CN113913401A - 经修饰的谷氨酸脱氢酶及其应用 - Google Patents

经修饰的谷氨酸脱氢酶及其应用 Download PDF

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CN113913401A
CN113913401A CN202110772483.7A CN202110772483A CN113913401A CN 113913401 A CN113913401 A CN 113913401A CN 202110772483 A CN202110772483 A CN 202110772483A CN 113913401 A CN113913401 A CN 113913401A
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谢新开
徐伟
范俊英
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Abstract

本发明涉及经修饰的谷氨酸脱氢酶(GluDH)。具体而言，本发明的经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L‑草铵膦的活性和/或改进的动力学性质。本发明还涉及编码本发明的经修饰的GluDH的多核苷酸、表达本发明的经修饰的GluDH的载体和宿主细胞以及使用本发明的经修饰的GluDH和宿主细胞生产L‑草铵膦的方法。

Description

经修饰的谷氨酸脱氢酶及其应用
技术领域
本发明涉及酶工程领域。具体而言，本发明涉及经修饰的谷氨酸脱氢酶(GluDH)及其在生产草铵膦中的应用。
背景技术
草铵膦(glufosinate，也称为4-[羟基(甲基)膦酰基]-D,L-高丙氨酸)是世界销量第二的转基因作物耐受的除草剂。草铵膦是一种广谱触杀型除草剂，通过抑制植物体内的L-谷氨酰胺合成酶的活性，导致植物体内氮代谢紊乱，最终杀死植物。与草甘膦相比，草铵膦具有显著优势，如应用范围广、见效快、持效期长、更低毒、安全等。因此，草铵膦的销量增长迅速，在未来一段时间内市场需求巨大，前景非常广阔。
但是，草铵膦的工艺路线复杂，导致产品生产技术难度高。高昂的价格阻碍其迅速取代草甘膦。目前市售的草铵膦是包含等量的两种光学异构体的外消旋混合物(D,L-草铵膦)，但其中只有L-构型具有生理活性。因此，通过D,L-草铵膦去消旋化制备手性纯的L-草铵膦具有重要现实意义，成为近年来合成L-草铵膦的热门方向。
近年来报道从D,L-草铵膦制备L-草铵膦的方法众多。传统的化学修饰拆分法因成本高且不能利用D-型草铵膦而不具备竞争性。目前报道了下列将D-草铵膦转化成为L-草铵膦的主要代表性技术路线：
将D,L-草铵膦转化成N-乙酰基草铵膦，再经羧肽酶催化，L型N-乙酰基草铵膦经选择性水解，获得L-草铵膦，而D型N-乙酰基草铵膦不水解，可经化学或酶法消旋化后再次循环进入水解步骤(参见例如中国专利申请CN108690854A)。该方法的缺点是需多步反应，还有需要将经水解获得的L-草铵膦与N-乙酰化的底物分离。
将D-草铵膦氧化成2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)，再对PPO进行还原或转氨生成L-草铵膦。在大部分文献中，使用D-氨基酸氧化酶(DAAO)催化将D-草铵膦氧化成PPO，而PPO可以在钯碳催化下被甲酸还原生成D,L-草铵膦。利用DAAO的立体选择性，将D,L-草铵膦逐渐转化为L-草铵膦(参见，例如，CN105567780A)。这个方案的缺点是钯碳催化剂用量大，并且浪费反应原料(如氧气和甲酸铵)。
还可以采用L-氨基酸转氨酶(L-TA)催化的立体选择性转氨反应将PPO转化成L-草铵膦(参见，例如，US20180030487A1)。这个方案的缺点在于，转氨步骤是平衡反应，需要提供过量的氨基供体(氨基酸或有机胺)来实现高转化率(例如提供3倍当量的氨基供体，转化率90％)，而过量的氨基供体和对应的副产物将严重影响后续的分离纯化步骤。
此外，可以通过L-氨基酸脱氢酶(L-AADH)催化的立体选择性还原反应，以无机铵盐为氨基供体，辅以NAD(P)H循环系统，消耗甲酸、葡萄糖或简单醇类，将PPO转化为L-草铵膦。L-AADH催化的反应并不需要大大过量的氢给体，可以实现高转化率。
有报道利用天然的或经修饰的谷氨酸脱氢酶(GluDH)催化PPO不对称还原胺化制备L-草铵膦(参见，例如，CN107630052B、CN106978453B、CN108588045B和CN109609474A)。
但是，本领域仍需要提供具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质(例如，提高的Vmax值、降低的Km值或增加的Vmax/Km)的GluDH。
发明内容
在第一方面，本发明提供一种经修饰的谷氨酸脱氢酶(GluDH)，与其起始GluDH相比，包含两个或多个位置的氨基酸取代，其中所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质(例如，提高的Vmax值、降低的Km值或增加的Vmax/Km)。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第173、175和182位的取代，其中所述位置参照SEQ ID NO:1进行编号。在一些实施方案中，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、242、263、339、420、431和437位的一或多个位置的氨基酸取代。优选地，第9位的氨基酸取代为S、L或Y。优选地，第22位的氨基酸取代为W或E。优选地，第23位的氨基酸取代为M。优选地，第25位的氨基酸取代为D。优选地，第31位的氨基酸取代为H。优选地，第56位的氨基酸取代为Q。优选地，第124位的氨基酸取代为L。优选地，第143位的氨基酸取代为E。优选地，第199位的氨基酸取代为W或Y。优选地，第216位的氨基酸取代为G。优选地，第263位的氨基酸取代为S。优选地，第339位的氨基酸取代为Q。优选地，第420位的氨基酸取代为R。优选地，第431位的氨基酸取代为S。优选地，第437位的氨基酸取代为K。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第22、56、173、175、182、199和420位的氨基酸取代。优选地，第22位的氨基酸取代为E。优选地，第56位的氨基酸取代为Q。优选地，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。优选地，第199位的氨基酸取代为Y。优选地，第420位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH还包含选自第31、124和216位的一或多个位置的氨基酸取代。优选地，第31位的氨基酸取代为H。优选地，第124位的氨基酸取代为L。优选地，第216位的氨基酸取代为G。在一些实施方案中，所述起始GluDH是野生型GluDH。在一些实施方案中，所述起始GluDH来源于芽胞杆菌科(Bacillaceae)的微生物，优选赖氨酸芽孢杆菌属(Lysinibacillus)或芽胞杆菌属(Bacillus)的微生物，更优选球形赖氨酸芽胞杆菌(Lysinibacillus sphaericus)或贝莱斯芽胞杆菌(Bacillus velezensis)。在优选的实施方案中，所述起始GluDH包含SEQ ID NO:1或2的氨基酸序列。
在一些实施方案中，所述经修饰的GluDH包含SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一的氨基酸序列。
在一些实施方案中，所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
在第二方面，本发明提供编码本发明的经修饰的GluDH的多核苷酸，以及包含本发明的多核苷酸的载体。
在第三方面，本发明提供包含本发明的的经修饰的GluDH、其编码多核苷酸或包含所述多核苷酸的载体的宿主细胞。
