CN115516102A - 合成的檀香烯合酶 - Google Patents
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Abstract
公开了具有改善产物特征的檀香烯合酶和用于改善檀香烯合酶的方法。本发明还涉及通过发酵产生的檀香烯组合物，其具有比α‑檀香烯含量更大的β‑檀香烯含量。
Description
檀香烯合酶是催化法呢基焦磷酸(FPP)转化成广泛类型化合物(包括檀香烯，例如α-檀香烯、β-檀香烯和表-β-檀香烯)的萜烯合酶。
式I是(-)-β-檀香烯(CAS编号511-59-1；下文称为β-檀香烯)的图示。
檀香烯合酶以底物法呢基焦磷酸为开始，但是一般产生倍半萜产物的混合物。一般地，檀香烯合酶将产生(-)-α-檀香烯(CAS编号512-61-8；下文称为α-檀香烯)作为主产物，随后是β-檀香烯(参见式I)和/或反式-α-香柠檬烯(CAS编号13474-59-4；下文也称为香柠檬烯)作为第二和第三最丰富的产物。产生的量取决于具体酶，并且还取决于β-檀香烯是否为第二最丰富者或香柠檬烯是占优势的，不过α-檀香烯在迄今可获得的油中占优势。
已经报道了数个编码檀香烯合酶的基因(参见例如国际专利申请WO2018/160066及其中参考文献)。另外，这些檀香烯合酶生产一系列檀香烯倍半萜(最特别地包括β-檀香烯、α-檀香烯、表-β-檀香烯、香柠檬烯和β-红没药烯)。
已知产生α-檀香烯作为主要产物的檀香烯合酶，例如从WO201100026和Jones等人,(2011)(“Sandalwood fragrance biosynthesis involves sesquiterpene synthasesof both the terpene synthase(TPS)-a and TPS-b subfamilies,including santalenesynthases(檀香木香精生物合成涉及萜烯合酶(TPS)-a亚家族和TPS-b亚家族的倍半萜合酶，包括檀香烯合酶)”已知。Jones C.G.,Moniodis J.,Zulak K.G.等人,The Journal ofbiological chemistry,第286卷,第20期,第17445-17454页,5/20/2011；DOI:10.1074/jbc.M111.231787)描述了来自三个不同檀香属(Santalum)物种(檀香(Santalum album)、新喀里多尼亚檀香(S.austrocaledonicum)和大果檀香(S.spicatum))的、同时产生α-檀香烯，α-反式-香柠檬烯，表-β-檀香烯和β-檀香烯的萜烯合酶。国际专利申请WO201100026在遭到可能错误解读的图1和表2数据中公开到：比α-檀香烯更多的β-檀香烯可能已存在，例如通过显示来自半定量GC-MS分析的数据公开，未指出这种方法相对于化合物的量而言定量不可靠。在该同一发明公开中，向本领域技术人员明确展示存在相反情况(α-檀香烯比β-檀香烯更多)–如显示GC-FID可靠定量数据的WO201100026表2第4列和报告天然檀香木油中α-檀香烯超过β-檀香烯两倍多的WO201100026表1中所示。
还已经在WO201100026之后研究者的以下出版物中对相同酶报道α-檀香烯而非β-檀香烯剩余，所示文献也在2011年发表并且因此假定地采用相同数据基础：“Sandalwoodfragrance biosynthesis involves sesquiterpene synthases of both the terpenesynthase(TPS)-a and TPS-b subfamilies,including santalene synthases(檀香木香精生物合成涉及萜烯合酶(TPS)-a亚家族和TPS-b亚家族的倍半萜合酶，包括檀香烯合酶).”Jones C.G.,Moniodis J.,Zulak K.G.等人,The Journal of biologicalchemistry,第286卷，第20期，第17445-17454页5/20/2011；DOI:10.1074/jbc.M111.231787。该论文的补充资料以及初始出版物附图补正(参见：Erratum：Sandalwood fragrance biosynthesis involves sesquiterpene synthases of boththe terpene synthase(TPS)-a and TPS-b subfamilies,including santalenesynthases(檀香木香精生物合成涉及萜烯合酶(TPS)-a亚家族和TPS-b亚家族的倍半萜合酶，包括檀香烯合酶)(Journal of Biological Chemistry(2011)286(17445-17454)),Journal of Biological Chemistry volume第287卷，第45期，第37713-37714页2012,DOI:10.1074/jbc.A111.231787s)确证以下事实：这些研究者观察到比β-檀香烯更多的α-檀香烯。这些研究者的后续出版物确认天然檀香木油并不具备相对于α-檀香烯过量的β-檀香烯(Moniodis等人,2017“Sesquiterpene Variation in West Australian Sandalwood(Santalum spicatum)(西澳大利亚檀香木(大花澳洲檀香(Santalum spicatum)中的倍半萜变动))”；Molecules 2017；22(6))。已知的檀香烯合酶产生比β-檀香烯更多的α-檀香烯(Diaz-Chavez等人,2013，“Biosynthesis of Sandalwood Oil:Santalum album CYP76FCytochromes P450 Produce Santalols and Bergamotol(檀香木油生物合成：檀香CYP76F细胞色素P450产生檀香醇和香柠檬醇)”,PLoS ONE,2013；8(9))，甚至烟草植物中异源表达时也如此(Yin JL，Wong WS(2019)“Production of santalenes and bergamotene inNicotiana tabacum plants(在普通烟草(Nicotiana tabacum)植物中产生檀香烯和香柠檬烯).”PLOS ONE 14(1):e0203249.https://doi.org/10.1371/journal.pone.0203249)。
作为WO2015153501公布的国际专利申请描述了修饰的檀香烯合酶，其衍生自檀香的檀香烯合酶，其相比檀香的天然檀香烯合酶时具有增加的萜烯合酶活性，然而α-檀香烯依旧相对β-檀香烯过量，并且已经发现α-檀香烯产物特征高的檀香烯合酶(Schalk,M.,2011.Method for Producing Alpha-Santalene(用于产生α檀香烯的方法)，美国专利2011/008836A1；作为WO2018160066公布的国际专利申请)。作为WO2010/067309公布的国际专利申请描述了一种使用来自檀香属(Santalum)的檀香烯合酶产生β-檀香烯的方法(Schalk，2014)(美国专利号8993284)，但α檀香烯仍然超过β-檀香烯。
因此，已知相比α-檀香烯总以更低量产生β-檀香烯的酶，并且不存在已知体内β-檀香烯产物特征比α-檀香烯更大的檀香烯合酶的例子。
檀香烯合酶的产物可以以生物合成方式或化学方式氧化，以产生其相应的檀香烯醇；α-檀香醇、β-檀香醇和表-β-檀香醇。檀香醇是檀香木油的主要组分，一种高度珍贵的天然存在香料，其为香精、化妆品，化妆用具、芳香疗法和药物中的重要成分。它具有从倍半萜醇类α-檀香醇和β-檀香醇赋予的柔和、甜美-木材和香脂气味。特别地，将β-檀香醇视作赋予最重要的檀香木嗅觉香调。需要一种对β-檀香烯具有更大特异性的合酶，因为产物可以氧化成β-檀香醇含量高的油。
当前已知的檀香烯合酶具有在檀香烯工业生产工艺应用时尤其不利的众多不同缺点，其中在孤立的反应中(在体外，例如使用分离的檀香烯合酶或(透化的)全细胞)或否则例如在作为最终导致β-檀香烯从糖产生的更长代谢途径的一部分的发酵过程中(体内)，从FPP制备檀香烯(和可能随后檀香醇和尤其β-檀香醇)。
如果该酶将产生更少α-檀香烯和更多的反式-α-香柠檬烯，也可能有利于一些应用。
能够根据特定需要引导檀香烯合酶的三种主产物的产物比率是有利的。
本发明公开了，出人意料地通过相对简单地改变檀香烯合酶的三级结构的某部分的灵活性，可以改善檀香烯合酶的产物特征。这些改善的檀香烯合酶中某些产生β-檀香烯及有时香柠檬烯超过α-檀香烯，其他檀香烯合酶具有相比野生型酶增加的α-檀香烯产量，并且它们可用于生产这些化合物，例如用于大规模工业过程中。
发明详述
与某属性或值相联系的情况下，术语“实质地”、“约”、“大致”、“基本上”等还分别具体地确切限定该属性或确切限定该值。在相同功能活性或基本上相同功能的语境下，术语“基本上”意指与参比功能相比，优选地在20％范围内、更优选地在10％范围内、最优选地在5％或更小范围内功能的差异。在制剂和组合物的语境下，术语“基本上”(例如，“基本上由化合物X组成的组合物”)可以在本文中如下使用：制剂和组合物中基本上含有具有给定效果的所指化合物、不含具有这类效果的其他化合物或按这类化合物的不显示出可度量或有意义效果的最大量含有所指化合物。在给定数值或范围的情况下，术语“约”特别设计处于给予的所述值或范围的20％以内、10％以内或5％以内的值或范围。如本文所用，术语“包含”也包括术语“由……组成”。
术语“分离的”意指材料基本上不含至少一种在其原始环境内部与之天然结合的其他组分。例如，活的动物中存在的天然存在的多核苷酸、多肽或酶不是分离的，然而与该天然系统中一些或全部共存物质分开的相同多核苷酸、多肽或酶是分离的。作为又一例子，一种分离的核酸分子，例如DNA或RNA分子是这样的分子，它并不紧密邻接于5′和3′侧翼序列，所述侧翼序列正常情况下在衍生该分子的生物的天然存在的基因组中存在时与所述分子紧密邻接。此类多核苷酸可能是载体的部分、并入具有不相关遗传背景的细胞的基因组中(或并入具有基本上相似遗传背景的细胞的基因组，但处在与其天然存在位点不同的位点)、或通过PCR扩增或限制性酶消化产生、或是通过体外转录产生的RNA分子，和/或这类多核苷酸、多肽或酶可以是组合物的一部分，并且仍可以是分离的，从而这种载体或组合物不是其天然环境的一部分。
“纯化”意指该物质处于相对纯的状态，例如，至少约90％纯度，至少约95％纯度或至少约98％或99％纯度。优选地，“纯化的”意指材料处于100％纯状态。
“合成的”或“人工的”化合物由体外化学合成或酶促合成产生。它包括但不限于用针对宿主生物(如酵母细胞宿主或其他选定表达宿主)的最佳密码子选择所产生的变体核酸或相比野生型蛋白质序列，具有氨基酸修饰(如例如取代)(例如以优化多肽特性)的变体蛋白序列。合成多肽因此应理解为作为一种合成的、非天然存在的“人造”蛋白质序列的多肽。优选地，合成的多肽在本发明时在至少一个氨基酸位置异于任何天然存在多肽。
术语“非天然存在的”指不存在于其原始环境或来源中的(多)核苷酸、氨基酸、(多)肽、酶蛋白、细胞、生物或其他材料，不过它可以最初衍生自其原始环境或来源并且随后通过其他手段加以繁殖。这类非天然存在的(多)核苷酸、氨基酸、(多)肽、酶蛋白、细胞、生物或其他材料可以在结构上和/或功能上与其天然副本相似或相同。
术语“天然”(或“野生型”或“内源”)细胞或生物和“天然”(或野生型或内源)多核苷酸或多肽指如自然界中存在的细胞或生物并且分别指如细胞中其自然形式和遗传环境下存在(即，无任何人类干预)的所讨论多核苷酸或多肽。
术语“异源”(或外源或外来或重组)多肽在本文中定义为：
(a)不是宿主细胞天然的多肽。这种异源多肽的蛋白质序列是合成的、非天然存在的“人造”蛋白质序列；
(b)宿主细胞天然的多肽，但包含作为通过改变该天然多肽的重组DNA技术操纵宿主细胞的DNA之结果的结构性修饰，例如，缺失、置换、和/或插入；或
(c)宿主细胞天然的多肽，其中作为通过重组DNA技术操作DNA宿主细胞(例如，更强启动子)的结果，其表达定量性改变或从异于天然宿主细胞的基因组位置受到指导。
上文b)和c)的描述指处于其天然形式但不天然地由用于产生它的细胞表达的序列。产生的多肽因此更精确地定义为“重组表达的内源多肽”，其不与上文定义矛盾但反映以下特殊情形：它不是合成性质或操作的蛋白质的序列，而是产生多肽分子的方式。
类似地，术语“异源”(或外源或外来或重组)多核苷酸指：
(a)不是宿主细胞天然的多核苷酸；
(b)宿主细胞天然的多核苷酸，但包含作为通过改变天然多核苷酸的重组DNA技术操纵宿主细胞的DNA之结果的结构性修饰，例如，缺失、置换、和/或插入；
(c)宿主细胞天然的多核苷酸，其中作为通过重组DNA技术操作多核苷酸的调节元件之结果(例如，更强启动子)，其表达定量性改变；或
(d)宿主细胞天然的多核苷酸，但作为通过重组DNA技术遗传操作之结果而未整合于其天然遗传环境内部。
对于两个或更多个多核苷酸序列或两个或更多个氨基酸序列，术语“异源”用来表征两个或更多个多核苷酸序列或两个或更多个氨基酸序列并非按照特定的彼此组合天然出现。
术语“多核苷酸”、“核酸序列”、“核苷酸序列”、“核酸”、“核酸分子”在本文中可互换地使用并且指任意长度的聚合非分支形式的核苷酸：核糖核苷酸或脱氧核糖核苷酸，或这二者的组合。
对于核苷酸序列(例如，共有序列)，使用IUPAC核苷酸命名法(国际生物化学联合会命名委员会(NC-IUB)(1984).“核酸序列中未完整指明碱基的命名法”)，连同对本发明有意义的以下核苷酸两可定义和核苷酸两可定义：A，腺嘌呤；C，胞嘧啶；G，鸟嘌呤；T，胸腺嘧啶；K，鸟嘌呤或胸腺嘧啶；R，腺嘌呤或鸟嘌呤；W，腺嘌呤或胸腺嘧啶；M，腺嘌呤或胞嘧啶；Y，胞嘧啶或胸腺嘧啶；D，不是胞嘧啶；N，任何核苷酸。
额外地，记号“N(3-5)”意指所示共有位置可以具有3至5个任意(N)核苷酸。例如，共有序列“AWN(4-6)”表示3种可能变体–在末尾处4、5或6个任意核苷酸：AWNNNN、AWNNNNN、AWNNNNNN。
术语“调节元件”和“调节序列”均在本文中可相互交换地使用并且应在广泛背景下上理解成意指能够实现与之连接的序列表达(包括但不限于编码多肽的多核苷酸表达)的调节性核酸序列。调节元件或调节序列可以包括单独具有功能或目的和/或处于某特定排列或该排列内部其他元件或序列分组内部的任何核苷酸序列。调节序列的例子包括但不限于，前导序列或信号序列(如5’-UTR)、开始信号、前肽序列、启动子、增强子、沉默子、多聚腺苷化序列、核糖体结合位点(RBS、SD序列)、终止信号、终止子、3’-UTR及其组合。调节元件或调节序列可以彼此或相对于待表达的核苷酸序列为天然的(即来自相同基因)或外来的(即来自不同基因)。
“有效连接”意指所述组件处于允许它们以其意图方式发挥功能的关系。例如，与编码序列有效连接的调节序列以这样的方式连接，从而在与所述调节序列相容的条件下实现该编码序列的表达。
可以修饰核酸和多肽以包括标签或结构域。标签可以用于多种目的，包括用于检测、纯化、增溶或固定化，并且可以包括例如生物素、荧光团、表位、交配因子或调节序列。结构域可以为任何尺寸并且提供想要的功能(例如，赋予增加的稳定性、溶解度、活性、简化纯化)并且可以包括例如结合结构域、信号序列、启动子序列、调节序列、N端扩展物或C30末端扩展物。也可以利用标签和/或结构域的组合。
术语“融合蛋白”指通过本领域已知的任意手段接合在一起的两种或多种多肽。这些手段包括化学合成法或通过重组工程剪接编码性核酸。
修饰核酸以在编码的蛋白质中引入变化的方法
·基因编辑
基因编辑或基因组编辑是一种基因工程，其中将DNA插入、替换或从基因组移除并且可以通过以下方式实现所述基因工程：使用如“基因改组(gene shuffling)”或由反复DNA改组组成“定向进化”的多种技术，随后进行适当筛选和/或选择，以产生编码具有修饰的生物学活性的蛋白质的变体核酸或其部分(Castle等人,(2004)Science304(5674):1151-4；美国专利5,811,238和6,395,547)，或借助“T-DNA活化”标签法(Hayashi等人,Science(1992)1350-1353)，其中所产生的转基因生物因接近于已引入启动子的基因的受调节表达而显示显性表型，或借助“TILLING”(基因组中定向引入的局部损害(TargetedInduced Local Lesions In Genomes))，并且指可用来生成和/或鉴定核酸编码的具有修饰的表达和/或活性的蛋白质的诱变技术。TILLING还允许选择携带这类突变变体的生物。本领域熟知用于TILLING的方法(McCallum等人,(2000)Nat Biotechnol 18:455-457；经Stemple(2004)Nat Rev Genet 5(2):145-50综述)。另一项技术使用人工工程化的核酸酶如锌指核酸酶、转录激活物样效应物核酸酶(TALENs)、CRISPR/Cas系统和工程化大范围核酸酶(meganucleases)，如再工程化的归巢核酸内切酶(Esvelt,KM.；Wang,HH.(2013),MolSyst Biol 9(1):641；Tan,WS.等人(2012),Adv Genet 80:37–97；Puchta,H.；Fauser,F.(2013),Int.J.Dev.Biol 57:629–637)。
·诱变
可以使用分子生物学中已知的多种技术，修饰DNA和它们编码的蛋白质以产生具有新特性或改变特性的变体蛋白或酶。例如，随机PCR诱变，参见，例如，Rice(1992)Proc.Natl.Acad.Sci.USA 89:5467-5471；或者组合式多盒诱变，参见，例如，Crameri(1995)Biotechniques 18:194-196。
备选地，核酸例如基因可以在随机或“随意”片段化后重装配，参见，例如，美国专利号6,291,242；6,287,862；6,287,861；5,955,358；5,830,721；5,824,514；5,811,238；5,605,793。
备选地，通过以下方法引入修饰、添加或缺失：易错PCR、改组、位点定向诱变、组装PCR、有性PCR诱变、体内诱变(噬菌体辅助连续进化、体内连续进化)、盒诱变、递归式整体诱变(recursive ensemble mutagenesis)、指数式整体诱变、位点特异性诱变、基因再组装、基因位点饱和诱变(GSSM)、合成性连接再组装(SLR)、重组、递归式序列重组、硫代磷酸酯修饰的DNA诱变、含尿嘧啶的模板诱变、缺口双链体诱变、点错配修复诱变、修复缺陷型宿主菌株诱变、化学诱变、放射诱变、缺失诱变、限制-选择诱变、限制-纯化诱变、人工基因合成、整体诱变、嵌合核酸多聚体创建、和/或这些方法和其他方法的组合。
备选地，“基因位点饱和诱变(gene site saturation mutagenesis)”或“GSSM”包括一种使用简并寡核苷酸引物向多核苷酸引入点突变的方法，如美国专利号6,171,820和6,764,835中详述。
备选地，合成性连接再组装(Synthetic Ligation Reassembly，SLR)包括将寡核苷酸结构单元非随意地连接在一起的方法(例如，如美国专利号6,537,776中公开)。
备选地，定制的多位点组合组装(Tailored multi-site combinatorialassembly，“TMSCA”)是一种通过在单一反应中使用至少两个致突变性非重叠寡核苷酸引物，产生在多个位点具有各种突变的不同组合的多个后代多核苷酸的方法(如PCT公开号WO2009/018449中所述)。
可以用众多软件工具生成序列比对，如：
-Needleman和Wunsch算法-Needleman,Saul B.&Wunsch,Christian D.(1970)."Ageneral method applicable to the search for similarities in the amino acidsequence of two proteins(适用于两种蛋白质的氨基酸序列中检索相似性的一般方法)".Journal of Molecular Biology.48(3):443–453。
将这种算法例如实施入执行两个序列全局比对的“NEEDLE”程序中。NEEDLE程序例如包含于欧洲分子生物学开放软件套件(EMBOSS)内部。
-EMBOSS-各种程序的集合：欧洲分子生物学开放软件套件(a collection ofvarious programs:The European Molecular Biology Open Software Suite(EMBOSS)),Trends in Genetics 16(6),276(2000)。
-BLOSUM(BLOcks SUbstitution Matrix，即，区块替换矩阵)-一般基于比对保守区域(例如蛋白质结构域)生成(Henikoff S,Henikoff JG:Amino acid substitutionmatrices from protein blocks(来自蛋白质区块的氨基酸替换矩阵).Proceedings ofthe National Academy of Sciences of the USA.1992Nov 15；89(22):10915-9)。众多BLOSUM之一是“BLOSUM62”，其往往是比对蛋白质序列时许多程序的“默认”设置。
-BLAST(基础局部比对检索工具)–由主要用来在庞大序列数据库中检索相似序列的若干个独立程序(BlastP、BlastN、…)组成。BLAST程序还产生局部比对。一般使用NCBI提供的“BLAST”接口，其为改进版本(“BLAST2”)。“初始”BLAST：Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.和Lipman,D.J.(1990)"基本局部比对检索工具(Basic localalignment search tool)"J.Mol.Biol.215:403-410；BLAST2：Altschul,Stephen F.,Thomas L.Madden,Alejandro A.Schaffer,Jinghui Zhang,Zheng Zhang,Webb Miller和David J.Lipman(1997),"Gapped BLAST and PSI-BLAST:a new generation of proteindatabase search programs(空位BLAST和PSI-BLAST：新一代蛋白质数据库检索程序)",Nucleic Acids Res.25:3389-3402。
酶变体可以通过它们与亲本酶相比时的序列同一性定义。序列同一性通常作为“序列同一性％”或“同一性％”提供。为了确定两个氨基酸序列之间的同一性百分数，在第一步骤中，在这两个序列之间生成配对序列比对，其中将两个序列在其整个长度范围内比对(即，配对的全局比对)。用实施Needlem和Wunsch算法(J.Mol.Biol.(1979)48,第443-453页)的程序，优选地通过使用程序“NEEDLE”(欧洲分子生物学开放软件套件(EuropeanMolecular Biology Open Software Suite)(EMBOSS))，采用程序默认参数(空位开口＝10.0、空位延伸＝0.5和矩阵＝EBLOSUM62)，生成比对。用于本发明的优选比对是从其可以确定最高序列同一性的比对。
以下实例意在阐述对两个核苷酸序列的计算，但是相同计算适用于蛋白质序列：
Seq A：AAGATACTG长度：9个碱基
Seq B：GATCTGA长度：7个碱基
因此，较短序列是序列B。
产生在其整个长度范围内显示两个序列的两两全局比对产生了
比对中的“I”符号表示相同的残基(其意指DNA的碱基或蛋白质的氨基酸)。相同残基的数目是6。
比对中的“-”符号指示空位。Seq B内部因比对引入的空位数是1。通过比对引入的空位的编号在Seq B的边界处是2，并且在Seq A的边界处为1。
在其整个长度范围内显示比对序列的比对长度是10。
根据本发明产生在其整个长度范围内显示较短序列的两两比对因此产生：
根据本发明产生在其整个长度范围内显示序列A的两两比对因此产生：
根据本发明产生在其整个长度范围内显示序列B的两两比对因此产生：
在其整个长度范围内显示较短序列的比对长度是8(存在一个在较短序列的比对长度中考虑的缺口)。
因此，在其整个长度范围内显示Seq A的比对长度将是9(意指Seq A是本发明的序列)。
因此，在其整个长度范围内显示Seq B的比对长度将是8(意指Seq B是本发明的序列)。
在比对两个序列后，在第二步骤中，从产生的比对确定同一性值。为了本说明书的目的，通过以下方式计算同一性百分数：同一性％＝(相同残基/在其整个长度范围内显示较短序列的比对区域的长度)*100。因此，根据这个实施方案，通过相同残基的数目除以其整个长度范围内显示较短序列的比对区域的长度，计算与比较两个氨基酸序列有关的序列同一性。这个值乘以100以得出“同一性％”。根据上文提供的该实例，同一性％是：(6/8)*100＝75％。
与全长多肽序列相比，檀香烯合酶的变体可以具有与相应亲本多肽分子的氨基酸序列相同至少百分之n的氨基酸序列，n是50和100之间的整数，优选地是50、55、60、65、70、75、80、85、90、91、92、93、94、95、96、97、98或99。
檀香烯合酶变体可以由它们与亲本酶相比时的序列相似性定义。序列相似性通常作为“序列相似性％”或“相似性％”提供。为了在第一步骤中计算序列相似性，必须如上文所述生成序列比对。在第二步骤中，必须计算相似性百分数，而序列相似性百分数考虑了定义的氨基酸集合共有相似的特性，例如，依据其大小、依据其疏水性、依据其电荷或依据其他特征共有相似的特性。在这里，一个氨基酸交换为一个相似氨基酸叫做“保守性突变”。包含保守性突变的酶变体似乎对蛋白质折叠影响最小，导致与亲本酶的酶特性相比时，基本上维持某些酶特性。
为了确定本发明的相似性％，以下适用，这还与例如为(如上文所参考的)“NEEDLE”程序所用的BLOSUM62矩阵一致，
所述矩阵是最常用于数据库检索和序列比对的氨基酸相似性矩阵之一。
氨基酸A类似于氨基酸S
氨基酸D类似于氨基酸E；N
氨基酸E类似于氨基酸D；K；Q
氨基酸F类似于氨基酸W；Y
氨基酸H类似于氨基酸N；Y
氨基酸I类似于氨基酸L、M；V
氨基酸K类似于氨基酸E；Q；R
氨基酸L类似于氨基酸I；M；V
氨基酸M类似于氨基酸I；L；V
氨基酸N类似于氨基酸D；H；S
氨基酸Q类似于氨基酸E；K；R
氨基酸R类似于氨基酸K；Q
氨基酸S类似于氨基酸A；N；T
氨基酸T类似于氨基酸S
氨基酸V类似于氨基酸I；L；M
氨基酸W类似于氨基酸F；Y
氨基酸Y类似于氨基酸F；H；W。
保守性氨基酸置换可以出现在功能蛋白质如酶的多肽序列的全长序列范围内。在一个实施方案中，这类突变不涉及酶的功能性结构域。在一个实施方案中，保守性突变不涉及酶的催化中心。
因此，根据本说明书，以下的相似性百分数计算适用：
相似性％＝[相同残基+相似残基/在其整个长度范围内显示更短序列的比对区域的长度]*100。因此这里，通过相同残基数目加相似残基数目除以其整个长度范围内显示更短序列的比对区域的长度，计算与根据这个实施方案比较两个氨基酸序列有关的序列相似性。这个值乘以100以得出“相似性％”。
包含保守性突变的变体酶预期具有基本上不变的酶特性，如酶活性，其中与全长多肽序列相比，变体酶至少百分之m相似于相应亲本序列，其中m是50和100之间的整数，优选地是50、55、60、65、70、75、80、85、90、91、92、93、94、95、96、97、98或99。
如本文所用的“构建体”、“遗传构建体”或“表达盒”(可以互换使用)是一种DNA分子，其由至少一个待表达的与如本文所述的一个或多个调节序列(至少与启动子)有效连接的目的序列组成。一般地，表达盒包含三种元件：启动子序列、可读框和3'非翻译区，所述非翻译区在真核生物中通常含有多聚腺苷化位点。额外的调节元件可以包括转录增强子以及翻译增强子。也可以将内含子序列添加至5'非翻译区(UTR)或在编码序列中，以增加胞质溶胶中积累的成熟信使的量。技术人员非常了解必须在表达盒中存在以便成功表达的遗传元件。优选地，形成表达盒的至少DNA部分或遗传元件的排列是人工的。表达盒可以是载体的部分或可以整合入宿主细胞的基因组并且随其宿主细胞的基因组一起复制。表达盒能够增加或减少目的DNA和/或蛋白质的表达。
如本文中所提及的术语“引入”或“转化”包括将外源性多核苷酸转移入宿主细胞，无论用于转化的方法是什么。即，如本文所用的术语“转化”独立于载体、穿梭系统或宿主细胞，并且它不仅涉及如本领域已知的转化多核苷酸的转移方法(参见，例如，Sambrook,J.等人,(1989)Molecular Cloning:A Laboratory Manual,第2版,Cold Spring HarborLaboratory Press,Cold Spring Harbor,NY)，而且它涵盖任何其他种类的多核苷酸转移方法，例如但不限于转导或转染。
术语“重组生物”指这样的真核生物(酵母、真菌、藻类、植物、动物)或原核微生物(例如，细菌)，所述生物已经如此以遗传方式改变、修饰或工程化，从而如与衍生所述生物的野生型生物相比，它显示出改变、修饰或不同的基因型。优选地，“重组生物”包含外源核酸。“重组生物”、“基因修饰生物”和“转基因生物”在本文中可互换地使用。外源核酸可以位于染色体外的DNA片段(如质粒)上或可以整合在生物的染色体DNA中。在重组真核生物的情况下，将其理解为意指所用的核酸不存在于或不源自所述生物的基因组，或存在于所述生物的基因组中但是不在所述生物的基因组中其天然基因座处，可能在一个或多个内源和/或外源调节元件的调节下表达该核酸。
“宿主细胞”可以是选自细菌细胞、酵母细胞、真菌细胞、藻细胞或蓝细菌细胞、非人类动物或哺乳动物细胞或植物细胞的任何细胞。技术人员非常了解必须在所述基因构建体上存在以便成功转化、选择和增殖含有目的序列的宿主细胞的遗传元件。宿主细胞可以选自这些生物中任一者：
细菌类
○革兰氏阳性：芽孢杆菌属(Bacillus)、链霉菌属(Streptomyces)
■有用的革兰氏阳性细菌包括但不限于芽孢杆菌细胞，例如，嗜碱芽孢杆菌(Bacillus alkalophius)、解淀粉芽孢杆菌(Bacillus amyloliquefacins)、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacillus circulans)、克劳氏芽孢杆菌(Bacillusclausii)、凝固芽孢杆菌(Bacillus coagulans)、坚强芽孢杆菌(Bacillus firmus)、灿烂芽孢杆菌(Bacillus Jautus)、迟缓芽孢杆菌(Bacillus lentus)、地衣芽孢杆菌(Bacilluslicheniformis)、巨大芽孢杆菌(Bacillus megaterium)、短小芽孢杆菌(Bacilluspumilus)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、枯草芽孢杆菌(Bacillussubtilis)和苏云金芽孢杆菌(Baccillus thuringiensis)。最优选地，原核生物是芽孢杆菌细胞，优选地是枯草芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌和迟缓芽孢杆菌的芽孢杆菌细胞。
■其他一些优选的细菌类包括放线菌目(Actinomycetales)、优选地链霉菌属(Streptomyces)的菌株，优选地类球形链霉菌(Streptomyces spheroides)(ATTC 23965)，热紫链霉菌(Streptomyces thermoviolaceus)(IFO 12382)、浅青紫链霉菌(Streptomyceslividans)或鼠灰链霉菌(Streptomyces murinus)或轮枝链轮丝菌轮枝物种(Streptoverticillum verticillium ssp.verticillium)。优选的其他细菌类包括类球红细菌(Rhodobacter sphaeroides)、Rhodomonas palustri、乳酸链球菌(Streptococcuslactis)。进一步优选的细菌类包括属于粘球菌属(Myxococcus)的菌株，例如，变绿粘球菌(M.virescens)。
○革兰氏阴性：大肠杆菌，假单胞菌属(Pseudomonas)
■优选的革兰氏阴性细菌为大肠杆菌(Escherichia coli)和假单胞菌属物种，优选地Pseudomonas purrocinia(ATCC 15958)或荧光假单胞菌(Pseudomonas fluorescens)(NRRL B-11)。
真菌类
○曲霉属(Aspergillus)、镰刀菌属(Fusarium)、木霉属(Trichoderma)
■微生物可以是真菌细胞。如本文所用的“真菌”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)和接合菌门(Zygomycota)以及卵菌门(Oomycota)和半知菌亚门(Deuteromycotina)和全部半知真菌。子囊菌门的代表性菌群例如包括脉孢菌属(Neurospora)、正青霉属(Eupenicillium)(＝青霉属(Penicillium))、翘孢霉属(Emericella)(＝曲霉属(Aspergillus))、散囊菌属(Eurotium)(＝曲霉属(Aspergillus))和下文所列的真酵母列。担子菌门的例子包括蘑菇、锈菌和黑粉菌。壶菌门的代表性菌群例如包括异水霉属(Allomyces)、芽枝霉属(Blastocladiella)、雕蚀菌属(Coelomomyces)和水生真菌。卵菌门的代表性菌群例如包括水霉属水生真菌(水霉菌)如绵霉(Achlya)。半知菌真菌的例子包括曲霉属、青霉属、假丝酵母属(Candida)和链格孢属(Alternaria)。接合菌的代表性菌群例如包括根霉属(Rhizopus)和毛霉属(Mucor)。
■一些优选的真菌包括属于半知菌亚门、丝孢纲的菌株，例如，镰刀菌属、腐质霉属(Humicola)、木霉属(Tricoderma)、漆斑菌属(Myrothecium)、轮枝菌属(Verticillum)、Arthromyces、Caldariomyces、细基格孢属(Ulocladium)、埃里格孢属(Embellisia)、分枝孢子菌属(Cladosporium)或Dreschlera、尤其尖孢镰刀菌(Fusarium oxysporum)(DSM2672)、特异腐质霉(Humicola insolens)、里氏木霉(Trichoderma resii)、疣孢漆斑菌(Myrothecium verrucana)(IFO 6113)、黄萎轮枝菌(Verticillum alboatrum)、大丽轮枝菌(Verticillum dahlie)、Arthromyces ramosus(FERM P-7754)、Caldariomyces fumago、纸细基格孢(Ulocladium chartarum)、大蒜艾氏链格孢(Embellisia alli)或Dreschlerahalodes。
■其他优选的真菌包括属于担子菌亚门，担子菌纲的菌株，例如鬼伞属(Coprinus)、平革菌属(Phanerochaete)、革盖菌属(Coriolus)或栓菌属(Trametes)，尤其灰盖鬼伞(Coprinus cinereus)f.microsporus(IFO 8371)、长根鬼伞(Coprinusmacrorhizus)、黄孢原毛平革菌(Phanerochaete chrysosporium)(例如NA-12)或栓菌属(Trametes)(前称猪苓)，例如杂色栓菌(T.versicolor)(例如PR4 28-A)。
■进一步优选的真菌包括属于接合菌亚门(Zygomycotina)、毛霉科的菌株，例如根霉属(Rhizopus)或毛霉属(Mucor)，尤其外皮毛霉(Mucor hiemalis)。
酵母
○毕赤酵母属(Pichia)
○酵母属(Saccharomyces)
■真菌宿主细胞可以是酵母细胞。如本文所用的“酵母”包括产子囊孢子酵母(内孢霉目(内孢霉目(Endomycetales)))、产担孢子酵母和属于半知菌类(Fungi Imperfecti)(芽孢纲(Blastomycetes))的酵母。产子囊孢子酵母分成蚀精霉科(Spermophthoraceae)和酵母科(Saccharomycetaceae)。后者由四个亚科组成：裂殖酵母亚科(Schizosaccharomycoideae)(例如，裂殖酵母属(Schizosaccharomyces))、拿逊酵母亚科(Nadsonioideae)、油脂酵母亚科(Lipomycoideae)和酵母亚科(Saccharomycoideae)(例如克鲁维酵母属(Kluyveromyces)、毕赤酵母属和酵母属)。产担孢子酵母包括白冬孢酵母属(Leucosporidim)，红冬孢酵母属(Rhodosporidium)、锁掷酵母属(Sporidiobolus)、线黑粉酵母属(Filobasidium)和拟线黑粉酵母属(Filobasidiella)。属于半知菌类的酵母分成两个科：掷孢酵母科(例如，掷孢酵母属(Sporobolomyces)和布勒掷孢酵母属(Bullera))和隐球菌科(例如假丝酵母属(Candida))。
真核生物
○非人类动物、非人类哺乳动物、鸟类、爬行动物、昆虫、植物、酵母、真菌
本文中术语“檀香烯合酶”用于具有从法呢基焦磷酸形成檀香烯和檀香烯样萜类如α-檀香烯、β-檀香烯、反式-α-香柠檬烯和表-β-檀香烯的催化活性的多肽，和用于包含这种多肽的其他部分。这类其他部分的例子包括所述多肽与一种或多种其他多肽的复合体、包含与肽或蛋白质标签序列融合的檀香烯合酶多肽的融合蛋白、所述多肽的其他复合体(例如金属蛋白复合体)、包含所述多肽和另一个有机部分的大分子化合物、与支撑材料结合的所述多肽等。檀香烯合酶可以在其天然环境下提供，即在产生该酶的细胞中提供，或在已经由产生该酶的细胞分泌至其中的培养基中提供，它也可以与已经产生该多肽的来源分开提供，并且可以通过接合至运载体、使用标记用部分标记等来操作。
