrII酶切位点已被成功去除，将其命名为pGAPzAm。利用引物PYC-F(SEQ ID NO:46):(GGGTTCGAAACG ATGGCCGAAGAAGACTACTCCC)/PYC-R(SEQ ID NO:47):(GGGGCGGCCGCTTACTCAGCCTTGACGATTTTGGCGATC),MDH-F(SEQ ID NO:48):(GGGTTCGAAACGATGGTTAAAGTCACAGTTTGCGGAG)/MDH-R(SEQ ID NO:49):(GGGGCGGCCGCTTAGTTGCCAGCAATGAAGGCAGTTCC)，以毕赤酵母GS115基因组为模板通过PCR扩增丙酮酸羧化酶PYC和苹果酸脱氢酶MDH基因,PCR反应条件为：先98℃30s；然后98℃10s，60℃30s，72℃3min(PYC)或1min(MDH),30个循环；最后72℃10min，4℃10min。PCR产物过纯化柱纯化后，采用BstBI和NotI进行双酶切，质粒pGAPzA和pGAPzAm采用同样的酶进行双酶切，然后将PYC与pGAPzA，MDH与pGAPzAm通过T4DNA连接酶22℃连接1h，取10μl连接产物转化大肠杆菌感受态细胞DH5α，涂布于25μg/mlZeocin的低盐LB培养基平板上筛选阳性克隆，进行测序，测序结果表明成功构建了pGAPzA-PYC(图1)和pGAPzAm-MDH(图2)质粒。进一步将pGAPzAm-MDH采用BglII和BamHI进行双酶切，获取MDH的表达框，连接于pGAPzA-PYC经过BamH1酶切的质粒，连接产物转化大肠杆菌感受态细胞DH5α，涂布于25μg/mlZeocin的低盐LB培养基平板上筛选阳性克隆，进行测序，构建出pGAPzA-PYC-pGAPzAm-MDH表达质粒，质粒示意图如图3所示。
二、过表达丙酮酸羧化酶和苹果酸脱氢酶毕赤酵母菌株构建：
1、将保存在-80℃的毕赤酵母GS115菌株划线于YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖，2％琼脂)平板，30℃培养待长出单菌落。
2、挑取单菌落于250ml装量为30ml的液体YPD培养基，200rpm，30℃培养活化2d。
3、将活化好的毕赤酵母GS115菌株二次转接于装有30ml液体YPD的培养基，接种量控制在千分之一，200rpm，30℃培养约16h-20h，待OD600长到1.0。
4、3000rpm，4℃离心，收集菌体，用预冷的无菌水洗涤2次。
5、然后用预冷的1M山梨醇洗涤1次，离心收集沉淀。
6、加入约400μl 1M山梨醇重悬菌体，然后以每支80μl分装于预冷的1.5ml离心管，制成毕赤酵母GS115感受态细胞，保存于-80℃备用。
7、提取pGAPzA-PYC-pGAPzAm-MDH质粒，对质粒采用AvrII进行线性化，然后通过电击转化于毕赤酵母GS115感受态细胞，之后加入1ml预冷1M山梨醇，30℃静置2h，然后涂布于含50或75μg/ml的YPD平板，待长出菌落后用于苹果酸生产，该菌株命名P0。
三、苹果酸合成能力测定
苹果酸合成菌株首先用YPD培养基活化48h，然后以初始OD600＝1.0，接种于产苹果酸培养基(100g/L glucose，1.24g/L KNO3，0.64g/L KH2PO4,0.04g/L ZnSO4·7H2O，0.25g/LMgSO4·7H2O,0.0005g/L FeSO4·7H2O 4g/L peptone,0.1g/LCaCl2，75g/L CaCO3，0.1g/Lhistine)，250ml三角瓶装液量30ml，200rpm，30℃培养，每隔24h，取样1ml，加入1ml 20％(V/V)H2SO4，80℃酸化处理30min，加入一定量的水进行稀释，然后离心，取上清用HPLCAminex 87H色谱柱进行苹果酸含量测定。HPLC参数条件如下；色谱柱温度35℃，检测器温度40℃，流动相5mM H2SO4，流速0.5ml/min，以苹果酸标品进行定量。结果如图7所示，和GS115菌株相比，P0菌株可产3.0g/l苹果酸,GS115产1.0g/L苹果酸。
实施例2二元羧酸转运蛋白突变库的构建筛选。
一、二元羧酸转运蛋白表达载体构建：
引物为：
Hph‐BamH1SEQ ID NO:50GGGGGATCCTGTACAGCTTGCCTCGTCCCC
Hph‐R SEQ ID NO:51GTCGACACTGGATGGCGGCGTTAG
以pAG34质粒为模板，PCR扩增潮霉素基因，PCR反应条件为：先98℃30s；然后98℃10s，60℃30s，72℃2min,30个循环；最后72℃10min，4℃10min。PCR反应结束后，将产物进行过纯化柱纯化，以BamH1进行酶切，连接于以BamH1和EcoRV酶切的pGAPzA质粒，连接产物转化大肠杆菌感受态细胞DH5α，单克隆测序正确，获得pGAP-hph。
引物为：
his‐BglII SEQ ID NO:52GGGAGATCTGTTGTAACACTGGCAGAGCATTACG
his‐BamH1SEQ ID NO:53GGGGGATCCGTCCCAGTTTCTCCATACGAACC)
以pPIC9K质粒为模板，PCR增his基因，PCR片段经过BglII和BamH1酶切与经BglII酶切的pGAP-hph相连接，获得pGAP-hph-his。
引物为：
SpMae‐ClaI SEQ ID NO:54GGGATCGATATGGGTGAACTCAAGGAAATCTTGSpMae‐NotISEQ ID NO:55GGGGCGGCCGCTTAAACGCTTTCATGTTCACTACTAGG
以Schizosaccharomyces pombe基因组DNA为模板，PCR扩增SpMae基因。
引物为：
AoMae‐BstBI SEQ ID NO:56GGGTTCGAAATGCTGACACCTCCCAAGTTTG
AoMae‐NotI SEQ ID NO:57GGGCGGCCGCCTAATCAGATACATCCTCATCTTTAC
以Aspergillus oryzae DSM1863(DSMZ，购自德国微生物和细胞培养有限公司)的cDNA为模板，PCR扩增AoMae基因；
引物为：
Kljen2‐EcoRI SEQ ID NO:58GGGGAATTCATGGCTGCAGAATCAATAGTGTCTC
Kljen2‐NotI SEQ ID NO:59GGGGCGGCCGCTTATGTTGAATTTCTTGCTTGAAATTTAGATC
以Kluyveromyces lactis基因组DNA为模板，PCR扩增获得Kljen2。SpMae,AoMae及Kljen2PCR产物分别经过ClaI/NotI，BstBI/NotI,和EcoRI/NotI酶切纯化后与载体pGAP-hph-his连接(用对应的酶进行酶切，(ClaI与BstBI是同尾酶)连接产物转化大肠杆菌感受态细胞DH5α，单克隆测序正确，获得pGAP-hph-his-SpMae，pGAP-hph-his-AoMae和pGAP-hph-his-Kljen2(图4)
二、二元羧酸转运蛋白候选蛋白产苹果酸能力比较：载体pGAP-hph-his-SpMae，pGAP-hph-his-AoMae和pGAP-hph-his-Kljen2分别经过AvrII线性化后，电击转化P0菌株，涂布于MD(YNB 13.4g/L,biotin 4×10-4g/L,琼脂2.0g/L,葡萄糖2.0g/L)，待长出均落后，以产酸培养基进行摇瓶培养，结果如图7所示，其中来源于A.oryzae的AoMae更利于苹果酸等有机酸合成，以100g/L葡萄糖为底物可以合成55g/L苹果酸，获得的菌株命名为P1，以此为出发蛋白进行蛋白质工程改造。
三、基于水解圈的高通量筛选方法构建：由于20-30g/L的苹果酸钙可溶的特性(本研究前期发现)，我们建立了水解圈的高通量筛选方法。具体方法是：当转运蛋白表达质粒转入P0感受态细胞，待长出菌落后，以无菌牙签将菌落挑取，然后穿刺于产苹果酸的固体培养基(液体中加入2.0g/L琼脂，CaCO3的浓度为3g/L)，每天检查水解圈的大小，以水解圈的直径与菌落直径的比值作为筛选标准，值越大，说明产苹果酸越高。
四、Aomae突变文库构建及产苹果酸突变株筛选：对来源于A.oryzae的AoMae进行蛋白质工程改造，以AoMae-BstBI和AoMae-NotI为引物，采用随机突变试剂盒GeneMorph IIRandom Mutagenesis(Agilent)进行AoMae的随机突变扩增，将PCR产物采用BstBI和NotI酶切后与pGAP-hph-his进行连接，连接产物转化大肠杆菌感受态细胞DH5α，涂布于含有125μg/ml潮霉素的LB固体平板培养基，37℃过夜培养，随机挑取20个单克隆进行测序，分析突变率，结果表明突变率在1％左右。对突变库用含有125μg/ml潮霉素的LB液体培养基洗脱下来，然后37℃复苏2h，后提取混合质粒，经过AvrII线性化后转化P0感受态细胞，涂布于MD平板，待长出单菌落后，穿刺于产苹果酸固体培养基，以AoMae野生型菌株P1为对照，突变库中比P1水解圈大的菌株(图8)，通过摇瓶进行复筛和HPLC苹果酸含量测定。
五、产苹果酸突变体中AoMae基因拷贝数的测定
提取产苹果酸突变体的基因组DNA。
引物为：
Actin‐RT1‐F SEQ ID NO:60CTGCGATTCTTATTCTTCCGATTG
Actin‐RT1‐R SEQ ID NO:61CAACCGAGGACAACCAACAG
扩增Actin基因为内参
引物为：
Aomae‐RT1‐F SEQ ID NO:62CATCATCTGCGGCTTGTC
Aomae‐RT1‐R SEQ ID NO:63TGAGAAGACGAACGAGTATTG
扩增AoMae基因，结果表明所获得的AoMae转化子都是单拷贝。
六、突变位点鉴定:对苹果酸产量提高的突变菌株，提取其基因组DNA，然后以AoMae-BstB1和AoMae-NotI为引物进行PCR扩增，扩增产物经过纯化柱回收后，连接于pJET1.2blunt载体，转化大肠杆菌感受态细胞DH5α，涂布于含100μg/ml氨苄的LB培养基，对单克隆进行测序获得突变位点I80F、V142D、R272P、E8G、I109V、S123R、I301K、P163Q、F101H、L140N、F97G、A191C、Y115I、A214G、F201K、H227F、M251Y和E222H。
七、定点饱和突变和组合突变AoMae提升苹果酸合成：将随机突变获得的位点和基于结构比对获得的关键氨基酸位点进行饱和突变，采取方法是：在引物中间引入突变位点，，然后PCR成环扩增pGAP-hph-his-AoMae，对PCR产物采用DpnI进行酶切，去除模板质粒，然后取5μl转化大肠杆菌感受态细胞DH5α，涂布于含125μg/ml潮霉素的LB培养基，对单克隆进行测序获得突变位点，对获得的AoMae突变体表达质粒进行AvrII线性化后，转化P0菌株，然后经液体苹果酸生产培养基摇瓶培养和HPLC分析。
I80F突变采用引物I80F(SEQ ID NO:64):
CCATGGCTATAAGGTTCTTCCTGCACGGCAACCTTCTG和I80-rev(SEQ ID NO:65):GAACCTTATAGCCATGGTAGAG,其他位点I80X突变(X代表除F外其他18种氨基酸)分别采用引物I80X(SEQ ID NO:66):
CCATGGCTATAAGGTTCXXXCTGCACGGCAACCTTCTG(XXX是不同氨基酸对应的密码子)和I80-rev(SEQ ID NO:65)引入。
V142D突变采用引物V142D(SEQ ID NO:67):GCGTCTGCACCTTACTCGACGCAATCATCCAATACTCG和V142-rev(SEQ ID NO:68):GAGTAAGGTGCAGACGCAGTAG,其他位点V142X突变(X代表除F外其他18种氨基酸)分别采用引物V142X(SEQ ID NO:69):GCGTCTGCACCTTACTCXXXGCAATCATCCAATACTCG(XXX是不同氨基酸对应的密码子)和V142-rev(SEQ ID NO:68)引入。
R272P突变采用引物R272P(SEQ ID NO:69):CACGCCCTGGAAGATGGCCCAATCATCGAGCTGCTGGC和R272-rev(SEQ ID NO:70):GCCATCTTCCAGGGCGTG,其他位点R272X突变(X代表除F外其他18种氨基酸)分别采用引物R272X(SEQ ID NO:71):CACGCCCTGGAAGATGGCXXXATCATCGAGCTGCTGGC(XXX是不同氨基酸对应的密码子)和R272-rev(SEQ ID NO:70)引入。
E8G突变采用引物E8G(SEQ ID NO:72):CTGACACCTCCCAAGTTTGGTGATGAGAAGCAGCTGGG和E8-rev(SEQ ID NO:73):AAACTTGGGAGGTGTCAGC,其他位点E8X突变(X代表除F外其他18种氨基酸)分别采用引物E8X(SEQ ID NO:74):CTGACACCTCCCAAGTTTXXXGATGAGAAGCAGCTGGG(XXX是不同氨基酸对应的密码子)和E8-rev(SEQ ID NO:73)引入。
I109V突变采用引物I109V(SEQ ID NO:75):TCTCCGTCGCAACCATCGTCTGCGGCTTGTCTCGCTAC和I109-rev(SEQ ID NO:76):GATGGTTGCGACGGAGAG,其他位点I109X突变(X代表除F外其他18种氨基酸)分别采用引物I109X(SEQ ID NO:77):TCTCCGTCGCAACCATCXXXTGCGGCTTGTCTCGCTAC(XXX是不同氨基酸对应的密码子)和I109-rev(SEQ ID NO:76)引入。
S123R突变采用引物S123R(SEQ ID NO:78):GTGAAGAATCGAATGAGAGATTCCAACTAGCCCTCGAAG和S123-rev(SEQ ID NO:79):CTCATTCGATTCTTCACCG,其他位点S123X突变(X代表除F外其他18种氨基酸)分别采用引物S123X(SEQ ID NO:80):GTGAAGAATCGAATGAGXXXTTCCAACTAGCCCTCGAAG(XXX是不同氨基酸对应的密码子)和S123-rev(SEQ ID NO:79)引入。
I301K突变采用引物I301K(SEQ ID NO:81):CGCCATTGTCGCCGTCAAGCGCTCGCCCCCCGAGGCC和I301-rev(SEQ ID NO:82):GACGGCGACAATGGCGATG,其他位点S123X突变(X代表除F外其他18种氨基酸)分别采用引物S123X(SEQ ID NO:83):CGCCATTGTCGCCGTCXXXCGCTCGCCCCCCGAGGCC(XXX是不同氨基酸对应的密码子)和I301-rev(SEQ ID NO:82)引入。
P163Q突变采用引物P163Q(SEQ ID NO:84):GCCTTCAAACCATGATGCAATCATGGATCCTTCCAGCC和P163Q-rev(SEQ ID NO:85):CATCATGGTTTGAAGGCC,其他位点P163X突变(X代表除F外其他18种氨基酸)分别采用引物P163X(SEQ ID NO:86):GCCTTCAAACCATGATGXXXTCATGGATCCTTCCAGCC(XXX是不同氨基酸对应的密码子)和P163-rev(SEQ ID NO:85)引入。