在第四方面，本发明还提供一种生产L-草铵膦的方法，包括使本发明的经修饰的GluDH或本发明的宿主细胞与PPO接触。
发明详述
本发明主要涉及经修饰的GluDH，用于催化PPO与氨基供体反应，以生产L-草铵膦。除非另有说明，本文中使用的术语具有本领域技术人员一般理解的含义。
一、经修饰的谷氨酸脱氢酶
如本文所用，术语“谷氨酸脱氢酶”和“GluDH”是指催化谷氨酸脱氢产生α-酮戊二酸的酶。此外，GluDH还具有催化PPO与氨基供体反应，生成L-草铵膦的活性。本发明提供经修饰的GluDH多肽，其具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质，包括但不限于提高的Vmax、降低的Km和提高的Vmax/Km。
如本文所用，术语“肽”表示通过肽键连接的至少两个氨基酸的链。术语“多肽”在本文中可以与术语“蛋白质”互换使用，是指含有十个或更多个氨基酸残基的链。本文中的所有肽和多肽化学式或序列均是从左至右书写的，表示从氨基末端至羧基末端的方向。
术语“氨基酸”包括蛋白质中天然存在的氨基酸和非天然氨基酸。蛋白质中天然存在的氨基酸的单字母和三字母命名采用本领域惯用名，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
Figure BDA0003154279360000041
Figure BDA0003154279360000051
如本文所用，术语“修饰”是指对由本发明的多肽或其同源序列组成的多肽的任何修饰，包括但不限于，取代、删除、插入和/或添加一或多个氨基酸。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含两个或多个位置的氨基酸取代，其中所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质，包括但不限于提高的Vmax、降低的Km和提高的Vmax/Km。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。
在本文中，在其基础上进行氨基酸修饰的GluDH多肽称为起始GluDH。所述起始GluDH可以是野生型GluDH，也可以是野生型GluDH的变体。例如，从SEQ ID NO:1的多肽开始进行修饰，则相对于经修饰的GluDH，SEQ ID NO:1的多肽是“起始GluDH”；而如果从SEQ IDNO:1的变体多肽(例如SEQ ID NO:3-30)开始进行修饰，则相对于经修饰的GluDH，所述变体多肽是“起始GluDH”。
如本文所用，术语“野生型GluDH”是指天然存在的GluDH。在一些实施方案中，所述起始GluDH来源于芽胞杆菌科的微生物。在一些实施方案中，所述野生型GluDH是来自赖氨酸芽孢杆菌属或芽胞杆菌属的微生物的GluDH。优选地，所述野生型GluDH是来自球形赖氨酸芽胞杆菌的GluDH(SEQ ID NO:2)或来自贝莱斯芽胞杆菌的GluDH(SEQ ID NO:1)。
对于本发明，为确定两个氨基酸序列或两个核酸序列的相同性百分比，以最佳比较为目的比对序列(例如在第一个氨基酸或核酸序列中可导入缺口，以与第二个氨基酸或核酸序列进行最佳比对)。然后比较在相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一个序列中的位置在第二个序列中相应位置由相同氨基酸残基或核苷酸占据时，则这些分子在这个位置是相同的。两个序列之间的相同性百分比是所述序列共有的相同位置的数量的函数(即相同性百分比＝相同位置的数量/位置(即重叠位置)的总数量×100)。优选地，这两个序列是相同长度的。
本领域技术人员知晓，可以使用不同的计算机程序确定两个序列之间的相同性。
“氨基酸相同性百分比”或者“氨基酸序列相同性百分比”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有大约指定的相同氨基酸百分比。例如，“95％的氨基酸相同性”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽有95％的氨基酸相同。
在一些实施方案中，本文所用的野生型GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含第173、175和182位的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、263、339、420、431和437位的一或多个位置的氨基酸取代。优选地，第9位的氨基酸取代为S、L或Y。优选地，第22位的氨基酸取代为W或E。优选地，第23位的氨基酸取代为M。优选地，第25位的氨基酸取代为D。优选地，第31位的氨基酸取代为H。优选地，第56位的氨基酸取代为Q。优选地，第124位的氨基酸取代为L。优选地，第143位的氨基酸取代为E。优选地，第199位的氨基酸取代为W或Y。优选地，第216位的氨基酸取代为G。优选地，第263位的氨基酸取代为S。优选地，第339位的氨基酸取代为Q。优选地，第420位的氨基酸取代为R。优选地，第431位的氨基酸取代为S。优选地，第437位的氨基酸取代为K。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。例如，所述起始GluDH包含SEQ ID NO:1或由SEQ ID NO:1组成。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第22、56、173、175、182、199和420位的氨基酸取代。优选地，第22位的氨基酸取代为E。优选地，第56位的氨基酸取代为Q。优选地，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。优选地，第199位的氨基酸取代为Y。优选地，第420位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH还包含选自第31、124和216位的一或多个位置的氨基酸取代。优选地，第31位的氨基酸取代为H。优选地，第124位的氨基酸取代为L。优选地，第216位的氨基酸取代为G。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。例如，所述起始GluDH包含SEQ ID NO:1或由SEQ IDNO:1组成。
在一些实施方案中，本发明的经修饰的GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案，所述起始GluDH与SEQ ID NO:1或2的区别在于，具有一或多个氨基酸的取代、删除、插入和/或添加。在一些实施方案中，所述起始GluDH与SEQ ID NO:1或2相比，具有一或多个氨基酸的保守取代。在一些实施方案中，所述起始GluDH与SEQ ID NO:1或2相比，具有一或多个氨基酸的插入或删除。
术语“保守取代”也称为由“同源”氨基酸残基取代，是指其中氨基酸残基由具有相似侧链的氨基酸残基置换的取代，例如，碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸，负电荷的谷氨酸、天冬氨酸，疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸，极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺，芳香性的苯丙氨酸、酪氨酸、色氨酸，化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。
蛋白质中氨基酸保守取代的例子包括：Ser取代Ala，Lys取代Arg，Gln或His取代Asn，Glu取代Asp，Ser取代Cys，Asn取代Gln，Asp取代Glu，Pro取代Gly，Asn或Gln取代His，Leu或Val取代Ile，Ile或Val取代Leu，Arg或Gln取代Lys，Leu或Ile取代Met，Met、Leu或Tyr取代Phe，Thr取代Ser，Ser取代Thr，Tyr取代Trp，Trp或Phe取代Tyr，及Ile或Leu取代Val。