檀香烯合酶的活性和产物特征可以用已知方法测量，例如，如国际专利申请WO2018160066中所公开那样。
下文中，术语“合成的檀香烯合酶”和“改善的檀香烯合酶”互换地用来指相比野生型檀香烯合酶在常见条件下产生β-檀香烯超过α-檀香烯或增加α-檀香烯量的檀香烯合酶合成性序列。
“改善的α檀香烯合酶”因此指常见条件下相比其来自自然界的对应物具有增加的α檀香烯产量的那些合成的檀香烯合酶。“改善的β檀香烯合酶”指常见条件下产生β-檀香烯超过α-檀香烯的檀香烯合酶合成序列。
术语“超过(in excess)”与术语“剩余(surplus)”互换使用并且应理解第一种所说物质比第二种所说物质存在更多。A超过B意指基于可以是摩尔或重量或百分数的相同基础，物质A比物质B更多存在。
在法呢基焦磷酸转化成萜烯产物中，焦磷酸经切割以产生活泼碳正离子过渡态，导致一系列潜在反应如氢负离子迁移和环化。参与支持某种潜在过渡态超过其他者的残基可以因此影响可能产物的终产物比率。
已知檀香烯合酶的主产物主要是α-檀香烯、香柠檬烯和/或β檀香烯。
檀香烯合酶是萜烯合酶家族酶类并且因为从相同底物产生的多种产物，划分喂属于酶类EC4.2.3.81、EC4.2.3.82和/或EC4.2.3.83、或EC4.2.3.50-后一类酶使用(2Z,6Z)-法呢基焦磷酸作为底物，而不是使用(2E,6E)-法呢基焦磷酸作为底物。它们包含活性部位和金属结合位点的N端PFAM结构域PF01397和C端PFAM结构域PF03936(使用32.0版PFAM分析，关于PFAM详情，请参见“The Pfam protein families database in 2019(Pfam:2019年蛋白质家族数据库)：S.El-Gebali,J.Mistry,A.Bateman,S.R.Eddy,A.Luciani,S.C.Potter,M.Qureshi,L.J.Richardson,G.A.Salazar,A.Smart,E.L.L.Sonnhammer,L.Hirsh,L.Paladin,D.Piovesan,S.C.E.Tosatto,R.D.Finn Nucleic Acids Research(2019)和http://pfam.xfam.org/)。它们需要二价阳离子(通常镁或锰)作为辅因子。在它们的功能状态下，它们一般具有由两个富含天冬氨酸的金属结合位点配位的三个Mg2+离子。这些位点之一称作DDxxD基序，其中该基序是两个天冬氨酸、后接任何氨基酸、后接另一个可变氨基酸，优选地为苯丙氨酸或酪氨酸、更优选地为酪氨酸、以及后接又一个天冬氨酸的序列。第二金属结合位点称作NSE/DTE三联体。这种位点是一个氨基酸序列，其始于天冬酰胺或天冬氨酸，后接第二个天冬氨酸，后接两个可变氨基酸，后接一个丝氨酸或苏氨酸，再次后接一个或两个可变氨基酸，后接一个赖氨酸或精氨酸，任选地后接一个可变氨基酸并且止于一个天冬氨酸或谷氨酸残基。在这些基序中，可变氨基酸优选地是允许基序的定义氨基酸采取为金属离子结合(一般为镁结合)所需的三级结构的那些氨基酸。
配位镁离子的这些保守结合位点中的一个保守结合位点，即DDxxD基序，位于α螺旋中。在称作CiCaSSy(作为SEQ ID NO:1提供)的樟树(Cinnamomum camphora)檀香烯合酶中，这个α螺旋从SEQ ID NO:1的位置278处脯氨酸或紧邻其之后伸展至位置302处的天冬氨酸并且命名为螺旋D。在其他檀香烯合酶中，存在包含DDxxD基序的等同α螺旋，尽管它们的命名可能不同，但该螺旋总是直接影响活性部位。在下文中，对任何螺旋D的提及是指给定檀香烯合酶的在对应于SEQ ID NO 1的氨基酸位置298至302的位置处包含DDxxD基序的α螺旋，无论该螺旋是否可以用字母D辨识或在相应的蛋白质序列中按不同方式辨识。归因于DDxxD基序和其他保守残基及结构性特征高度保守，这些螺旋为本领域已知并且可以在檀香烯合酶的新序列中轻易地辨识。
发明人意识到，螺旋D对檀香烯合酶的产物特征至关重要，然而改变它可能过度扰动活性部位的敏感区域内的酶结构和/或危及酶作用必备的镁离子结合。
发明人发现可以通过更微妙的变化实现酶的产物特征改变。在檀香烯合酶中，螺旋D前置有另一个α螺旋。在CiCaSSy中，这个螺旋称作螺旋C并且从SEQ ID NO:1中位置263伸展至位置272。某些预测使这个α-螺旋延伸至位置276，然而核心是从位置263至272。
SEQ ID NO:1的位置263中该螺旋起始处存在精氨酸残基，其为SEQ ID NO:1的位置261至263中存在的精氨酸–天冬氨酸-精氨酸三联体的一部分。这个三联体在N末端含有檀香烯合酶中保守的精氨酸残基。
螺旋C与螺旋D在它们的对面侧(facing sides)相互作用。螺旋D的特定相关氨基酸位置处在与SEQ ID NO:1的位置291对应的区域中。与螺旋C氨基酸的侧链可能出现侧链相互作用的其他位置在SEQ ID NO:1、2和3中位置287和288处(分别是异亮氨酸和苏氨酸)上游，及SEQ ID NO:1、2和3中位置294和295处(分别是甲硫氨酸和苏氨酸)下游。
发明人发现，操纵螺旋C为酶提供更多灵活性，这将影响产生的产物，与此同时不以负向方式过度扰动酶结构或酶的镁结合过程或底物结合过程。他们从一级结构发现许多檀香烯合酶似乎原则上适应于想要的变化并且选择了CiCaSSy(SEQ ID NO:1)展示本发明的效果。CiCaSSy与β-檀香烯产量相对高(尽管仍然少于产生的α檀香烯)的檀香烯合酶共享螺旋C元件，这也是CiCaSSy关于这两种檀香烯的产物特征。仅次于CiCaSSy的这类已知酶的例子是SaSSy(SEQ ID NO:4)、SaSSy14(SEQ ID NO:5)、SspiSSy(SEQ ID NO:6)或SauSSy(SEQ ID NO:7)或SaSSy134(SEQ ID NO:9)。然而，CiCaSSy还与少量β-檀香烯生产者且α-檀香烯强生产者的檀香烯合酶如ClaSSy(SEQ ID NO:8)共有元件。归因于这些类群之间的这种居中地位，选择CiCaSSy作为操纵螺旋C的起点，旨在以正向方式影响酶结构(例如螺旋D和其他下游部件)的灵活性。
在深度研究后，选择CiCaSSy的残基267用于突变。这个残基预计以其侧链与螺旋D的面侧相互作用(参见图3)。与其他檀香烯合酶相比，CiCaSSy毗邻于这个残基具有一些更少见的氨基酸，预计使其更适应导致产物特征变化。在与SEQ ID NO:1的天冬酰胺267(称作N267)对应的位置，许多其他檀香烯合酶具有丝氨酸或亮氨酸(参见图1的比对)。然而，在这个位置具有丝氨酸或亮氨酸的这些檀香烯合酶在其物特征方面具有所述缺点，类似于SEQID NO:1的未突变CiCaSSy。然而，发明人意识到，SEQ ID NO:1中N267的周围环境如此有利，从而发明人选择将位置267处的不常见天冬酰胺替换为丝氨酸和亮氨酸，尽管这些氨基酸存在于其他已知性能匮乏的檀香烯合酶中相应的位置处。称为N267S和N267L的改善的檀香烯合酶的所得合成性蛋白质序列分别在SEQ ID NO:2和SEQ ID NO:3中给出。令人惊讶地，这个位置处回复突变成更常见氨基酸导致酶的催化部分(例如两个相邻α螺旋)的空间灵活度变化和新的有利的产物特征变化。另外，这种有利的产物特征变化还可能用针对与SEQID NO:1的267对应的位置的其他巧妙替换(例如用下文中所示的甘氨酸或丙氨酸)实现。
编码野生型CiCaSSy、N267S和N267L的DNA序列分别列为SEQ ID NO:10、11和12。
与SEQ ID NO:1的位置267对应的位置处携带丝氨酸的额外合成性蛋白质序列作为SEQ ID NO:13至20显示，并且与SEQ ID NO:1的位置267对应的位置处携带亮氨酸的额外改善的蛋白质序列作为SEQ ID NO:21至28显示。
在一个实施方案中，本发明因此涉及一种合成的β檀香烯合酶，其在一般导致α-檀香烯和β-檀香烯二者产生的条件下从法呢基焦磷酸产生β-檀香烯超过α-檀香烯，尽管已知的檀香烯合酶一般在这类条件下产生α-檀香烯超过β-檀香烯，其中本发明的合成的β檀香烯合酶特征在于以下事实：与产生超过β-檀香烯的过量α-檀香烯的天然存在的檀香烯合酶中的相同三级结构相比，对应于从SEQ ID NO:1的氨基酸位置272延伸至位置291、优选至位置284的α螺旋的三级结构的灵活性增加。可以例如通过以下方式测定灵活性：相同条件下按pH 8.0、300K、1atm、水环境、无底物情况下离子存在的设置使用模拟持续500ns的均方根波动分析并且依照模拟的最后450ns评估每种酶结构，并且其中在已经使用gmx trjconv对每个轨迹框架进行蛋白质结构的结构性叠加并且使用已平衡体系的蛋白质Cα作为参考后，通过GROMACS软件2018版的gmx rmsf工具进行计算。
在一个实施方案中，本发明的多肽是一种具有β檀香烯合酶的酶促功能的合成的多肽并且相比其天然存在的对应物，意在增加螺旋D的灵活性、优选地三级结构的灵活性，所述三级结构对应于从对应于SEQ ID NO:1中位置272至位置291的氨基酸位置伸出的α螺旋，并进一步特征在于，在适于产生β檀香烯的条件下从FPP产生β檀香烯超过α檀香烯。
在本发明的一个方面，对应于从SEQ ID NO:1的氨基酸位置272延伸至位置291、优选至位置284的三级结构的灵活性相比产生超过β-檀香烯的过量α-檀香烯的天然存在的檀香烯合酶中的相同三级结构增加，其中通过以下方式测定灵活性：相同条件下按pH 8.0、300K、1atm、水环境、无底物情况下离子存在的设置使用模拟持续500ns的均方根波动分析并且依照模拟的最后450ns评估每种酶结构，并且其中在已经使用gmx trjconv对每个轨迹框架进行蛋白质结构的结构性叠加并且使用已平衡体系的蛋白质Cα作为参考后，通过GROMACS软件2018版的gmx rmsf工具进行计算。与产生超过β-檀香烯的过量α-檀香烯的天然存在的檀香烯合酶的相应三级结构的灵活性相比，灵活性增加至少5％、优选地至少10％、更优选地至少15％。
在又一个实施方案中，与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸填充。在本发明的另一个方面，合成的檀香烯合酶还包含两个结合Mg2+的天冬氨酸丰富基序，优选地DDxxD基序和NSE/DTE三联体。
在一个实施方案中，改善的β檀香烯合酶包含一段氨基酸，其起自与SEQ ID NO:1的位置261对应的精氨酸(R261)至两个与SEQ ID NO:1的位置298和299对应的天冬氨酸残基(D298和D299)，后接两个氨基酸，优选地两个氨基酸中第二者是酪氨酸，并且后接一个与SEQ ID NO:1的位置302对应的第三天冬氨酸(D302)，其中这五个氨基酸优选地参与酶的金属结合，并且优选地，与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸填充。在一个优选的实施方案中，合成的檀香烯合酶包含这样的区段，其中进一步所述的区段始于与SEQ ID NO:1的R261对应的精氨酸并且止于与SEQ ID NO:1的D302对应的天冬氨酸；并且此外按渐增优选性顺序相对于SEQ ID NO:2、3、13至53的氨基酸261至302的全长、优选地来自SEQ ID NO:2、3、14至17、21至52的那些氨基酸的全长具有至少50％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％或97％序列同一性，其中更优选地，这个区段中如图1中所述的全部强烈保守型氨基酸均存在于该区段中。
在本发明的一个方面，本发明的和可用于本发明方法与宿主细胞中的改善的檀香烯合酶携带接近于其N端起点的R(R/K)xxxxxxxxW基序(精氨酸后接一个精氨酸或赖氨酸，随后是任何类型的八个氨基酸，随后是一个精氨酸，参见SEQ ID NO:55)、优选地RRxxxxxxxxW基序(RRX8W，参见SEQ ID NO:54)。在一个实施方案中RRX8W基序始于与SEQ IDNO:2、3、29、57或58中位置7对应的位置并且止于与SEQ ID NO:2、3或29的位置17对应的位置。在另一个实施方案中，存在于本发明改善的檀香烯合酶中和可用于本发明方法与宿主细胞中的RRX8W基序在与SEQ ID NO:2、3、29、57或58的位置7至17对应的位置具有与SEQ IDNO:2、3或29的以下位置：位置7、8和12至17中那些SEQ ID NO:2、3、29、57或58氨基酸相同的氨基酸。
在又一个实施方案中，本发明的和可用于本发明方法与宿主细胞中的改善的檀香烯合酶接近于其N端起点含有与如SEQ ID NO:2、3或29中存在的RRX8W基序至少80％或90％同一的RRX8W基序。在另一个方面，本发明的和可用于本发明方法与宿主细胞中的改善的檀香烯合酶的这种基序与SEQ ID NO:2、3或29的RRX8W基序相同。
在本发明的一个方面，本发明的和可用于本发明方法与宿主细胞中的改善的檀香烯合酶包含PFAM结构域PF01397“Terpene_synth”和C端PFAM结构域PF03936“Terpene_synth_C”。
在本发明的另一个方面，本发明的和可用于本发明方法与宿主细胞中的改善的檀香烯合酶包含通过InterPro软件鉴定的以下特征：
结构域“萜烯合酶，金属结合结构域”IPR005630、“萜烯环化酶样1，C端结构域”IPR034741和“萜烯合酶，N端结构域”IPR001906
和同源超家族“类异戊二烯合酶结构域超家族”IPR008949，
“类萜环化酶/蛋白质异戊二烯基转移酶α-α环”IPR008930和
“萜烯合酶，N端结构域超家族”IPR036965。
如所示，螺旋C的关键区中仅一个或几个氨基酸改变为必备，以提供改善的产物特征的所需效果。归因于螺旋C区短，一个或几个氨基酸改变迅速导致用于螺旋C区的两种序列的序列同一性差异相对大。
又一个优选的实施方案涉及一种相对于野生型酶如此改善的合成的檀香烯合酶，从而它从法呢基焦磷酸产生β-檀香烯超过α-檀香烯，其中使用与SEQ ID No.2或3的位置261处精氨酸对应的精氨酸残基和与SEQ ID NO:2、3、29、57或58的位置278处脯氨酸对应的脯氨酸残基对齐两个蛋白质序列以确定序列同一性时，檀香烯合酶相对于SEQ ID NO:2、3至29的氨基酸位置261至278的全长、优选地与SEQ ID NO:2、3至29的位置261至位置272具有至少50％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性，并且更优选地，与SEQ ID NO:2、3、29、57或58的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸填充，并且与SEQ ID NO:2、3、29、57或58的位置291对应的位置用除了组氨酸或亮氨酸之外的氨基酸填充；优选地，这个位置用以下这些氨基酸中任一者填充：异亮氨酸、缬氨酸、丝氨酸、半胱氨酸、苯丙氨酸或苏氨酸。在本发明的一个方面，所述合成的β檀香烯合酶在适于产生β-檀香烯和α-檀香烯的条件下以β-檀香烯与α-檀香烯等于或大于1、优选地至少1.1和更优选地至少1.2和甚至更优选地1.3的比率产生这些檀香烯。
本发明的另一个方面是一种从法呢基焦磷酸产生β-檀香烯超过α-檀香烯的合成的β檀香烯合酶，其中檀香烯合酶与SEQ ID NO:2、3、29至40、57或58的氨基酸位置261至302具有至少50％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性，其中与SEQ ID No.2或3的位置261对应的位置是与SEQ ID No.2或3的位置261处精氨酸对应的精氨酸残基并且三个天冬氨酸残基存在于与SEQ ID NO:2或3或29至40的位置298、299和302处天冬氨酸对应的位置处，并且其中所述合成的β檀香烯合酶在适于产生β-檀香烯和α-檀香烯的条件下以β-檀香烯与α-檀香烯等于或大于1、优选地至少1.1及更优选地至少1.2以及甚至更优选地1.3的比率产生这些檀香烯。
在一个优选的实施方案中，改善的β檀香烯合酶中与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、甘氨酸、丙氨酸或苏氨酸填充并且与SEQ ID NO:1的位置282对应的位置用具有极性不带电荷侧链或带正电荷侧链的氨基酸、优选地谷氨酰胺或天冬酰胺或精氨酸或赖氨酸填充。
在另一个优选的实施方案中，改善的檀香烯合酶中与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸填充并且它还在与SEQ ID NO 1中氨基酸名称后括号里所提供位置对应的位置具有亮氨酸以下氨基酸：精氨酸(261)、天冬氨酸或天冬酰胺(262)、精氨酸或天冬酰胺(263)、亮氨酸或异亮氨酸或缬氨酸或甲硫氨酸(264)亮氨酸或异亮氨酸或缬氨酸或甲硫氨酸(265)、谷氨酸或谷氨酰胺(266)、组氨酸或酪氨酸(268)和谷氨酰胺或精氨酸或赖氨酸(282)。
更优选地，这些氨基酸是与括号里所提供SEQ ID NO 1中位置对应的位置处的以下氨基酸：精氨酸(261)、天冬氨酸(262)、精氨酸(263)、亮氨酸(264)、亮氨酸(265)、谷氨酸(266)、组氨酸(268)、亮氨酸(269)、苯丙氨酸(270)和谷氨酰胺或精氨酸(282)。
在本发明的一个方面，除了如先前段落中限定的氨基酸之外，本发明的改善的β檀香烯合酶中与SEQ ID NO:2,3,29,57或58的位置291对应的位置以除了组氨酸或亮氨酸之外的氨基酸填充，优选地，该位置用以下这些氨基酸中任一者填充：异亮氨酸、缬氨酸、丝氨酸、半胱氨酸、苯丙氨酸或苏氨酸。
在又一个优选的实施方案中，改善的檀香烯合酶额外地在与SEQ ID NO:1的位置271对应的位置处包含丝氨酸或苏氨酸、优选地丝氨酸，并且在与SEQ ID NO:1的位置273对应的位置处包含丙氨酸、异亮氨酸、缬氨酸或半胱氨酸、优选地丙氨酸。
进一步优选地，改善的檀香烯合酶是在与SEQ ID NO:1的位置267对应的位置携带丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸的那些酶，并且SEQ ID NO:1中位置对应的位置用下表A、B或C中针对SEQ ID NO:1的相应位置所列的氨基酸填充。
表A
表B_
表C_
SEQ ID NO:1的位置298处的天冬氨酸标示SEQ ID NO:1中DDXXD基序的起点。
在另一个优选的实施方案中，改善的檀香烯合酶包含与SEQ ID NO:1的位置268对应的位置处的组氨酸、与SEQ ID NO:1的位置269对应的位置处的亮氨酸和与SEQ ID NO:1的位置270对应的位置处的苯丙氨酸，并且优选地，与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸或亮氨酸填充。更优选地，改善的檀香烯合酶还在与表A、B或C中对SEQ ID NO:1列出的位置对应的位置包含表A、B或C中列出的氨基酸。
在一个优选的实施方案中，改善的β檀香烯合酶在与SEQ ID NO:1的位置291对应的位置具有另一个除了组氨酸、甘氨酸或亮氨酸之外的氨基酸。
发明人应用进一步的方案增加螺旋C和螺旋D周围的灵活性。SEQ ID NO:1、2、3、29、57或58中位置291是作为螺旋D面向螺旋C的部分的位置。在SEQ ID NO:1的野生型CiCassy中，该位置以异亮氨酸填充。令人吃惊地，发明人发现，将SEQ IDNO:1的位置291处的异亮氨酸替换为苏氨酸、丝氨酸、缬氨酸、苯丙氨酸或半胱氨酸对β-檀香烯/α-檀香烯比率具有积极影响，同时维持比N267S或N267L突变体中更高的α-檀香烯水平。在本发明的另一个方面，与SEQ ID NO:1的位置291对应的位置具有苏氨酸、丝氨酸、甲硫氨酸、缬氨酸、苯丙氨酸或半胱氨酸、优选地苏氨酸、丝氨酸、缬氨酸、苯丙氨酸或半胱氨酸的合成的β檀香烯合酶还包含两个结合Mg2+的天冬氨酸丰富基序，优选地DDxxD基序和NSE/DTE三联体。
进一步地，发明人产生了一种合成的檀香烯合酶序列，其中将与SEQ ID NO:1的位置291对应的位置处的氨基酸替换为亮氨酸，并且α-檀香烯产量与SEQ ID NO:1的产量相比增加。
本发明的又一个方面涉及一种与SEQ ID NO:1的267和/或291对应的位置处存在有利突变的合成的檀香烯合酶，其中该檀香烯合酶包含结合Mg2+的天冬氨酸丰富基序DDxxD，同时酪氨酸或苯丙氨酸在第四位置处，更优选地，该结合基序具有N末端始自两个天冬氨酸、苯丙氨酸、酪氨酸并且后接又一天冬氨酸的序列。
进一步地，对SEQ ID NO:1的位置291对应的位置处优选的异亮氨酸氨基酸替换，在优选的实施方案中，改善的檀香烯合酶还在与SEQ ID NO:1中位置287对应的位置具有异亮氨酸或亮氨酸、优选地异亮氨酸并且在与其位置288对应的位置具有苏氨酸、丝氨酸或缬氨酸、优选地苏氨酸或丝氨酸、更优选地苏氨酸。本发明另外一个优选的方面涉及一种在与SEQ ID NO:1的位置286对应的位置具有丙氨酸、在与SEQ ID NO:1的位置287对应的位置具有异亮氨酸、在与SEQ ID NO:1的位置288对应的位置具有苏氨酸、在与SEQ ID NO:1的位置289对应的位置具有赖氨酸、在与SEQ ID NO:1的位置290对应的位置具有丙氨酸的改善的檀香烯合酶。
除了优选的氨基酸替换与SEQ ID NO:1的位置291对应的位置处的异亮氨酸外，在优选的实施方案中，改善的檀香烯合酶还在与SEQ ID NO:1中位置294对应的位置具有甲硫氨酸或亮氨酸或谷氨酸残基、优选地甲硫氨酸或谷氨酸残基、更优选地甲硫氨酸。
本发明的一个方面涉及一种从法呢基焦磷酸产生β-檀香烯超过α-檀香烯的合成的β檀香烯合酶，其中檀香烯合酶具有与SEQ ID NO:1至少50％同一的氨基酸序列；并且在对应于以下的氨基酸位置：
a.SEQ ID NO:1的位置267处具有以下氨基酸中任一者
丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸；或
b.SEQ ID NO:1的位置291处具有以下氨基酸中任一者
苏氨酸、半胱氨酸、丝氨酸、苯丙氨酸或缬氨酸；或
c.以上a.和b.的组合；或
d.SEQ ID NO:1的位置267处具有天冬酰胺并且与SEQ ID NO:1的位置291对应的位置是以下氨基酸中任一者：
苏氨酸、半胱氨酸、丝氨酸、苯丙氨酸或缬氨酸；或
e.SEQ ID NO:1的位置267处具有以下氨基酸中任一者
丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸并且与SEQ ID NO:1的位置291对应的位置是异亮氨酸。
在一个其他方面，本发明因此涉及从法呢基焦磷酸产生β-檀香烯超过α-檀香烯的合成的β檀香烯合酶，其中檀香烯合酶具有与SEQ ID NO:1至少60％同一的氨基酸序列；并且在对应于以下的氨基酸位置：a)SEQ ID NO:1位置267处具有以下氨基酸中任一者：丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸或丙氨酸，优选地丝氨酸或苏氨酸；和/或b)SEQ ID NO:1的位置291处具有以下氨基酸中任一者：异亮氨酸、丝氨酸、半胱氨酸、缬氨酸、苯丙氨酸或苏氨酸、优选地苏氨酸、苯丙氨酸或缬氨酸；或当与SEQ ID NO:1的位置267对应的位置是天冬酰胺时，与SEQ ID NO:1的位置291对应的位置处具有以下氨基酸中任一者：丝氨酸、半胱氨酸、缬氨酸、苯丙氨酸或苏氨酸、优选地苏氨酸、苯丙氨酸或缬氨酸；在本发明的另一个方面，除先前句子的特征之外，合成的β檀香烯合酶还使得与SEQ ID NO:1的位置285对应的位置用缬氨酸填充、使得与SEQ ID NO:1的位置282对应的位置用谷氨酰胺或精氨酸填充、使得与SEQ ID NO:1的位置271对应的位置用丝氨酸填充、使得与SEQ IDNO:1的位置273对应的位置用丙氨酸填充和/或使得与SEQ ID NO:1的位置274对应的位置用缬氨酸填充。
另外，改善的檀香烯合酶是在与SEQ ID NO:1的位置291对应的位置携带异亮氨酸、缬氨酸、甲硫氨酸、半胱氨酸、丝氨酸、苯丙氨酸或苏氨酸，优选地缬氨酸、半胱氨酸、丝氨酸、苯丙氨酸或苏氨酸、更优选地半胱氨酸、苏氨酸或缬氨酸或备选地对改善的α檀香烯合酶而言携带亮氨酸的那些酶，并且另外，与SEQ ID NO:1中位置对应的位置用下表A’、B’或C’中针对SEQ ID NO:1的相应位置所列的氨基酸填充。
表A’
表B‘
表C‘
在本发明的一个方面，改善的檀香烯合酶在与SEQ ID NO:1的位置267对应的位置具有在以下SEQ ID No:2、3或29任一者的多肽的位置267处存在的氨基酸及在与SEQ IDNO:1的位置291对应的位置具有在以下SEQ ID No:30、31、32、33或34任何者的多肽的位置291处存在的氨基酸以改善β檀香烯合酶，或在与SEQ ID NO:53的多肽的291处存在的氨基酸以改善α檀香烯合酶，并且相对于SEQ ID NO:2、3、29至40或53中任一多肽的全长具有至少50％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性。
在本发明的又一个方面，改善的檀香烯合酶在与表D提供的SEQ ID NO:1中位置对应的位置具有表D中列出的以下氨基酸残基，并且优选地，与SEQ ID NO:1的位置267对应的位置用丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、优选地丝氨酸、苏氨酸或亮氨酸填充，并且更优选地，与SEQ ID NO:1的位置255对应的位置用具有疏水性侧链或极性不带电荷侧链的氨基酸、优选地丝氨酸、苏氨酸、丙氨酸或缬氨酸、更优选地丙氨酸填充。
表D
在本发明的又一个优选的方面，除与SEQ ID NO:1的位置267和291对应的位置处的有利氨基酸之外，改善的檀香烯合酶还在与紧邻此处所列的每个氨基酸在括号里提供的SEQ ID NO 1位置对应的位置具有以下氨基酸：丝氨酸(271)、丙氨酸(273)、缬氨酸(274)、谷氨酰胺(282)、缬氨酸(285)、丙氨酸(286)、缬氨酸(292)、甲硫氨酸(294)、丙氨酸(296)和苯丙氨酸(300)。
在本发明的另一个优选方面，除上文所列位置处的有利氨基酸之外，改善的檀香烯合酶还在与SEQ ID NO:1中位置232处对应的位置具有精氨酸。
表1显示一些改善的檀香烯合酶和对照中的β-檀香烯/α-檀香烯比率：
名称：受测的檀香烯合酶；
比率b/a：β-檀香烯/α-檀香烯比率w％/w％
巧妙的改善导致改善的檀香烯合酶的产物中β-檀香烯/α-檀香烯比率升高或α檀香烯增加，如表1中所显示。斜体条目用于SEQ ID NO:1的未修饰酶(“野生型”)，及用于产生过量α檀香烯的I291L。后者显示在给定位置处巧妙修饰将导致想要的β-檀香烯/α-檀香烯比率或α-檀香烯产量改善，因为I291L修饰允许比SEQ ID NO:1的未修饰酶产生更大量的α-檀香烯，如通过I291L的较低β-檀香烯/α-檀香烯比率所示。
增加或减少产生的β-檀香烯要求在位置267处和/或291处作出本发明的氨基酸选择。例如，发明人用亮氨酸替换与SEQ ID NO:1的位置291对应的位置处的异亮氨酸(参见SEQ ID NO:53)，以增加α-檀香烯产量超过SEQ ID NO:1，但代价是β-檀香烯和香柠檬烯的产量未改善，反而减少。本发明的一个方面因此是一种在与SEQ ID NO:1的位置291对应的位置具有亮氨酸的合成的α檀香烯合酶，其相比未修饰酶具有改善的α-檀香烯产量。
在SEQ ID NO:1的位置291处引入组氨酸时，檀香烯合酶的活性遭摧毁并且α-檀香烯、β-檀香烯和香柠檬烯未产生。在本发明的一个方面，本发明的改善的檀香烯合酶在291位置处具有除了组氨酸之外的氨基酸。
在一个方面，本发明的改善的檀香烯合酶在与SEQ ID NO:1的位置291对应的位置没有组氨酸残基或甘氨酸残基，而在与SEQ ID NO:1的位置291对应的位置具有异亮氨酸、缬氨酸、苏氨酸、半胱氨酸、苯丙氨酸或丝氨酸、优选地半胱氨酸、缬氨酸、丝氨酸、苯丙氨酸或苏氨酸；或在想要α檀香烯/β檀香烯比率升高的情况下亮氨酸。在本发明的另一个方面，与SEQ ID NO:1的位置267对应的位置用丝氨酸、苏氨酸或亮氨酸填充时，异亮氨酸存在于与SEQ ID NO:1的位置291对应的位置处、或与SEQ ID NO:1的位置267对应的位置用天冬酰胺填充时，在位置291存在缬氨酸、半胱氨酸、丝氨酸、苯丙氨酸或苏氨酸。
在另一个优选的实施方案中，改善的檀香烯合酶在与紧邻此处所列的每个氨基酸在括号里提供的SEQ ID NO 1位置对应的位置处包含精氨酸(261)、亮氨酸(264)、亮氨酸(265)、丝氨酸(271)、丙氨酸(273)、脯氨酸(278)、精氨酸(284)、异亮氨酸(287)、天冬氨酸(298)、天冬氨酸(299)和天冬氨酸(302)、并且优选地，与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸或亮氨酸填充。对于改善的β檀香烯合酶，在又一个实施方案中，这个位置用天冬酰胺填充并且与SEQ ID NO:1的位置291对应的位置用缬氨酸、半胱氨酸、丝氨酸、苯丙氨酸或苏氨酸填充。
在又一个优选的实施方案中，改善的檀香烯合酶额外地具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、更优选地至少91％、92％、93％、94％、95％、96％、97％、98％或99％和甚至更优选地100％在图1中以黑背景底纹标记作为强烈保守的全部那些氨基酸。
在另一个优选的实施方案中，改善的檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为亮氨酸、丝氨酸或苏氨酸，并且位置291处的氨基酸替换为苏氨酸、丝氨酸、半胱氨酸、苯丙氨酸或缬氨酸，或在想要产生增加的α檀香烯量的情况下，替换为亮氨酸。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Leu，并且位置291处的氨基酸替换为Thr。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Leu，并且位置291处的氨基酸替换为Ser。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Leu，并且位置291处的氨基酸替换为Cys或Phe。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Leu，并且位置291处的氨基酸替换为Val。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Ser，并且位置291处的氨基酸替换为Thr。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Ser，并且位置291处的氨基酸替换为Ser。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Ser，并且位置291处的氨基酸替换为Cys或Phe。
在另一个优选的实施方案中，改善的β檀香烯合酶包含SEQ ID NO:1、其变体、衍生物、直向同源物、旁系同源物或同源物的序列，其中位置267处的氨基酸替换为Ser，并且位置291处的氨基酸替换为Val。
改善的檀香烯合酶一般在无任何标签、添加的结构域或与其他蛋白质部分融合的情况下具有60和70kDa之间、优选地61和66kDa之间的分子量。
在一个优选的实施方案中，改善的檀香烯合酶相对于SEQ ID NO:1的全长具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％，例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％和例如100％序列同一性。在又一个优选的实施方案中，改善的檀香烯合酶相对于SEQ ID NO:2、3、14至17、21至52中任一者、优选地SEQ ID NO:2、3、29至40中任一者的全长蛋白质序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％和例如100％序列同一性，用于改善的β檀香烯合酶；或者如果想要增加α檀香烯产量，相对于SEQ ID NO:13、18、19、20或53、优选地SEQ IDNO:53的全长蛋白质序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％和例如100％序列；并且更优选地额外具有图1中以黑背景底纹标记作为强烈保守的全部那些氨基酸。
在β-檀香烯/α-檀香烯比率升高的檀香烯合酶中，优选地a)与SEQ ID NO:1的位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸或亮氨酸填充，或与SEQ ID NO:1的位置291对应的位置用缬氨酸、苏氨酸、半胱氨酸、苯丙氨酸或丝氨酸、更优选地用Thr、Val、Cys或Ser填充，或b)与SEQ ID NO:1中位置267对应的位置是天冬酰胺并且与SEQ ID NO:1的位置291对应的位置用缬氨酸、苏氨酸、半胱氨酸、苯丙氨酸或丝氨酸、更优选地用Thr、Val、Cys或Ser填充；或c)与SEQ ID NO:1中位置267对应的位置用丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸、更优选地丝氨酸、甘氨酸、丙氨酸或亮氨酸填充并且与SEQ ID NO:1的位置291对应的位置用异亮氨酸、缬氨酸、苏氨酸或甲硫氨酸填充；或d)a)、b)或c)与SEQ ID NO:1的位置255对应的位置处为丙氨酸残基的组合；或e)a)、b)或c)与SEQ ID NO:1的位置268对应的位置处为组氨酸的组合。
在α-檀香烯/β-檀香烯比率升高的檀香烯合酶中，与SEQ ID NO:1的位置291对应的位置用亮氨酸填充，并且与SEQ ID NO:1中位置267对应的位置是天冬酰胺、丝氨酸、苏氨酸或亮氨酸、优选地天冬酰胺。
本发明的一个方面涉及一种从法呢基焦磷酸产生α-檀香烯超过β-檀香烯的合成的檀香烯合酶，其中使用与SEQ ID No:1、2、3、29、57或58的位置261处精氨酸对应的精氨酸残基和三个与SEQ ID NO:1、2、3、29、57或58的位置298、299和302处天冬氨酸对应的天冬氨酸残基对齐两个蛋白质序列以确定序列同一性时，该檀香烯合酶与SEQ ID NO:1、2、3、29、57或58中任一者的氨基酸位置261至302具有至少50％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性，并且其中与SEQ ID NO:2或3中位置291对应的位置是甘氨酸或亮氨酸、优选地亮氨酸。在一个方面，这些改善的α檀香烯合酶在与SEQ ID NO:1的位置267对应的位置具有天冬酰胺。
优选地，本发明的改善的β檀香烯合酶与SEQ ID NO:2、3、14至17、21至52、57或58中任一者、优选地SEQ ID NO:2、3、29至40、57或58中任一者在以下蛋白质部分中具有至少50％、优选地至少60％、至少70％、至少80％、至少90％或至少95％序列同一性，所述蛋白质部分始于与SEQ ID NO:2、3、29、57或58的位置261中精氨酸对应的位置中的精氨酸并且伸展至与SEQ ID NO:2、3或29至40、57或58的位置298、299和302处天冬氨酸对应的位置中的三个天冬氨酸并且在与SEQ ID NO:2、3、29、57或58的位置267对应的位置具有丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸，和/或在与SEQ ID NO:2、3、29、57或58的位置291对应的位置处具有缬氨酸、半胱氨酸、丝氨酸、苯丙氨酸或苏氨酸、优选地缬氨酸、丝氨酸、苯丙氨酸或苏氨酸，或在与SEQ ID NO:2、3、29、57或58的位置267对应的位置存在丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸情况下，在与SEQ ID NO:2、3或29的位置291对应的位置具有异亮氨酸。
在另一个优选的方面，改善的β檀香烯合酶与SEQ ID NO:2、3、14至17、21至52、57或58、优选地SEQ ID NO:2、3或29至40、57或58中任一者在以下蛋白质部分中具有至少50％、优选地至少60％、至少70％或80％序列同一性，所述蛋白质部分始于与SEQ ID NO:2、3、29、57或58的位置261中精氨酸对应的位置中的精氨酸并且伸展至与SEQ ID NO:2、3、29、57或58的位置298、299和302处天冬氨酸对应的位置中的三个天冬氨酸并且在与SEQ IDNO:2、3、29、57或58的位置267对应的位置具有天冬酰胺、丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地天冬酰胺、丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸，和/或在与SEQ ID NO:2、3、29、57或58的位置291对应的位置具有缬氨酸、丝氨酸、半胱氨酸、苯丙氨酸或苏氨酸、优选地丝氨酸、缬氨酸、苯丙氨酸或苏氨酸，并且优选地在与SEQ ID NO:2、3、29、57或58中位置268对应的位置具有组氨酸。
通过可靠的定量方法、优选地带FID探测器的气相色谱法测定β-檀香烯和α-檀香烯的量。实施例部分中详述用于测定α-檀香烯，β-檀香烯和香柠檬烯的量的优选方法。
改善的β檀香烯合酶产生β-檀香烯超过α-檀香烯，这意指在适于产生这些檀香烯的条件下，该酶按大于1.0的β-檀香烯/α-檀香烯摩尔比产生β-檀香烯和α-檀香烯。改善的α檀香烯合酶产生α-檀香烯超过β-檀香烯，这意指在适于产生这些檀香烯的条件下，该酶按小于1.0的β-檀香烯/α-檀香烯摩尔比产生β-檀香烯和α-檀香烯。
