改良的丙氨酸2,3－氨基变位酶和相关多核苷酸的制作方法

专利名称：改良的丙氨酸2,3－氨基变位酶和相关多核苷酸的制作方法
技术领域：
本发明涉及酶学领域，且尤其涉及丙氨酸2，3-氨基变位酶(AAM)酶学领域。更特定来说，本发明涉及具有改良酶活性(意即高底物转换率)和稳定性的丙氨酸2，3-氨基变位酶多肽，和编码改良的丙氨酸2，3-氨基变位酶多肽的多核苷酸序列。本发明是适用的，这是由于丙氨酸2，3-氨基变位酶多肽可与其他酶偶合以产生高产率的合成有机化学制品，诸如泛酸或3-羟基丙酸。
背景技术：
诸如有机酸、酯和多元醇的有机化学制品可用于合成塑料材料和其他产品。为了满足对有机化学制品的增加的需要，正在开发更有效且节约成本的生产方法，其利用基于碳水化合物而非碳氢化合物的原材料。例如，已将某些细菌用于生产大量聚乳酸生产中所用的乳酸。
3-羟基丙酸(3-HP)为有机酸。已描述产生3-HP的若干化学合成途径，且也已揭露生物催化途径(Suthers等人的WO 01/16346)。3-HP具有用于特殊合成的效用且可通过化学工业中已知的方法转化成商业上重要的中间物，例如，通过脱水作用转化成丙烯酸，通过氧化作用转化成丙二酸，通过与醇的酯化反应转化成酯和通过还原作用转化成1，3-丙二醇。
化合物3-HP可通过关键的β-丙氨酸中间物，自PEP或丙酮酸盐经生物催化产生(图1)。β-丙氨酸可在细胞中由肌肽、β-丙氨酰基精氨酸、β-丙氨酰基赖氨酸、经由5，6-二氢尿嘧啶和N-氨甲酰基-β-丙氨酸的尿嘧啶、N-乙酰基-β-丙氨酸、鹅肌肽或天冬氨酸产生。然而，这些途径在商业上不能获得，因为其需要比3-HP更有价值的少见的前驱物或起始化合物。因此，如果α-丙氨酸可直接转化成β-丙氨酸(图1)，那么使用生物催化途径的3-HP的产生将更有效。不幸的是，还未鉴别出使α-丙氨酸与β-丙氨酸互相转化的天然产生的酶。如果鉴别出进行α-丙氨酸向β-丙氨酸转化的酶活性(例如，丙氨酸2，3-氨基变位酶)，那么将为有利的。因此，本发明的一目标为鉴别具有改良的丙氨酸2-3-氨基变位酶活性的酶。
催化赖氨酸与β-赖氨酸的厌氧相互转化的赖氨酸2，3-氨基变位酶(KAM)首先由Barker描述于催化赖氨酸发酵中的第一步的梭菌(Clostridium)SB4(现称为近端梭菌(C.subterminale))中。KAM已由近端梭菌纯化出，其为于大肠杆菌(E.coli)中克隆和表达的基因。参见，例如由Frey等人于2001年7月19日颁布的美国专利6,248,874，其全文以引用的方式并入本文中。来自近端梭菌SB4细胞的经纯化KAM的比活性据报导为30-40单位/mg(Lieder等人，Biochemistry 372578(1998))，其中将一单位定义为微摩尔赖氨酸/min。相应的经纯化重组产生的KAM具有相当的酶活性(34.5±1.6微摩尔赖氨酸/min/mg蛋白质)。参见由Frey等人于2003年7月19日公开的美国专利申请公开案第2003/0113882 A1号，其全文以引用的方式并入本文中。
基于来自近端梭菌的KAM序列，KAM基因已经注解于其他有机体的基因组中。然而，在多数情况下，未证实由这些基因编码的多肽的酶活性。例外的是枯草杆菌(B.subtilis)基因(Chen，D.，Ruzicka，F.J.，and Frey，P.A.(2000)Biochem.J.348539-549))，和牙龈卟啉菌(Porphyromonas gingivalis)与具核梭杆菌(F.nucleatum)基因。由yodO基因编码的枯草杆菌KAM比近端梭菌KAM更耐O2，但其活性明显较低。如Frey所报导，枯草杆菌KAM仅具有0.62U/mg的比活性。
近端梭菌SB4 KAM据报导与L-丙氨酸具有一些交叉反应性，将其转化成β-丙氨酸。参见美国专利申请公开案第2003/0113882 A1号。WO 03/062173和WO 02/42418揭露基于kam基因修饰的AAM活性的最先报导。在这些申请案中，合成的aam基因具有如由ΔpanD大肠杆菌菌株的互补所检测到的AAM活性。然而，由于丙氨酸并非此酶的天然底物，所以此转化的活性实质上比赖氨酸(其天然底物)转化活性要小。也具有AAM活性的枯草杆菌KAM变异体的AAM活性为大约0.001U/mg。本发明的一目标为提供编码具有实质上比于野生型酶中发现的AAM更强的AAM活性的多肽的多核苷酸。

发明内容
本发明具有多个方面。在一方面中，本发明涉及催化图1的反应的多肽。在此第一方面的一实施例中，本发明涉及具有优选地如由实例8的检定所测量的丙氨酸2，3-氨基变位酶(AAM)活性和具有以下条件的多肽(a)具有选自由SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51组成的群组的多肽；(b)具有氨基酸序列，其具有与选自由SEQ ID NO2、22、28、32和36组成的群组的氨基酸序列至少98％的同源性；(c)具有氨基酸序列，其具有与选自由SEQ ID NO4、6、8、12、16、24、26、30、34和40组成的群组的氨基酸序列至少99％的同源性；
(d)为由核酸序列编码的多肽，所述核酸序列在高严格条件下与任一以下各物杂交(i)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49的核苷酸序列，(ii)至少100个核苷酸的(i)的子序列，或(iii)(i)或(ii)的互补链(J.Sambrook，E.F.Fritsch，and T.Maniatis，1989，MolecularCloning，A Laboratory Manual，第2版，Cold Spring Harbor，N.Y.)；或(e)为(c)的多肽的变异体，其包含由此取代、缺失和/或插入1至6个氨基酸且具有于pH 7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD的AAM活性。
总体来说，上述(b)和(c)的多肽在本文中是指“同源多肽”。出于本发明的目的，将两个氨基酸序列之间的同源程度表达为“同源性百分比”、“一致性百分比”、“一致性％”、“相同百分比”和“相同％”，其在本发明中可互换使用，表示通过ClustalW分析(版本W 1.8，获自European Bioinformatics Institute，Cambridge，UK)获得的氨基酸序列一致性百分比，ClustalW分析计数对准的相同配对的数量且以参考序列的长度划分所述数量的相同配对，且使用以下默认的ClustalW参数来实现慢/精确成对最佳对准—空隙开放罚分10；空隙延伸罚分0.10；蛋白质重量矩阵Gonnet系列；DNA重量矩阵IUB；Toggle慢/快成对对准＝慢或全对准。
在一实施例中，本发明也涉及呈分离和纯化形式的如本文所述的AAM多肽。
在另一实施例中，本发明也涉及呈冻干形式的如本文所述的AAM多肽。
在又一实施例中，本发明涉及包含如本文所述的AAM多肽和适合载剂的组合物，所述载剂通常为缓冲溶液，更通常为具有介于6.0与8.0之间的pH值的含水缓冲溶液。所述组合物也可呈冻干形式。