F101H突变采用引物F101H(SEQ ID NO:87):GGTCTCTTCTTCCCGACCCACTGGCTCTCCGTCGCAACC和F101-rev(SEQ ID NO:88):GGTCGGGAAGAAGAGACCC,其他位点F101X突变(X代表除F外其他18种氨基酸)分别采用引物F101X(SEQ ID NO:89):GGTCTCTTCTTCCCGACCXXXTGGCTCTCCGTCGCAACC(XXX是不同氨基酸对应的密码子)和F101-rev(SEQ ID NO:88)引入。
L140N突变采用引物L140N(SEQ ID NO:90):TCTACTGCGTCTGCACCAACCTCGTCGCAATCATCC和L140-rev(SEQ ID NO:91):GGTGCAGACGCAGTAGATC,其他位点L140X突变(X代表除F外其他18种氨基酸)分别采用引物L140X(SEQ ID NO:92):TCTACTGCGTCTGCACCXXXCTCGTCGCAATCATCC(XXX是不同氨基酸对应的密码子)和L140-rev(SEQ ID NO:91)引入。
F97G突变采用引物F97G(SEQ ID NO:93):CATGACCGCGAGGGTCTCGGGTTCCCGACCTTCTGGCTC和F97-rev(SEQ ID NO:94):GAGACCCTCGCGGTCATG,其他位点F97X突变(X代表除F外其他18种氨基酸)分别采用引物F97X(SEQ ID NO:95):CATGACCGCGAGGGTCTCXXXTTCCCGACCTTCTGGCTC(XXX是不同氨基酸对应的密码子)和F97-rev(SEQ ID NO:94)引入。
A191C突变采用引物A191C(SEQ ID NO:96):CAACAACCCGCTCGCGCATGTCTCCCCATCATCGGCGC和A191-rev(SEQ ID NO:97):TGCGCGAGCGGGTTGTTG,其他位点A191X突变(X代表除F外其他18种氨基酸)分别采用引物F97X(SEQ ID NO:98):CAACAACCCGCTCGCGCATGTCTCCCCATCATCGGCGC(XXX是不同氨基酸对应的密码子)和A191-rev(SEQ ID NO:97)引入。
Y115I突变采用引物Y115I(SEQ ID NO:99):TCTGCGGCTTGTCTCGCATCTTCGGTGAAGAATCGAATG和Y115-rev(SEQ ID NO:100):GCGAGACAAGCCGCAGATG,其他位点Y115X突变(X代表除F外其他18种氨基酸)分别采用引物F97X(SEQ ID NO:101):TCTGCGGCTTGTCTCGCXXXTTCGGTGAAGAATCGAATG(XXX是不同氨基酸对应的密码子)和Y115-rev(SEQ ID NO:100引入。
A214G突变采用引物A214G(SEQ ID NO:102):TCAGCTTCATGATGTACGGGCACTACATCGGCCGACTG和A214-rev(SEQ ID NO:103):GTACATCATGAAGCTGATG,其他位点A214X突变(X代表除F外其他18种氨基酸)分别采用引物A214X(SEQ ID NO:104):TCAGCTTCATGATGTACXXXCACTACATCGGCCGACTG(XXX是不同氨基酸对应的密码子)和A214X-rev(SEQ ID NO:103)引入。
F201K突变采用引物F201K(SEQ ID NO:105):CGGCGCCGGCGTCACCAAGCAGGGCCTCGGCTTCTCC和F201-rev(SEQ ID NO:106):GGTGACGCCGGCGCCGATG,其他位点F201X突变(X代表除F外其他18种氨基酸)分别采用引物A214X(SEQ ID NO:107):CGGCGCCGGCGTCACCXXXCAGGGCCTCGGCTTCTCC(XXX是不同氨基酸对应的密码子)和F201-rev(SEQ ID NO:106)引入。
H227F突变采用引物H227F(SEQ ID NO:108):ATGGAGTCCGGCCTCCCCTTCAGCGACCACAGACCAGGC和H227-rev(SEQ ID NO:109):GGGGAGGCCGGACTCCATC,其他位点H227X突变(X代表除F外其他18种氨基酸)分别采用引物A214X(SEQ ID NO:110):ATGGAGTCCGGCCTCCCCXXXAGCGACCACAGACCAGGC(XXX是不同氨基酸对应的密码子)和H227-rev(SEQ ID NO:109)引入。
M251Y突变采用引物M251Y(SEQ ID NO:111):CCCTCGCCCTCGTCGGCTACAGCAAAGGCCTCCCCGAAG和M251-rev(SEQ ID NO:112):GCCGACGAGGGCGAGGGC,其他位点M251X突变(X代表除F外其他18种氨基酸)分别采用引物M251X(SEQ ID NO:113):CCCTCGCCCTCGTCGGCXXXAGCAAAGGCCTCCCCGAAG(XXX是不同氨基酸对应的密码子)和M251-rev(SEQ ID NO:112)引入。
E222H突变采用引物E222Y(SEQ ID NO:114):TACATCGGCCGACTGATGCACTCCGGCCTCCCCCAC和E222-rev(SEQ ID NO:115):CATCAGTCGGCCGATGTAG,其他位点E222X突变(X代表除F外其他18种氨基酸)分别采用引物M251X(SEQ ID NO:116):TACATCGGCCGACTGATGXXXTCCGGCCTCCCCCAC(XXX是不同氨基酸对应的密码子)和E222-rev(SEQ ID NO:115)引入。
各突变体转化P0菌株，筛选获得苹果酸的结果如图9-图26所示：
选取4个最优的点E8Q、I80K、V142I和R272A进行组合突变(分别可以提升苹果酸合成能力15％、14％、21％和21％)，采用和饱和突变同样的方法，同样对组合突变转化P0菌株，产苹果酸结果如图27，其中的I80K/R272A(对应质粒pGAP-hph-his-AoMaeKA)突变合成苹果酸能力最好，比出发菌株提升30％，达到71.5g/L。
实施例3丙酮酸羧化酶筛选平台菌株构建
一、二元羧酸转运蛋白表达载体构建，引物如下：
Hph-BamH1SEQ ID NO:117GGGGGATCCTGTACAGCTTGCCTCGTCCCC
Hph-R SEQ ID NO:118GTCGACACTGGATGGCGGCGTTAG
以pAG34质粒为模板，PCR扩增潮霉素基因，PCR反应条件为：先98℃30s；然后98℃10s，60℃30s，72℃2min,30个循环；最后72℃10min，4℃10min。PCR反应结束后，将产物进行过纯化柱纯化，以BamH1进行酶切，连接于以BamH1和EcoRV酶切的pGAPzA质粒，连接产物转化大肠杆菌感受态细胞DH5α，单克隆测序正确，获得pGAP-hph。来源于Aspergillus oryzae的AoMae从基因组DNA直接PCR扩增；AoMae经BstBI和NotI酶切后与载体pGAP-hph连接，连接产物转化大肠杆菌感受态细胞DH5α，单克隆测序正确，获得pGAP-hph-AoMae(图5)。
二、过表达二元羧酸转运蛋白毕赤酵母菌株构建
1、将保存在-80℃的毕赤酵母GS115菌株划线于YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖，2％琼脂)平板，30℃培养待长出单菌落。
2、挑取单菌落于250ml装量为30ml的液体YPD培养基，200rpm，30℃培养活化2d。
3、将活化好的毕赤酵母GS115菌株二次转接于装有30ml液体YPD的培养基，接种量控制在千分之一，200rpm，30℃培养约16h-20h，待OD600长到1.0。
4、3000rpm，4℃离心，收集菌体，用预冷的无菌水洗涤2次
5、然后用预冷的1M山梨醇洗涤1次，离心收集沉淀
6、加入约400μl 1M山梨醇重悬菌体，然后以每支80μl分装于预冷的1.5ml离心管，制成毕赤酵母GS115感受态细胞，保存于-80℃备用。
7、提取pGAP-hph-AoMae质粒，对质粒采用AvrII进行线性化，然后通过电击转化于毕赤酵母GS115感受态细胞，之后加入1ml预冷1M山梨醇，30℃静置2h，然后涂布于含300μg/ml的YPD平板，待长出菌落后用于苹果酸生产，该菌株命名P2。
三、苹果酸合成能力测定
苹果酸合成菌株首先用YPD培养基活化48h，然后以初始OD600＝1.0，接种于产苹果酸培养基(100g/L glucose，1.24g/L KNO3，0.64g/L KH2PO4,0.04g/L ZnSO4·7H2O，0.25g/LMgSO4·7H2O,0.0005g/L FeSO4·7H2O.4g/L peptone,0.1g/LCaCl2，75g/L CaCO3，0.1g/Lhistine，biotin 4×10-4g/L)，250ml三角瓶装液量30ml，200rpm，30℃培养，每隔24h，取样1ml，加入1ml 20％(V/V)H2SO4，80℃酸化处理30min，加入一定量的水进行稀释，然后离心，取上清用HPLC Aminex87H色谱柱进行苹果酸含量测定。HPLC参数条件如下；色谱柱温度35℃，检测器温度40℃，流动相5mM H2SO4，流速0.5ml/min，以苹果酸标品进行定量。结果表明P2菌株可产5.0/l苹果酸,GS115产1.0g/L苹果酸。
实施例4丙酮酸羧化酶突变库的构建筛选
一、丙酮酸羧化酶表达载体构建，引物如下：
his‐BglII SEQ ID NO:119GGGAGATCTGTTGTAACACTGGCAGAGCATTACG
his‐BamH1SEQ ID NO:120GGGGGATCCGTCCCAGTTTCTCCATACGAACC
以pPIC9K质粒为模板，PCR增his基因，PCR片段经过BglII和BamH1酶切与经BglII酶切的pGAPzA相连接，获得pGAPzA-his。青霉、根瘤菌来源的丙酮酸羧化酶由金唯智合成，引物如下：
PvPyc‐F SEQ ID NO:121GGGTTCGAAATGGCCGCGCCGCAACGCCAACCCG
PvPyc‐R SEQ ID NO:122GGGGCGGCCGCTCATCAGGCCTTGCCGATCTTAC
RePyc‐F SEQ ID NO:123GGGTTCGAAATGCCCATTAGCAAGATCCTCGTGGC
RePyc‐R SEQ ID NO:124GGGGCGGCCGCTCAGCCGCCGTAGACCGCGAGGAG
然后采用采用BstBI和NotI酶切后与同样酶切的pGAPzA-his进行连接，转化大肠杆菌DH5α，转化子测序正确，命名为pGAPzA-his-PvPyc和pGAPzA-his-RePyc(图6)。曲霉、米根霉、谷氨酸棒杆菌和毕赤酵母来源的丙酮酸羧化酶，引物如下：
AoPyc‐F SEQ ID NO:125GGGTTCGAAATGGCGGCTCCGTTTCGTCAGCC
AoPyc‐R SEQ ID NO:126GGGGCGGCCGCCTATTACGCTTTGACGATCTTGC
RoPyc‐F SEQ ID NO:127AATTGAACAACTATTTCGAAATGCCTGCTGCACC
RoPyc‐R SEQ ID NO:128AGAAAGCTGGCGGCCGCTTAGGCTTCCTCTTTGAC
CgPyc‐F SEQ ID NO:129CAATTGAACAACTATTTCGAAATGTCGACTCACAC
CgPyc‐R SEQ ID NO:130CTAGAAAGCTGGCGGCCGCCTATTAGGAAACGAC
PpPyc‐F SEQ ID NO:131GGGTTCGAAATGGCCGAAGAAGACTACTCCCCGC
PpPyc‐R SEQ ID NO:132GGGGCGGCCGCTTACTCAGCCTTGACGATTTTGGC
以各自的基因组DNA为模板，进行PCR扩增，PCR反应体系为：5×phusionHF buffer10μl 10mM dNTPs 1μl，AvrII-F 1μl，AvrII-R 1μl，pGAPzA 1μl，Phusion DNA polymerase0.5μl，水35.5μl。PCR反应条件为：先98℃30s；然后98℃10s，60℃30s，72℃3min,30个循环；最后72℃10min，4℃10min。PCR产物经过纯化柱纯化后，以BstBI和NotI进行酶切(对基因内部含有的酶切位点通过突变消除)，与载体pGAPzA-his进行连接，转化大肠杆菌转化大肠杆菌DH5α，转化子测序正确，命名为pGAPzA-his-AoPyc、pGAPzA-his-RoPyc、pGAPzA-his-CgPyc和pGAPzA-his-PpPyc(图6)。
二、丙酮酸羧化酶候选基因比较
对以上构建的6个不同来源的丙酮酸羧化酶表达载体采用AvrII进行酶切后，电击转入含有二元羧酸转运蛋白AoMae的毕赤酵母P2菌株，涂布与MD平板，待长出菌落后，挑取单菌落，采用YPD液体培养基活化48h，以产酸培养基进行摇瓶培养，结果如图28所示，其中来源于谷氨酸棒杆菌的丙酮酸羧化酶CgPyc更利于苹果酸等有机酸合成，以100g/L葡萄糖为底物可以合成53.2g/L苹果酸，以此为出发蛋白进行蛋白质工程改造。
三、基于水解圈的高通量筛选方法构建：由于20-30g/L的苹果酸钙可溶的特性(本研究前期发现)，我们建立了水解圈的高通量筛选方法。具体方法是：丙酮酸羧化酶转化P2感受态细胞，待长出菌落后，以无菌牙签将菌落挑取，然后穿刺于产苹果酸的固体培养基(液体中加入2.0g/L琼脂，CaCO3的浓度为3g/L)，每天检查水解圈的大小，以水解圈的直径与菌落直径的比值作为筛选标准，值越大，说明产苹果酸越高(图29)。
四、PYC突变文库构建及产苹果酸突变株筛选：对来源于谷氨酸棒杆菌的丙酮酸羧化酶CgPyc进行蛋白质工程改造。首先对CgPyc内含有的BstBI和NotI进行突变，采用引物如下:
BstBI-mut-F SEQ ID NO:133TGGGCCTTGCGGATCGTTTCGAGCTC
BstBI-mut-R SEQ ID NO:134ACGATCCGCAAGGCCCAGTGCGGTGGC
NotI-mut-F SEQ ID NO:135TGCTGCTTCGCGGTCGCAAC
NotI-mut-R SEQ ID NO:136CGAAGCAGCATCTGAATGTTTAC
所采用方法同实施例2同的点突变，获得质粒pGAPZA-his-CgPyc(BstBI NotImut)用作模板进行CgPyc随机突变和定点突变。引物如下：
CgPyc‐ep‐F SEQ ID NO:137GGGTTCGAAATGTCGACTCACACATCTTC
CgPyc‐ep‐R SEQ ID NO:138GGGGCGGCCGCTTAGGAAACGACGACGATCAAG