在一些实施方案中，经修饰的GluDH，包含SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一的氨基酸序列或由SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一的氨基酸序列组成，或者所述经修饰的GluDH与SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一相比包含1-10个氨基酸取代(例如，保守取代)，其中所述取代位于位置9、22、23、25、31、56、104、124、132、133、143、173、175、181、182、199、203、216、263、339、420、431和437之外的位置，其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性，和/或所述经修饰的GluDH具有改进的动力学性质，例如提高的Vmax值、降低的Km值或增加的Vmax/Km。在一些实施方案中，所述经修饰的GluDH与与SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一相比，包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代(例如，保守取代)，其中所述取代位于位置9、22、23、25、31、56、104、124、132、133、143、173、175、181、182、199、203、216、263、339、420、431和437之外的位置。在一些实施方案中，本发明的经修饰的GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
如本文所用，酶的活性指在一定条件下，在单位质量的酶催化的化学反应中，单位时间内底物的减少量或产物的增加量。例如，本发明的经修饰的GluDH的活性，用一定条件下，在单位质量的经修饰的GluDH催化下，单位时间内PPO减少的量或L-草铵膦增加的量来表示。
在本文中，酶的活性也可以指酶的相对活性，以感兴趣的酶的活性与催化相同反应的给定的酶的活性的比值表示，如百分比相对活性。
在一些实施方案中，本发明的经修饰的GluDH的活性以与SEQ ID NO:3相比的百分比相对活性表示。在一些实施方案中，所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
在一些实施方案中，本发明的经修饰的GluDH的活性以与SEQ ID NO:30相比的百分比相对活性表示。在一些实施方案中，所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:30的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
如本文所用，术语“氨基供体”是指提供氨基的化合物，包括无机化合物和有机化合物。“氨基供体”包括但不限于铵盐(例如NH4Cl、NH4NO3、(NH4)2SO4、醋酸铵等)、氨基酸或有机胺。在一些实施方案中，所述氨基供体是铵盐，例如NH4Cl。
在生产中，除了酶活性之外，还需要考虑酶的动力学性质。在本文中，酶的动力学性质包括但不限于酶的Vmax、Km和Vmax/Km。在本文中，改进的动力学性质包括，例如但不限于，提高的Vmax、降低的Km和提高的Vmax/Km。
如本文所用，术语“Vmax”是指在一定酶浓度下，所能达到的催化反应的最大速度。具体而言，在酶浓度不变的条件下，当底物浓度在一定范围内时，反应速度通常会随着底物浓度的提升而提升；而当底物浓度达到一定值时，反应速度达到最大值(即Vmax)，不再随着底物浓度的提升而提升。
如本文所用，术语“Km”是指在一定酶浓度下，当催化速度达到最大催化速度(即Vmax)的一半时的底物浓度。
二、编码经修饰的GluDH的多核苷酸。
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，优选双链DNA。所述核酸的合成可以使用核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)。这种核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。
本发明还提供编码本发明的经修饰的GluDH的多核苷酸。因此，在本发明中，术语修饰还包括对编码本发明的GluDH多肽的多核苷酸的遗传操作。所述修饰可以是取代、删除、插入和/或添加一或多个核苷酸。
如本文所用，术语“编码”是指多核苷酸直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
此外，涵盖本发明的全部或部分核酸序列的核酸分子可以通过聚合酶链反应(PCR)分离，所述PCR使用基于所述序列中包含的序列信息设计合成的寡核苷酸引物。
本发明的多核苷酸可以使用cDNA、mRNA或者基因组DNA作为模板及合适的寡核苷酸引物根据标准PCR扩增技术进行扩增。如此扩增的核酸可以克隆进合适的载体中，并通过DNA序列分析进行表征。
本发明的多核苷酸可以通过标准的合成技术制备，例如使用自动化DNA合成仪制备。
本发明还涉及本文描述的核酸分子的互补链。与其它核苷酸序列互补的核酸分子是与该核苷酸序列充分互补的分子，使得其可以与其他核苷酸序列杂交，从而形成稳定双链体。
如本文所用，术语“杂交”是在给定的严格杂交和洗涤条件下，彼此至少大约90％、优选至少大约95％、更优选至少大约96％、更优选至少98％同源的核苷酸序列通常保持彼此杂交。
本领域技术人员知道各种用于杂交的条件，如严格杂交条件和高度严格杂交条件。参见，例如，Sambrook et al.,1989,Molecular Cloning,A Laboratory Manual,ColdSpring Harbor Press,N.Y.；和Ausubel et al.(eds.),1995,Current Protocols inMolecular Biology,John Wiley&Sons,N.Y.。
当然，本发明的多核苷酸不包括仅与poly A序列(如mRNA的3’末端poly(A))或者与互补的一段poly T(或U)残基杂交的多核苷酸。
三、表达和生产经修饰的GluDH
为表达本发明的经修饰的GluDH，还提供包含本发明的多核苷酸的核酸构建体和载体，如表达载体。
如本文所用，术语“表达”包括多肽生产中包含的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。
术语“核酸构建体”是指单链或双链的核酸分子，其分离自天然存在的基因或者被修饰为含有天然不存在的核酸区段。当所述核酸构建体含有表达本发明编码序列所需的控制序列时，术语核酸构建体与术语“表达盒”同义。
术语“表达载体”在本文是指线性或环形DNA分子，其包含编码本发明多肽的多核苷酸，所述多核苷酸与为所述多核苷酸表达而提供的另外的核苷酸，例如，控制序列，可操纵地连接。所述表达载体包括病毒载体或质粒载体。
术语“控制序列”在本文是指包括表达编码本发明多肽的多核苷酸所需或有利的所有元件。各控制序列对于编码多肽的核苷酸序列可以是天然的或者是外来的，或者彼此是天然或者外来的。这种控制序列包括但不限于前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列及转录终止子。最低限度，控制序列包括启动子和转录及翻译终止信号。
例如，所述控制序列可以是合适的启动子序列，一种由宿主细胞识别以表达编码本发明多肽的多核苷酸的核苷酸序列。所述启动子序列含有介导所述多肽的表达的转录控制序列。所述启动子可以是在所选择的宿主细胞中表现出转录活性的任何核苷酸序列，例如，大肠杆菌(Escherichia coli)lac操纵子。所述启动子还包括突变的、截短的和杂合的启动子，并且可以从与宿主细胞同源或异源的编码胞外或胞内多肽的基因获得。
术语“可操纵地连接”在本文是指这样的构型，其中控制序列置于相对于多核苷酸序列的编码序列的适当位置，由此所述控制序列指导多肽编码序列的表达。