本发明的新颖AAM多肽具有比野生型KAM多肽显著增强的AAM活性，所述新颖多肽最终是由野生型KAM多肽衍生而来。显著增强的AAM活性意谓本发明的AAM多肽具有在约1至约32μM所产生的β-丙氨酸/小时/1细胞OD(单位)的范围内的AAM活性，更优选为约20至约32单位；最优选为约25至约32单位。
本发明的优选的AAM多肽具有SEQ ID NOs2、6、12、16、20、24、28、30、32、34、38、44、46或48的氨基酸序列；更优选地，其具有SEQ ID NOs6、12、28、34、46或48的氨基酸序列；最优选地，其具有SEQ ID NOs28或34的氨基酸序列。
祖辈亲本分子中的一者为不具有可检测的AAM活性的枯草杆菌的KAM。如WO03/062173中所述来修饰编码此祖辈亲本分子(将其命名为“丙氨酸2，3-氨基变位酶”)的DNA以产生具有可检测的丙氨酸2，3-氨基变位酶活性的多肽。
在本申请案中，申请者利用SEQ ID NO58的多核苷酸序列作为一亲本分子，其编码SEQ ID NO59的471个残基多肽且呈现大约.001U/mg(单位/mg细胞质量)的AAM活性。SEQ ID NO59的分子与不具有可检测的AAM活性的野生型枯草杆菌KAM的不同之处在于其具有四(4)个氨基酸取代L103M、M136V、Y140H和D339H。
在又一实施例中，本发明涉及具有约1至约32单位的AAM活性且通常与SEQ ID NO59的多肽有1-7个氨基酸残基的变化，更通常有1-6个氨基酸残基的变化，甚至更通常有1-5个氨基酸残基的变化且最通常有1-4个氨基酸残基的变化的多肽。
在其第二方面中，本发明涉及编码相应的参考AAM多肽的多核苷酸序列。倘若有遗传密码的简并，本发明也涉及编码上文本发明的参考AAM多肽的任何多核苷酸。在另一优选实施例中，本发明涉及分别编码SEQ ID NOS2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51的新颖AAM多肽的SEQ ID NOS1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47和49的某些特定多核苷酸。优选的多核苷酸编码SEQID NO2、6、12、16、20、24、28、30、32、34、38、44、46或48的多肽；更优选地，其编码SEQ ID NO6、12、28、34、46或48的多肽；最优选地，其具有SEQ ID NO28或34的序列的多肽。
在第三方面中，本发明涉及核酸构筑体、载体或宿主细胞，其包含编码本发明的AAM多肽的可操作地连接至启动子的多核苷酸序列。
在第四方面中，本发明涉及制造本发明的AAM多肽的方法，其包含(a)在适于产生所述多肽的条件下培养经编码本发明的AAM多肽的核酸序列转化的宿主细胞；和(b)在适于产生β-丙氨酸的条件下向所培养的宿主细胞提供葡萄糖。β-丙氨酸可视情况由细胞回收。
在第五方面中，本发明涉及产生b-丙氨酸的方法，其包含(a)在适于产生所述多肽的条件下培养经编码本发明的AAM多肽的核酸序列转化的宿主细胞；和(b)在适于产生b-丙氨酸的条件下向所培养的宿主细胞提供葡萄糖。b-丙氨酸可视情况由细胞回收。


图1说明由丙氨酸2，3-氨基变位酶催化的α-丙氨酸(意即，L-丙氨酸或2-氨基丙酸)与β-丙氨酸(3-氨基丙酸)之间的可逆反应。
图2为经由作为中间物的β-丙氨酸自α-丙氨酸进行的3-羟基丙酸(3-HP)合成路径。
图3为本发明的4036bp表达载体(pCK110900-I Bla)，其包含P15A复制起点(P15Aori)、lacI阻遏物、CAP结合位点、lac启动子(lac)、T7核糖体结合位点(T7g10 RBS)和抗氯霉素基因(camR)。
图4A-4J组合提供4个用于产生本发明的AAM的亲本多肽的氨基酸序列的对准图。所述亲本多肽为非天然产生的且部分分别是由斯氏梭菌(Clostrisium stricklandii)(SEQID NO53)、牙龈卟啉菌(SEQ ID NO55)、具核梭杆菌(SEQ ID NO57)和枯草杆菌(SEQ ID NO59)的KAM衍生而来。两个野生型KAM的序列揭露于SEQ ID NOS60(P GI2529467_G8_AAB81159.1)和61(P_GI2634361_EMB_CAB13860.1)中。也提供作为SEQ ID NO62的一致序列。
当与附图联系起来看时，将更好地理解以上概述和以下本发明的某些实施例的详细描述。出于说明本发明的目的，于图中显示某些实施例。然而，应了解本发明不受限于附图中所示的配置和手段。
具体实施例方式
本发明具有多个方面。在一方面中，本发明涉及具有优选地如由实例8的检定所测量的丙氨酸2，3-氨基变位酶(AAM)活性和具有以下条件的多肽(a)具有选自由SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51组成的群组的多肽；(b)具有氨基酸序列，其具有与选自由SEQ ID NO2、22、28、32和36组成的群组的氨基酸序列至少98％的同源性；(c)具有氨基酸序列，其具有与选自由SEQ ID NO4、6、8、12、16、24、26、30、34和40组成的群组的氨基酸序列至少99％的同源性；(d)为由核酸序列编码的多肽，所述核酸序列在高严格条件下与任一以下各物杂交(i)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49的核苷酸序列，(ii)至少100个核苷酸的(i)的子序列，或(iii)(i)或(ii)的互补链(J.Sambrook，E.F.Fritsch，and T.Maniatis，1989，MolecularCloning，A Laboratory Manual，第2版，Cold Spring Harbor，N.Y.)；或(e)为(d)的多肽的变异体，其包含由此取代、缺失和/或插入1至6个氨基酸且具有于pH 7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD的AAM活性。
总体来说，上述(b)和(c)的多肽在本文中是指“同源多肽”。出于本发明的目的，将两个氨基酸序列之间的同源程度表达为“同源性百分比”、“一致性百分比”、“一致性％”、“相同百分比”和“相同％”，其在本发明中可互换使用，表示通过ClustalW分析(版本W 1.8，获自European Bioinformatics Institute，Cambridge，UK)获得的氨基酸序列一致性百分比，ClustalW分析计数对准的相同配对的数量且以参考序列的长度划分所述数量的相同配对，且使用以下默认的ClustalW参数来实现慢/精确成对最佳对准—空隙开放罚分10；空隙延伸罚分0.10；蛋白质重量矩阵Gonnet系列；DNA重量矩阵IUB；Toggle慢/快成对对准＝慢或全对准。