采用随机突变试剂盒GeneMorph II Random Mutagenesis(Agilent)进行CgPyc的随机突变扩增，将PCR产物采用BstB1和NotI酶切后与pGAPzA-his进行连接，连接产物转化大肠杆菌感受态细胞DH5α，涂布于含有25μg/ml Zeocin的LB固体平板培养基，37℃过夜培养，随机挑取20个单克隆进行测序，分析突变率，结果表明突变率在1％左右。对突变库用含有25μg/ml Zeocin的LB液体培养基洗脱下来，然后37℃复苏2h，后提取混合质粒，经过AvrII线性化后转化P2感受态细胞，涂布于MD平板，待长出单菌落后，穿刺于产苹果酸固体培养基，以CgPyc野生型菌株为对照，突变库中比野生型CgPyc水解圈大的菌株，通过摇瓶进行复筛和HPLC苹果酸含量测定。
五、突变位点鉴定:对苹果酸产量提高的突变菌株，提取其基因组DNA，然后以CgPyc-F和CgPyc-R为引物进行PCR扩增，扩增产物经过纯化柱回收后，连接于pJET1.2blunt载体，转化大肠杆菌感受态细胞DH5α，涂布于含100μg/ml氨苄的LB培养基，对单克隆进行测序获得突变位点H51E、F453Q、A254N、G837M、A762T、P824L、P826T、P1031S。
六、定点饱和突变和组合突变CgPyc提升苹果酸合成：将随机突变获得的位点进行饱和突变，采取方法是：在引物中间引入突变位点，然后PCR成环扩增pGAPzA-his-CgPyc，对PCR产物采用DpnI进行酶切，去除模板质粒，然后取5μl转化大肠杆菌感受态细胞DH5α，涂布于含25μg/mlZeocin的LB培养基，对单克隆进行测序获得突变位点，对获得的CgPyc突变体表达质粒进行AvrII线性化后，转化P2菌株，然后经过液体苹果酸生产培养基进行摇瓶培养和HPLC分析。突变所用引入如下：
H51E突变采用引物H51E(SEQ ID NO:139):GAAGATCGGGGATCATTCGAGCGCTCTTTTGCTTCTG和H51-rev(SEQ ID NO:140):GAATGATCCCCGATCTTC,其他位点H51X突变(X代表除F外其他18种氨基酸)分别采用引物H51X(SEQ ID NO:141):GAAGATCGGGGATCATTCXXXCGCTCTTTTGCTTCTG(XXX是不同氨基酸对应的密码子)和H51-rev(SEQ ID NO:140)引入。
F453Q突变采用引物F453Q(SEQ ID NO:142):AGCGCATCGCCACCGGACAAATTGCCGATCACCCGCAC和F453-rev(SEQ ID NO:143):TCCGGTGGCGATGCGCTTG,其他位点F453X突变(X代表除F外其他18种氨基酸)分别采用引物F453X(SEQ ID NO:144):AGCGCATCGCCACCGGAXXXATTGCCGATCACCCGCAC(XXX是不同氨基酸对应的密码子)和F453-rev(SEQ ID NO:143)引入。
A254N突变采用引物A254N(SEQ ID NO:145):AGTTGTCGAAATTGCGCCAAACCAGCATTTGGATCCAG和A254-rev(SEQ ID NO:146):TGGCGCAATTTCGACAAC,其他位点A254X突变(X代表除F外其他18种氨基酸)分别采用引物A254X(SEQ ID NO:147):AGTTGTCGAAATTGCGCCAAACCAGCATTTGGATCCAG(XXX是不同氨基酸对应的密码子)和A254X-rev(SEQ ID NO:146)引入。
G837M突变采用引物G837M(SEQ ID NO:148):TACCGCCACGAAATCCCAATGGGACAGTTGTCCAACCTG和G837-rev(SEQ ID NO:149):TGGGATTTCGTGGCGGTAG,其他位点G837X突变(X代表除F外其他18种氨基酸)分别采用引物G837X(SEQ ID NO:150):TACCGCCACGAAATCCCAXXXGGACAGTTGTCCAACCTG(XXX是不同氨基酸对应的密码子)和G837-rev(SEQ ID NO:149)引入。
A762T突变采用引物A762T(SEQ ID NO:151):GCTCAAGCTGGTACCGATGCTGTTGACGGTGCTTC和A762-rev(SEQ ID NO:152):ACCAGCTTGAGCTGCAGC,其他位点A762X突变(X代表除F外其他18种氨基酸)分别采用引物A762X(SEQ ID NO:153):GCTCAAGCTGGTXXXGATGCTGTTGACGGTGCTTC(XXX是不同氨基酸对应的密码子)和A762-rev(SEQ ID NO:152)引入。
P824L突变采用引物P824L(SEQ ID NO:154):TTGAGTCTGGAACCTTGGGCCCAACCGGTCGC和P824-rev(SEQ ID NO:155):GGTTCCAGACTCAAATGGCAGG,其他位点P824X突变(X代表除F外其他18种氨基酸)分别采用引物P824X(SEQ ID NO:156):TTGAGTCTGGAACCXXXGGCCCAACCGGTCGC(XXX是不同氨基酸对应的密码子)和P824-rev(SEQ ID NO:155)引入。
P826T突变采用引物P826T(SEQ ID NO:157):CTGGAACCCCAGGCACCACCGGTCGCGTCTAC和P826-rev(SEQ ID NO:158):GCCTGGGGTTCCAGACTC,其他位点P826X突变(X代表除F外其他18种氨基酸)分别采用引物G837X(SEQ ID NO:159):CTGGAACCCCAGGCXXXACCGGTCGCGTCTAC(XXX是不同氨基酸对应的密码子)和P826-rev(SEQ ID NO:158)引入。
P1031S突变采用引物P1031S(SEQ ID NO:160):TGCGATCTCTGAGTCCGACGATAAGGGTATGC和P1031-rev(SEQ ID NO:161):CTCAGAGATCGCATCCAGGC,其他位点P1031X突变(X代表除F外其他18种氨基酸)分别采用引物P1031X(SEQ ID NO:162):TGCGATCTCTGAGXXXGACGATAAGGGTATGC(XXX是不同氨基酸对应的密码子)和P1031-rev(SEQ ID NO:161)引入。将所有突变体转化P2感受态细胞，筛选获得苹果酸的结果如图30-图37所示：对其中的正突变效果较好的突变点，选取A762D、P824V、P826E和P1031E进行组合突变(这四个点分可以提升苹果酸35.7％、29.4％、28.3％和34.2％)，采用和饱和突变同样的方法，同样对组合突变转化P2菌株，产苹果酸结果图38，其中A726D/P826E和A762D/P824E/P1031E分别可以提升苹果酸41.4％和42.3％，最终苹果酸合成能力达到75.2 g/L和75.5 g/L。
实施例5 Aomae在酿酒酵母合成苹果酸中的应用
一、pyc、mdh和AoMae共表达质粒载体构建，引物如下：
Figure BDA0001835380250000311
以酿酒酵母CEN PK.2-1C DNA和米曲霉cDNA为模板，分别PCR扩增ScPYC2,AoMae和MDH3(去掉线粒体定位序列SKL)，三个基因片段通过gibson方法连接于Prs426-pgk-CYCtt载体的EcoRI和HindIII，三基因通过2A肽(raegrgslltcgdveenpg)构成单一顺反子，获得表达质粒Prs426-PGK-ScPYC2-Aomae-MDH3ΔSKL。
二、产苹果酸酿酒酵母构建
将质粒Prs426-PGK-ScPYC2-Aomae-MDH3ΔSKL通过LiAc/SS Carrier DNA的方法(Methods Mol Biol.2014；1205:1-12.)转化入酿酒酵母CEN.PK2-1C,采用和毕赤酵母产苹果酸相同的培养基进行苹果酸合成，结果如图39所示，结果表明100 g/l葡萄糖可以产22g/l苹果酸。
实施例6改造C4-二羧酸转运蛋白编码基因mae提高嗜热毁丝霉苹果酸合成能力
一、mae过表达载体的构建
从米曲霉DSM1863(DSMZ，购自德国微生物和细胞培养有限公司)的cDNA和质粒pGAP-hph-his-AoMaeKA，分别扩增出C4-二羧酸转运蛋白编码基因mae(XM_001820829.2，SEQ ID NO:1)及其突变后的基因maeKA，经BglII酶切后，连接入经BglII及EcoRV酶切后线性化载体pAN52-hph，将连接产物用限制性内切酶进行酶切鉴定，得到过表达载体，命名为pAN52-Wtmae和pAN52-maeKA。引物如下所示：
mae-F SEQ ID NO:169GGAAGATCTTAATTAACTCGAGCGGCCGCGTTTAAACACTAGTATGCTGACACCTCCCAAGTTTG
mae-R SEQ ID NO:170ATCCTAATCAGATACAT CCTCATCTTTA
以出发菌株嗜热毁丝霉ATCC42464(购自美国模式培养物集存库，American typeculture collection)基因为模板，PCR扩增出翻译延长因子编码阅读框(MYCTH_2298136)上游1.4kb的启动子(命名为Ptef启动子)，引物如下所示：
tef-F SEQ ID NO:171CCTTAATTAACATGTACCTTGACGTCCTCCGAG
tef-R SEQ ID NO:172GGACTAGTTCTGAAGAACGAAACTGGC GACT
PCR反应结束后，经PacI和SpeI酶切，连接入经同样酶双酶切的线性化载体pAN52-hph-mae，将连接产物用限制性内切酶进行酶切鉴定，得到在启动子tef调控下mae基因表达载体，命名为pAN52-Ptef-Wtmae和pAN52-Ptef-maeKA。
二、将表达载体导入嗜热毁丝霉
A.将嗜热毁丝霉ATCC42464在MM斜面培养基[50×Vogel’s盐20ml，蔗糖20g，琼脂15g，组氨酸(50mg/ml)20ml，定容体积到1l，高压灭菌。50×Vogel’s盐(1l)：柠檬酸三钠(1/2H2O)150g，无水KH2PO4 250g，无水NH4NO3 100g，MgSO4·7H2O 10g，CaCl2·2H2O 5g，微量元素盐溶液5mL，生物素(0.1mg/ml)2.5mL，定容体积到1l。
B.嗜热毁丝霉原生质体转化
1)菌丝体准备
将成熟的嗜热毁丝霉孢子，用0.05％吐温80灭菌水收集，经擦镜纸过滤出去菌丝后，涂布于铺有玻璃纸的MM平板，45℃培养14h。
2)原生质体制备
将带有菌丝的玻璃纸放置于30mL裂解液(配方：0.15g裂解酶，无菌操作加入30mL溶液A，过滤除菌；溶液A:1.0361g磷酸二氢钾，21.864g山梨醇，溶于90ml去离子水，氢氧化钾调PH到5.6，定量至100ml，高温灭菌)中，28℃裂解2h，每隔20min轻轻摇动。
而后经过玻璃纸过滤后，2000rpm 4℃离心10min，弃上清，加入4mL溶液B(0，735g氯化钙，18，22g山梨醇，1ml Tris HCl 1M pH 7.5，溶于90ml去离子水，盐酸调PH到7.6，定量至100ml，高温灭菌)，2000rpm 4℃离心10min；弃上清，按200μl/质粒加入一定体积溶液B。
3)原生质体转化
预冷的15ml离心管，依次加入50ul预冷PEG(12.5g PEG6000，0，368g氯化钙，500μlTris HCl 1M pH 7.5。)，10μl用HindIII线性化的质粒，200μl原生质体。放置冰上20min后加入2ml预冷PEG，室温5min，加入4mL溶液B，轻轻混匀。取3ml上述溶液加入12ml融化的含相应抗生素MM培养基中，置于平板中，45℃培养，2d-4d后于体式显微镜下挑取单个菌丝体于相应抗性平板生长，而后用PCR进行验证。
三、嗜热毁丝霉转化子生产苹果酸能力测定
将上述验证的转化子全部接种至250ml三角瓶中的50mL以结晶纤维素(Avicel)为碳源培养基中(配方：碳源75g/l，蛋白胨6.0g/l，0.15g/l KH2PO4，0.15g/l K2HPO4，0.10g/lCaCl2·2H2O，0.10g/l MgSO4·7H2O，碳酸钙80.0g/l，1mL/L0.5g/l生物素，1ml/l微量元素液；微量元素配方(100ml)：5g C6H8O·7H2O，5g ZnSO4·7H2O，1g Fe(NH4)2(SO4)·6H2O，0.25g CuSO4·5H2O，0.05g MnSO4·H2O，0.05g H3BO3，0.05g NaMoO4·2H2O，溶于水中，定容至100ml)，接种量为2.5*105个/ml，45℃，150rpm培养，第八天取样测定苹果酸含量。
1)样品处理：
取1ml发酵液于15ml离心管中，并添加1ml 1M H2SO4，而后80℃下放置30min，每个隔10min进行充分震荡。之后将2ml双蒸水添加至离心管中，充分震荡后，取1ml液体于1.5ml离心管中，12000rpm离心10min，取上清液测定苹果酸含量。
2)苹果酸含量测定
处理后的样品用高效液相色谱测定苹果酸含量，其中检测器为紫外检测器，5mMH2SO4为流动相，流速为0.5ml/min。结果显示苹果酸转运蛋白经过改造后，相比野生型转运蛋白，能显著促进苹果酸的生产。第八天时，转化子MaeKA的苹果酸产量为46g/l，相比对照菌株WtMae(42g/l)提高9.5％。实验说明C4-二羧酸转运蛋白经过改造后，能够显著提高嗜热真菌嗜热毁丝霉苹果酸生产能力。
实施例7改造还原型TCA关键酶丙酮酸羧化酶提高嗜热毁丝霉苹果酸合成能力
一、过表达载体的构建
从谷氨酸棒状杆菌ATCC13032基因组和和质粒pGAPzA-his-CgPycDE分别扩增出丙酮酸羧化酶基因Cgpyc及其突变后的基因CgpycA726D/P826E，经SpeI和EcoRV酶切后，连接入经相同内切酶酶切后线性化载体pAN52-C1pgdA-neo，将连接产物用限制性内切酶进行酶切鉴定，得到过表达载体，命名为pAN52-Wtpyc和pAN52-pycA726D/P826E。引物如下所示
CgPYC-F SEQ ID NO:173GGACTAGTATGTCGACTCACACATCTTCA
CgPYC-R SEQ ID NO:174AAAAAGATATCTTAGGAAACGACGACGATCAAG
二、嗜热毁丝霉转化子生产苹果酸能力的测定
将过表达载体pAN52-Wtpyc和pAN52-pycA726D/P826E经BglII线性化后整合至嗜热毁丝霉WtMae菌株基因组，以终浓度为100μg/ml G418为筛选抗生素，其方法见实施例1步骤2。利用引物CgPYC-F和CgPYC-R，验证得到转化子，命名为WtPYC和PYCA726D/P826E。
将验证的转化子全部接种至250ml三角瓶中的50ml以结晶纤维素(Avicel)为碳源培养基中(配方见实施例1步骤3)，接种量为2.5*105个/ml，45℃，150rpm培养，第八天取样。样品经实施例1步骤3.2所述方法进行处理后，测定发酵液中苹果酸含量。
结果显示相比野生型丙酮酸羧化酶，其突变后能够显著提高转化子的苹果酸产量。第八天时，转化子WtPYC的苹果酸产量为49.9g/l，而过表达突变pyc转化子的苹果酸产量达到57.9g/l，提高16％。实验说明经过蛋白改造提高丙酮酸活性后，能够显著提高嗜热毁丝霉苹果酸合成能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 提高苹果酸产量的新突变蛋白