编码本发明多肽的多核苷酸可以进行各种操作，以使得多肽表达。在将其插入载体之前，根据表达载体对多核苷酸的操作是可取的或必需的。利用重组DNA方法修饰多核苷酸序列的技术为本领域熟知。
为了鉴定和选择包含本发明的表达载体的宿主细胞，本发明的载体优选含有一或多个可选择标记，其使得可以对转化、转染、转导等的细胞进行简单的选择。可选择标记是一种基因，其产物提供生物杀灭剂或病毒抗性、重金属抗性、补充营养缺陷型等。例如，细菌的可选择标记是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或者赋予抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记。
本发明的载体可整合进宿主细胞基因组中或者在细胞中不依赖于基因组而自主复制。为了整合进宿主细胞基因组中或者自主复制所需的元件是本领域已知的(参见例如前述Sambrook et al.,1989)。
载体DNA可以通过常规转化或转染技术导入原核或真核细胞中。如本文所用，术语“转化”和“转染”是指将外源核酸(例如DNA)导入宿主细胞中的各种本领域公认的技术，可见于例如前述Sambrook et al.,1989；Davis et al.,Basic Methods in MolecularBiology(1986)及其它实验室手册。
本发明还涉及重组宿主细胞，其包含本发明的多核苷酸，所述多核苷酸有利地用于GluDH多肽的重组产生中。包含本发明多核苷酸的载体被导入宿主细胞中，由此所述载体作为染色体整合体或作为自身复制染色体外载体被保留。本领域技术人员知晓表达蛋白质的常规载体和宿主细胞。
在一些实施方案中，本发明的宿主细胞是大肠杆菌细胞，如大肠杆菌BL21(DE3)。在一些实施方案中，所述表达载体是pET-30a(+)。
本发明的经修饰的GluDH可以与非-GluDH多肽(例如异源氨基酸序列)可操纵地连接，形成融合蛋白。例如，在一个实施方案中，所述融合蛋白是GST-GluDH融合蛋白，其中GluDH序列与GST序列的C-末端融合。这种融合蛋白可帮助重组GluDH的纯化。在另一实施方案中，所述融合蛋白是在其N末端含有异源信号序列的GluDH蛋白。在某些宿主细胞中(例如哺乳动物和酵母宿主细胞)，可以通过使用异源信号序列增加GluDH的表达和/或分泌。
四、生产L-草铵膦
此外，本发明提供一种制备L-草铵膦的方法，包括使本发明的经修饰的GluDH或宿主细胞与PPO接触。
在一些实施方案中，本发明的制备L-草铵膦的方法包括如下步骤：
(a)向包含PPO和氨基供体的反应介质提供本发明的经修饰的GluDH的活性，任选地，所述反应介质包含NADPH/NADP循环系统，及
(b)温育所述反应介质以生成L-草铵膦。
在一些实施方案中，使用无细胞催化方法生产L-草铵膦，在步骤(a)中，提供本发明的经修饰的GluDH。在一些实施方案中，可以使用游离或固定化的本发明的修饰的GluDH。
在一些实施方案中，所述氨基供体是铵盐，例如NH4Cl。
在一些实施方案中，所述反应介质包含NADPH/NADP。适用于本发明的NADPH/NADP循环系统是本领域已知的，其包含，包括但不限于，醇脱氢酶、葡萄糖脱氢酶(GDH)或葡糖-6-磷酸脱氢酶(G6PD)。在一些实施方案中，所述NADPH/NADP循环系统包含醇脱氢酶。在一些实施方案中，所述醇脱氢酶也可以被固定化。
在一些实施方案中，所述温育在20-50℃，优选25-40℃，更优选28-35℃，例如30℃进行。
在一些实施方案中，所述介质包含缓冲液，例如PBS、Tris-HCl缓冲液。在一个实施方案中，所述介质包含PBS，例如100mM的PBS。在一些实施方案中，所述反应介质的pH值为7.5-8。
在一些实施方案中，反应介质是部分或全部由细胞培养基组成的介质，本发明的经修饰的GluDH的活性由本发明的宿主细胞提供，所述宿主细胞在所述反应介质中培养。
在一些实施方案中，所述反应介质是部分或全部由细胞培养基组成的介质，且所述NADPH/NADP循环系统，如醇脱氢酶活性由本发明的宿主细胞或者由第二种宿主细胞提供，所述宿主细胞在所述反应介质中培养。
在一些实施方案中，将本发明的宿主细胞和/或所述第二宿主细胞在细胞培养基中培养并扩增，然后从细胞培养基分离经扩增的宿主细胞，使用缓冲液或水使生物量重悬浮。在加入所述经扩增的宿主细胞之前、期间或之后向所述缓冲液或水提供PPO。
在一些实施方案中，可以使用细菌细胞，例如大肠杆菌细胞。
实施例
通过以下实施例，本领域技术人员会更清楚地理解本发明。应理解，实施例只是用于说明，而非限制本发明的范围
实施例1、材料和方法
如无特别说明，本发明中使用的实验方法均为常规方法，基因克隆操作具体可参见前述Sambrook et al.,1989。
i)试剂：DNA聚合酶(PrimeSTAR Max DNA Polymerase)和DpnI内切酶购自TaKaRa公司，质粒提取试剂盒购自Axygen公司，PPO是申请人根据现有技术(参见J.Org.Chem.1991,56,1783-1788)合成的，NH4Cl购自国药集团化学试剂北京有限公司，NADP+/NADPH购自阿拉丁，醇脱氢酶是大肠杆菌重组表达的来自高加索酸奶乳杆菌的脱氢酶(NCBI登录号WP_054768785.1)。
ii)载体和菌株：所使用的表达载体为pET-30a(+)，质粒购自Novagen公司，所使用的宿主细胞为大肠杆菌BL21(DE3)，购自天根生化科技(北京)有限公司。
iii)测序与引物合成由苏州泓迅生物科技股份有限公司完成。
iv)定点突变：
设计特异性引物对，在所需突变的氨基酸位置对应的碱基引入所需的取代。用提取的突变前质粒(包含野生型GluDH编码序列，pET-30a(+)骨架)为模版，利用Quickchange技术(Nucleic Acids Research,2004,32(14):e115)通过PCR引入突变。PCR扩增结束后，扩增产物用Dpn I消化4h去除模版质粒。将消化产物转化至大肠杆菌BL21(DE3)感受态细胞中，涂布于LB琼脂培养基(含有50mg/L的卡那霉素)、挑单菌落至LB液体培养基(含有50mg/L的卡那霉素)中培养，测序验证突变正确性。经验证的克隆置于-80℃保藏备用。
v)蛋白质表达及粗酶液的制备：
在LB琼脂培养基上将保藏的克隆活化。然后，将单菌落接种至LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育12h。将1mL培养物转接至50mL新鲜的LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育至OD600达到0.6左右，加入IPTG(终浓度为0.4mM)在25℃温育16h以诱导蛋白质表达。
温育后，将培养物以4,000g在4℃离心10min，弃上清，收集大肠杆菌细胞。将收集的大肠杆菌细胞重悬于预冷的15mL pH 7.0的50mM PBS中，在4℃超声破碎大肠杆菌细胞。细胞破碎液以6,000g在4℃离心15min去除沉淀，得到的上清为含重组酶的粗酶液。
vi)酶活性测定
向PPO的PBS(100mM)溶液，中加入NH4Cl、NADP+，并用氨水调节溶液pH值至8，溶液中PPO的终浓度为100mM，NH4Cl的终浓度为50mM，而NADP+的终浓度为0.2g/L。向上述溶液加入如v)中描述的方法制备的GluDH粗酶液和醇脱氢酶，GluDH终浓度为0.02g/L，而醇脱氢酶的终浓度为0.2g/L。在30℃，于振荡器上持续震荡(400rpm)2小时，取样并用OPA柱前衍生化高效液相色谱检测L-草铵膦的生成量，从而测定催化反应初始速度。
vii)酶动力学测定
在96孔酶标板上配置多个200μL反应体系，其中含100mM pH 7.5PBS(用氨水调pH)、0.15mM的NADPH(还原型)、50mM的NH4Cl，10％体积稀释后的粗酶液(稀释500倍)、不同浓度底物PPO(5-100mM)。在30℃检测340nm紫外吸光强度的变化，记录并计算吸收随时间的变化率mA/min。所得参数带入Michaelis-Menten方程，其中的反应速度用吸光度变化率计。
实施例2、制备和检测来自贝莱斯芽胞杆菌的GluDH(BvGluDH)的突变体