AAM多肽对氧敏感且优选地维持和使用于缺氧环境中。如果AAM多肽由于暴露于氧而变得失活，那么其可根据Chirpich，等人，Journal Biol.Chem.，245(7)1778-1789(1970)中所述的方法，通过在37℃下与硫氢基化合物一起厌氧培养而活化，所述文献全文以引用的方式并入本文中。本发明的AAM多肽优选地以全细胞形式(意即呈经AAM多核苷酸转化的全细胞，AAM多核苷酸在一定条件下使用以便经编码的AAM多肽表达于细胞中)使用或者在缺氧条件下分离和利用。根据Petrovich，等人，Journal Biol.Chem.，266(12)7656-7660(1991)中所述的方法，本发明的AAM多肽可在厌氧条件下(例如在氮气氛下)经分离，且视情况经纯化，所述文献描述赖氨酸-2，3-氨基变位酶的分离和纯化且其全文以引用的方式并入本文中。如本文中所用，术语“缺氧”是指缺乏氧。呈全细胞形式或呈经分离酶的AAM多肽可经冻干。在又一实施例中，本发明涉及包含如本文所述的AAM多肽(例如，呈全细胞形式或呈经分离的多肽)和适合载剂的组合物，所述载剂通常为缓冲液，更通常为具有介于6.0与8.0之间的pH值的含水缓冲溶液。经冻干以提供冻干形式组合物的含水缓冲组合物也在本发明的范围内，其中所述组合物通过添加基于水的组合物来复原。
在一实施例中，本发明也涉及呈分离和纯化形式的如本文所述的AAM多肽。
在另一实施例中，本发明涉及呈冻干形式的如本文所述的AAM多肽。冻干法是使用标准冻干设备来执行。通常，将含有多肽的溶液分散于适当大小的瓶中，冷冻且置于减压下以引起水蒸发，留下冻干(冷冻干燥)的多肽。使用前，将冻干的多肽用蒸馏水或适当缓冲溶液复原。
在又一实施例中，本发明涉及含有如本文所述的AAM多肽和适合载剂的组合物，所述载剂通常为缓冲溶液，更通常为具有介于6.0与8.0之间的pH值的含水缓冲溶液。所述组合物也可呈冻干形式。
本发明的新颖AAM多肽具有相对于野生型KAM多肽的显著增强的AAM活性，所述新颖多肽最终是由野生型KAM多肽衍生而来。显著增强的AAM活性意谓本发明的AAM多肽具有在约1至约32μM所产生的β-丙氨酸/小时/1细胞OD(单位)的范围内的AAM活性，更优选为约20至约32单位；最优选为约25至约32单位。
表1提供显示本发明的各种AAM多肽的AAM活性的图表，本发明的AAM多肽是由其克隆数量和SEQ ID NO来鉴别。在表1中，OD600nm是在培养5小时(t＝5)之后于收集时报告。表1也报告5小时之后每1细胞OD所产生的β-丙氨酸的总μM数。最后，表1的最后一列报告所产生的β-丙氨酸(μM)/小时/1细胞OD的速率。
表1

本发明的优选的AAM多肽具有SEQ ID NOs2、6、12、16、20、24、28、30、32、34、38、44、46或48的氨基酸序列；更优选地，其具有SEQ ID NOs6、12、28、34、46或48的氨基酸序列；最优选地，其具有SEQ ID NOs28或34的氨基酸序列。
基本的祖辈亲本分子为不具有可检测的AAM活性的枯草杆菌的KAM。如WO03/062173中所述来修饰编码此祖辈亲本分子(将其命名为“丙氨酸2，3-氨基变位酶”)的DNA以产生具有可检测的丙氨酸2，3-氨基变位酶活性的多肽。
在本申请案中，申请者利用SEQ ID NO58的多核苷酸作为一亲本分子，其编码SEQID NO59的471个残基多肽且其呈现大约.001U/mg(单位/mg细胞质量)的AAM活性。SEQ ID NO59的分子与不具有可检测的AAM活性的野生型枯草杆菌KAM(SEQ ID NO60)的不同之处在于其具有四(4)个氨基酸取代L103M、M136V、Y140H和D339H。
在本发明中用作起始材料的其他祖辈亲本分子为编码KAM多肽的来自其他微生物(例如，牙龈卟啉菌、具核梭杆菌和斯氏梭菌(Clostridium sticklandii))的DNA序列。这些DNA序列是使用标准技术来修饰以引入最终产生KAM多肽的点取代，所述KAM多肽也具有与α-丙氨酸的可检测的交叉反应性。衍生自牙龈卟啉菌的一所述亲本分子为SEQ ID NO54的多核苷酸，其编码SEQ ID NO55的416个残基多肽。SEQ ID NO55的亲本多肽与野生型牙龈卟啉菌KAM的不同之处在于其具有以下七(7)个氨基酸取代N19Y、E30K、L53P、H85Q、I192V、D331G和M342T。衍生自具核梭杆菌的另一所述亲本分子为编码SEQ ID NO57的425个残基多肽的SEQ ID NO56多核苷酸。
另一亲本多核苷酸是通过对编码KAM的斯氏梭菌的多核苷酸进行修饰衍生而来。具有与α-丙氨酸的可检测的交叉反应性的所得亲本多核苷酸为编码SEQ ID NO53的416个残基多肽的SEQ ID NO52的多核苷酸。
在图4的对准图表中对比上述SEQ ID NOs53、55、57和58的亲本多肽。由对准图表可见，来自牙龈卟啉菌、斯氏梭菌和具核梭杆菌的KAM相对于来自枯草杆菌的KAM在N-末端处和在C-末端处截短，而在这四个物种之间，在KAM多肽的中心部分有约40％残基位置是保守的。基于图4的对准图表中经截短的物种，可推断出在SEQ ID NO58N末端处的前8个氨基酸残基和在SEQ ID NO58 C末端处的后40个残基不是KAM活性或由KAM活性所衍生出的AAM活性所必需的。在图4中，也提供一致序列。
具有增强的AAM活性的本发明的AAM多肽分子是通过将定向进化技术直接应用于上述亲本分子而制得。这些技术将在本文中进一步详细描述。
在又一方面中，本发明涉及在偶合反应中具有增强的活性的AAM多肽。
在另一实施例中，本发明涉及由核酸序列编码的AAM多肽，所述核酸序列在高严格条件下与下列各物杂交(i)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49的核苷酸序列；(ii)至少100个核苷酸的(i)的子序列；或(iii)(i)或(ii)的互补链(J.Sambrook，E.F.Fritsch，和T.Maniatis，1989，Molecular Cloning，A Laboratory Manual，第2版，Cold Spring Harbor，N.Y.)。对于长度至少100个核苷酸的多核苷酸，根据标准南方印迹程序(Southern blottingprocedure)，将低至极高严格条件定义为在42℃下于5×SSPE、0.3％SDS、200μg/ml经剪切和变性的鲑鱼精子DNA中预杂交和杂交，且对于低严格性为25％甲酰胺，对于中等和中高严格性为35％甲酰胺，或对于高和极高严格性为50％甲酰胺。
对于长度至少100个核苷酸的多核苷酸，最后使用2×SSC，0.2％SDS在至少50℃下(低严格性)，至少55℃下(中等严格性)，至少60℃下(中高严格性)，至少65℃下(高严格性)和至少70℃下(极高严格性)将载剂材料洗涤三次，每次15分钟。