可以例如通过在宿主细胞中表达编码改善的檀香烯合酶的DNA来提供用于产生这些檀香烯的合适条件，所述宿主细胞提供有活性的改善的檀香烯合酶并且提供全部底物和辅因子例如法呢基焦磷酸和镁离子，以便改善的酶进行生成α-檀香烯和β-檀香烯的反应。
迄今，已知的檀香烯合酶产生其中β-檀香烯/α-檀香烯的摩尔比低于1的组合物。本发明的改善的β檀香烯合酶按等于或大于1的β-檀香烯/α-檀香烯摩尔比产生β-檀香烯和α-檀香烯，优选地通过GC-FID测量；例如，该比率是至少1.05、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9或至少2。β-檀香烯/α-檀香烯的比率可以是至少3:1、优选地至少4:1、更优选地至少5:1、甚至更优选地6:1，然而甚至更优选地是至少7:1、最优选地至少8:1和甚至至少9:1。在本发明的一个方面，该比率不大于100:1。
本发明的一个方面涉及编码本发明合成的檀香烯合酶中任一者的合成的核酸，所述合成的檀香烯合酶为β-檀香烯/α檀香烯产量升高的檀香烯合酶(例如但不限于SEQ IDNO:2、3、14至17、21至52的多肽或其变体)，或与修饰前的天然檀香烯合酶相比α檀香烯产量改善的那些檀香烯合酶，例如但不限于SEQ ID NO:53的多肽或其变体。本发明的又一个部分是包含本发明合成性核酸的表达盒。
又一个优选的实施方案是一种用于使用本文公开的改善的β檀香烯合酶产生β-檀香烯超过α-檀香烯的组合物的方法、优选地一种适于大规模生产的方法，所示方法包括步骤：i)提供一种或多种处于活性形式且具有全部必备辅因子(例如但不限于金属离子如镁离子)的改善的β檀香烯合酶，ii)使法呢基焦磷酸与一种或多种改善的β檀香烯合酶在允许产生檀香烯的条件下接触，iii)从法呢基焦磷酸产生β-檀香烯和α檀香烯及任选地香柠檬烯以及任选地其他檀香烯，其中所产生β-檀香烯的量大于所产生α-檀香烯的量并且任选地纯化产物，例如旨在将它们与檀香烯合酶和任何剩余底物和不利的化合物分离。优选地，这些方法产生包含β-檀香烯多于α檀香烯的组合物，其中β-檀香烯与α檀香烯这两者的摩尔比是至少1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9或至少2；β-檀香烯/α-檀香烯的比率可以是至少3:1、优选地至少4:1、更优选地至少5:1、甚至更优选地6:1，然而甚至更优选地是至少7:1、最优选地至少8:1和甚至至少9:1。在本发明的一个方面，该比率不大于100:1。
执行用于产生β-檀香烯超过α-檀香烯的组合物的方法将会特别有益，所述方法包括提供改善的β檀香烯合酶的发酵步骤并且使法呢基焦磷酸与该酶接触并产生檀香烯。例如尽管体外使用本发明已分离的檀香烯合酶的方法是可能的，但特别优选用于产生包含β-檀香烯超过α-檀香烯的组合物的发酵方法，所述方法包括以下步骤：
a)提供以适合在宿主中表达的方式编码改善的β檀香烯合酶的核酸，
b)将a)的核酸引入能够给待有活性的檀香烯合酶提供法呢基焦磷酸和全部必要辅因子的宿主细胞中，
c)以宿主细胞按活性形式产生由核酸编码的檀香烯合酶并且提供法呢基焦磷酸和全部必要辅因子给檀香烯合酶的方式培育宿主细胞，
d)借助改善的β檀香烯合酶从法呢基焦磷酸产生β-檀香烯和α-檀香烯及任选地香柠檬烯，其中产生的β-檀香烯的量大于产生的α-檀香烯的量，
e)当已经产生所需量的这些化合物时，收获产生的β-檀香烯和α-檀香烯及任选地香柠檬烯，
f)任选地纯化β-檀香烯和α-檀香烯和任选的香柠檬烯。
相比同一条件下用未修饰的檀香烯合酶产生的那些量，用改善的β檀香烯合酶和通过本发明方法产生的β-檀香烯的量基于重量/重量以增加的优选顺序包含至少10％、20％、30％、40％、50％、60％、70％、80％、90％或95％更多的β-檀香烯。任选地，相比同一条件下用未修饰的檀香烯合酶产生的那些量，用改善的β檀香烯合酶和通过本发明方法产生的香柠檬烯的量基于重量/重量以增加的优选顺序包含至少10％、20％、30％、40％、50％、60％、70％、80％、90％或95％更多的香柠檬烯。在本发明的一个方面，用本发明的改善的檀香烯合酶和方法产生至少12％(w/w)、18％(w/w)或20％(w/w)的香柠檬烯。甚至更优选地，至少两倍量的β-檀香烯和任选地香柠檬烯存在于产生的组合物中。
在本发明的一个方面，本发明进一步涉及借助改善的β檀香烯合酶产生的檀香烯组合物，其具有比α-檀香烯含量更高的β-檀香烯含量。在本发明的一个方面，通过本发明的一种或多种合成的β檀香烯合酶、方法或宿主细胞产生本发明的组合物，其中组合物包含相对于α-檀香烯过量的β-檀香烯。
一个优选的实施方案是这样的组合物，其包含借助改善的β檀香烯合酶产生的β-檀香烯和α-檀香烯及香柠檬烯，优选地基本上由其组成，其中组合物具有超过α-檀香烯的β-檀香烯。在本发明的一个具体方面，组合物包含比香柠檬烯更多的β-檀香烯和比α-檀香烯更多的香柠檬烯。
本发明组合物优选地按大于β-檀香烯与α檀香烯这两者的比率包含比α檀香烯更多的β-檀香烯，例如，该比率是至少1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9或至少2。β-檀香烯/α-檀香烯的比率可以是至少3:1、优选地至少4:1、更优选地至少5:1、甚至更优选地6:1，然而甚至更优选地是至少7:1、最优选地至少8:1和甚至至少9:1。在本发明的一个方面，该比率不大于1000:1。
本发明还涉及：借助改善的β檀香烯合酶产生的组合物，其具有比α-檀香烯含量更高的香柠檬烯含量。这类组合物按大于香柠檬烯与α檀香烯这两者的比率包含比α檀香烯更多的香柠檬烯，优选地，该比率是至少1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9或至少2。香柠檬烯/α-檀香烯的比率可以是至少3:1、优选地至少4:1、更优选地至少5:1、甚至更优选地6:1，然而甚至更优选地是至少7:1、最优选地至少8:1和甚至至少9:1。在本发明的一个方面，该比率不大于1000:1。
在本发明的一个方面，借助改善的檀香烯合酶产生的组合物包含至少12％(w/w)、18％(w/w)或20％(w/w)的香柠檬烯。
通过改善的檀香烯合酶产生并且在本发明组合物中存在的香柠檬烯/β-檀香烯的比率可以高于1(香柠檬烯过量)或低于1(β-檀香烯过量)。前者对于例如借助N267S(SEQ IDNO:2)或α檀香烯过量生产者I291L(SEQ ID NO:53)产生的组合物是这样，后者为借助N267L(Seq ID NO.3)或SEQ ID NO:30至34或36或37中任一者产生的组合物所例举，如可见于图4和图5。取决于想要的产品和对组合物的进一步加工，有利的是使用香柠檬烯过量类型的改善的β檀香烯合酶，或β-檀香烯过量类型的改善的β檀香烯合酶。改善的β-檀香烯合酶N267G(SEQ ID NO:57)和N267A(SEQ ID NO:58)显示几乎处于β-檀香烯水平的香柠檬烯量，同时α-檀香烯产量强烈降低，这对于一些用途也可能合乎需要。
在本发明的一个方面，香柠檬烯/β-檀香烯的比率低于1.0，例如等于或低于0.95、0.9、0.85、0.8或0.75，例如等于或低于0.70，但高于0.28，例如高于0.30。
在一个实施方案中，借助改善的β檀香烯合酶产生的组合物中香柠檬烯/β-檀香烯的比率是至少1:1。在本发明的一个方面，该比率不高于5.5:1，例如不高于5:1或4.5至1、或4:1、或3.5至1或3至1、或2.5至1、或2:1。
在另一个实施方案中，借助改善的β檀香烯合酶产生的组合物中香柠檬烯/β-檀香烯的比率是1:2、1:3、1:4、1:5或1:10或更低。
因此，在一个方面，本发明涉及改善的β檀香烯合酶、本发明的宿主细胞或本发明的方法，其中该檀香烯合酶除产生比α-檀香烯更多的β-檀香烯之外，还产生超过α-檀香烯的过量反式-α-香柠檬烯。
又一个实施方案涉及一种通过改善的β檀香烯合酶、本发明宿主细胞或本发明方法可产生的组合物，其包含比β-檀香烯更多的香柠檬烯和比α-檀香烯更多的β-檀香烯。优选地，产生该组合物，包括产生改善的β檀香烯合酶或产生组合物的发酵步骤。
在一个优选的实施方案中，通过培养一个或多个类型宿主细胞，优选地细菌细胞、植物细胞或真菌细胞(包括酵母细胞)、更优选地细菌、甚至更优选地大肠杆菌(Escherichia coli)、拟无枝酸菌属物种(Amycolatopsis sp)或类球红细菌获得β-檀香烯比α-檀香烯多的组合物。
在又一个优选的实施方案中，本发明涉及包含从前体组合物产生的β-檀香醇((2Z)-2-甲基-5-[2-甲基-3-亚甲基-双环并[2.2.1]庚-2-基]戊-2-烯-1-醇；CAS编号77-42-9)和α-檀香醇((Z)-5-(2,3-二甲基三环并[2.2.1.02,6]庚-3-基)-2-甲基戊-2-烯-1-醇，CAS编号115-71-9)的组合物，所示前体组合物包含通过本发明方法产生的β-檀香烯和α檀香烯，其中β-檀香醇(本文中也称作贝塔-檀香醇)以基于w/w高于α-檀香醇(本文中也称作阿尔法檀香醇)的量存在，原因在于前体组合物中超出的β檀香烯含量。这些组合物中β-檀香醇/α檀香醇比率大于1，优选地，该比率是至少1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9或至少2。β-檀香醇/α-檀香醇的比率可以是至少3:1、优选地至少4:1、更优选地至少5:1、甚至更优选地6:1，然而甚至更优选地是至少7:1、最优选地至少8:1和甚至至少9:1。在本发明的一个方面，该比率不大于100:1。
又一个优选的实施方案是一种无需a)在转化成α-檀香醇之前削减α-檀香烯含量和/或b)从檀香烯转化后通过蒸馏或其他手段增加β-檀香醇含量的情况下产生β-檀香醇超过α-檀香醇的组合物的方法，其中该方法包括通过本发明方法产生β-檀香烯超过α-檀香烯的组合物的步骤并且在一个或多个后续步骤中使β-檀香烯氧化成β-檀香醇及使α-檀香烯氧化成α-檀香醇。这种檀香烯转化可以按生物合成和/或化学方式完成至其相应的醇。在转化成檀香醇后，可以纳入移除其他化合物的纯化步骤如蒸馏，并且如果需要，可以通过蒸馏改变β-檀香醇/α-檀香醇比率，但通过利用改善的β檀香烯合酶提供β-檀香烯超过α-檀香烯的组合物后，可以在不进一步改变β-檀香醇/α-檀香醇比率的情况下获得β-檀香醇比α-檀香醇多的组合物。本发明的一个方面因此是一种用于产生一种相对于α-檀香醇包含过量β-檀香醇的组合物的方法，其中该方法包括通过本发明方法产生β-檀香烯超过α-檀香烯的组合物的步骤并且在一个或多个后续步骤中使β-檀香烯氧化成β-檀香醇及使α-檀香烯氧化成α-檀香醇并且其中在氧化檀香烯后进行檀香醇蒸馏，以纯化檀香醇，同时相对于α-檀香醇含量不大幅度增加β-檀香醇含量。
另外，本发明涉及采用本发明改善的β檀香烯合酶或采用本发明宿主细胞，通过本发明方法中任一者产生的包含相对于α-檀香醇过量的β-檀香醇的组合物，任选地伴以组合物中香柠檬醇的总量小于10％(w/w)或甚至小于8％(w/w)。在另一个方面，采用本发明的改善的β檀香烯合酶或采用本发明的宿主细胞，通过本发明方法中任一者产生的包含相对于α-檀香醇过量的β-檀香醇的本发明组合物包含低于3％的表-β-檀香醇。
本发明的一个方面涉及产生β-檀香烯超过α-檀香烯的合成的檀香烯合酶、编码这种酶的核酸、包含这类核酸的表达盒、包含这类表达盒的宿主细胞、本发明的方法和用本发明酶和方法产生的组合物，所述组合物分别以至少等于或大于1.3、1.5或2的β-檀香烯/α-檀香烯比率或β-檀香醇/α-檀香醇比率包含β-檀香烯和α-檀香烯和/或β-檀香醇和α檀香醇。
优选地，本发明的组合物是亲脂组合物。
通过本发明方法或本发明组合物产生的β-檀香烯、α檀香烯或香柠檬烯可以用于香料应用或香精应用中、用于化妆品用途中、用作驱虫剂(insect repellent)或昆虫诱引剂或用于农业中，例如用于作物防护或动物饲养。
本发明的一个方面是适合从一种或多种编码一种或多种改善的檀香烯合酶的核酸产生所述改善的檀香烯合酶并且适合为改善的檀香烯合酶提供法呢基焦磷酸和对其活性必需的全部辅因子的宿主细胞，其中宿主细胞包含这类核酸。
又一个优选的实施方案因此是包含本发明改善的檀香烯合酶的宿主细胞。能够产生β-檀香烯比α-檀香烯多的组合物的微生物可以是真菌细胞(包括酵母)或细菌或植物细胞或动物细胞，例如，其来自以下属所组成的群组：埃希氏菌属(Escherichia)、克雷伯氏菌属(Klebsiella)、螺杆菌属(Helicobacter)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、链球菌属(streptococcus)、拟无枝酸菌属(Amycolatopsis)、红杆菌属(Rhodobacter)、乳球菌属(Lactococcus)、毕赤酵母属(Pichia)、酵母属(Saccharomyces)和克鲁维酵母属(Kluyveromyces)。在一个优选的实施方案中，适合产生β-檀香烯比α-檀香烯多的组合物的一种或多种宿主细胞是细菌细胞，其选自a)革兰氏阴性细菌群组，如红杆菌属(Rhodobacter)(例如类球红细菌(R.sphaeroides)、荚膜红细菌(R.capsulatus))、农杆菌属(Agrobacterium)、副球菌属(Paracoccus)(例如产类胡萝卜素副球菌(P.carotinifaciens)、产玉米黄素副球菌(P.zeaxanthinifaciens))或埃希氏菌属(Escherichia)；b)选自革兰氏阳性细菌群组的细菌细胞，如芽孢杆菌属(Bacillus)、棒状杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)、拟无枝酸菌属(Amycolatopis)；c)选自群组的真菌细胞：曲霉属(Aspergillus)、布拉霉属(Blakeslea)、青霉属(Peniciliium)、法夫酵母(Phaffia)(法夫酵母属(Xanthophyllomyces))、毕赤酵母属(Pichia)、酵母属(Saccharamoyces)、克鲁维酵母属(Kluyveromyces)、耶罗维亚酵母属(Yarrowia)和汉逊酵母属(Hansenula)；或d)转基因植物或培养物，包括转基因植物细胞，其中细胞属以下转基因植物的细胞：烟草属物种(Nicotiana spp)、菊苣(Cichorumintybus)、莴苣(lacuca sativa)、薄荷属物种(Mentha spp)、黄花蒿(Artemisia annua)、块茎形成植物、油料作物和树；e)或转基因蘑菇或培养物，包括转基因蘑菇细胞，其中微生物选自裂褶菌属(Schizophyllum)、伞菌属(Agaricus)和侧耳属(Pleurotisi)。更优选的生物是属于埃希氏菌属，酵母属、毕赤酵母属、拟无枝酸菌属、红杆菌属或副球菌属的微生物，并且甚至更优选地是以下物种的那些生物：大肠杆菌、酿酒酵母(S.cerevisae)、类球红细菌或拟无枝酸菌属物种。
又一个实施方案是包含编码改善的檀香烯合酶的合成核酸的表达盒。这些核酸可以是作为SEQ ID NO:11或12列出的那些，或是编码SEQ ID NO:2、3、14至17、21至52中任一多肽的那些，或对于增加的α檀香烯产量，是编码SEQ ID NO:53的多肽的核酸。表达盒中适合改变宿主细胞中檀香烯产量的其他核酸是编码改善的檀香烯合酶的那些，如但不限于在SEQ ID NO:2、3、13至53之任一者中公开的那些。对于编码α-檀香烯产量增加的檀香烯合酶的核酸，编码SEQ ID NO:53的多肽的核酸可以用于这类表达盒和宿主细胞中。
所述表达盒可以包含于载体、胞核、质粒、人工染色体或允许在宿主细胞中按所需强度和方式表达的任何其他工具中。
本发明的又一个方面是一种通过改变三级结构的灵活性有目的地改变檀香烯合酶的产物特征的方法，所述三级结构与SEQ ID NO:1的螺旋C并且与SEQ ID NO:1的螺旋D及与SEQ ID NO:1中连接这两个螺旋的多肽链对应。例如，这种方法涉及步骤：改变编码檀香烯合酶的核酸，从而与SEQ ID NO:1的位置267对应的位置处的氨基酸是丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸、优选地丝氨酸、苏氨酸、色氨酸、甘氨酸、丙氨酸或亮氨酸，例如丝氨酸或亮氨酸，和/或按以下方式改变编码檀香烯合酶的核酸的密码子的步骤：与SEQ ID NO:1的位置291的密码子对应的密码子现在编码亮氨酸、缬氨酸、苏氨酸、半胱氨酸或丝氨酸，例如Thr、Val、Cys、Phe或Ser；后接在适于表达本发明合成的檀香烯合酶的宿主细胞中表达经修饰核酸的步骤。
附图说明
图1显示已知的檀香烯合酶CiCaSSy野生型(SEQ ID NO:1)、SaSSY、SaSSy14、SspiSSy、SauSSy、ClaSSy和SaSSy134(分别是SEQ ID NO:4至9)的比对。在这个比对中SaSSY134以SEQ 280标示。还包括两种改善的β檀香烯合酶N267S和N267L突变体(SEQ IDNO:2和3)。用clustalw软件使用常见设置进行该比对。黑背景底纹标出强烈保守的残基，灰色背景底纹标出在至少50％所比对序列中保守的残基，白色底纹标出非保守氨基酸。
图2显示用PyrMol软件生成的CiCaSSy SEQ ID NO:1的3D模型。显示了α螺旋，并且黑色标出CiCaSSy的两个螺旋：螺旋C(黑色短螺旋)和螺旋D(黑色较长螺旋)。
图3显示螺旋C和螺旋D在野生型CiCaSSy(A)和N267S突变体(B)中相互作用的图示。图中心的α螺旋表示螺旋D，向左的α螺旋表示螺旋C。位置267中，两个氨基酸的侧链用深灰色标出。
图4显示通过改善檀香烯合酶SEQ ID NO:1(“野生型”)所致三种主产物α-檀香烯、β-檀香烯和香柠檬烯的变化。对这三种主产物，值已经作了归一化；未显示次要产物。黑色填充条形代表α-檀香烯，空心条代表香柠檬烯，并且划对角线的条代表β-檀香烯。将位置267替换为如SEQ ID NO:2中所示的丝氨酸(“N267S”)或如SEQ ID NO:3中所示的亮氨酸残基(“N267L”)允许酶产生比α-檀香烯更多的β-檀香烯和更多的香柠檬烯(N267S)，或更多香柠檬烯且α-檀香烯仍然可观，但是β-檀香烯更少(N267L)。显示了本领域已知的两种檀香烯合酶的数据用于比较(图4中称作“SaSSy”和“SaSSY-134”)。数据取自本领域的报告值，参见WO2015153501。已知的檀香烯合酶(野生型CICassy、SaSSY和SaSSy-134)显示α-檀香烯产量比其他两种化合物高。改进形式N267S和N267L显示怎样可以根据如N267L所致β-檀香烯单独超过α-檀香烯的所需优势或如N267S所致β-檀香烯和香柠檬烯二者均超过α-檀香烯的所需优势改变这个产物特征。
图5显示通过在与SEQ ID NO:1的位置291对应的位置处单独或联合与SEQ ID NO:1的位置267对应的位置修饰时改善檀香烯合酶所致的三种产物α-檀香烯、香柠檬烯和β-檀香烯的变化。为改善清晰度，未显示次要产物。黑色填充条形代表α-檀香烯，空心条代表香柠檬烯并且划对角线的条代表β-檀香烯。野生型(SEQ ID NO:1)和修饰的酶“I291L”作为对照显示。如SEQ ID NO:53中那样将位置291替换为亮氨酸残基(“I291L”)并不改变与β-檀香烯和香柠檬烯相比产生过量α-檀香烯的事实，相反，这种修饰提高α-檀香醇产量胜过一种野生型酶，这一点从附图可见。
将位置291替换为缬氨酸、丝氨酸、苏氨酸或半胱氨酸(分别“I291V”、“I291S”、“I291T”和“I291C”)，允许酶产生比α-檀香烯更多的β-檀香烯，然而维持比改善的β檀香烯合酶的N267S形式中高得多的α-檀香烯水平。改善形式I291V、I291S、I291C和I291T显示怎样可以根据如I291T、I291S和I291C所致β-檀香烯单独超过α-檀香烯的所需优势或如I291V所致β-檀香烯超过α-檀香烯且香柠檬烯水平类似于或高于那些α-檀香烯水平的所需优势改变这个产物特征，然而与N267S改善相比维持较高的α-檀香烯水平。这种具有更多剩余α-檀香烯的特征可以有利于一些应用。
最后两组条显示采用与SEQ ID NO:1的位置267和291对应的位置被修饰的两种双突变体的结果。对“I291T/N267S”显示的数据来自其中位置267用丝氨酸填充且位置291用苏氨酸填充的酶。对“I291T/N267T”显示的数据来自其中已在这两个位置引入苏氨酸的一种酶。如可以从图5中所示的突变体见到，双突变体“I291T/N267S”产生最高百分数的β-檀香烯。“I291T/N267S”酶的α-檀香烯和香柠檬烯的量属于两种单突变体的这些值的居间类型，在这种组合中突变N267S比I291T更多影响这些值。其他双突变体的数据显示，位置267处的苏氨酸相比这个位置处的丝氨酸对α-檀香烯、香柠檬烯和β-檀香烯具有相似影响，然而并非那么强烈。
实施例
可公开获得的电子序列信息用来利用标准软件工具分析檀香烯合酶结构。生成了CiCaSSy(SEQ ID NO:1)(作为WO2018160066公布的国际专利申请中当作SEQ ID NO 3披露的具有α-檀香烯/β-檀香烯正常比率的樟树檀香烯合酶)的3D模型。用于这种分析的共同工具例如是结构比对软件：DALI、CE、STAMP；参见http：//www.rcsb.org/pdb/home/home.do以便选择。
与其他檀香烯合酶相比，称作CiCaSSy的酶具有略微不常见的氨基酸定位。例如，它与许多其他檀香烯合酶共有少于50％的序列同一性，然而在某些区段中合并来自许多其他檀香烯合酶的元件。鉴定了活性部位空穴，并且瞄准这个空穴中的残基进行诱变。特别地，优先处理可能影响产物特征(product profile)的残基。选择一个在氨基酸序列中部包含空间上靠近的两个α-螺旋的区域用于突变。与已知的每种檀香烯合酶相比，CiCaSSy在这个蛋白质区域具有某种氨基酸差异，然而与此同时，许多元件以仅存在于CiCaSSy中的组合为不同组的檀香烯合酶所共享。如果这个蛋白质区域对于想要的产物特征变化而言是关键部分，则转换成他檀香烯序列轻易可行，即便它们在剩余部分以大的程度具有差异。
突变测试
在深度研究后，选择CiCaSSy的残基267来突变。发明人意识到，SEQ ID NO:1中N267的周围环境如此有利，从而发明人选择将位置267处的不常见天冬酰胺也替换为丝氨酸和亮氨酸，尽管这些氨基酸存在于其他已知性能匮乏的檀香烯合酶中相应的位置处。合成了编码CiCaSSy蛋白连同位置267处两个目的突变的DNA序列。命名为N267S和N267L的所得蛋白质序列分别在SEQ ID NO:2和SEQ ID NO:3中给出。
用这两个新的蛋白质序列进行原子位置均方根偏差(RMSD)和均方根波动(RSMF)分析。将每种酶在相同条件(pH 8.0、300K、1atm、水环境、无底物情况下离子存在)下模拟500ns。RMSD提供蛋白质总体的运动和灵活性指标，而RSMF提示给定位置处的平均运动和灵活性。RSMF显示，N267S显示出在螺旋C与螺旋D之间环区域和螺旋D的与SEQ ID NO:1至3的位置267的侧链相互作用的部分中预测的波动及因此灵活性增加超过野生型CiCaSSy。观察到与野生型CiCaSSy中的灵活性相比，N267S中与位置272至291对应的区段的灵活性(其为螺旋D的侧链位于其中的区域，所述侧链将与位置267处氨基酸的侧链相互作用)增加。从位置272至284的区段中这种增加的幅度较高，所述区段含有螺旋C与螺旋D之间的环，当然预计所述环的刚性比螺旋更小。N267S和N267L均在位置380至500的区域内具有波动增长的其他区段，如进一步下游的灵活性提示。当这与过量产生α-檀香烯的其他檀香烯合酶的RSMF分析比较时，在所分析的SEQ ID NO:4、5、8或9的序列中未观察到这种模式。RSMD分析显示对于N267S，在30000皮秒后，按nm计的偏离从起初平衡增长约五分之一。在分析的其余檀香烯序列之任一者中未观察到这种结构灵活性。
WO2018160066的实施例6至19中(第44页第19行至第50页第22行；通过引用方式并入本文作为参考)对野生型CiCaSSy所述的流程适用于采用编码蛋白质N267S和N267L的已突变CiCaSSy序列的实验。通过作为WO2018160066公布的国际专利申请中对CiCaSSy(本发明的SEQ ID NO:1)公开的流程，将编码SEQ ID NO:2和3的CiCaSSy檀香烯合酶的已突变DNA序列引入类球红细菌，WO2018160066中SEQ ID NO:3使用基于质粒的系统来表达异源DNA序列并形成突变酶。如WO2018160066中那样进行类球红细菌发酵用于产生、提取和分析宿主细胞产生的α-檀香烯、β-檀香烯和香柠檬烯。
用带FID检测器的气相色谱法测定α-檀香烯、β-檀香烯和香柠檬烯：
在配备Restek RTX-SSil MS毛细管柱(30m x 0.25mm，0.5pm)的ShimadzuGC2010Plus进行气相色谱。将上样器温度和FID检测器温度分别设置成280℃和300℃。过柱气流量设置成40mL/分钟。炉初始温度是160℃，以2℃/分钟的速率增至180℃，进一步以50℃/分钟的速率增至300℃，并且在该温度保持3分钟。上样的标本量是1μL，分流比1:50，并且氮气尾吹气流量细胞术是30ml/分钟。
CiCaSSy的267位置处的两个酶突变体——N267S和N267L明显影响α-檀香烯、β-檀香烯和香柠檬烯的产物比率。两种突变均导致与野生型CiCaSSy相比，β-檀香烯产量增加，甚至首次产生比α-檀香烯更多的β-檀香烯，及β-檀香烯/α-檀香烯产物比率升高(图4)。令人吃惊地，N267S突变体还产生显著更少的α-檀香烯，这意图该突变体对β-檀香烯具有超过α-檀香烯的高特异性——首次观察到这种现象。N267L突变具有甚至更大的产物比率变化，并且产生β-檀香烯作为其主产物，同时产生相对更少的α-檀香烯和反式-α香柠檬烯，如图4中所示。
将额外的突变体用上文描述的相同实验设置测试。例如，将与序列267对应的位置替换为甘氨酸、丙氨酸或色氨酸还产生改善的檀香烯合酶。
进一步地，发现在野生型中替换与SEQ ID NO:1的位置291对应的位置处的异亮氨酸导致α-檀香烯水平更高，并且在这个位置引入组氨酸摧毁作为檀香烯合酶的活性。这说明该位置重要的，不过如何改变它也重要。
I291V、I29S、I291C、I291F和I291T突变体也受测试并且显示—如N267S或N267L那样—β-檀香烯过量，但是与N267S相比，仍有更多α-檀香烯，尽管相比野生型对照α-檀香烯更少(参见图5和表1)。在宿主细胞中表达SEQ ID NO:34时，存在β-檀香烯的最高百分数值。
与SEQ ID NO:1的位置267对应的位置处具有N267S或N267T变化且与SEQ ID NO:1的位置291对应的位置处有苏氨酸而非亮氨酸的双突变体也产生β-檀香烯过量的改善的β檀香烯合酶：不过与单突变体改善的β檀香烯合酶相比，它们显示居间产物特征(参见图5)。
对这些突变体的建模在RSMF图中显示，N257S单突变体以及其在SEQ ID NO:1的291对应的位置处具有丝氨酸、半胱氨酸或苏氨酸的双突变体显示螺旋C和螺旋D的灵活性增加，这与N267S和其在SEQ ID NO:1的位置291对应的位置处具有苏氨酸的双突变体的实验结果一致(参见图5)。有趣地，进一步下游的一些其他区域内灵活性似乎也改善，如RSMF数据显示。
使用的软件工具
同源性模型
使用
Prime软件包(www.schrodinger.com/prime；
发行版2020-2:Prime,
LLC,New York,NY,2020；M Jacobson等人,Proteins,2004,55,351-367)生成同源性模型。模板结构下载自PDB-蛋白质数据库(HM Breman等人,NucleicAcid Research,2000,28,235-242)，表2中指示每次同源性模型生成的模板结构。
| 同源性模型_ | 模板结构(PDB代码) |
| CiCaSSy | 6A1I |
| CiCaSSy N267L | 6A1I |
| CiCaSSy N267S | 6A1I |
| SaSSy | 5ZZJ |
| SaSSy14 | 5ZZJ |
| ClaSSy | 6A1I |
| SaSSy134 | 5ZZJ |
表2.用于生成同源性模型的模板结构。对于每个建成的同源性模型，指示所用模板结构的蛋白质数据库代码。
MD模拟
通过使用2018版软件GROMACS(www.gromacs.org；D van Der Spoel等人,JComput Chem,2005,26,1701-1718)进行MD模拟。全部酶均在OPLS-AA力场中限定(WLJorgensen和J Tirado-Rives,J Am Chem Soc,1988,110,1657-1666)，将酶质子化在pH8.0限定并且使用pdb2pqr工具(TJ Dolinsky等人,Nucleic Acids Res,2007 35,W522-W525)加以计算；如MW van der Kamp等人,Biochemistry,2013,52,8094-8105中所述，通过以下方式在模型中纳入3个金属离子(Mg2+)：将它们的相对位置固定至其配位性氨基酸残基。将每种酶置于1000nm3立方晶系的中心并且用TIP4P水明确溶剂化(WL Jorgensen等人,J Chem Phys,1983,79,926-935)，通过添加适当量的Na+或Cl-离子中和晶系的总电荷系统。使用最陡递降算法，将每个晶系对10000个步骤最小化，并且随后平衡10ns。在平衡后，对每个晶系模拟500ns。使用v-rescale算法(G Bussi等人,J Chem Phys,2007,126,014101)保持温度在300K恒定，使用Parrinello-Rahman算法(M Parrinello和A Rahman,Phys RevLett,1980,45,1196-1198)保持压力在1atm恒定并且通过扩展的粒子网片Ewald算法(UEssmann等人,J Chem Phys,1995,103,8577-8593)模拟静电相互作用。每5天保存模拟框架。
RMSD
均方根依赖模拟完整时长(500ns)评价每种酶结构的偏差(RMSD)。在已经使用已平衡晶系作为参考对每个轨迹框架(gmx trjconv)进行蛋白质结构的结构性叠加后，通过GROMACS软件包的gmx rms工具进行计算。
RMSF
均方根依赖模拟最后450ns评价每种酶结构的波动(RMSF)。在已经对每个轨迹框架(gmx trjconv)进行蛋白质结构的结构性叠加并且使用已平衡晶系的蛋白质Cα作为参考后，通过GROMACS软件包的gmx rmsf工具进行计算。
图片
通过使用PyMOL(pymol.org)软件生成图2和图3的蛋白质图片。通过使用针对Python 3.6版(python.org)的Matplotlib库(matplotlib.org)生成RMSD图片和RMSF图片。
PFAM结构域分析
将PFAM结构域PF01397“Terpene_synth”和C端PFAM结构域PF03936“Terpene_synth_C”使用2020年5月29日32.0版PFAM软件鉴定并用2020年6月11日发行的33.1版PFAM软件确认；关于PFAM的详情，参见“The Pfam protein families database in 2019(2019年Pfam蛋白质家族数据库)：S.El-Gebali,J.Mistry,A.Bateman,S.R.Eddy,A.Luciani,S.C.Potter,M.Qureshi,L.J.Richardson,G.A.Salazar,A.Smart,E.L.L.Sonnhammer,L.Hirsh,L.Paladin,D.Piovesan,S.C.E.Tosatto,R.D.Finn Nucleic Acids Research(2019)和http://pfam.xfam.org/及“Pfam:The protein families database in2021(Pfam:2021年蛋白质家族数据库):J.Mistry,S.Chuguransky,L.Williams,M.Qureshi,G.A.Salazar,E.L.L.Sonnhammer,S.C.E.Tosatto,L.Paladin,S.Raj,L.J.Richardson,R.D.Finn,A.Bateman Nucleic Acids Research(2020)doi:10.1093/nar/gkaa913”
Interpro基序
以下结构域
用2020年12月发布的83.0版InterPro扫描软件鉴定；InterPro的更多细节参见：Blum M,Chang H,Chuguransky S,Grego T,Kandasaamy S,Mitchell A,Nuka G,Paysan-Lafosse T,Qureshi M,Raj S,RichardsonL,Salazar GA,Williams L,Bork P,Bridge A,Gough J,Haft DH,Letunic I,Marchler-Bauer A,Mi H,Natale DA,Necci M,Orengo CA,Pandurangan AP,Rivoire C,Sigrist CJA,Sillitoe I,Thanki N,Thomas PD,TosattoSCE,Wu CH,Bateman A和Finn RD,The InterPro protein families and domainsdatabase:20years on(InterPro蛋白质家族和结构域数据库：20年续).Nucleic AcidsResearch,Nov 2020,(doi:10.1093/nar/gkaa977)
序列表
<110> 艾瑟拜奥尼克斯公司
<120> 合成的檀香烯合酶
<130> 201746WO01
<160> 58
<170> BiSSAP 1.3.6
<210> 1
<211> 553
<212> PRT
<213> 樟树（Cinnamomum camphora）
<220>
<223> 野生型CiCaSSy
<400> 1
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 2
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >CiCaSSy_N267S
<400> 2
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 3
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >CiCaSSy_N267L
<400> 3
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Leu His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 4
<211> 569
<212> PRT
<213> 檀香（Santalum album）
<220>
<223> >SaSSy
<400> 4
Met Asp Ser Ser Thr Ala Thr Ala Met Thr Ala Pro Phe Ile Asp Pro
1 5 10 15
Thr Asp His Val Asn Leu Lys Thr Asp Thr Asp Ala Ser Glu Asn Arg
20 25 30
Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln
35 40 45
Ser Leu Ala Thr His His Asn Ile Val Glu Glu Arg His Leu Lys Leu
50 55 60
Ala Glu Lys Leu Lys Gly Gln Val Lys Phe Met Phe Gly Ala Pro Met
65 70 75 80
Glu Pro Leu Ala Lys Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly
85 90 95
Leu Asn His Leu Phe Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Ile
100 105 110
Tyr Lys Asp Gly Ser Asn Gly Trp Trp Phe Gly His Leu His Ala Thr
115 120 125
Ser Leu Arg Phe Arg Leu Leu Arg Gln Cys Gly Leu Phe Ile Pro Gln
130 135 140
Asp Val Phe Lys Thr Phe Gln Asn Lys Thr Gly Glu Phe Asp Met Lys
145 150 155 160
Leu Cys Asp Asn Val Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser Tyr
165 170 175
Leu Gly Trp Lys Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Thr
180 185 190
Thr Lys Cys Leu Lys Ser Ala Trp Glu Asn Ile Ser Glu Lys Trp Leu
195 200 205
Ala Lys Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val
210 215 220
Pro Arg Ile Glu Ala Arg Trp Phe Ile Glu Ala Tyr Glu Gln Glu Ala
225 230 235 240
Asn Met Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met
245 250 255
Val Gln Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp
260 265 270
Val Thr Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu
275 280 285
Gln Ser Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys
290 295 300
Leu Ala Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val
305 310 315 320
Asp Asp Gly Tyr Asp Val Tyr Gly Ser Ile Asp Glu Leu Asp Leu Tyr
325 330 335
Thr Ser Ser Val Glu Arg Trp Ser Cys Val Glu Ile Asp Lys Leu Pro
340 345 350
Asn Thr Leu Lys Leu Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu
355 360 365
Val Gly Leu Arg Val Gln His Glu Arg Gly Tyr Asn Ser Ile Pro Thr
370 375 380
Phe Ile Lys Ala Trp Val Glu Gln Cys Lys Ser Tyr Gln Lys Glu Ala
385 390 395 400
Arg Trp Phe His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu
405 410 415
Asn Gly Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr
420 425 430
Val Ala Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val His Pro Leu
435 440 445
Pro Asp Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp
450 455 460
Ile Gly Thr Ser Pro Asp Glu Met Ala Arg Gly Asp Asn Leu Lys Ser
465 470 475 480
Ile His Cys Tyr Met Asn Glu Thr Gly Ala Ser Glu Glu Val Ala Arg
485 490 495
Glu His Ile Lys Gly Val Ile Glu Glu Asn Trp Lys Ile Leu Asn Gln
500 505 510
Cys Cys Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Ile Thr Phe Asn
515 520 525
Leu Asn Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly
530 535 540
Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu
545 550 555 560
Ile Asp Pro Ile Pro Leu Gly Glu Glu
565
<210> 5
<211> 569
<212> PRT
<213> 檀香
<220>
<223> >SaSSy14
<400> 5
Met Asp Ser Ser Thr Ala Thr Ala Met Arg Ala Pro Phe Ile Asp His
1 5 10 15
Thr Asp His Val Asn Leu Arg Thr Asp Asn Asp Ser Ser Glu Asn Arg
20 25 30
Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln
35 40 45
Ser Leu Ala Thr Arg His Asn Ile Met Glu Glu Arg His Leu Lys Leu
50 55 60
Ala Glu Lys Leu Lys Gly Gln Val Lys Phe Met Phe Gly Ala Pro Met
65 70 75 80
Glu Pro Leu Ala Lys Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly
85 90 95
Leu Asn His Arg Phe Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Ile
100 105 110
Tyr Lys Asp Glu Ser Asn Gly Trp Trp Phe Gly His Leu His Ala Thr
115 120 125
Ser Leu Arg Phe Arg Leu Leu Arg Gln Cys Gly Leu Phe Ile Pro Gln
130 135 140
Asp Val Phe Lys Thr Phe Gln Ser Lys Thr Gly Glu Phe Asp Met Lys
145 150 155 160
Leu Cys Asp Asn Val Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser Phe
165 170 175
Leu Gly Trp Arg Asp Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Ala
180 185 190
Thr Lys Tyr Leu Lys Asn Ala Trp Glu Asn Ile Ser Gln Lys Trp Leu
195 200 205
Ala Lys Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val
210 215 220
Pro Arg Ile Glu Ala Arg Trp Phe Val Glu Ala Tyr Gly Glu Glu Glu
225 230 235 240
Asn Met Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met
245 250 255
Val Gln Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp
260 265 270
Val Thr Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu
275 280 285
Gln Ser Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys
290 295 300
Leu Ala Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val
305 310 315 320
Asp Asp Ala Tyr Asp Val Tyr Gly Ser Met Asp Glu Leu Asp Leu Tyr
325 330 335
Thr Asn Ser Val Glu Arg Trp Ser Cys Thr Glu Ile Asp Lys Leu Pro
340 345 350
Asn Thr Leu Lys Leu Ile Phe Met Ala Met Phe Asn Lys Thr Asn Glu
355 360 365
Val Gly Leu Arg Val Gln His Glu Arg Gly Tyr Ser Gly Ile Thr Thr
370 375 380
Phe Ile Lys Ala Trp Val Glu Gln Cys Lys Ser Tyr Gln Lys Glu Ala
385 390 395 400
Arg Trp Tyr His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu
405 410 415
Asn Gly Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr
420 425 430
Val Ala Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val His Pro Leu
435 440 445
Pro Asp Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp
450 455 460
Met Gly Thr Ser Ser Asp Glu Leu Glu Arg Gly Asp Asn Leu Lys Ser
465 470 475 480
Ile Gln Cys Tyr Met Asn Gln Thr Gly Ala Ser Glu Lys Val Ala Arg
485 490 495
Glu His Ile Lys Gly Ile Ile Glu Glu Asn Trp Lys Ile Leu Asn Glu
500 505 510
Cys Cys Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Val Thr Phe Asn
515 520 525
Leu Asn Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly
530 535 540
Phe Gly Val Thr Asn Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu
545 550 555 560
Ile Asp Pro Ile Pro Leu Asp Glu Glu
565
<210> 6
<211> 569
<212> PRT
<213> 大果檀香（Santalum spicatum）
<220>
<223> >SpiSSy
<400> 6
Met Asp Ser Ser Thr Ala Thr Ala Thr Thr Ala Pro Phe Ile Asp His
1 5 10 15
Thr Asp His Val Asn Leu Lys Ile Asp Asn Asp Ser Ser Glu Ser Arg
20 25 30
Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln
35 40 45
Ser Leu Ala Ile His His Asn Ile Val Glu Glu Lys His Leu Lys Leu
50 55 60
Ala Glu Lys Leu Lys Gly Gln Val Met Ser Met Phe Gly Ala Pro Met
65 70 75 80
Glu Pro Leu Ala Lys Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly
85 90 95
Leu Asn His Gln Phe Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Val
100 105 110
Tyr Lys Asp Gly Ser Asn Gly Trp Trp Phe Gly His Leu His Ala Thr
115 120 125
Ser Leu Arg Phe Arg Leu Leu Arg Gln Cys Gly Leu Phe Ile Pro Gln
130 135 140
Asp Val Phe Lys Thr Phe Gln Ser Lys Thr Asp Glu Phe Asp Met Lys
145 150 155 160
Leu Cys Asp Asn Ile Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser Phe
165 170 175
Leu Gly Trp Lys Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Ala
180 185 190
Thr Lys Tyr Leu Lys Asn Ala Trp Glu Asn Ile Ser Gln Lys Trp Leu
195 200 205
Ala Lys Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val
210 215 220
Pro Arg Ile Glu Ala Arg Trp Phe Ile Glu Ala Tyr Glu Gln Glu Glu
225 230 235 240
Asn Met Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met
245 250 255
Val Gln Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp
260 265 270
Val Thr Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu
275 280 285
Gln Ser Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys
290 295 300
Leu Ala Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val
305 310 315 320
Asp Asp Ala Tyr Asp Val Tyr Gly Ser Met Asp Glu Leu Asp His Tyr
325 330 335
Thr Tyr Ser Val Glu Arg Trp Ser Cys Val Glu Ile Asp Lys Leu Pro
340 345 350
Asn Thr Leu Lys Leu Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu
355 360 365
Val Gly Leu Arg Val Gln His Glu Arg Gly Tyr Asn Gly Ile Pro Thr
370 375 380
Phe Ile Lys Ala Trp Val Glu Gln Cys Lys Ala Tyr Gln Lys Glu Ala
385 390 395 400
Arg Trp Tyr His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu
405 410 415
Asn Gly Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr
420 425 430
Ile Ala Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val His Pro Leu
435 440 445
Pro Asp Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp
450 455 460
Met Gly Thr Ser Pro Asp Glu Met Ala Arg Gly Asp Asn Leu Lys Ser
465 470 475 480
Ile His Cys Tyr Met Asn Glu Thr Gly Ala Ser Glu Glu Val Ala Arg
485 490 495
Glu His Ile Lys Gly Ile Ile Glu Glu Asn Trp Lys Ile Leu Asn Gln
500 505 510
Cys Cys Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Ile Thr Phe Asn
515 520 525
Leu Asn Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly
530 535 540
Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu
545 550 555 560
Ile Asp Pro Ile Pro Leu Gly Glu Glu
565
<210> 7
<211> 569
<212> PRT
<213> 新喀里多尼亚檀香（Santalum austrocaledonicum）
<220>
<223> >SauSSy
<400> 7
Met Asp Ser Ser Thr Ala Thr Ala Met Thr Ala Pro Phe Ile Asp Pro
1 5 10 15
Thr Asp His Val Asn Leu Lys Thr Asp Thr Asp Ala Ser Glu Asn Arg
20 25 30
Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln
35 40 45
Ser Leu Ala Thr His His Asn Ile Val Glu Glu Arg His Leu Lys Leu
50 55 60
Ala Glu Lys Leu Lys Gly Gln Val Lys Phe Met Phe Gly Ala Pro Met
65 70 75 80
Glu Pro Leu Ala Lys Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly
85 90 95
Leu Asn His Arg Phe Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Ile
100 105 110
Tyr Lys Asp Glu Ser Asn Gly Trp Trp Phe Gly His Leu His Ala Thr
115 120 125
Ser Leu Arg Phe Arg Leu Leu Arg Gln Cys Gly Leu Phe Ile Pro Gln
130 135 140
Asp Val Phe Lys Thr Phe Gln Asn Lys Thr Gly Glu Phe Asp Met Lys
145 150 155 160
Leu Cys Asp Asn Val Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser Tyr
165 170 175
Leu Gly Trp Lys Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Ala
180 185 190
Thr Lys Tyr Leu Lys Ser Ala Trp Glu Asn Ile Ser Glu Lys Trp Leu
195 200 205
Ala Lys Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val
210 215 220
Pro Arg Ile Glu Ala Arg Trp Phe Ile Glu Ala Tyr Glu Gln Glu Ala
225 230 235 240
Asn Met Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met
245 250 255
Val Gln Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp
260 265 270
Val Thr Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu
275 280 285
Gln Ser Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys
290 295 300
Leu Ala Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val
305 310 315 320
Asp Asp Ala Tyr Asp Val Tyr Gly Ser Met Asp Glu Leu Asp Leu Tyr
325 330 335
Thr Ser Ser Val Glu Arg Trp Ser Cys Val Glu Ile Asp Lys Leu Pro
340 345 350
Asn Thr Leu Lys Leu Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu
355 360 365
Val Gly Leu Arg Val Gln His Glu Arg Gly Tyr Asn Ser Ile Pro Thr
370 375 380
Phe Ile Lys Ala Trp Val Gln Gln Cys Lys Ser Tyr Gln Lys Glu Ala
385 390 395 400
Arg Trp Phe His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu
405 410 415
Asn Gly Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr
420 425 430
Val Ala Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val His Pro Leu
435 440 445
Pro Asp Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp
450 455 460
Ile Gly Thr Ser Pro Asp Glu Met Ala Arg Gly Asp Asn Leu Lys Ser
465 470 475 480
Ile His Cys Tyr Met Asn Gly Thr Gly Ala Ser Glu Glu Val Ala Arg
485 490 495
Glu His Ile Lys Gly Val Ile Glu Glu Asn Trp Lys Ile Leu Asn Gln
500 505 510
Cys Cys Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Ile Thr Phe Asn
515 520 525
Leu Asn Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly
530 535 540
Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu
545 550 555 560
Ile Asp Pro Ile Pro Leu Gly Glu Glu
565
<210> 8
<211> 551
<212> PRT
<213> 黄皮（Clausena lansium）
<220>
<223> >ClaSSy
<400> 8
Met Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala Ala
1 5 10 15
Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro Ser
20 25 30
Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys Glu
35 40 45
Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln Arg
50 55 60
Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His Phe
65 70 75 80
Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro Phe
85 90 95
Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe Lys
115 120 125
Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln Gly
130 135 140
Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu Asp
145 150 155 160
Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser Thr
165 170 175
Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg His
180 185 190
Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser Arg
195 200 205
Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr Leu
210 215 220
Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His Gln
225 230 235 240
Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe Leu
245 250 255
Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe Trp
260 265 270
Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg Ile
275 280 285
Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr Phe
290 295 300
Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala Val
305 310 315 320
Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met Lys
325 330 335
Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln Glu
340 345 350
Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg Ser
355 360 365
Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu Asn
370 375 380
Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu Arg
385 390 395 400
Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met Gly
405 410 415
Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro Lys
420 425 430
Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile Ala
435 440 445
Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile Glu
450 455 460
Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys Ala
465 470 475 480
Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu Leu
485 490 495
Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile Leu
500 505 510
Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp Arg
515 520 525
Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu Lys
530 535 540
Asp Pro Val Lys Leu Asp Asp
545 550
<210> 9
<211> 567
<212> PRT
<213> 人工序列
<220>
<223> >SaSSY134
<400> 9
Met Asp Ser Ser Thr Ala Thr Ala Thr Thr Ala Pro Phe Ile Asp Pro
1 5 10 15
Thr Asn His Val Asn Leu Lys Ile Asp Asn Asp Ser Ser Glu Asn Arg
20 25 30
Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln
35 40 45
Ser Leu Ala Thr His His Asn Ile Val Glu Glu Arg His Leu Lys Leu
50 55 60
Ala Glu Lys Leu Lys Gly Gln Val Arg Ile Leu Leu Lys Glu Lys Met
65 70 75 80
Glu Pro Leu Ala Gln Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly
85 90 95
Leu Asn His Leu Leu Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Ile
100 105 110
Tyr Lys Asp His Ile Asp Ser Asp Lys Ala Asp Leu His Ala Thr Ser
115 120 125
Leu Arg Phe Arg Leu Leu Arg Gln Gln Gly Ile Lys Ile Ser Cys Asp
130 135 140
Val Phe Glu Gln Phe Lys Asp Asp Glu Asp Arg Phe Lys Ser Ser Leu
145 150 155 160
Ile Asn Asp Ile Gln Gly Met Leu Ser Leu Tyr Glu Ala Ser Phe Leu
165 170 175
Gly Trp Lys Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Ala Thr
180 185 190
Lys Tyr Leu Lys Ala Met Val Glu Ser Leu Gly Gly His Leu Ala Lys
195 200 205
Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val Pro Arg
210 215 220
Ile Glu Ala Arg Trp Phe Ile Glu Ala Tyr Glu Gln Glu Glu Asn Met
225 230 235 240
Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met Val Gln
245 250 255
Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp Val Thr
260 265 270
Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu Gln Ser
275 280 285
Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys Leu Ala
290 295 300
Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val Asp Asp
305 310 315 320
Ala Tyr Asp Val Tyr Gly His Met Asp Glu Leu Asp Leu Tyr Thr Ser
325 330 335
Ser Val Glu Gly Trp Ser Cys Ala Glu Ile Asp Arg Leu Pro Asp Thr
340 345 350
Leu Lys Leu Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu Val Gly
355 360 365
Leu Arg Val Gln His Glu Arg Gly Tyr Asn Ser Ile Pro Thr Phe Ile
370 375 380
Lys Ala Trp Val Glu Gln Cys Lys Ser Tyr Gln Lys Glu Ala Arg Trp
385 390 395 400
Phe His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu Asn Gly
405 410 415
Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr Ile Ala
420 425 430
Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val Arg Pro Leu Pro Asp
435 440 445
Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp Met Gly
450 455 460
Thr Ser Pro Asp Glu Met Ala Arg Gly Asp Asn Leu Lys Ser Ile His
465 470 475 480
Cys Tyr Met Asn Glu Thr Gly Ala Ser Glu Glu Val Ala Arg Glu His
485 490 495
Ile Lys Gly Ile Ile Glu Glu Asn Trp Lys Ile Leu Asn Gln Cys Cys
500 505 510
Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Ile Thr Phe Asn Leu Asn
515 520 525
Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly Phe Gly
530 535 540
Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu Ile Asp
545 550 555 560
Pro Ile Pro Leu Gly Glu Glu
565
<210> 10
<211> 1662
<212> DNA
<213> 樟树
<220>
<223> >CiCaSSy_wt
<400> 10
atggacagca tggaagtccg gcggtcggcg atctaccaca gcacgttctg ggacatcgac
60
agcatccggg cgctcctggc gcggcgggac tgcacggcgg ccgcggccct ctcgcccgac
120
caccataagc gcctgaagga gcgcatccag cgccgcctcc aggacatcac ccagccccac
180
catctgctcg gcctcatcga cgccgtgcag cgcctgggcg tggcctacca gttcgaggaa
240
gagatctcgg acgcgctgca cggcctccat tcggagaaca ccgagcacgc catcaaggac
300
tcgctgcacc atacgtcgct ctatttccgc ctgctccgcc agcatggctg caacctgtcg
360
tcggacatct tcaacaagtt caagaaggaa ggcggcggct tcaaggcctc gctctgcgag
420
gacgccatgg gcctgctctc gctgtatgag gccgtgcgcc tctcggtgaa gggcgaggcc
480
atcctggagg aagcccaggt gttctcgatc gccaacctga agatcctcat ggagcgcgtg
540
gagcgcaagc tcgccgaccg catcgagcat gccctggaga tcccgctcta ttggcgcgcc
600
ccgcgtctgg aggcccgctg gtacatcgac gtgtatgaga aggaagacgg ccgcatcgac
660
gacctgctcg acttcgcgaa gctggacttc aaccgcgtgc agatgctcta tcagaccgag
720
ctgaaggagc tctcgatgtg gtgggagctg ctgggcctgc ccgccaagat gggcttcttc
780
cgcgaccgcc tgctcgagaa ccacctcttc tcgatcgccg tggtggtgga gccccagtac
840
tcgcagtgcc gcgtggccat caccaaggcg atcgtgctga tgacggcgat ggacgacttc