<130> P2018-1764
<160> 176
<170> PatentIn version 3.5
<210> 1
<211> 1140
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 1
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
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Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
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Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
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Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
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Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
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Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
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Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
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Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
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Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
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Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
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Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
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Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
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Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
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Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
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Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
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Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
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Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
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Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
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Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
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Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
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Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
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Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
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Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
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Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
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Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
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Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
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Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
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Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
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Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 2
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 2
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 3
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 3
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 4
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 4
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 5
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 5
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 6
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 6
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Asp Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 7
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 7
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Lys Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 8
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 8
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe Asn Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 9
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 9
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Glu Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 10
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 10
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 11
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 11
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 12
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 12
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 13
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 13
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 14
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 14
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 15
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 15
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 16
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 16
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 17
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 17
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 18
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 18
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 19
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 19
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 20
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 20
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Asp Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Val Gly Glu Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Glu Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 21
<211> 3423
<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 21
atgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540
gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600
gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660
gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac 900
ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960
gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020
atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080
ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140
cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200
aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgagttca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg 1320
gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca cccgcacctc 1380
cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500
cctaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740
gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800
ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860
ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacactgc gggtggccag ctggcaacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400
gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460
tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagttg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700
gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ccgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420
taa 3423
<210> 22
<211> 380
<212> PRT
<213> 米曲霉(Aspergillus oryzae)
<400> 22
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 23
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 23
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Lys
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 24
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 24
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Ala
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 25
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 25
Met Leu Thr Pro Pro Lys Phe Gln Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 26
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 26
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Ile Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 27
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 27
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Val Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 28
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 28
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Tyr Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 29
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 29
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Glu Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 30
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 30
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Lys Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 31
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 31
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Trp Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 32
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 32
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Asn Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 33
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 33
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Ile Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 34
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 34
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Arg Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 35
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 35
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Gln Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 36
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 36
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Ser Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 37
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 37
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro Phe Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 38
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 38
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Met Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 39
<211> 213
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 39
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr
210
<210> 40
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 40
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Ile
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Glu Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Arg
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 41
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 41
Met Leu Thr Pro Pro Lys Phe Glu Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Lys
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Ala
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 42
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白(mutant protein)
<400> 42
Met Leu Thr Pro Pro Lys Phe Gln Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Lys
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Phe Phe Pro Thr Phe Trp Leu Ser Val Ala Thr Ile Ile Cys Gly Leu
100 105 110
Ser Arg Tyr Phe Gly Glu Glu Ser Asn Glu Ser Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Leu Leu Val Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Pro Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Ala Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Phe Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Glu Ser Gly
210 215 220
Leu Pro His Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Met Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Ala
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Ile Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380
<210> 43
<211> 1143
<212> DNA
<213> 米曲霉(Aspergillus oryzae)
<400> 43
atgctgacac ctcccaagtt tgaggatgag aagcagctgg gccccgtggg tatccgggag 60
aggcttcgcc atttcacttg ggcctggtac acattaacga tgagtggagg agggctggcc 120
gtcctcatca tcagccagcc ctttgggttc cgcggattga gagagatcgg catcgctgtc 180
tatatcctca acctgatcct cttcgccctt gtctgctcta ccatggctat aaggttcatc 240
ctgcacggca accttctgga gtccctccgt catgaccgcg agggtctctt cttcccgacc 300
ttctggctct ccgtcgcaac catcatctgc ggcttgtctc gctacttcgg tgaagaatcg 360
aatgagtcct tccaactagc cctcgaagcc ctcttctgga tctactgcgt ctgcacctta 420
ctcgtcgcaa tcatccaata ctcgttcgtc ttctcatccc acaagtacgg ccttcaaacc 480
atgatgcctt catggatcct tccagccttc cccatcatgc tcagcggcac catcgcctcc 540
gtcatcggtg aacaacaacc cgctcgcgca gccctcccca tcatcggcgc cggcgtcacc 600
ttccagggcc tcggcttctc catcagcttc atgatgtacg cccactacat cggccgactg 660
atggagtccg gcctccccca cagcgaccac agaccaggca tgttcatctg cgtcggaccc 720
cccgccttca cagccctcgc cctcgtcggc atgagcaaag gcctccccga agacttcaag 780
ctgctccacg acgcccacgc cctggaagat ggccgcatca tcgagctgct ggccatctcc 840
gccggcgtct tcctctgggc cctgagtctc tggttcttct gcatcgccat tgtcgccgtc 900
atccgctcgc cccccgaggc cttccacctc aactggtggg ccatggtctt ccccaacacc 960
ggcttcaccc tggccaccat caccctgggc aaggctctca acagtaacgg tgtgaagggc 1020
gtcggttccg ccatgtctat ctgcatcgtg tgcatgtata tcttcgtctt cgtcaacaat 1080
gtccgcgccg ttatccggaa ggatatcatg tacccgggta aagatgagga tgtatctgat 1140
tag 1143
<210> 44
<211> 43
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 44
ccaccgcccg ttaccgtccg aaggaaattt tactctgctg gag 43