在另一实施例中，本发明涉及(d)的多肽的变异体，其包含由此取代、缺失和/或插入1至6个氨基酸且具有诸如由实例8的方法所测定的于pH 7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD的AAM活性。优选地，氨基酸变化是微小的，即不显著影响蛋白质的折叠和/或活性的保守氨基酸取代；小缺失，通常1至6个氨基酸的缺失；小的氨基或羧基末端延伸；小连接肽；或小延伸，其通过改变净电荷或另一功能诸如聚-组氨酸径(poly-histidine tract)、抗原决定部位或结合域来促进纯化。
保守取代的实例是在基本氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷酰胺和天冬酰胺)、疏水性氨基酸(亮氨酸、异亮氨酸和缬氨酸)、芳族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸、丝氨酸、苏氨酸、脯氨酸、半胱氨酸和蛋氨酸)的群组内。一般不改变比活性的氨基酸取代为此项技术已知的且描述于例如H.Neurath and R.L.Hill，1979，In，The Proteins，Academic Press，New York中。最常发生的交换为Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu和Asp/Gly以及反之亦然。
在另一实施例中，本发明涉及如上文实施方式第一段中所述的(a)、(b)或(c)的片段，其具有诸如由实例8的方法所测定的于pH 7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD。术语“片段”意谓在N末端有1至8个氨基酸残基缺失或在C末端有1至40个残基缺失或两者都有的多肽。优选地，在C末端缺失1至20个残基，更优选地，在C末端缺失1至10个残基。
多核苷酸在其第二方面中，本发明涉及编码本发明的AAM多肽的多核苷酸序列。倘若有遗传密码的简并，本发明也涉及编码上文本发明的参考AAM多肽的任何多核苷酸。在其第二方面中，本发明涉及编码相应的参考AAM多肽的多核苷酸序列。倘若有遗传密码的简并，本发明也涉及编码上文本发明的参考AAM多肽的任何多核苷酸。在一优选实施例中，本发明涉及分别编码SEQ ID NOS2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51的新颖AAM多肽的SEQID NOS1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47和49的某些特定多核苷酸。优选的多核苷酸编码SEQ ID NO2、6、12、16、20、24、28、30、32、34、38、44、46或48的多肽；更优选地，其编码SEQ IDNO6、12、28、34、46或48的多肽；最优选地，其具有SEQ ID NO28或34的序列的多肽。
为了制造本发明的改良的AAM多肽，我们由一种或一种以上编码KAM多肽的野生型多核苷酸开始。术语“野生型”多核苷酸意谓核酸片段不包含来自由天然分离的形式的任何突变。术语“野生型”蛋白质意谓蛋白质将具有天然所发现的活性水平下的活性且通常将包含如天然所发现的氨基酸序列。因此，术语“野生型”或“祖辈亲本序列”表示在本发明的操控之前的起始或参考序列。
待改良的作为起始材料的野生型KAM的适合来源易于通过对于KAM活性筛检基因组文库而鉴别出。KAM的尤其适合来源为天然所发现的芽孢杆菌属(Bacillus sp.)菌的yodO基因。使用枯草杆菌的公开KAM基因序列(例如，WO 03 0623173 A2)，使用常规技术来制造用于扩增来自其各自基因文库的基因的引物。用于分离枯草杆菌的KAM的一种如此技术揭露于Chen等人，″A novel lysine 2，3-aminomutase encoded by the yodOgene of Bacillus subtilischaracterization on observation of organic radical intermediates，″Biochem J.348539-549(2000)中，所述文献以引用的方式并入本文中。
使用WO 03 0623173 A2中揭露的技术获得SEQ ID NOs52、54、56和58的起始多核苷酸，为了揭露本文实例中所列举的那些技术，所述文献以引用的方式并入本文中。特定来说，WO 03 0623173 A2揭露枯草杆菌野生型赖氨酸2，3-氨基变位酶(KAM)和其突变形式，其编码丙氨酸2，3-氨基变位酶(AAM)。此外，WO 03 0623173 A2也揭露牙龈卟啉菌野生型赖氨酸2，3-氨基变位酶(KAM)和其突变形式，其编码丙氨酸2，3-氨基变位酶(AAM)。
由SEQ ID NO58的多核苷酸起始，使用熟知的突变发生或直接进化方法中的任何一种来产生具有未知AAM活性的非天然产生和突变和/或进化的酶。参见，例如Ling，等人，″Approaches to DNA mutagenesisan overview，″Anal.Biochem.，254(2)157-78(1997)；Dale，等人，″Oligonucleotide-directed random mutagenesis using thephosphorothioate method，″Methods Mol.Biol.，57369-74(1996)；Smith，″In vitromutagenesis，″Ann.Rev.Genet.，19423-462(1985)；Botstein，等人，″Strategies andapplications of in vitro mutagenesis，″Science，2291193-1201(1985)；Carter，″Site-directedmutagenesis，″Biochem.J.，2371-7(1986)；Kramer，等人，″Point Mismatch Repair，″Cell，38879-887(1984)；Wells，等人，″Cassette mutagenesisan efficient method for generation ofmultiple mutations at defined sites，″Gene，34315-323(1985)；Minshull，等人，″Proteinevolution by molecular breeding，″Current Opinion in Chemical Biology，3284-290(1999)；Christians，等人，″Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling，″Nature Biotechnology，17259-264(1999)；Crameri，等人，″DNAshuffling of a family of genes from diverse species accelerates directed evolution，″Nature，391288-291；Crameri，等人，″Molecular evolution of an arsenate detoxification pathway byDNA shuffling，″Nature Biotechnology，15436-438(1997)；Zhang，等人，″Directed evolutionof an effective fucosidase from a galactosidase by DNA shuffling and screening，″Proceedingsof the National Academy of Sciences.U.S.A.