以BvGluDH(SEQ ID NO:1)的编码核酸为模板，根据实施例1的方法制备突变体，并测量酶活性和动力学参数Vmax和Km。所得突变体及其活性和动力学参数如表1所示，相对酶活是指突变体的活性vs.SEQ ID NO:3的突变体的活性的百分比。
表1
Figure BDA0003154279360000191
Figure BDA0003154279360000201
实施例3、制备和检测来自球形赖氨酸芽胞杆菌的GluDH(LsGluDH)的突变体
以LsGluDH(SEQ ID NO:2)的编码核酸为模板，根据实施例1的方法制备突变体，并测量酶活性和动力学参数Vmax和Km。所得突变体及其活性和动力学参数如表2所示，其中SEQ ID NO:30的突变体是CN108588045B中报道的突变体(LsGluDH A175G)，相对酶活是指突变体的活性vs.SEQ ID NO:30的突变体的活性的百分比。
表2
突变体中引入的取代 SEQ ID NO: 相对酶活 Vmax(mA/min) Km(mM)
A175G 30 100％ 39 49
A175G-P132L 31 127％ 45 35
A175G-I133V 32 106％ 39 35
A175G-V173S 32 185％ 106 62
A175G-V173G 34 132％ 64 49
A175G-A182R 35 102％ 46 38
A175G-V173G-A182R 36 169％ 58 31
A175G-I133V-V173S-A182R 37 131％ 38 26
实施例4、制备和检测BvGluDH的突变体
根据实施例1的方法制备突变体，在SEQ ID NO:21的突变体的基础上引入额外的突变，并测量酶活性。所得突变体及其活性如表3所示，初始相对酶活是指未经热处理时，突变体的活性vs.SEQ ID NO:21的突变体的活性的百分比；热处理后相对酶活是指在45℃温育30min之后，突变体的活性vs.SEQ ID NO:21的突变体的活性的百分比。
表3
Figure BDA0003154279360000202
Figure BDA0003154279360000211
实施例5、制备和检测BvGluDH的突变体
根据实施例1的方法制备突变体，在SEQ ID NO:62的突变体的基础上引入额外的突变，并测量酶活性。所得突变体及其活性如表4所示，初始相对酶活是指未经热处理时，突变体的活性vs.SEQ ID NO:62的突变体的活性的百分比。
表4
突变体中的突变 SEQ ID NO: 相对酶活
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R 62 100％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H 63 165％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-F124L 64 106％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A216G 65 117％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-F124L 66 143％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-A216G 67 132％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-F124L-A216G 68 97％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-F124L-A216G 69 103％
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20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 4
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 4
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Cys Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 5
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 5
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 6
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 6
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 7
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 7
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 8
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 8
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 9
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 9
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Lys Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 10
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 10
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Arg Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 11
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 11
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 12
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 12
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Ile Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 13
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 13
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 14
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 14
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 15
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 15
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 16
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 16
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 17
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 17
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 18