<210> 45
<211> 43
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 45
ctccagcaga gtaaaatttc cttcggacgg taacgggcgg tgg 43
<210> 46
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 46
gggttcgaaa cgatggccga agaagactac tccc 34
<210> 47
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 47
ggggcggccg cttactcagc cttgacgatt ttggcgatc 39
<210> 48
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 48
gggttcgaaa cgatggttaa agtcacagtt tgcggag 37
<210> 49
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 49
ggggcggccg cttagttgcc agcaatgaag gcagttcc 38
<210> 50
<211> 30
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 50
gggggatcct gtacagcttg cctcgtcccc 30
<210> 51
<211> 24
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 51
gtcgacactg gatggcggcg ttag 24
<210> 52
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 52
gggagatctg ttgtaacact ggcagagcat tacg 34
<210> 53
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 53
gggggatccg tcccagtttc tccatacgaa cc 32
<210> 54
<211> 33
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 54
gggatcgata tgggtgaact caaggaaatc ttg 33
<210> 55
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 55
ggggcggccg cttaaacgct ttcatgttca ctactagg 38
<210> 56
<211> 31
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 56
gggttcgaaa tgctgacacc tcccaagttt g 31
<210> 57
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 57
ggggcggccg cctaatcaga tacatcctca tctttac 37
<210> 58
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 58
ggggaattca tggctgcaga atcaatagtg tctc 34
<210> 59
<211> 43
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 59
ggggcggccg cttatgttga atttcttgct tgaaatttag atc 43
<210> 60
<211> 24
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 60
ctgcgattct tattcttccg attg 24
<210> 61
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 61
caaccgagga caaccaacag 20
<210> 62
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 62
catcatctgc ggcttgtc 18
<210> 63
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 63
tgagaagacg aacgagtatt g 21
<210> 64
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 64
ccatggctat aaggttcttc ctgcacggca accttctg 38
<210> 65
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 65
gaaccttata gccatggtag ag 22
<210> 66
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 66
ccatggctat aaggttcnnn ctgcacggca accttctg 38
<210> 67
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 67
gcgtctgcac cttactcgac gcaatcatcc aatactcg 38
<210> 68
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 68
gagtaaggtg cagacgcagt ag 22
<210> 69
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 69
gcgtctgcac cttactcnnn gcaatcatcc aatactcg 38
<210> 70
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 70
cacgccctgg aagatggccc aatcatcgag ctgctggc 38
<210> 71
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 71
cacgccctgg aagatggcnn natcatcgag ctgctggc 38
<210> 72
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 72
ctgacacctc ccaagtttgg tgatgagaag cagctggg 38
<210> 73
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 73
aaacttggga ggtgtcagc 19
<210> 74
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 74
ctgacacctc ccaagtttnn ngatgagaag cagctggg 38
<210> 75
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 75
tctccgtcgc aaccatcgtc tgcggcttgt ctcgctac 38
<210> 76
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 76
gatggttgcg acggagag 18
<210> 77
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 77
tctccgtcgc aaccatcnnn tgcggcttgt ctcgctac 38
<210> 78
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 78
gtgaagaatc gaatgagaga ttccaactag ccctcgaag 39
<210> 79
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 79
ctcattcgat tcttcaccg 19
<210> 80
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 80
gtgaagaatc gaatgagnnn ttccaactag ccctcgaag 39
<210> 81
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 81
cgccattgtc gccgtcaagc gctcgccccc cgaggcc 37
<210> 82
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 82
gacggcgaca atggcgatg 19
<210> 83
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (17)..(19)
<223> n is a, c, g,或t
<400> 83
cgccattgtc gccgtcnnnc gctcgccccc cgaggcc 37
<210> 84
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 84
gccttcaaac catgatgcaa tcatggatcc ttccagcc 38
<210> 85
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 85
catcatggtt tgaaggcc 18
<210> 86
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 86
gccttcaaac catgatgnnn tcatggatcc ttccagcc 38
<210> 87
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 87
ggtctcttct tcccgaccca ctggctctcc gtcgcaacc 39
<210> 88
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 88
ggtcgggaag aagagaccc 19
<210> 89
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 89
ggtctcttct tcccgaccnn ntggctctcc gtcgcaacc 39
<210> 90
<211> 36
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 90
tctactgcgt ctgcaccaac ctcgtcgcaa tcatcc 36
<210> 91
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 91
ggtgcagacg cagtagatc 19
<210> 92
<211> 36
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 92
tctactgcgt ctgcaccnnn ctcgtcgcaa tcatcc 36
<210> 93
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 93
catgaccgcg agggtctcgg gttcccgacc ttctggctc 39
<210> 94
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 94
gagaccctcg cggtcatg 18
<210> 95
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 95
catgaccgcg agggtctcnn nttcccgacc ttctggctc 39
<210> 96
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 96
caacaacccg ctcgcgcatg tctccccatc atcggcgc 38
<210> 97
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 97
tgcgcgagcg ggttgttg 18
<210> 98
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 98
caacaacccg ctcgcgcatg tctccccatc atcggcgc 38
<210> 99
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 99
tctgcggctt gtctcgcatc ttcggtgaag aatcgaatg 39
<210> 100
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 100
gcgagacaag ccgcagatg 19
<210> 101
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 101
tctgcggctt gtctcgcnnn ttcggtgaag aatcgaatg 39
<210> 102
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 102
tcagcttcat gatgtacggg cactacatcg gccgactg 38
<210> 103
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 103
gtacatcatg aagctgatg 19
<210> 104
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 104
tcagcttcat gatgtacnnn cactacatcg gccgactg 38
<210> 105
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 105
cggcgccggc gtcaccaagc agggcctcgg cttctcc 37
<210> 106
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 106