，9445-4-4509；Crameri，等人，″Improved greenfluorescent protein by molecular evolution using DNA shuffling，″Nature Biotechnology＜14315-319(1996)；Stemmer，″Rapid evolution of a protein in vitro by DNA shuffling，″Nature，370389-391(1994)；Stemmer，″DNA shuffling by random fragmentation andreassemblyIn vitro recombination for molecular evolution，″Proceedings of the NationalAcademy of Sciences，U.S.A.，9110747-10751(1994)；WO 95/22625；WO 97/0078；WO97/35966；WO 98/27230；WO 00/42651；WO 01/75767和于2003年3月25日颁布给Arnold等人且标题为“Method for creating polynucleotide and polypeptide sequences.”的美国专利6,537,746。
可应用这些方法的任一者产生AAM多核苷酸。为了最大化任何多样性，可相继使用上述技术的若干种。通常，通过一种诱变或进化技术来制造改组多核苷酸(shuffledpolynucleotide)的文库且筛检其表达产物以找出具有最大AAM活性的多肽。接着，应用第二种诱变或进化技术来编码最大活性多肽的多核苷酸以制造第二文库，随后对于AAM活性通过相同技术来筛检文库。按需要，包括点突变插入的突变和筛检过程可重复多次以得到编码具有所要活性、热稳定性或辅因子优先的多肽的多核苷酸。
或者，可根据已知合成方法，通过标准固相方法来制备本发明的多核苷酸和寡核苷酸。通常，将高达约100个碱基的片段个别合成，随后接合(例如，通过酶促或化学连接方法，或聚合酶介导的方法)以基本上形成任何所要的连续序列。例如，可使用例如由Beaucage等人(1981)Tetrahedron Letters 221859-69所述的经典亚磷酰胺方法或例如由Matthes等人(1984)EMBO J.3801-05所述的方法(其通常在自动合成方法中实行)，通过化学合成来制备本发明的多核苷酸和寡核苷酸。根据亚磷酰胺方法，例如在自动DNA合成器中合成寡核苷酸，将其纯化、退火、连接且于适当载体中克隆。
此外，基本上任何核酸可由诸如The Midland Certified Reagent Company，Midland，TX、The Great American Gene Company，Ramona，CA、ExpressGen Inc.，Chicago，IL、OperonTechnologies Inc.，Alameda，CA(上述所有的都具有互联网网址)和许多其他公司的多种商业来源的任一者定制。类似地，肽和抗体可由多种来源的任一者定制，所述来源为诸如PeptidoGenic、HTI Bio-products，Inc.、BMA Biomedicals Ltd.(U.K.)、Bio.Synthesis，Inc.和许多其他公司。
也可通过如专业文献中所述的熟知技术来合成多核苷酸。参见，例如Carruthers等人，Cold Spring Harbor Symp.Quant.Biol.47411-418(1982)和Adams等人，J.Am.Chem.Soc.105661(1983)。随后可通过合成互补链且在适当条件下将链在一起退火，或通过使用DNA聚合酶用适当引物序列来添加互补链而获得双链DNA片段。
描述适用于本文的包括突变发生的分子生物技术的一般文章包括Berger和Kimmel，Guide to Molecalar Cloning Techniques，Methods in Enzymology，第152卷Academic Press，Inc.，San Diego，CA(″Berger″)；Sambrook等人，Molecular Cloning-A Laboratory Manual(第二版)，第1-3卷，Cold Spring Harbor Laboratory，Cold Spring Harbor，New York，1989(″Sambrook″)；和Current Protocols in Molecular Biology，F.M.Ausubel等人，编，CurrentProtocols，a joint venture between Greene Publishing Associates，Inc.and John Wiley&Sons，Inc.(在2000年中增刊)(″Ausubel″)。包括聚合酶链反应(PCR)、连接酶链反应(LCR)、Qβ-复制酶扩增和其他RNA聚合酶介导的技术(例如NASBA)的足以通过活体外扩增方法来指导所属领域技术人员的技术的实例是在以下文献中找到Berger，Sambrook，和Ausubel，以及Mullis等人，(1987)美国专利第4,683,202号；PCR Protocols A Guided toMethods and Applications(Innis等人编)Academic Press Inc.San Diego，CA(1990)；Arnheim和Levinson(1990年10月1日)Chemical and Engineering News36-47；The JournalOf NIH Research(1991)381-94；Kwoh等人(1989)Proc.Natl.Acad.Sci.USA861173；Guatelli等人(1990)Proc.Natl.Acad.Sci.USA871874；Lomell等人(1989)LClin.Chem.351826；Landegren等人，(1988)Science2411077-1080；Van Brunt(1990)Biotechnology8291-294；Wu和Wallace，(1989)Gene4560；Barringer等人(1990)Gene89117，以及Sooknanan和Malek(1995)Biotechnology13563-564。活体外克隆扩增的核酸的改良方法描述于Wallace等人的美国专利第5,426,039号中。通过PCR扩增大核酸的改良方法概述于Cheng等人(1994)Nature369684-685和本文的参考文献中，其中产生高达40kb的PCR扩增子。所属领域技术人员将会了解可使用逆转录酶和聚合酶基本上将RNA转化成适用于限制性消化、PCR延伸和定序的双链DNA。参见上述Ausubel、Sambrook和Berger。