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 18
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 19
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 19
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 20
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 20
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 21
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 21
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 22
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 22
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 23
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 23
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 24
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 24
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 25
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 25
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 26
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 26
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 27
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 27
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 28
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 28
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 29
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 29
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Val Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 30
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 30
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 31
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 31
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 32
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 32
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Leu Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 33
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 33
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 34
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 34
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 35
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 35
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 36
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 36
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 37
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 37
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Val Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Ser Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Arg Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 38
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 38
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 39
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 39
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 40
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 40
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Met Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 41
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 41
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 42
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 42
Met Thr Thr Thr Thr Val Ser Asn Ser Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 43
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 43
Met Thr Thr Thr Thr Val Ser Asn Leu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 44
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 44
Met Thr Thr Thr Thr Val Ser Asn Tyr Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 45
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 45
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asp Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 46
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 46
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Trp Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 47
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 47
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 48
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 48
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Glu Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 49
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 49
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ser Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 50
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 50
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Gln Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 51
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 51
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 52
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 52
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Ser Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 53
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 53
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Asn Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Lys Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 54
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 54
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Trp Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 55
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 55
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Trp Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 56
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 56
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 57
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 57
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 58
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 58
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 59
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 59
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 60
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 60
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 61
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 61
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Trp Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Ala Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 62
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 62
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 63
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 63
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln His Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 64
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 64
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Leu Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 65
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 65
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Gly Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 66
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 66
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln His Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Leu Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 67
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 67