ggtgacgccg gcgccgatg 19
<210> 107
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (17)..(19)
<223> n is a, c, g,或t
<400> 107
cggcgccggc gtcaccnnnc agggcctcgg cttctcc 37
<210> 108
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 108
atggagtccg gcctcccctt cagcgaccac agaccaggc 39
<210> 109
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 109
ggggaggccg gactccatc 19
<210> 110
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 110
atggagtccg gcctccccnn nagcgaccac agaccaggc 39
<210> 111
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 111
ccctcgccct cgtcggctac agcaaaggcc tccccgaag 39
<210> 112
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 112
gccgacgagg gcgagggc 18
<210> 113
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 113
ccctcgccct cgtcggcnnn agcaaaggcc tccccgaag 39
<210> 114
<211> 36
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 114
tacatcggcc gactgatgca ctccggcctc ccccac 36
<210> 115
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 115
catcagtcgg ccgatgtag 19
<210> 116
<211> 36
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 116
tacatcggcc gactgatgnn ntccggcctc ccccac 36
<210> 117
<211> 30
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 117
gggggatcct gtacagcttg cctcgtcccc 30
<210> 118
<211> 24
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 118
gtcgacactg gatggcggcg ttag 24
<210> 119
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 119
gggagatctg ttgtaacact ggcagagcat tacg 34
<210> 120
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 120
gggggatccg tcccagtttc tccatacgaa cc 32
<210> 121
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 121
gggttcgaaa tggccgcgcc gcaacgccaa cccg 34
<210> 122
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 122
ggggcggccg ctcatcaggc cttgccgatc ttac 34
<210> 123
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 123
gggttcgaaa tgcccattag caagatcctc gtggc 35
<210> 124
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 124
ggggcggccg ctcagccgcc gtagaccgcg aggag 35
<210> 125
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 125
gggttcgaaa tggcggctcc gtttcgtcag cc 32
<210> 126
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 126
ggggcggccg cctattacgc tttgacgatc ttgc 34
<210> 127
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 127
aattgaacaa ctatttcgaa atgcctgctg cacc 34
<210> 128
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 128
agaaagctgg cggccgctta ggcttcctct ttgac 35
<210> 129
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 129
caattgaaca actatttcga aatgtcgact cacac 35
<210> 130
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 130
ctagaaagct ggcggccgcc tattaggaaa cgac 34
<210> 131
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 131
gggttcgaaa tggccgaaga agactactcc ccgc 34
<210> 132
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 132
ggggcggccg cttactcagc cttgacgatt ttggc 35
<210> 133
<211> 26
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 133
tgggccttgc ggatcgtttc gagctc 26
<210> 134
<211> 27
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 134
acgatccgca aggcccagtg cggtggc 27
<210> 135
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 135
tgctgcttcg cggtcgcaac 20
<210> 136
<211> 23
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 136
cgaagcagca tctgaatgtt tac 23
<210> 137
<211> 29
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 137
gggttcgaaa tgtcgactca cacatcttc 29
<210> 138
<211> 33
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 138
ggggcggccg cttaggaaac gacgacgatc aag 33
<210> 139
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 139
gaagatcggg gatcattcga gcgctctttt gcttctg 37
<210> 140
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 140
gaatgatccc cgatcttc 18
<210> 141
<211> 37
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 141
gaagatcggg gatcattcnn ncgctctttt gcttctg 37
<210> 142
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 142
agcgcatcgc caccggacaa attgccgatc acccgcac 38
<210> 143
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 143
tccggtggcg atgcgcttg 19
<210> 144
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (18)..(20)
<223> n is a, c, g,或t
<400> 144
agcgcatcgc caccggannn attgccgatc acccgcac 38
<210> 145
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 145
agttgtcgaa attgcgccaa accagcattt ggatccag 38
<210> 146
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 146
tggcgcaatt tcgacaac 18
<210> 147
<211> 38
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 147
agttgtcgaa attgcgccaa accagcattt ggatccag 38
<210> 148
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 148
taccgccacg aaatcccaat gggacagttg tccaacctg 39
<210> 149
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 149
tgggatttcg tggcggtag 19
<210> 150
<211> 39
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (19)..(21)
<223> n is a, c, g,或t
<400> 150
taccgccacg aaatcccann nggacagttg tccaacctg 39
<210> 151
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 151
gctcaagctg gtaccgatgc tgttgacggt gcttc 35
<210> 152
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 152
accagcttga gctgcagc 18
<210> 153
<211> 35
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (13)..(15)
<223> n is a, c, g,或t
<400> 153
gctcaagctg gtnnngatgc tgttgacggt gcttc 35
<210> 154
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 154
ttgagtctgg aaccttgggc ccaaccggtc gc 32
<210> 155
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 155
ggttccagac tcaaatggca gg 22
<210> 156
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (15)..(17)
<223> n is a, c, g,或t
<400> 156
ttgagtctgg aaccnnnggc ccaaccggtc gc 32
<210> 157
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 157
ctggaacccc aggcaccacc ggtcgcgtct ac 32
<210> 158
<211> 18
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 158
gcctggggtt ccagactc 18
<210> 159
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (15)..(17)
<223> n is a, c, g,或t
<400> 159
ctggaacccc aggcnnnacc ggtcgcgtct ac 32
<210> 160
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 160
tgcgatctct gagtccgacg ataagggtat gc 32
<210> 161
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 161
ctcagagatc gcatccaggc 20
<210> 162
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<220>
<221> misc_feature
<222> (14)..(16)
<223> n is a, c, g,或t
<400> 162
tgcgatctct gagnnngacg ataagggtat gc 32
<210> 163
<211> 57
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 163
caactagtgg atcccccggg ctgcaggaat tcatgagcag tagcaagaaa ttggccg 57
<210> 164
<211> 68
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 164