900
tatgacgtgc atggcctgcc ggacgagctc aaggtgttca ccgacacggt gaaccgctgg
960
gacctggagg gcatcgacca gctccccgag tacatgaagc tgtactatct ggcgctctac
1020
aacaccacga acgagacggc ctatatcatc ctgaaggaga agggcttcaa cgccacgcat
1080
tacctgaaga agctctgggc catgcagtcg aacgcgtatt tccgcgaggc ccagtggttc
1140
aactcgggct acatcccgaa gttcgacgag tatctggaca acgccctcgt gtcggtgggc
1200
gccccgttcg tgctgggcct ctcgtatccc atgatccagc agcagatctc gaaggaagag
1260
atcgacctga tccccgagga cctcaacctg ctccgctggg cctcgatcat cttccgcctg
1320
tacgacgacc tggccacctc gaaggccgag cagcagcgcg gcgacgtgcc caagtcgatc
1380
cagtgctata tgcatgagac gggctcgtcg gaggaagtgg cggccaacca tatccgcgac
1440
ctgatctcgg acgcgtggaa ggaagtgaac gccgagtgcc tgaagccgac ctcgctctcg
1500
aagcactacg tgggcgtggc ccccaactcg gcccgctcgg gcgtgctcat gtatcaccat
1560
gacttcgacg gcttcgcgtc gccccatggc cgcacgaacg cccacatcac gagcatcttc
1620
ttcgagccgg tccccctcaa ggagagcatc aacctgggct ga
1662
<210> 11
<211> 1662
<212> DNA
<213> 人工序列
<220>
<223> >CiCaSSy_N267S
<400> 11
atggacagca tggaagtccg gcggtcggcg atctaccaca gcacgttctg ggacatcgac
60
agcatccggg cgctcctggc gcggcgggac tgcacggcgg ccgcggccct ctcgcccgac
120
caccataagc gcctgaagga gcgcatccag cgccgcctcc aggacatcac ccagccccac
180
catctgctcg gcctcatcga cgccgtgcag cgcctgggcg tggcctacca gttcgaggaa
240
gagatctcgg acgcgctgca cggcctccat tcggagaaca ccgagcacgc catcaaggac
300
tcgctgcacc atacgtcgct ctatttccgc ctgctccgcc agcatggctg caacctgtcg
360
tcggacatct tcaacaagtt caagaaggaa ggcggcggct tcaaggcctc gctctgcgag
420
gacgccatgg gcctgctctc gctgtatgag gccgtgcgcc tctcggtgaa gggcgaggcc
480
atcctggagg aagcccaggt gttctcgatc gccaacctga agatcctcat ggagcgcgtg
540
gagcgcaagc tcgccgaccg catcgagcat gccctggaga tcccgctcta ttggcgcgcc
600
ccgcgtctgg aggcccgctg gtacatcgac gtgtatgaga aggaagacgg ccgcatcgac
660
gacctgctcg acttcgcgaa gctggacttc aaccgcgtgc agatgctcta tcagaccgag
720
ctgaaggagc tctcgatgtg gtgggagctg ctgggcctgc ccgccaagat gggcttcttc
780
cgcgaccgcc tgctcgagtc gcacctcttc tcgatcgccg tggtggtgga gccccagtac
840
tcgcagtgcc gcgtggccat caccaaggcg atcgtgctga tgacggcgat ggacgacttc
900
tatgacgtgc atggcctgcc ggacgagctc aaggtgttca ccgacacggt gaaccgctgg
960
gacctggagg gcatcgacca gctccccgag tacatgaagc tgtactatct ggcgctctac
1020
aacaccacga acgagacggc ctatatcatc ctgaaggaga agggcttcaa cgccacgcat
1080
tacctgaaga agctctgggc catgcagtcg aacgcgtatt tccgcgaggc ccagtggttc
1140
aactcgggct acatcccgaa gttcgacgag tatctggaca acgccctcgt gtcggtgggc
1200
gccccgttcg tgctgggcct ctcgtatccc atgatccagc agcagatctc gaaggaagag
1260
atcgacctga tccccgagga cctcaacctg ctccgctggg cctcgatcat cttccgcctg
1320
tacgacgacc tggccacctc gaaggccgag cagcagcgcg gcgacgtgcc caagtcgatc
1380
cagtgctata tgcatgagac gggctcgtcg gaggaagtgg cggccaacca tatccgcgac
1440
ctgatctcgg acgcgtggaa ggaagtgaac gccgagtgcc tgaagccgac ctcgctctcg
1500
aagcactacg tgggcgtggc ccccaactcg gcccgctcgg gcgtgctcat gtatcaccat
1560
gacttcgacg gcttcgcgtc gccccatggc cgcacgaacg cccacatcac gagcatcttc
1620
ttcgagccgg tccccctcaa ggagagcatc aacctgggct ga
1662
<210> 12
<211> 1662
<212> DNA
<213> 人工序列
<220>
<223> >CiCaSSy_N267L
<400> 12
atggacagca tggaagtccg gcggtcggcg atctaccaca gcacgttctg ggacatcgac
60
agcatccggg cgctcctggc gcggcgggac tgcacggcgg ccgcggccct ctcgcccgac
120
caccataagc gcctgaagga gcgcatccag cgccgcctcc aggacatcac ccagccccac
180
catctgctcg gcctcatcga cgccgtgcag cgcctgggcg tggcctacca gttcgaggaa
240
gagatctcgg acgcgctgca cggcctccat tcggagaaca ccgagcacgc catcaaggac
300
tcgctgcacc atacgtcgct ctatttccgc ctgctccgcc agcatggctg caacctgtcg
360
tcggacatct tcaacaagtt caagaaggaa ggcggcggct tcaaggcctc gctctgcgag
420
gacgccatgg gcctgctctc gctgtatgag gccgtgcgcc tctcggtgaa gggcgaggcc
480
atcctggagg aagcccaggt gttctcgatc gccaacctga agatcctcat ggagcgcgtg
540
gagcgcaagc tcgccgaccg catcgagcat gccctggaga tcccgctcta ttggcgcgcc
600
ccgcgtctgg aggcccgctg gtacatcgac gtgtatgaga aggaagacgg ccgcatcgac
660
gacctgctcg acttcgcgaa gctggacttc aaccgcgtgc agatgctcta tcagaccgag
720
ctgaaggagc tctcgatgtg gtgggagctg ctgggcctgc ccgccaagat gggcttcttc
780
cgcgaccgcc tgctcgagct ccacctcttc tcgatcgccg tggtggtgga gccccagtac
840
tcgcagtgcc gcgtggccat caccaaggcg atcgtgctga tgacggcgat ggacgacttc
900
tatgacgtgc atggcctgcc ggacgagctc aaggtgttca ccgacacggt gaaccgctgg
960
gacctggagg gcatcgacca gctccccgag tacatgaagc tgtactatct ggcgctctac
1020
aacaccacga acgagacggc ctatatcatc ctgaaggaga agggcttcaa cgccacgcat
1080
tacctgaaga agctctgggc catgcagtcg aacgcgtatt tccgcgaggc ccagtggttc
1140
aactcgggct acatcccgaa gttcgacgag tatctggaca acgccctcgt gtcggtgggc
1200
gccccgttcg tgctgggcct ctcgtatccc atgatccagc agcagatctc gaaggaagag
1260
atcgacctga tccccgagga cctcaacctg ctccgctggg cctcgatcat cttccgcctg
1320
tacgacgacc tggccacctc gaaggccgag cagcagcgcg gcgacgtgcc caagtcgatc
1380
cagtgctata tgcatgagac gggctcgtcg gaggaagtgg cggccaacca tatccgcgac
1440
ctgatctcgg acgcgtggaa ggaagtgaac gccgagtgcc tgaagccgac ctcgctctcg
1500
aagcactacg tgggcgtggc ccccaactcg gcccgctcgg gcgtgctcat gtatcaccat
1560
gacttcgacg gcttcgcgtc gccccatggc cgcacgaacg cccacatcac gagcatcttc
1620
ttcgagccgg tccccctcaa ggagagcatc aacctgggct ga
1662
<210> 13
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS1
<400> 13
Met Asp Ser Met Glu Val Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Ile Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Ile Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Gly Ile Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Leu Asp Arg Leu Asp Arg Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Asp Leu Pro Leu Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Leu Tyr Glu Lys Asp Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Val Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Ser His Leu Phe Thr Val
260 265 270
Ala Ile Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Leu Ala Ile Thr
275 280 285
Lys Ala Leu Ile Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Asp Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Glu Ala Met Glu Gln Leu Pro Glu Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Val Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Val Thr Tyr Pro Val Ile Gln Gln Gln Leu
405 410 415
Ser Lys Glu Glu Leu Asp Leu Val Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Met Ile Ser Asp Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Leu Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Arg Thr Asn Gly His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 14
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS2
<400> 14
Met Asp Ser Val Asp Met Arg Lys Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Glu His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Glu Val Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Glu Asn Thr Glu His
85 90 95
Gly Val Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Lys Gly Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Leu Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Gln Leu Arg Ile Leu
165 170 175
Met Glu Arg Val Glu Lys Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Asp Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Lys Glu Glu Gly Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Ser Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Met Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Met
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Gln Arg Trp
305 310 315 320
Asp Val Glu Gly Met Asp Gln Leu Pro Asp Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Val Ile Leu Arg
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Lys Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Ile Gly
385 390 395 400
Gly Pro Phe Val Leu Ala Leu Ser Tyr Pro Leu Leu Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Glu Leu Ile Pro Glu Asp Leu Asn Leu Ile Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Thr Lys
435 440 445
Ala Glu Gln Asn Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Ser Ser Leu Ser Lys His Tyr Leu Gly Val Ala Pro Asn Thr Ala Arg
500 505 510
Thr Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Met Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 15
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS3
<400> 15
Met Asp Ser Met Glu Val Arg Lys Ser Ala Met Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Ile Leu Ala Lys Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Glu His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Asp Val Thr Asn Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Gln Ser Asp His
85 90 95
Gly Leu Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Lys Ile Ile
165 170 175
Ile Glu Arg Leu Glu Arg Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Ile Tyr Glu Lys Asp Asp Gly Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Met Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Thr His Leu Phe Ser Ile
260 265 270
Ala Met Ile Val Glu Pro Gln Tyr Ser Asn Cys Arg Ile Gly Ile Thr
275 280 285
Lys Ala Ile Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Val Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Asp Tyr Met Lys Val Tyr Tyr
325 330 335
Leu Gly Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Val Val Lys
340 345 350
Asp Lys Gly Tyr Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Thr Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Val Gly Leu Thr Tyr Pro Leu Leu Gln Gln Gln Leu
405 410 415
Thr Lys Glu Glu Ile Glu Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Thr Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Met Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Thr Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Ser Ser Val Ser Lys His Tyr Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Met Val Ile Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Ile Lys Asp Ser Ile Asn Leu Gly
545 550
<210> 16
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS4
<400> 16
Met Asp Ser Val Glu Ile Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Ile Ala Arg Arg Asp Cys Ser
20 25 30
Gly Gly Gly Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Lys
35 40 45
Ile Gln Arg Arg Leu Asn Asp Val Thr Asn Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Met His Ser Glu Asn Ser Asp His
85 90 95
Gly Val Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Gln Arg Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Ser Ile Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Leu Phe Ser Ile Ala Asn Leu Lys Leu Val
165 170 175
Val Asp Lys Val Glu Lys Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Asp Leu Pro Ile Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Ile Tyr Glu Arg Asp Glu Ala Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Asp Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Thr His Leu Phe Ser Leu
260 265 270
Ala Leu Ile Val Glu Pro Gln Phe Ser Asn Cys Arg Met Ala Met Thr
275 280 285
Lys Ala Val Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Ile Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Val Glu Gly Val Asp Gln Leu Pro Glu Tyr Met Lys Ile Tyr Tyr
325 330 335
Val Ala Leu Phe Asn Thr Thr Asn Glu Thr Ala Tyr Leu Met Leu Lys
340 345 350
Asp Lys Gly Tyr Asn Ala Thr His Phe Leu Lys Lys Ile Trp Ala Ile
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Val Gly
385 390 395 400
Gly Pro Phe Leu Val Gly Leu Ser Tyr Pro Val Met Gln Asn Asn Ile
405 410 415
Thr Lys Glu Asp Ile Asp Leu Val Pro Glu Asp Leu Asn Leu Leu Lys
420 425 430
Trp Ala Ser Met Ile Phe Arg Leu Trp Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Thr Asp Ala Trp Lys Asp Val Asn Gly Glu Cys Leu Lys Pro
485 490 495
Thr Thr Leu Ser Arg His Tyr Met Gly Ile Ala Pro Asn Ser Ala Arg
500 505 510
Thr Ala Val Val Val Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Gly Arg Ser Asn Ala His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Ile Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 17
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS5
<400> 17
Met Asp Ser Ile Asp Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Glu Cys Ser
20 25 30
Ala Ala Gly Ala Met Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Leu Thr Gln Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Asp Asn Ser Glu His
85 90 95
Ala Ile Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Thr Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Arg Ile Val
165 170 175
Ile Glu Lys Ile Glu Arg Lys Leu Ala Asp Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Ile Tyr Glu Lys Asp Glu Gly Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Val
260 265 270
Ala Met Leu Val Glu Pro Asn Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Val Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Leu Asp Ala Leu Asp Gln Leu Pro Glu Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Met Leu Lys
340 345 350
Asp Lys Gly Phe Asn Ala Ser His Tyr Leu Lys Lys Val Trp Ala Leu
355 360 365
Gln Ser Asn Gly Tyr Phe Arg Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Ile Ser Val Gly
385 390 395 400
Gly Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Leu
405 410 415
Thr Lys Glu Glu Met Glu Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Phe Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Asn Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Met Ala Ala Gln His Ile Lys Asp
465 470 475 480
Ile Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Ser Val Ser Arg His Phe Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Ile Val Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Arg Ser Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 18
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS6
<400> 18
Met Asp Ser Met Glu Ile Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Glu Ser Ile Lys Ala Leu Leu Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Gly Ala Val Ser Pro Glu His His Lys Lys Leu Lys Glu Arg
35 40 45
Ile Gln Lys Lys Leu Gln Asp Val Thr Gln Pro His His Val Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Asp Glu
65 70 75 80
Glu Ile Thr Glu Ala Leu His Gly Ile His Ser Glu Asn Thr Asp His
85 90 95
Gly Val Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Thr Leu Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Leu Glu Arg Met Asp Lys Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Glu Val Pro Ile Phe Phe Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Glu Ile Tyr Glu Arg Asp Glu Gly Arg Met Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Val Ile Tyr Gln Glu Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Val Leu Glu Thr His Leu Phe Thr Val
260 265 270
Ala Leu Met Val Glu Pro Asn Phe Ser Asn Cys Arg Ile Gly Leu Thr
275 280 285
Lys Ala Leu Ile Leu Ile Thr Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Glu Ala Val Asp Asn Leu Pro Asp Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Gln Glu Thr Ala Tyr Ile Leu Leu Lys
340 345 350
Asp Lys Gly Trp Asn Ala Thr His Phe Leu Lys Lys Ile Trp Ala Ile
355 360 365
Gln Ser Gln Gly Tyr Tyr Lys Glu Ala Gln Trp Tyr Gln Thr Gly Tyr
370 375 380
Leu Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Met Leu Ala Leu Ser Tyr Pro Leu Leu Gln Gln Gln Leu
405 410 415
Thr Arg Glu Glu Val Asp Leu Val Pro Glu Asp Leu Asn Leu Ile Lys
420 425 430
Trp Gly Ser Leu Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Asn Arg Gly Glu Met Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Asp
465 470 475 480
Leu Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Thr Thr Val Ser Lys His Phe Leu Ala Leu Ala Pro Asn Thr Ala Arg
500 505 510
Ser Ala Val Leu Leu Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Glu His Ile Ser Ser Ile Phe Tyr Asp Pro Val
530 535 540
Pro Ile Lys Asp Ser Ile Asn Leu Gly
545 550
<210> 19
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS7
<400> 19
Met Asp Ser Val Glu Leu Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Val Asp Ser Ile Arg Ala Leu Leu Ala Lys Lys Asp Cys Ser
20 25 30
Ala Ala Gly Ala Ile Ser Pro Glu His His Lys Lys Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Glu Leu Thr Asn Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Glu Gln Thr Asp His
85 90 95
Gly Val Arg Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Ser Val Cys Glu Asp Ala Val Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Met Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Asn Leu Phe Ser Val Ala Asn Leu Arg Leu Met
165 170 175
Leu Asp Lys Ile Asp Lys Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Asp Leu Pro Leu Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Tyr Tyr
195 200 205
Ile Glu Ile Tyr Glu Arg Asp Glu Ala Arg Ile Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Lys Leu Gln Ile Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Asp Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Val Leu Glu Ser His Leu Phe Thr Leu
260 265 270
Ala Leu Ile Val Glu Pro Gln Phe Ser Gln Cys Arg Leu Gly Leu Thr
275 280 285