所属领域技术人员将会了解由于遗传密码的简并，可制造许多编码本发明的AAM多肽的核苷酸序列，其中的一些与本文中所明确揭露的核酸序列具有大体上的一致性。编码本发明的AAM多肽的多核苷酸可为经最优化以用于自为表达所选出的宿主有机体的最佳产生的密码子，上述实情也在本发明的范围之内。所属领域技术人员将认识到易于获得提供各种有机体的密码子优先信息的表格和其他参考文献。参加，例如Henaut和Danchin，″Escherichia coli and Salmonella，″Neidhardt，等人编，ASM Press，WashingtonD.C.，第2047-2066页(1996)。
应注意在大肠杆菌中的表达与在其他有机体中的不同。例如，在本发明中，密码子(tgg)编码SEQ ID NO59的亲本多肽中的残基位置31的Trp(W)。然而，残基位置31的相应密码子在分别编码SEQ ID NOs2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46和48的AAM多肽的SEQ ID NOs1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45和47的子代多核苷酸中的每一者中为“tga”。所属领域技术人员认识到密码子“tga”通常为终止(无义)密码子。然而，在ΔpanD大肠杆菌菌株中所用的本发明的表达系统中，且在所施加的选择条件下，此密码子是通过大肠杆菌作为有义密码子连读且可能表达为Trp(W)。其他人报导“tga”为大肠杆菌最弱的终止密码子且常常作为Trp(W)的有义密码子以高表达连读。参见，例如Parker，J.，″Errors and Alternatives in Reading theuniversal Genetic Code，″Microbiological Reviews，53(3)273-298(1989)；Roth，J.，″UGANonsense Mutations in Salmonella typhimurium，″J.of Bacteriology，102(2)467-475(1970)；和McBeath，G.和Kast，P.，″UGA Read-Through Artifacts-When Popular Gene ExpressionSystems Need a Patch，″BioTechniques，24789-794(May 1998)，该等文献以引用的方式并入本文中。因此，对于非大肠杆菌系统中的表达，将残基位置31处的密码子(tga)改变为“tgg”将为有利的，“tgg”为Trp(W)的通用有义密码子。
在SEQ ID NO49中，编码残基72的密码子为“tag”，其是作为终止密码子读出。然而，产生两个片段。具有SEQ ID NO50的残基1-71的第一片段不具有任何可检测的AAM活性。第二片段是由残基73(Val)起始而非通常的Met起始来产生。此第二片段具有399个残基(SEQ ID NO51)且基于实例8的检定，其确实具有显著的AAM活性(参见表2)。因此，在AAM多肽N末端处的最前72个残基(基于来自枯草杆菌的一致序列或亲本KAM序列)并非AAM活性所绝对必需的。
在本发明的情况下，通过对SEQ ID NOs52、54、56和58的多核苷酸应用多种突变发生技术来制造若干圆形No.1文库。
在其第三方面中，本发明涉及表达载体和宿主细胞，其包含可操作地连接至控制序列的本发明的多核苷酸。为了获得编码AAM多肽的变异基因的表达，首先将变异基因可操作地连接至一个或一个以上控制基因表达的异源调节序列以产生核酸构筑体，诸如表达载体或表达盒。其后，将诸如表达载体或表达盒的所得核酸构筑体插入适当的宿主细胞中用于最终表达由改组基因编码的AAM多肽。在本文中将“核酸构筑体”定义为单链或双链核酸分子，其是由天然产生的基因分离出或其已经修饰以含有以非天然存在的方式组合和并置的核酸区段。因此，在一方面中，本发明涉及包含编码本发明的AAM多肽的多核苷酸的核酸构筑体。
当核酸构筑体含有表达本发明的编码序列所需的所有控制序列时，术语“核酸构筑体”与术语“表达盒”同义。在本文中将术语“编码序列”定义为直接指定其蛋白质产物的氨基酸序列的核酸序列。编码序列可包括(但不限于)DNA、cDNA和重组核酸序列。
编码本发明的AAM多肽的经分离的多核苷酸可以多种方式来操控以供多肽表达之用。在将经分离的多核苷酸插入载体之前对其的操控可视表达载体而为需要或必需的。利用重组DNA方法来修饰多核苷酸和核酸序列的技术为此项技术中熟知的。
在本文中将术语“控制序列”定义为包括所有组件，其为本发明多肽的表达所必需或有利的。每个控制序列可为天然的或对于编码多肽的核酸序列为外来的。所述控制序列包括(但不限于)前导子、多聚腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。控制序列至少包括启动子和转录与翻译终止信号。出于引入特定限制位点以促进控制序列与编码多肽的核酸序列的编码区的连接的目的，可向控制序列提供连接子。
在本文中将术语“可操作地连接”定义为其中控制序列适当地位于相对于DNA序列的编码序列的位置以便控制序列指导多肽表达的配置。
控制序列可为适当的启动子序列。“启动子序列”为相对短的核酸序列，其由宿主细胞识别而用于随后更长编码区的表达。启动子序列含有转录控制序列，其介导多肽的表达。启动子可为任何核酸序列，其在选出的宿主细胞中显示转录活性，包括突变、截短和杂交启动子，且其可由编码与宿主细胞同源或异源的细胞外或细胞内多肽的基因而获得。
对于细菌宿主细胞，用于指导本发明的核酸构筑体转录的适合启动子包括由大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因(dagA)、枯草杆菌6-果聚糖蔗糖酶基因(sacB)、地衣形芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(arnyL)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽基因淀粉酶基因(amyM)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、地衣形芽孢杆菌青霉素酶基因(penP)、枯草杆菌xylA与xylB基因和原核β-内酰胺酶基因(Villa-Kamaroff等人，1978，Proceedings of the National Academy of Sciences USA 753727-3731)获得的启动子，以及tac启动子(DeBoer等人，1983，Proceedings of the National Academy of SciencesUSA 8021-25)。其他启动子描述于“Useful proteins from recombinant bacteria”in ScientificAmerican，1980，24274-94；和上文Sambrook等人，1989中。