ctcaacgtca ccgcaagtaa gcaaagaacc tctaccctca gctctctttt tttgggatgg 60
gggtaggg 68
<210> 165
<211> 66
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 165
ctttgcttac ttgcggtgac gttgaggaaa acccaggtcc aatgctgaca cctcccaagt 60
ttgagg 66
<210> 166
<211> 64
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 166
catctccgca ggtcaaaagg gatccacgtc cttcggcacg atcagataca tcctcatctt 60
tacc 64
<210> 167
<211> 76
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 167
gacgtggatc ccttttgacc tgcggagatg tcgaagagaa tcctggacct atggtcaaag 60
tcgcaattct tggcgc 76
<210> 168
<211> 57
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 168
cccctcgagg tcgacggtat cgataagctt tcaagagtct aggatgaaac tcttgcc 57
<210> 169
<211> 65
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 169
ggaagatctt aattaactcg agcggccgcg tttaaacact agtatgctga cacctcccaa 60
gtttg 65
<210> 170
<211> 28
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 170
atcctaatca gatacatcct catcttta 28
<210> 171
<211> 33
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 171
ccttaattaa catgtacctt gacgtcctcc gag 33
<210> 172
<211> 31
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 172
ggactagttc tgaagaacga aactggcgac t 31
<210> 173
<211> 29
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 173
ggactagtat gtcgactcac acatcttca 29
<210> 174
<211> 33
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> 引物 (primer)
<400> 174
aaaaagatat cttaggaaac gacgacgatc aag 33
<210> 175
<211> 1140
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白 (mutant protein)
<220>
<221> misc_feature
<222> (51)..(51)
<223> Xaa=N,或G
<220>
<221> misc_feature
<222> (254)..(254)
<223> Xaa=K, E, R,或F
<220>
<221> misc_feature
<222> (453)..(453)
<223> Xaa=E,或P
<220>
<221> misc_feature
<222> (762)..(762)
<223> Xaa=D, P, V,或T
<220>
<221> misc_feature
<222> (824)..(824)
<223> Xaa=V, A, I, Y, L, M, N, R,或T
<220>
<221> misc_feature
<222> (826)..(826)
<223> Xaa=E, Q, Y, T, D,或I
<220>
<221> misc_feature
<222> (837)..(837)
<223> Xaa=D,或H
<220>
<221> misc_feature
<222> (1031)..(1031)
<223> Xaa=E, N, S,或G
<400> 175
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe Xaa Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Xaa Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Xaa Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Xaa Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Xaa Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Xaa Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Xaa Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 176
<211> 380
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 突变蛋白 (mutant protein)
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa=Q, Y, R,或P
<220>
<221> misc_feature
<222> (80)..(80)
<223> Xaa=K, W,或C
<220>
<221> misc_feature
<222> (97)..(97)
<223> Xaa=V,或R
<220>
<221> misc_feature
<222> (101)..(101)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> misc_feature
<222> (109)..(109)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> misc_feature
<222> (115)..(115)
<223> Xaa=E,或L
<220>
<221> misc_feature
<222> (123)..(123)
<223> Xaa=K, H, R,或D
<220>
<221> misc_feature
<222> (140)..(140)
<223> Xaa=N, K,或H
<220>
<221> misc_feature
<222> (142)..(142)
<223> Xaa=I, S,或C
<220>
<221> misc_feature
<222> (163)..(163)
<223> Xaa=I, V, F, L, Q,或Y
<220>
<221> misc_feature
<222> (191)..(191)
<223> Xaa=R,或K
<220>
<221> misc_feature
<222> (201)..(201)
<223> Xaa=Q, G, N,或K
<220>
<221> misc_feature
<222> (222)..(222)
<223> Xaa=S,或H
<220>
<221> misc_feature
<222> (227)..(227)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> misc_feature
<222> (251)..(251)
<223> Xaa=E,或G
<220>
<221> misc_feature
<222> (272)..(272)
<223> Xaa=A,或F
<220>
<221> misc_feature
<222> (301)..(301)
<223> Xaa=W, F, H, K,或Y
<400> 176
Met Leu Thr Pro Pro Lys Phe Xaa Asp Glu Lys Gln Leu Gly Pro Val
1 5 10 15
Gly Ile Arg Glu Arg Leu Arg His Phe Thr Trp Ala Trp Tyr Thr Leu
20 25 30
Thr Met Ser Gly Gly Gly Leu Ala Val Leu Ile Ile Ser Gln Pro Phe
35 40 45
Gly Phe Arg Gly Leu Arg Glu Ile Gly Ile Ala Val Tyr Ile Leu Asn
50 55 60
Leu Ile Leu Phe Ala Leu Val Cys Ser Thr Met Ala Ile Arg Phe Xaa
65 70 75 80
Leu His Gly Asn Leu Leu Glu Ser Leu Arg His Asp Arg Glu Gly Leu
85 90 95
Xaa Phe Pro Thr Xaa Trp Leu Ser Val Ala Thr Ile Xaa Cys Gly Leu
100 105 110
Ser Arg Xaa Phe Gly Glu Glu Ser Asn Glu Xaa Phe Gln Leu Ala Leu
115 120 125
Glu Ala Leu Phe Trp Ile Tyr Cys Val Cys Thr Xaa Leu Xaa Ala Ile
130 135 140
Ile Gln Tyr Ser Phe Val Phe Ser Ser His Lys Tyr Gly Leu Gln Thr
145 150 155 160
Met Met Xaa Ser Trp Ile Leu Pro Ala Phe Pro Ile Met Leu Ser Gly
165 170 175
Thr Ile Ala Ser Val Ile Gly Glu Gln Gln Pro Ala Arg Ala Xaa Leu
180 185 190
Pro Ile Ile Gly Ala Gly Val Thr Xaa Gln Gly Leu Gly Phe Ser Ile
195 200 205
Ser Phe Met Met Tyr Ala His Tyr Ile Gly Arg Leu Met Xaa Ser Gly
210 215 220
Leu Pro Xaa Ser Asp His Arg Pro Gly Met Phe Ile Cys Val Gly Pro
225 230 235 240
Pro Ala Phe Thr Ala Leu Ala Leu Val Gly Xaa Ser Lys Gly Leu Pro
245 250 255
Glu Asp Phe Lys Leu Leu His Asp Ala His Ala Leu Glu Asp Gly Xaa
260 265 270
Ile Ile Glu Leu Leu Ala Ile Ser Ala Gly Val Phe Leu Trp Ala Leu
275 280 285
Ser Leu Trp Phe Phe Cys Ile Ala Ile Val Ala Val Xaa Arg Ser Pro
290 295 300
Pro Glu Ala Phe His Leu Asn Trp Trp Ala Met Val Phe Pro Asn Thr
305 310 315 320
Gly Phe Thr Leu Ala Thr Ile Thr Leu Gly Lys Ala Leu Asn Ser Asn
325 330 335
Gly Val Lys Gly Val Gly Ser Ala Met Ser Ile Cys Ile Val Cys Met
340 345 350
Tyr Ile Phe Val Phe Val Asn Asn Val Arg Ala Val Ile Arg Lys Asp
355 360 365
Ile Met Tyr Pro Gly Lys Asp Glu Asp Val Ser Asp
370 375 380

Claims (9)

1.一种丙酮酸羧化酶突变蛋白，其特征在于，所述丙酮酸羧化酶突变蛋白基于SEQ IDNO:1所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
A762D、A762P、A762V或A762T；和/或
P826E、P826Q、P826Y、P826T、P826D或P826I；
且所述的丙酮酸羧化酶突变蛋白具有将丙酮酸羧化合成苹果酸前体草酰乙酸的活性。
2.一种苹果酸转运蛋白突变蛋白，其特征在于，所述苹果酸转运蛋白突变蛋白基于SEQID NO:22所示的序列，具有选自以下一个或多个位点的核心氨基酸突变：
I80K、I80W、或I80C；和
R272A、或R272F；
且所述的苹果酸转运蛋白突变蛋白具有将苹果酸从细胞内转运至细胞外的活性。
3.一种分离的多核苷酸或其组合，其特征在于，所述多核苷酸或其组合编码权利要求1所述的丙酮酸羧化酶突变蛋白和/或权利要求2所述的苹果酸转运蛋白突变蛋白。
4.一种载体，其特征在于，所述载体含有权利要求3所述的分离的多核苷酸。
5.一种宿主细胞，其特征在于，所述宿主细胞含有权利要求4所述的载体，或所述宿主细胞的核酸中整合有权利要求3所述的分离的多核苷酸。
6.一种制备权利要求1所述丙酮酸羧化酶突变蛋白和/或权利要求2所述苹果酸转运蛋白突变蛋白的方法，其特征在于，包括步骤：
在适合表达的条件下，培养权利要求5所述的宿主细胞，从而表达权利要求1所述丙酮酸羧化酶突变蛋白和/或权利要求2所述苹果酸转运蛋白突变蛋白；和
分离表达产物，从而获得权利要求1所述丙酮酸羧化酶突变蛋白和/或权利要求2所述苹果酸转运蛋白突变蛋白。
7.一种提高苹果酸产量的方法，其特征在于，包括步骤：
i)将权利要求2所述的多核苷酸或其组合导入合适的宿主细胞；
ii)在碳源的存在下，培养所述的宿主细胞，从而提高苹果酸产量。
8.一种突变蛋白组合，其特征在于，所述突变蛋白组合含有权利要求1所述的丙酮酸羧化酶突变蛋白，和权利要求2所述的苹果酸转运蛋白突变蛋白。
9.权利要求1所述丙酮酸羧化酶突变蛋白和/或权利要求2所述苹果酸转运蛋白突变蛋白、权利要求3所述分离的多核苷酸、权利要求4所述载体、权利要求5所述宿主细胞、或权利要求8所述突变蛋白组合的应用，其特征在于，用于提高苹果酸产量和/或用于制备苹果酸。
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