Lys Ala Leu Ile Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Glu Ala Leu Glu Gln Leu Pro Glu Tyr Met Lys Ile Phe Tyr
325 330 335
Ile Gly Leu Tyr Asn Thr Thr Asn Glu Thr Ala Phe Met Val Met Arg
340 345 350
Glu Lys Gly Tyr Asn Ala Thr His Tyr Leu Arg Lys Ile Trp Gly Met
355 360 365
Gln Ser Asn Gly Tyr Phe Lys Glu Ala Gln Trp Tyr Gln Thr Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Trp Leu Leu Gly Leu Ser Tyr Pro Leu Met Gln Asn Gln Leu
405 410 415
Thr Lys Glu Asp Leu Glu Ile Val Pro Asp His Leu Gln Leu Val Lys
420 425 430
Trp Gly Ser Val Ile Phe Arg Leu Trp Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Asn Arg Gly Glu Met Pro Lys Thr Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Thr Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Gly Trp Lys Asp Val Asn Gly Glu Cys Leu Arg Pro
485 490 495
Ser Thr Val Ser Arg His Tyr Leu Ala Met Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Val Ile Tyr His His Glu Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Lys Ser Asn Gly His Ile Ser Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Val Arg Asp Ser Ile Asn Leu Gly
545 550
<210> 20
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarS8
<400> 20
Met Asp Ser Val Glu Val Arg Lys Thr Gly Met Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Val Asp Ser Ile Lys Ala Ile Leu Ala Lys Lys Glu Cys Ser
20 25 30
Ala Gly Gly Ala Met Thr Pro Glu His His Lys Lys Leu Lys Asp Arg
35 40 45
Ile Gln Lys Arg Leu Asn Glu Val Ser Gln Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Asp Asn Ser Asp His
85 90 95
Gly Val Lys Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Gln Leu Ser Ser Asp Ile Phe Gln Arg Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Ser Met Cys Glu Asp Ala Leu Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Gly
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Gly Asn Leu Arg Ile Met
165 170 175
Leu Asp Lys Ile Asp Lys Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Asp Val Pro Val Tyr Trp Lys Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Asp Ile Tyr Glu Lys Asp Glu Ala Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Asp Leu Ile Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Met Leu Glu Ser His Leu Phe Thr Met
260 265 270
Ala Ile Ile Met Glu Pro Asn Tyr Ser Asn Cys Arg Ile Ala Leu Thr
275 280 285
Lys Ala Leu Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Asp Ala Met Glu Asn Leu Pro Asp Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Phe Asn Thr Ser Asn Glu Thr Ala Tyr Ile Leu Val Lys
340 345 350
Asp Lys Gly Trp Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Ile
355 360 365
Gln Ser Asn Ala Tyr Tyr Lys Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Ile Ile Ser Ile Gly
385 390 395 400
Gly Pro Tyr Leu Met Ala Val Ser Tyr Pro Val Met Gln Asn Gln Val
405 410 415
Ser Lys Glu Asp Val Glu Leu Leu Pro Glu Glu Leu Asn Leu Val Lys
420 425 430
Tyr Ala Thr Met Ile Phe Arg Leu Trp Asp Asp Leu Gly Thr Thr Lys
435 440 445
Ala Glu Asn Asn Arg Gly Asp Val Pro Lys Thr Ile Gln Cys Tyr Met
450 455 460
His Glu Ser Gly Thr Ser Glu Glu Ile Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Asp Cys Leu Arg Pro
485 490 495
Ser Thr Leu Ser Arg His Phe Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Thr Ala Val Val Val Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Arg Ser Asn Ala His Ile Ser Thr Ile Phe Tyr Asp Pro Val
530 535 540
Pro Leu Arg Asp Ser Ile Asn Leu Gly
545 550
<210> 21
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL1
<400> 21
Met Asp Ser Met Glu Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Met Leu Ala Arg Arg Asp Cys Ser
20 25 30
Ala Gly Ala Ala Val Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Val Glu Lys Leu Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Glu Ile Tyr Glu Lys Glu Asp Gly Lys Ile Asp Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Ile Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Leu His Leu Phe Ser Met
260 265 270
Ala Val Ile Val Glu Pro Gln Phe Ser Gln Cys Arg Leu Ala Leu Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Met Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Leu Val Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Arg Val Trp Ala Met
355 360 365
Gln Ser Asn Gly Tyr Tyr Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Leu Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Leu Leu Gly Leu Ser Tyr Pro Met Met Gln Gln Gln Ile
405 410 415
Thr Lys Glu Glu Leu Asp Leu Ile Pro Glu Glu Leu Asn Leu Val Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Ile Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Val Ser Lys His Phe Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Val Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Asp Ser Ile Asn Leu Gly
545 550
<210> 22
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL2
<400> 22
Met Asp Ser Val Glu Leu Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Lys Lys Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Glu Asn Thr Asp His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Val
165 170 175
Met Glu Arg Ile Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Asp Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Leu His Leu Phe Ser Ile
260 265 270
Ala Met Leu Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Val Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Glu Gln Leu Pro Asp Tyr Met Lys Val Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Thr Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Ile
405 410 415
Ser Lys Glu Glu Leu Glu Leu Leu Pro Glu Asp Leu Asn Leu Ile Arg
420 425 430
Trp Ala Ser Met Ile Phe Arg Leu Trp Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Thr Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Val Ser Arg His Tyr Val Gly Val Ala Pro Asn Thr Ala Arg
500 505 510
Ser Gly Leu Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 23
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL3
<400> 23
Met Asp Ser Val Glu Val Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Val Ala Arg Arg Asp Cys Ser
20 25 30
Ala Gly Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Ala Ile His Thr Asp Gln Ser Asp His
85 90 95
Ala Ile Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Gln Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Leu
165 170 175
Leu Glu Arg Ile Glu Arg Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Lys Asp Asp Ala Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Met Met Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Leu His Leu Phe Ser Ile
260 265 270
Ala Met Val Val Glu Pro Asn Tyr Ser Gln Cys Arg Ile Ala Leu Thr
275 280 285
Lys Ala Ile Ile Leu Val Ser Ala Met Asp Asp Tyr Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Ile Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Glu Gln Leu Pro Asp Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Leu Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Ser His Tyr Leu Arg Lys Leu Trp Ala Val
355 360 365
Gln Ser Asn Ala Tyr Phe Lys Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Val Ser Ile Gly
385 390 395 400
Ala Pro Phe Ile Val Ala Val Thr Tyr Pro Met Ile Gln Asn Asn Val
405 410 415
Thr Lys Glu Glu Ile Glu Leu Leu Pro Glu Asp Leu Gln Leu Leu Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Thr Thr Leu Ser Arg His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Val Ile Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Lys Thr Gln Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 24
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL4
<400> 24
Met Asp Ser Met Glu Val Arg Arg Ser Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Ala Gly Leu Ser Pro Glu His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Asn Glu Ile Ser Asn Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Asp Asn Thr Asp His
85 90 95
Ala Val Lys Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Gln Leu Ser Ser Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Ser Leu Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Leu Phe Ser Ile Ala Asn Leu Lys Ile Met
165 170 175
Leu Glu Arg Leu Glu Lys Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Asp Leu Pro Val Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Asp Ile Tyr Glu Arg Asp Asp Ala Arg Leu Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Met Met Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ile His Leu Phe Thr Ile
260 265 270
Ala Val Leu Met Glu Pro Gln Phe Ser Gln Cys Arg Ile Ala Leu Thr
275 280 285
Lys Gly Ile Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Ile Glu Ala Met Glu Gln Leu Pro Glu Tyr Met Lys Val Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Thr Ser Gln Glu Thr Ala Tyr Ile Val Leu Lys
340 345 350
Asp Lys Gly Tyr Asn Ala Ser His Tyr Leu Lys Lys Ile Trp Gly Met
355 360 365
Gln Ser Asn Ala Tyr Phe Lys Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Val Ser Leu Gly
385 390 395 400
Gly Pro Phe Val Leu Ala Leu Ser Tyr Pro Leu Met Gln Gln Gln Ile
405 410 415
Thr Lys Glu Asp Leu Glu Leu Val Pro Glu Asp Leu Asn Leu Met Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Trp Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Leu Pro Lys Ser Ile Asn Cys Tyr Met
450 455 460
His Glu Thr Gly Thr Ser Glu Glu Leu Ala Ala Asn His Ile Lys Asp
465 470 475 480
Leu Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Val Ser Lys His Phe Met Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Thr Ala Val Leu Ile Tyr His His Glu Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Ser Thr Ile Phe Phe Asp Pro Val
530 535 540
Pro Ile Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 25
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL5
<400> 25
Met Asp Ser Leu Asp Leu Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Lys Ala Leu Ile Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Arg Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Met His Thr Asp Gln Thr Asp His
85 90 95
Ala Val Lys Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Arg
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Ile Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Leu Glu Lys Val Glu Lys Lys Leu Ala Asp Arg Ile Asp His Ala Leu
180 185 190
Glu Leu Pro Ile Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Arg Asp Glu Gly Arg Ile Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Val Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Val Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Ile His Leu Phe Ser Leu
260 265 270
Ala Ile Leu Val Glu Pro Gln Tyr Ser Asn Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Ile Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Asp Ala Ile Asp Asn Leu Pro Glu Tyr Met Lys Leu Phe Tyr
325 330 335
Val Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Leu Ile Val Lys
340 345 350
Glu Arg Gly Phe Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Gln Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Ile Leu Gly Leu Ser Tyr Pro Leu Ile Gln Asn Gln Val
405 410 415
Thr Lys Glu Glu Ile Asp Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Gln Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Ile Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Ala His Ile Thr Ser Ile Phe Tyr Asp Pro Val
530 535 540
Pro Leu Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 26
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL6
<400> 26
Met Asp Ser Val Glu Val Arg Arg Thr Gly Met Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Glu Cys Ser
20 25 30
Ala Gly Gly Ala Met Ser Pro Glu His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Lys Leu Asn Asp Val Ser Gln Pro His His Ile Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Asp Glu
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Glu Gln Thr Asp His
85 90 95
Gly Met Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Gln Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Val Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Leu Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Gln Leu Arg Leu Met
165 170 175
Leu Asp Arg Ile Asp Lys Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Ile Tyr Tyr Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Glu Ile Tyr Asp Lys Asp Glu Gly Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Ile Gln Met Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Leu His Leu Phe Ser Leu
260 265 270
Ala Leu Leu Val Glu Pro Asn Phe Ser Asn Cys Arg Met Gly Leu Thr
275 280 285
Lys Gly Ile Met Leu Val Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Ile Asp Ala Leu Glu Gln Leu Pro Asp Tyr Met Lys Met Phe Tyr
325 330 335
Val Ala Leu Phe Asn Thr Thr Asn Glu Thr Ala Tyr Met Leu Leu Arg
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Phe Leu Lys Lys Leu Trp Ala Leu
355 360 365
Gln Ser Asn Gly Tyr Phe Lys Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Ile Met Ser Ile Gly
385 390 395 400
Gly Pro Phe Leu Leu Ala Leu Ser Tyr Pro Met Val Gln Asn Gln Val
405 410 415
Thr Arg Glu Asp Leu Glu Met Val Pro Glu Glu Leu Asn Leu Ile Lys
420 425 430
Trp Ala Ser Leu Ile Phe Arg Leu Trp Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Ser Gly Thr Ser Asp Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Glu Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Met Ala Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Leu Val Val Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Lys Thr Asn Gly His Ile Ser Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Val Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 27
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL7
<400> 27
Met Asp Ser Leu Asp Ile Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Val Glu Ser Ile Lys Ala Ile Ile Ala Lys Lys Glu Cys Thr
20 25 30
Ala Gly Gly Ala Ile Ser Pro Glu His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Arg Leu Asn Glu Val Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Asp Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Asp Asn Ser Asp His
85 90 95
Gly Leu Arg Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Ser Ile Cys Glu Asp Ala Val Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Gly
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Ile
165 170 175
Ile Asp Lys Met Asp Lys Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Tyr Tyr
195 200 205
Ile Glu Ile Tyr Glu Arg Asp Glu Gly Lys Leu Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Met Val Tyr Gln Thr Glu
225 230 235 240
Leu Arg Asp Leu Thr Met Trp Trp Asp Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Ala Phe Phe Arg Asp Arg Ile Leu Glu Val His Leu Phe Thr Leu
260 265 270
Ala Ile Leu Val Glu Pro Gln Phe Ser Gln Cys Arg Ile Gly Val Thr
275 280 285
Lys Ala Val Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Met His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Met Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Val Asp Ala Val Glu Asn Leu Pro Glu Tyr Met Lys Val Tyr Tyr
325 330 335
Met Ala Leu Tyr Asn Ser Thr Asn Glu Thr Ala Phe Ile Val Met Lys
340 345 350
Asp Lys Gly Leu Asn Ala Thr His Tyr Leu Arg Lys Ile Trp Ala Met
355 360 365
Gln Ser Asn Gly Tyr Tyr Lys Glu Ala Gln Trp Tyr Gln Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Val Val Gly Met Thr Tyr Pro Leu Met Gln Asn Asn Val
405 410 415
Thr Lys Glu Glu Leu Glu Leu Val Pro Asp Glu Leu Asn Leu Val Lys
420 425 430
Trp Ala Thr Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Thr Lys
435 440 445
Ala Glu Asn Gln Arg Gly Glu Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Ser Gly Thr Ser Glu Glu Ile Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Ala Leu Ala Pro Asn Thr Ala Arg
500 505 510
Thr Ala Val Leu Leu Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Gly His Ile Ser Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Met Arg Asp Ser Ile Asn Leu Gly
545 550
<210> 28
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> >VarL8
<400> 28
Met Asp Ser Val Asp Ile Arg Lys Thr Gly Met Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Glu Ser Ile Lys Ala Leu Met Ala Arg Arg Glu Cys Ser
20 25 30
Ala Gly Ala Ala Leu Ser Pro Glu His His Lys Lys Leu Lys Asp Lys
35 40 45
Ile Gln Lys Arg Leu Asn Glu Leu Ser Asn Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Glu Asn Thr Asp His
85 90 95
Ala Leu Arg Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Ser Leu Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Leu Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Asn Leu Phe Ser Ile Ala Gln Leu Arg Ile Ile
165 170 175
Leu Glu Lys Ile Asp Lys Lys Leu Ala Glu Lys Ile Asp His Ala Leu
180 185 190
Asp Leu Pro Ile Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Glu Ile Tyr Glu Arg Asp Glu Gly Lys Leu Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Val Ile Tyr Gln Thr Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Val Leu Glu Val His Leu Phe Ser Met