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln His Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Gly Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 68
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 68
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Leu Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Gly Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455
<210> 69
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 69
Met Thr Thr Thr Thr Val Ser Asn Asp Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Glu Leu Lys Asn Gln Asn Ser His Gln His Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Ala Gln His Pro Glu Tyr Ile Gln Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp Asn
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Leu Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asn Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Gly Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Ile Thr Lys Ala Tyr Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Gly Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Glu Glu Met Leu Lys Glu Val Asn Asp Ser Phe
225 230 235 240
Glu Asp Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Ala Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Ala Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Asp Tyr Arg Pro Asn Ala Thr Phe Thr Glu Gly Cys Thr Gly Ile Trp
305 310 315 320
Ser Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Asp Ala Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Thr Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asn Ile Tyr Arg Asp Ser Lys Ala Ala Ala Asp Lys Tyr Gly Tyr Pro
420 425 430
Gly Asn Leu Val Val Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Ala Glu Gly Ile Tyr
450 455

Claims (17)

1.一种经修饰的谷氨酸脱氢酶(GluDH)，与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号，且其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)与氨基供体反应生成L-草铵膦的活性，和/或所述经修饰的GluDH具有提高的Vmax、降低的Km或增加的Vmax/Km。
2.权利要求1的经修饰的GluDH，其中所述起始GluDH是野生型GluDH。
3.权利要求1或2的经修饰的GluDH，其中所述起始GluDH来源于芽胞杆菌科的微生物，优选赖氨酸芽孢杆菌属或芽胞杆菌属的微生物，更优选球形赖氨酸芽胞杆菌(Lysinibacillus sphaericus)或贝莱斯芽胞杆菌(Bacillus velezensis)。
4.权利要求1-3任一项的经修饰的GluDH，其中所述起始GluDH包含SEQ ID NO:1或2的氨基酸序列。
5.一种经修饰的GluDH，包含SEQ ID NO:4-14、16-19、21、22、24-29和31-69之一的氨基酸序列。
6.一种经修饰的GluDH，与其起始GluDH相比，包含第173、175和182位的取代，其中第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，其中所述位置参照SEQ ID NO:1进行编号，且其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性。
7.权利要求6的经修饰的GluDH，其中所述起始GluDH是野生型GluDH。
8.权利要求6或7的经修饰的GluDH，其中所述起始GluDH来源于芽胞杆菌科的微生物，优选赖氨酸芽孢杆菌属或芽胞杆菌属的微生物，更优选球形赖氨酸芽胞杆菌或贝莱斯芽胞杆菌。
9.权利要求6-8任一项的经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、242、263、339、420、431和437位的一或多个位置的氨基酸取代，其中第9位的氨基酸取代为S、L或Y，第22位的氨基酸取代为W或E，第23位的氨基酸取代为M，第25位的氨基酸取代为D，第31位的氨基酸取代为H，第56位的氨基酸取代为Q，第124位的氨基酸取代为L，第143位的氨基酸取代为E，第199位的氨基酸取代为W或Y，第216位的氨基酸取代为G，第263位的氨基酸取代为S，第339位的氨基酸取代为Q，第420位的氨基酸取代为R，第431位的氨基酸取代为S，第437位的氨基酸取代为K。
10.权利要求6-8任一项的经修饰的GluDH，还包含第22、56、199和420位的氨基酸取代，其中第22位的氨基酸取代为E，第56位的氨基酸取代为Q，第199位的氨基酸取代为Y，第420位的氨基酸取代为R。
11.权利要求10的经修饰的GluDH，还包含选自第31、124和216位的一或多个位置的氨基酸取代，其中第31位的氨基酸取代为H，第124位的氨基酸取代为L，第216位的氨基酸取代为G。
12.权利要求6-11任一项的经修饰的GluDH，其中所述起始GluDH具有SEQ ID NO:1的氨基酸序列。
13.权利要求12的经修饰的GluDH，其中所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少130％。
14.一种多核苷酸，编码权利要求1-13任一项的经修饰的GluDH。
15.一种表达载体，包含权利要求14的多核苷酸。
16.一种宿主细胞，包含权利要求1-13任一项的经修饰的GluDH、权利要求14多核苷酸或权利要求15的载体。
17.一种生产L-草铵膦的方法，包括使1-13任一项的经修饰的GluDH或权利要求16的宿主细胞与PPO接触。
CN202110772483.7A 2020-07-09 2021-07-08 经修饰的谷氨酸脱氢酶及其应用 Pending CN113913401A (zh)

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