对于丝状真菌宿主细胞，用于指导本发明的核酸构筑体转录的适合启动子包括由稻属曲霉(Aspergillus oryzae)TAKA淀粉酶、米黑根霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉菌(Aspergillus niger)中性α-淀粉酶、黑曲霉菌酸稳定的α-淀粉酶、黑曲霉菌或泡盛曲霉菌(Aspergillus awamori)葡糖淀粉酶(glaA)、米黑根霉脂肪酶、稻属曲霉碱性蛋白酶、稻属曲霉磷酸丙糖异构酶、沟巢曲霉(Aspergillus nidulans)乙酰胺酶和尖镰孢(Fusarium oxysporum)胰酶样蛋白酶(WO 96/00787)的基因获得的启动子，以及NA2-tpi启动子(一种来自黑曲霉菌中性α-淀粉酶和稻属曲霉磷酸丙糖异构酶的基因的杂交启动子)，和其突变、截短和杂交启动子。
在酵母宿主中，适用的启动子是由酿酒酵母(Saccharomyces cerevisiae)烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)和酿酒酵母3-磷酸甘油酸激酶的基因获得。酵母宿主细胞的其他适用启动子是由Romanos等人，1992，Yeast 8423-488描述。
控制序列也可为适合的转录终止序列，一种由宿主细胞所识别以终止转录的序列。终止序列可操作地连接至编码多肽的核酸序列的3′末端。在选出的宿主细胞中有功能的任何终止子可用于本发明中。
用于丝状真菌宿主细胞的优选终止子是由稻属曲霉TAKA淀粉酶、黑曲霉菌葡糖淀粉酶、沟巢曲霉氨基苯甲酸合成酶、黑曲霉菌α-葡糖苷酶和尖镰孢胰酶样蛋白酶获得。
用于酵母宿主细胞的优选终止子是由酿酒酵母烯醇酶、酿酒酵母细胞色素C(CTYC1)和酿酒酵母甘油醛-3-磷酸脱氢酶的基因获得。用于酵母宿主细胞的其他适用终止子是由上文Romanos等人，1992描述。
控制序列也可为适合的前导子序列，在由宿主细胞进行的翻译中重要的mRNA的非翻译区。前导序列可操作地连接至编码多肽的核酸序列的5′末端。在选出的宿主细胞中有功能的任何前导序列可用于本发明中。用于丝状真菌宿主细胞的优选前导子是由稻属曲霉TAKA淀粉酶和稻属曲霉磷酸丙糖异构酶的基因获得。用于酵母宿主细胞的优选前导子是由酿酒酵母烯醇酶(ENO-1)、酿酒酵母3-磷酸甘油酸激酶、酿酒酵母α-因子和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)的基因获得。
控制序列也可为多聚腺苷酸化序列，一种可操作地连接至核酸序列的3′末端的序列且当其转录时，是由宿主细胞作为信号来识别以将聚腺苷残基添加至经转录的mRNA中。在选出的宿主细胞中有功能的任何多聚腺苷酸化序列可用于本发明中。用于丝状真菌宿主细胞的优选多聚腺苷酸化序列是由稻属曲霉TAKA淀粉酶、黑曲霉菌葡糖淀粉酶、沟巢曲霉氨基苯甲酸合成酶、尖镰孢胰酶样蛋白酶和黑曲霉菌α-葡糖苷酶的基因获得。对于酵母宿主细胞适用的多聚腺苷酸化序列是由Guo和Sherman，1995，Molecular CellularBiology 155983-5990描述。
控制序列也可为信号肽编码区，其编码连接至多肽的氨基末端的氨基酸序列且指导经编码的多肽进入细胞分泌途径。核酸序列的编码序列的5′末端可固有地含有于翻译阅读框中与编码分泌多肽的编码区的区段天然地连接的信号肽编码区。或者，编码序列的5′末端可含有对于编码序列为外来的信号肽编码区。当编码序列天然地不含信号肽编码区时，可需要外来信号肽编码区。
或者，外来信号肽编码区可仅仅替代天然信号肽编码区以增强多肽的分泌。然而，指导表达多肽进入选出的宿主细胞分泌途径的任何信号肽编码区可用于本发明中。
用于细菌宿主细胞的有效信号肽编码区为由芽孢杆菌NCIB 11837麦芽基因淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣形芽孢杆菌枯草杆菌蛋白酶、地衣形芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草杆菌prsA的基因获得的信号肽编码区。其他信号肽是由Simonen和Palva，1993，Microbiological Reviews 57109-137描述。
用于丝状真菌宿主细胞的有效信号肽编码区为由稻属曲霉TAKA淀粉酶、黑曲霉菌中性α-淀粉酶、黑曲霉菌葡糖淀粉酶、米黑根霉天冬氨酸蛋白酶、特异腐质霉(Humicolainsolens)纤维素酶和腐质霉(Humicola lanuginosa)脂肪酶的基因获得的信号肽编码区。
用于酵母宿主细胞的适用信号肽是由酿酒酵母α-因子和酿酒酵母转化酶的基因获得。其他适用的信号肽编码区是由上文Romanos等人，1992描述。
控制序列也可为编码位于多肽氨基末端的氨基酸序列的前肽编码区。所得多肽已知为酶原(proenzyme)或前多肽(或在某些情况下为酶原(zymogen))。前多肽通常不具有活性且可通过催化或自身催化前肽由前多肽的裂解而转化为成熟活性多肽。前肽编码区可由枯草杆菌碱性蛋白酶(aprE)、枯草杆菌中性蛋白酶(nprT)、酿酒酵母α-因子、米黑根霉天冬氨酸蛋白酶和蚀丝霉属嗜热甲烷八叠球菌(Myceliophthora thermophila)乳糖酶(WO 95/33836)的基因获得。
当信号肽区和前肽区两者都存在于多肽的氨基末端处时，前肽区紧接于多肽的氨基末端且信号肽区紧接于前肽区的氨基末端。
也可能需要添加调节序列，其允许多肽表达相对于宿主细胞生长的调节。调节系统的实例为那些响应化学或物理刺激而引起基因表达的开启或关闭的调节系统，包括调节化合物的存在。在原核宿主细胞中，适合的调节序列包括lac、tac和trp操纵子系统。在酵母宿主细胞中，适合的调节系统包括ADH2系统或GAL1系统。在丝状真菌中，适合的调节序列包括TAKA α-淀粉酶启动子、黑曲霉菌葡糖淀粉酶启动子和稻属曲霉葡糖淀粉酶启动子。
调节序列的其他实例为那些允许基因扩增的调节序列。在原核系统中，这些调节序列包括二氢叶酸还原酶基因，其在甲氨蝶呤存在下扩增；和金属硫因基因，其是用重金属来扩增。在这些情况下，编码本发明的AAM多肽的核酸序列将与调节序列可操作地连接。