260 265 270
Ala Leu Ile Leu Glu Pro Gln Tyr Ser Asn Cys Arg Leu Gly Val Thr
275 280 285
Lys Ala Val Met Leu Ile Ser Ala Met Asp Asp Tyr Tyr Asp Met His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Val Glu Ala Met Glu Gln Leu Pro Asp Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Ser Ser Asn Glu Thr Ala Phe Ile Met Leu Arg
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Arg Lys Ile Trp Ala Val
355 360 365
Gln Ser Gln Gly Tyr Phe Arg Glu Ala Gln Trp Phe Gln Thr Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Leu Ile Ala Val Ser Tyr Pro Leu Met Gln Asn Asn Leu
405 410 415
Thr Arg Glu Asp Val Glu Ile Leu Pro Glu Glu Leu Gln Leu Ile Lys
420 425 430
Tyr Ala Thr Val Ile Phe Arg Leu Trp Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Asn Arg Gly Glu Leu Pro Lys Ser Ile Asn Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Leu Ala Ala Asn His Ile Arg Glu
465 470 475 480
Ile Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Asp Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Gly Ile Ala Pro Asn Thr Ala Arg
500 505 510
Thr Gly Val Val Val Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Arg Thr Gln Ala His Ile Ser Thr Ile Phe Phe Asp Pro Val
530 535 540
Pro Met Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 29
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Cicassy N267T
<400> 29
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Thr His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 30
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> I291V
<400> 30
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Val Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 31
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> I291T
<400> 31
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Thr Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 32
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> I291S
<400> 32
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ser Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 33
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> I291C
<400> 33
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Cys Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 34
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> I291F
<400> 34
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Phe Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 35
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267L_I291V
<400> 35
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Leu His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Val Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 36
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267S_I291T
<400> 36
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Thr Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 37
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267S_I291T
<400> 37
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Thr His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Thr Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 38
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267S_I291S
<400> 38
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ser Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 39
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267S_I291C
<400> 39
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Cys Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 40
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267L_I291S
<400> 40
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Leu His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ser Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 41
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var1_291T
<400> 41
Met Asp Ser Val Glu Leu Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Lys Lys Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Glu Asn Thr Asp His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Arg Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Val
165 170 175
Met Glu Arg Ile Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Asp Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Met Leu Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Thr Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Glu Gln Leu Pro Asp Tyr Met Lys Val Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Thr Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Ile
405 410 415
Ser Lys Glu Glu Leu Glu Leu Leu Pro Glu Asp Leu Asn Leu Ile Arg
420 425 430
Trp Ala Ser Met Ile Phe Arg Leu Trp Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Thr Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Val Ser Arg His Tyr Val Gly Val Ala Pro Asn Thr Ala Arg
500 505 510
Ser Gly Leu Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 42
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var2_291T
<400> 42
Met Asp Ser Met Glu Val Arg Lys Ser Ala Met Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Ile Leu Ala Lys Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Glu His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Asn Asp Val Thr Asn Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Gln Ser Asp His
85 90 95
Gly Leu Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Lys Ile Ile
165 170 175
Ile Glu Arg Leu Glu Arg Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Ile Tyr Glu Lys Asp Asp Gly Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Met Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Asp Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Met Ile Val Glu Pro Gln Tyr Ser Asn Cys Arg Ile Gly Ile Thr
275 280 285
Lys Ala Thr Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Val Phe Thr Glu Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Asp Tyr Met Lys Val Tyr Tyr
325 330 335
Leu Gly Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Val Val Lys
340 345 350
Asp Lys Gly Tyr Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Thr Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Val Gly Leu Thr Tyr Pro Leu Leu Gln Gln Gln Leu
405 410 415
Thr Lys Glu Glu Ile Glu Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Thr Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Met Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Thr Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Ser Ser Val Ser Lys His Tyr Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Met Val Ile Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Gly Lys Thr Asn Ala His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Ile Lys Asp Ser Ile Asn Leu Gly
545 550
<210> 43
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var3_291T
<400> 43
Met Asp Ser Leu Asp Leu Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Lys Ala Leu Ile Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Arg Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Met His Thr Asp Gln Thr Asp His
85 90 95
Ala Val Lys Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Arg
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Ile Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Leu Glu Lys Val Glu Lys Lys Leu Ala Asp Arg Ile Asp His Ala Leu
180 185 190
Glu Leu Pro Ile Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Arg Asp Glu Gly Arg Ile Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Val Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Val Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Asn His Leu Phe Ser Leu
260 265 270
Ala Ile Leu Val Glu Pro Gln Tyr Ser Asn Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Thr Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Asp Ala Ile Asp Asn Leu Pro Glu Tyr Met Lys Leu Phe Tyr
325 330 335
Val Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Leu Ile Val Lys
340 345 350
Glu Arg Gly Phe Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Gln Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Ile Leu Gly Leu Ser Tyr Pro Leu Ile Gln Asn Gln Val
405 410 415
Thr Lys Glu Glu Ile Asp Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Gln Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Ile Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Ala His Ile Thr Ser Ile Phe Tyr Asp Pro Val
530 535 540
Pro Leu Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 44
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var1_291S
<400> 44
Met Asp Ser Met Glu Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Met Leu Ala Arg Arg Asp Cys Ser
20 25 30
Ala Gly Ala Ala Val Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Val His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Val Glu Lys Leu Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Phe Tyr
195 200 205
Ile Glu Ile Tyr Glu Lys Glu Asp Gly Lys Ile Asp Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Ile Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Met
260 265 270
Ala Val Ile Val Glu Pro Gln Phe Ser Gln Cys Arg Leu Ala Leu Thr
275 280 285
Lys Ala Ser Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Met Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Leu Val Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Arg Val Trp Ala Met
355 360 365
Gln Ser Asn Gly Tyr Tyr Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Leu Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Leu Leu Gly Leu Ser Tyr Pro Met Met Gln Gln Gln Ile
405 410 415
Thr Lys Glu Glu Leu Asp Leu Ile Pro Glu Glu Leu Asn Leu Val Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Ile Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Val Ser Lys His Phe Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Val Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Asp Ser Ile Asn Leu Gly
545 550
<210> 45
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var2_291S
<400> 45
Met Asp Ser Val Glu Val Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Val Ala Arg Arg Asp Cys Ser
20 25 30
Ala Gly Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Ala Ile His Thr Asp Gln Ser Asp His
85 90 95
Ala Ile Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Gln Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Leu
165 170 175
Leu Glu Arg Ile Glu Arg Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Lys Asp Asp Ala Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Met Met Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Met Val Val Glu Pro Asn Tyr Ser Gln Cys Arg Ile Ala Leu Thr
275 280 285
Lys Ala Ser Ile Leu Val Ser Ala Met Asp Asp Tyr Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Ile Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Glu Gln Leu Pro Asp Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Leu Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Ser His Tyr Leu Arg Lys Leu Trp Ala Val
355 360 365
Gln Ser Asn Ala Tyr Phe Lys Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Val Ser Ile Gly
385 390 395 400
Ala Pro Phe Ile Val Ala Val Thr Tyr Pro Met Ile Gln Asn Asn Val
405 410 415
Thr Lys Glu Glu Ile Glu Leu Leu Pro Glu Asp Leu Gln Leu Leu Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Thr Thr Leu Ser Arg His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Val Ile Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Lys Thr Gln Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 46
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var3_291S
<400> 46
Met Asp Ser Leu Asp Leu Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Lys Ala Leu Ile Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Arg Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Met His Thr Asp Gln Thr Asp His
85 90 95
Ala Val Lys Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Arg
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Ile Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Leu Glu Lys Val Glu Lys Lys Leu Ala Asp Arg Ile Asp His Ala Leu
180 185 190
Glu Leu Pro Ile Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Arg Asp Glu Gly Arg Ile Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Val Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Val Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Asn His Leu Phe Ser Leu
260 265 270
Ala Ile Leu Val Glu Pro Gln Tyr Ser Asn Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Ser Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Asp Ala Ile Asp Asn Leu Pro Glu Tyr Met Lys Leu Phe Tyr
325 330 335
Val Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Leu Ile Val Lys
340 345 350
Glu Arg Gly Phe Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Gln Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Ile Leu Gly Leu Ser Tyr Pro Leu Ile Gln Asn Gln Val
405 410 415
Thr Lys Glu Glu Ile Asp Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Gln Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Ile Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Ala His Ile Thr Ser Ile Phe Tyr Asp Pro Val
530 535 540
Pro Leu Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 47
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var1_291C
<400> 47
Met Asp Ser Met Glu Val Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Ile Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Ile Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Lys Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Gly Ile Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Asp Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Leu Asp Arg Leu Asp Arg Lys Leu Ala Glu Arg Ile Glu His Ala Leu
180 185 190
Asp Leu Pro Leu Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Leu Tyr Glu Lys Asp Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Val Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Asn His Leu Phe Thr Val
260 265 270
Ala Ile Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Leu Ala Ile Thr
275 280 285
Lys Ala Cys Ile Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Asp Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Glu Ala Met Glu Gln Leu Pro Glu Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Val Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Val Thr Tyr Pro Val Ile Gln Gln Gln Leu
405 410 415
Ser Lys Glu Glu Leu Asp Leu Val Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Met Ile Ser Asp Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Leu Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Arg Thr Asn Gly His Ile Thr Ser Ile Phe Phe Asp Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 48
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var2_291C
<400> 48
Met Asp Ser Val Glu Val Arg Arg Thr Gly Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Val Ala Arg Arg Asp Cys Ser
20 25 30
Ala Gly Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Ala Ile His Thr Asp Gln Ser Asp His
85 90 95
Ala Ile Arg Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Gln Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Leu
165 170 175
Leu Glu Arg Ile Glu Arg Lys Leu Ala Glu Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Lys Asp Asp Ala Lys Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Leu Gln Met Met Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Met Val Val Glu Pro Asn Tyr Ser Gln Cys Arg Ile Ala Leu Thr
275 280 285
Lys Ala Cys Ile Leu Val Ser Ala Met Asp Asp Tyr Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Ile Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Glu Gln Leu Pro Asp Tyr Met Lys Ile Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Leu Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Ser His Tyr Leu Arg Lys Leu Trp Ala Val
355 360 365
Gln Ser Asn Ala Tyr Phe Lys Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Leu Val Ser Ile Gly
385 390 395 400
Ala Pro Phe Ile Val Ala Val Thr Tyr Pro Met Ile Gln Asn Asn Val
405 410 415
Thr Lys Glu Glu Ile Glu Leu Leu Pro Glu Asp Leu Gln Leu Leu Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Glu Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Ile Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Thr Thr Leu Ser Arg His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Val Ile Tyr His His Asp Phe Asp Gly Phe Ala Thr Pro
515 520 525
His Ala Lys Thr Gln Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 49
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var3_291C
<400> 49
Met Asp Ser Ile Asp Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Glu Cys Ser
20 25 30
Ala Ala Gly Ala Met Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Leu Thr Gln Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Asp Asn Ser Glu His
85 90 95
Ala Ile Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Thr Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Arg Ile Val
165 170 175
Ile Glu Lys Ile Glu Arg Lys Leu Ala Asp Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Ile Tyr Glu Lys Asp Glu Gly Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Val
260 265 270
Ala Met Leu Val Glu Pro Asn Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Cys Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Leu Asp Ala Leu Asp Gln Leu Pro Glu Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Met Leu Lys
340 345 350
Asp Lys Gly Phe Asn Ala Ser His Tyr Leu Lys Lys Val Trp Ala Leu
355 360 365
Gln Ser Asn Gly Tyr Phe Arg Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Ile Ser Val Gly
385 390 395 400
Gly Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Leu
405 410 415
Thr Lys Glu Glu Met Glu Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Phe Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Asn Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Met Ala Ala Gln His Ile Lys Asp
465 470 475 480
Ile Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Ser Val Ser Arg His Phe Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Ile Val Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Arg Ser Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 50
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var_267S_291T
<400> 50
Met Asp Ser Ile Asp Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Glu Cys Ser
20 25 30
Ala Ala Gly Ala Met Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Leu Thr Gln Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Asp Asn Ser Glu His
85 90 95
Ala Ile Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Thr Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Arg Ile Val
165 170 175
Ile Glu Lys Ile Glu Arg Lys Leu Ala Asp Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Ile Tyr Glu Lys Asp Glu Gly Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Val
260 265 270
Ala Met Leu Val Glu Pro Asn Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Thr Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Leu Asp Ala Leu Asp Gln Leu Pro Glu Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Met Leu Lys
340 345 350
Asp Lys Gly Phe Asn Ala Ser His Tyr Leu Lys Lys Val Trp Ala Leu
355 360 365
Gln Ser Asn Gly Tyr Phe Arg Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Ile Ser Val Gly
385 390 395 400
Gly Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Leu
405 410 415
Thr Lys Glu Glu Met Glu Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Phe Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Asn Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Met Ala Ala Gln His Ile Lys Asp
465 470 475 480
Ile Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Ser Val Ser Arg His Phe Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Ile Val Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Arg Ser Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 51
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var_267S_291S
<400> 51
Met Asp Ser Ile Asp Ile Arg Arg Thr Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Lys Arg Glu Cys Ser
20 25 30
Ala Ala Gly Ala Met Ser Pro Asp His His Lys Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Leu Thr Gln Pro His His Leu Leu Ala
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Glu Ala Leu His Gly Ile His Ser Asp Asn Ser Glu His
85 90 95
Ala Ile Arg Glu Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Thr Asp Ile Phe Asn Lys Phe Arg
115 120 125
Lys Asp Gly Gly Gly Phe Lys Ala Thr Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Arg Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Ile Phe Ser Ile Ala Asn Leu Arg Ile Val
165 170 175
Ile Glu Lys Ile Glu Arg Lys Leu Ala Asp Lys Ile Glu His Ala Leu
180 185 190
Glu Leu Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Ile Tyr Glu Lys Asp Glu Gly Arg Ile Glu Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Leu Ile Tyr Gln Thr Glu
225 230 235 240
Leu Arg Glu Leu Thr Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ser His Leu Phe Ser Val
260 265 270
Ala Met Leu Val Glu Pro Asn Tyr Ser Gln Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Ser Val Leu Val Ser Ala Met Asp Asp Phe Tyr Asp Ile His
290 295 300
Gly Leu Pro Asp Glu Leu Arg Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Leu Asp Ala Leu Asp Gln Leu Pro Glu Tyr Met Lys Ile Phe Tyr
325 330 335
Met Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Leu Met Leu Lys
340 345 350
Asp Lys Gly Phe Asn Ala Ser His Tyr Leu Lys Lys Val Trp Ala Leu
355 360 365
Gln Ser Asn Gly Tyr Phe Arg Glu Ala Gln Trp Tyr Asn Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Val Ile Ser Val Gly
385 390 395 400
Gly Pro Phe Leu Leu Gly Val Ser Tyr Pro Leu Leu Gln Asn Gln Leu
405 410 415
Thr Lys Glu Glu Met Glu Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Phe Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Asn Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Met Ala Ala Gln His Ile Lys Asp
465 470 475 480
Ile Ile Ser Glu Gly Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Ser Val Ser Arg His Phe Leu Gly Leu Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Ile Val Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Arg Ser Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Ile Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 52
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> Var_267L_291S
<400> 52
Met Asp Ser Leu Asp Leu Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Lys Ala Leu Ile Ala Lys Arg Asp Cys Ser
20 25 30
Ala Ala Ala Ala Val Ser Pro Glu His His Arg Lys Leu Lys Glu Lys
35 40 45
Ile Gln Lys Lys Leu Gln Glu Ile Ser Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Asp
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Met His Thr Asp Gln Thr Asp His
85 90 95
Ala Val Lys Glu Ser Leu His His Thr Ser Leu Trp Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Arg Phe Arg
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Ile Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Ile Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Arg Ile Met
165 170 175
Leu Glu Lys Val Glu Lys Lys Leu Ala Asp Arg Ile Asp His Ala Leu
180 185 190
Glu Leu Pro Ile Phe Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Glu Val Tyr Glu Arg Asp Glu Gly Arg Ile Glu Glu Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Val Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Thr Met Trp Trp Glu Leu Val Gly Leu Pro Ala Arg
245 250 255
Met Gly Phe Phe Arg Asp Arg Ile Leu Glu Leu His Leu Phe Ser Leu
260 265 270
Ala Ile Leu Val Glu Pro Gln Tyr Ser Asn Cys Arg Val Ala Leu Thr
275 280 285
Lys Ala Ser Ile Leu Ile Ser Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Leu Phe Thr Glu Thr Val Gln Arg Trp
305 310 315 320
Asp Ile Asp Ala Ile Asp Asn Leu Pro Glu Tyr Met Lys Leu Phe Tyr
325 330 335
Val Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Trp Leu Ile Val Lys
340 345 350
Glu Arg Gly Phe Asn Ala Thr His Tyr Leu Arg Lys Val Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Gln Ser Gly Tyr
370 375 380
Val Pro Lys Phe Asp Glu Tyr Leu Glu Asn Ala Val Ile Ser Ile Gly
385 390 395 400
Gly Pro Phe Ile Leu Gly Leu Ser Tyr Pro Leu Ile Gln Asn Gln Val
405 410 415
Thr Lys Glu Glu Ile Asp Leu Leu Pro Glu Asp Leu Asn Leu Val Lys
420 425 430
Trp Ala Ser Val Ile Phe Arg Leu Tyr Asp Asp Leu Gly Thr Ser Lys
435 440 445
Ala Glu Asn Gln Arg Gly Asp Leu Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Lys Glu
465 470 475 480
Met Ile Ser Glu Ala Trp Lys Asp Val Asn Ala Glu Cys Leu Arg Pro
485 490 495
Ser Thr Ile Ser Arg His Phe Leu Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Ala Val Ile Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Ala Lys Thr Asn Ala His Ile Thr Ser Ile Phe Tyr Asp Pro Val
530 535 540
Pro Leu Arg Glu Ser Ile Asn Leu Gly
545 550
<210> 53
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> CiCassy I291L
<400> 53
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Asn His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Leu Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 54
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> RRX8W基序
<220>
<221> 变体
<222> 3
<223> 任意氨基酸
<220>
<221> 变体
<222> 4
<223> 任意氨基酸
<220>
<221> 变体
<222> 5
<223> 任意氨基酸
<220>
<221> 变体
<222> 6
<223> 任意氨基酸
<220>
<221> 变体
<222> 7
<223> 任意氨基酸
<220>
<221> 变体
<222> 8
<223> 任意氨基酸
<220>
<221> 变体
<222> 9
<223> 任意氨基酸
<220>
<221> 变体
<222> 10
<223> 任意氨基酸
<400> 54
Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg
1 5 10
<210> 55
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> R(R/K)X8W基序
<220>
<221> 变体
<222> 2
<223> Xaa是精氨酸或赖氨酸
<220>
<221> 变体
<222> 3
<223> 任意氨基酸
<220>
<221> 变体
<222> 4
<223> 任意氨基酸
<220>
<221> 变体
<222> 5
<223> 任意氨基酸
<220>
<221> 变体
<222> 6
<223> 任意氨基酸
<220>
<221> 变体
<222> 7
<223> 任意氨基酸
<220>
<221> 变体
<222> 8
<223> 任意氨基酸
<220>
<221> 变体
<222> 9
<223> 任意氨基酸
<220>
<221> 变体
<222> 10
<223> 任意氨基酸
<400> 55
Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg
1 5 10
<210> 56
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267W
<400> 56
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Trp His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 57
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267G
<400> 57
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Gly His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
530 535 540
Pro Leu Lys Glu Ser Ile Asn Leu Gly
545 550
<210> 58
<211> 553
<212> PRT
<213> 人工序列
<220>
<223> N267A
<400> 58
Met Asp Ser Met Glu Val Arg Arg Ser Ala Ile Tyr His Ser Thr Phe
1 5 10 15
Trp Asp Ile Asp Ser Ile Arg Ala Leu Leu Ala Arg Arg Asp Cys Thr
20 25 30
Ala Ala Ala Ala Leu Ser Pro Asp His His Lys Arg Leu Lys Glu Arg
35 40 45
Ile Gln Arg Arg Leu Gln Asp Ile Thr Gln Pro His His Leu Leu Gly
50 55 60
Leu Ile Asp Ala Val Gln Arg Leu Gly Val Ala Tyr Gln Phe Glu Glu
65 70 75 80
Glu Ile Ser Asp Ala Leu His Gly Leu His Ser Glu Asn Thr Glu His
85 90 95
Ala Ile Lys Asp Ser Leu His His Thr Ser Leu Tyr Phe Arg Leu Leu
100 105 110
Arg Gln His Gly Cys Asn Leu Ser Ser Asp Ile Phe Asn Lys Phe Lys
115 120 125
Lys Glu Gly Gly Gly Phe Lys Ala Ser Leu Cys Glu Asp Ala Met Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Val Arg Leu Ser Val Lys Gly Glu Ala
145 150 155 160
Ile Leu Glu Glu Ala Gln Val Phe Ser Ile Ala Asn Leu Lys Ile Leu
165 170 175
Met Glu Arg Val Glu Arg Lys Leu Ala Asp Arg Ile Glu His Ala Leu
180 185 190
Glu Ile Pro Leu Tyr Trp Arg Ala Pro Arg Leu Glu Ala Arg Trp Tyr
195 200 205
Ile Asp Val Tyr Glu Lys Glu Asp Gly Arg Ile Asp Asp Leu Leu Asp
210 215 220
Phe Ala Lys Leu Asp Phe Asn Arg Val Gln Met Leu Tyr Gln Thr Glu
225 230 235 240
Leu Lys Glu Leu Ser Met Trp Trp Glu Leu Leu Gly Leu Pro Ala Lys
245 250 255
Met Gly Phe Phe Arg Asp Arg Leu Leu Glu Ala His Leu Phe Ser Ile
260 265 270
Ala Val Val Val Glu Pro Gln Tyr Ser Gln Cys Arg Val Ala Ile Thr
275 280 285
Lys Ala Ile Val Leu Met Thr Ala Met Asp Asp Phe Tyr Asp Val His
290 295 300
Gly Leu Pro Asp Glu Leu Lys Val Phe Thr Asp Thr Val Asn Arg Trp
305 310 315 320
Asp Leu Glu Gly Ile Asp Gln Leu Pro Glu Tyr Met Lys Leu Tyr Tyr
325 330 335
Leu Ala Leu Tyr Asn Thr Thr Asn Glu Thr Ala Tyr Ile Ile Leu Lys
340 345 350
Glu Lys Gly Phe Asn Ala Thr His Tyr Leu Lys Lys Leu Trp Ala Met
355 360 365
Gln Ser Asn Ala Tyr Phe Arg Glu Ala Gln Trp Phe Asn Ser Gly Tyr
370 375 380
Ile Pro Lys Phe Asp Glu Tyr Leu Asp Asn Ala Leu Val Ser Val Gly
385 390 395 400
Ala Pro Phe Val Leu Gly Leu Ser Tyr Pro Met Ile Gln Gln Gln Ile
405 410 415
Ser Lys Glu Glu Ile Asp Leu Ile Pro Glu Asp Leu Asn Leu Leu Arg
420 425 430
Trp Ala Ser Ile Ile Phe Arg Leu Tyr Asp Asp Leu Ala Thr Ser Lys
435 440 445
Ala Glu Gln Gln Arg Gly Asp Val Pro Lys Ser Ile Gln Cys Tyr Met
450 455 460
His Glu Thr Gly Ser Ser Glu Glu Val Ala Ala Asn His Ile Arg Asp
465 470 475 480
Leu Ile Ser Asp Ala Trp Lys Glu Val Asn Ala Glu Cys Leu Lys Pro
485 490 495
Thr Ser Leu Ser Lys His Tyr Val Gly Val Ala Pro Asn Ser Ala Arg
500 505 510
Ser Gly Val Leu Met Tyr His His Asp Phe Asp Gly Phe Ala Ser Pro
515 520 525
His Gly Arg Thr Asn Ala His Ile Thr Ser Ile Phe Phe Glu Pro Val
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Pro Leu Lys Glu Ser Ile Asn Leu Gly
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Claims (15)
1.一种合成的β檀香烯合酶，其特征在于以下事实：所述合成的β檀香烯合酶中与SEQID NO：1之氨基酸位置272至位置291的区段对应的部分的三级结构相比天然存在的檀香烯合酶中相同三级结构的灵活性而言具有增加的灵活性，其中所述灵活性是通过均方根波动分析确定的，其使用这些设定：pH 8.0、300K、1atm、水环境、无底物下离子存在，对合成的和天然存在的檀香烯合酶二者模拟500ns，和依据最后450ns模拟评价每种酶结构；并且其中所述合成的β檀香烯合酶进一步的特征在于，其在适合产生β-檀香烯和α-檀香烯这两种檀香烯的常见条件下以等于或大于1的比率产生β-檀香烯和α-檀香烯的能力。
2.从法呢基焦磷酸产生β-檀香烯和α-檀香烯的合成的β檀香烯合酶，其中所述檀香烯合酶
a.与SEQ ID NO：2、3、29、57或58的氨基酸位置261至278、优选地位置261至位置272具有至少50％序列同一性，其中与SEQ ID NO：2、3、29、57或58的位置261对应的位置是精氨酸残基并且与SEQ ID NO：2、3、29、57或58的位置278对应的位置是脯氨酸残基并且所述精氨酸和所述脯氨酸用来对齐两个蛋白质序列以确定序列同一性；或
b.与SEQ ID NO：2、3或40的氨基酸位置261至302具有至少50％序列同一性，其中与SEQID NO：2或3的位置261对应的位置是精氨酸残基并且三个天冬氨酸残基存在于与SEQ IDNO：2或3或29至40的位置298、299和302处天冬氨酸对应的位置处；或
c.以上a.和b.的组合；或
d.与SEQ ID NO：1的全长具有至少50％序列同一性；
e.以上a.至c.中任一者和d.的组合；
并且其中所述合成的β檀香烯合酶在适于产生β-檀香烯和α-檀香烯的条件下以β-檀香烯与α-檀香烯等于或大于1的二者比率产生这些檀香烯。
3.根据前述权利要求中任一项所述的合成的β檀香烯合酶，其中它进一步在与SEQ IDNO 1中括号里所提供位置对应的位置处具有以下氨基酸：
精氨酸(261)、天冬氨酸(262)、精氨酸(263)、亮氨酸或异亮氨酸或缬氨酸或甲硫氨酸(264)、亮氨酸或异亮氨酸或缬氨酸(265)、谷氨酸或谷氨酰胺(266)和组氨酸或酪氨酸(268)。
4.根据前述权利要求中任一项所述的合成的β檀香烯合酶，其
a.在对应于SEQ ID NO：1的位置267具有任一以下氨基酸：
丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸；或
b.在对应于SEQ ID NO：1的位置291具有任一以下氨基酸：
苏氨酸、半胱氨酸、丝氨酸、苯丙氨酸或缬氨酸；或
c.以上a.和b.的组合；或
d.在对应于SEQ ID NO：1的位置267是天冬酰胺，和在与SEQ ID NO：1的位置291对应的位置是任一以下氨基酸：
苏氨酸、半胱氨酸、丝氨酸、苯丙氨酸或缬氨酸；或
e.在对应于SEQ ID NO：1的位置267具有任一以下氨基酸：
丝氨酸、亮氨酸、苏氨酸、半胱氨酸、异亮氨酸、缬氨酸、色氨酸、甘氨酸或丙氨酸并且在与SEQ ID NO：1的位置291对应的位置是异亮氨酸。
5.合成的核酸，其编码根据前述权利要求中任一项所述的任一合成的檀香烯合酶。
6.表达盒，其包含根据前述权利要求所述的合成的核酸。
7.一种用于产生包含β-檀香烯超过α-檀香烯的组合物的方法，包括以下步骤：
I.以活性形式提供一种或多种根据前述权利要求中任一项所述的改善的β檀香烯合酶并提供全部所需要的辅因子，
II.使法呢基焦磷酸与一种或多种改善的β檀香烯合酶在允许产生檀香烯的条件下接触，
III.从法呢基焦磷酸产生β-檀香烯和α檀香烯及任选地香柠檬烯，其中产生的β-檀香烯的量大于产生的α-檀香烯的量，
IV.任选地纯化产物。
8.非人宿主细胞，其适于从编码根据前述权利要求中任一项所述的檀香烯合酶的核酸产生所述檀香烯合酶，并且适于为所述檀香烯合酶提供法呢基焦磷酸和对其活性所需要的全部辅因子，其中所述宿主细胞包含编码根据前述权利要求中任一项所述的檀香烯合酶的核酸。
9.根据前述权利要求中任一项所述的檀香烯合酶、非人宿主细胞或方法，其中除产生比α-檀香烯更多的β-檀香烯之外，所述檀香烯合酶还产生超过α-檀香烯的反式-α-香柠檬烯。
10.通过根据前述权利要求中任一项所述的合成的檀香烯合酶、方法或非人宿主细胞产生的组合物，其中所述组合物包含超过α-檀香烯的β-檀香烯。
11.根据权利要求10所述的组合物，其中所述组合物包含比香柠檬烯更多的β-檀香烯和比α-檀香烯更多的香柠檬烯。
12.根据前述权利要求中任一项所述的组合物，其中所述组合物包含至少12％(w/w)的反式-α-香柠檬烯。
13.一种用于产生包含β-檀香醇超过α-檀香醇的组合物的方法，包括步骤：
I.通过前述权利要求中任一项所述的方法或用前述权利要求中任一项所述的宿主细胞或檀香烯合酶产生包含β-檀香烯超过α-檀香烯的组合物；
II.将a)产生的组合物中的至少一些β-檀香烯和α-檀香烯氧化成成它们相应的醇以产生包含β-檀香醇超过α-檀香醇的组合物；和
III.任选地纯化产物。
14.通过权利要求13所述的方法产生的组合物，其包含β-檀香醇超过α-檀香醇。
15.SEQ ID NO：2、3、13至53、56至58的任一合成的檀香烯合酶的用途，用于产生包含α-檀香烯、β-檀香烯和反式-α-香柠檬烯的组合物。
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| PCT/EP2021/064642 WO2021245064A1 (en) | 2020-06-04 | 2021-06-01 | Synthetic santalene synthases |
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| DE3546807C2 (zh) | 1985-03-30 | 1991-03-28 | Marc Genf/Geneve Ch Ballivet | |
| ATE177782T1 (de) | 1990-12-20 | 1999-04-15 | Ixsys Inc | Optimierung von bindenden proteinen |
| US6117679A (en) | 1994-02-17 | 2000-09-12 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
| US6395547B1 (en) | 1994-02-17 | 2002-05-28 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
| US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
| US6537776B1 (en) | 1999-06-14 | 2003-03-25 | Diversa Corporation | Synthetic ligation reassembly in directed evolution |
| US6171820B1 (en) | 1995-12-07 | 2001-01-09 | Diversa Corporation | Saturation mutagenesis in directed evolution |
| US6764835B2 (en) | 1995-12-07 | 2004-07-20 | Diversa Corporation | Saturation mutageneis in directed evolution |
| US6326204B1 (en) | 1997-01-17 | 2001-12-04 | Maxygen, Inc. | Evolution of whole cells and organisms by recursive sequence recombination |
| JP6014805B2 (ja) | 2007-07-31 | 2016-10-26 | ビーエーエスエフ エンザイムズ エルエルシー | テイラードマルチサイトコンビナトリアルアセンブリー |
| CN103923947B (zh) | 2008-03-06 | 2017-12-12 | 弗门尼舍有限公司 | 用于生产α‑檀香萜的方法 |
| ES2626456T3 (es) | 2008-12-11 | 2017-07-25 | Firmenich Sa | Procedimiento enzimático para producir beta-santaleno y la enzima correspondiente |
| WO2011000026A1 (en) | 2009-06-29 | 2011-01-06 | The University Of Western Australia | Terpene synthases from santalum |
| EP3126492A2 (en) | 2014-03-31 | 2017-02-08 | Evolva, Inc. | Modified santalene synthase polypeptides, encoding nucleic acid molecules and uses thereof |
| NL2018457B1 (en) | 2017-03-02 | 2018-09-21 | Isobionics B V | Santalene Synthase |
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| BR112022024475A2 (pt) | 2022-12-27 |
| WO2021245064A1 (en) | 2021-12-09 |
| MX2022015338A (es) | 2023-01-16 |
| US20230235310A1 (en) | 2023-07-27 |
| AU2021285040A1 (en) | 2023-01-05 |
| JP2023533154A (ja) | 2023-08-02 |
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