表达载体在另一方面中，本发明也涉及包含本发明的多核苷酸(其编码本发明的AAM多肽)和一种或一种以上表达调节区的重组表达载体。表达调节区包括启动子、终止子、复制起点等，这将取决于引入其的宿主的类型。可将上述各种核酸序列与对照序列接合在一起以产生重组表达载体，其可包括一个或一个以上便利的限制位点以允许编码多肽的核酸序列在所述位点处的插入或取代。或者，本发明的核酸序列可通过将核酸序列或包含所述序列的核酸构筑体插入用于表达的适当载体中来表达。在制造表达载体过程中，编码序列位于载体中以便编码序列与用于表达的适当控制序列可操作地连接。
重组表达载体可为任何载体(例如，质粒或病毒)，其可便利地经受重组DNA程序且可引起多核苷酸序列的表达。载体的选择通常将取决于载体与引入载体的宿主细胞的相容性。载体可为线性或闭合环形质粒。
表达载体可为自主复制载体，意即作为染色体外实体存在的载体，其复制与染色体复制无关，例如质粒(一种染色体外元件)、微小染色体或人造染色体。载体可含有用于确保自身复制的任何构件。或者，载体可为如此的载体当将其引入宿主细胞中时，其将整合至基因组中且与其所整合至的染色体一起复制。此外，可使用单一载体或质粒或两种或两种以上载体或质粒，其共同含有待引入宿主细胞基因组中的总DNA；或转位子。
本发明的表达载体优选含有一种或一种以上可选择标记物，其允许经转化细胞的简便选择。可选择标记物为一种基因，其产物提供抗杀虫剂性或抗病毒性、对重金属的抗性、对营养缺陷型的原营养和其类似性质。细菌的可选择标记物的实例为来自枯草杆菌或地衣形芽孢杆菌的dal基因，或给予诸如氨苄青霉素(ampicillin)、卡那徽素(kanamycin)、氯霉素(chloramphenicol)(实例1)或四环素(tetracycline)抗性的抗生素抗性的标记物。用于酵母宿主细胞的适合标记物为ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。
用于丝状真菌宿主细胞中的可选择标记物包括(但不限于)amdS(乙酰胺酶)、argB(鸟氨酸氨基甲酰转移酶)、bar(草酊磷(phosphinothricin)乙酰基转移酶)、hph(潮霉素(hygromycin)磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清苷-5′-磷酸脱羧酶)、(硫酸腺苷转移酶)和trpC(氨基苯甲酸合成酶)，以及其等效物。用于曲霉菌细胞中的优选可选择标记物为沟巢曲霉或稻属曲霉的amdS和pyrG基因和吸水链霉菌(Streptomyceshygroscopicus)的bar基因。
本发明的载体优选含有允许载体整合至宿主细胞基因组中且载体在细胞中不依赖于基因组进行自主复制的元件。对于整合至宿主细胞基因组中，载体可依靠编码多肽的核酸序列或用于通过同源或非同源重组将载体整合至基因组中的载体的任何其他元件。
或者，载体可含有用于指导通过同源重组而整合至宿主细胞基因组中的其他核酸序列。其他核酸序列使得载体能够在染色体中于精确位置处整合至宿主细胞基因组中。为了增加于精确位置处整合的可能性，整合元件应优选含有足够数量的核酸，诸如100至10,000个碱基对，优选地400至10,000个碱基对且最优选地800至10,000个碱基对，所述核酸与对应的靶序列高度同源以增加同源重组的可能性。整合元件可为与宿主细胞基因组中的靶序列同源的任何序列。此外，整合元件可为非编码或编码核酸序列。另一方面来说，可通过非同源重组将载体整合至宿主细胞的基因组中。
对于自主复制，载体可进一步包含使得载体能够在相关宿主细胞中自主复制的复制起点。细菌的复制起点的实例为P15A、pSC101、pMB1和ColE1。质粒pBR322(其具有pMB1复制起点)、pUC19(其具有ColE1复制起点)、pACYC177和pACYC184(其具有P15A复制起点)的复制起点允许在大肠杆菌中复制；质粒pUB110、pE194、pTA1060或pAM.β.1的复制起点允许在芽孢杆菌中复制。用于酵母宿主细胞中的复制起点的实例为2微米复制起点，ARS1、ARS4，ARS1与CEN3的组合，和ARS4与CEN6的组合。复制起点可为具有突变的复制起点，这使得其在宿主细胞中为温度敏感的(参见，例如Ehrlich，1978，Proceedings of the National Academy of Sciences USA 751433)。
可将本发明的核酸序列的一个以上拷贝插入宿主细胞中以增加基因产物的产生。核酸序列拷贝数量的增加可通过将序列的至少一个另外拷贝整合至宿主细胞基因组中或通过使核酸序列包括可扩增的可选择标记物基因来获得，其中含有可选择标记物基因的经扩增拷贝的细胞可通过在适当的可选择试剂存在下培育细胞而经选择，且从而使得核酸序列的另外拷贝经选择。
用于连接上述元件以构建重组核酸构筑体和本发明的表达载体的程序为所属领域技术人员熟知的(参见，例如J.Sambroolc，E.F.Fritsch，and T.Maniatis，1989，MolecularCloning，A Laboratory Manual，第二版，Cold Spring Harbor，N.Y.)。
本发明中使用的许多表达载体可购得。适合的商业表达载体包括来自Sigma-AldrichChemicals，St.Louis MO.的p3xFLAGTMTM表达载体，其包括用于在哺乳动物宿主细胞中表达的CMV启动子与hGH多聚腺苷酸化位点和用于在大肠杆菌中扩增的pBR322复制起点与氨苄青霉素抗性标记物。其他适合的表达载体为由Stratagene，LaJolla CA购得的pBluescriptII SK(-)与pBK-CMV和由pBR322(Gibco BRL)、pUC(Gibco BRL)、pREP4、pCEP4(Invitrogene)或pPoly(Lathe等人，1987，Gene 57，193-201)衍生而来的质粒。
如图3的载体图中所示，本文中的实例6揭露表达载体pCK110900-I Bla的用途。
宿主细胞用于表达本发明的表达载体的宿主细胞包括(但不限于)细菌细胞，例如大肠杆菌、链霉菌和鼠伤寒沙门氏菌(Salmonella typhimurium)细胞；真菌细胞，例如酵母细胞(例如，酿酒酵母或甲醇酵母(Pichia pastoris)(ATCC寄存编号201178))；昆虫细胞，例如果蝇S2和夜蛾Sf9细胞；动物细胞，例如CHO、COS、293和黑色素瘤细胞；和植物细胞。用于上述宿主细胞的适当培养基和条件为此项技术熟知的。
举例来说，通过表达载体来转化大肠杆菌W3110以表达本发明的改组基因。通过在lacI阻遏物基因的控制下将本发明的变异基因可操作地连接至lac启动子来制造表达载体。表达载体也含有P15A复制起点和氯霉素抗性基因。在含有氯霉素的适当培养基下培养经转化的大肠杆菌W3110以便表达表达载体的仅转化的大肠杆菌细胞存活。参见，例如实例1.纯化。
一旦通过大肠杆菌中的变异基因表达AAM多肽之后，那么使用任何一种或一种以上用于蛋白质纯化的已知技术由细胞和/或培养基纯化多肽，所述技术包括溶菌酶处理、超声降解法、过滤、用盐处理、超速离心、亲和色谱法以及在严格缺氧条件下的类似处理。用于由诸如大肠杆菌的细菌高效提取蛋白质的适合溶液可以商品名CelLytic BTM由Sigma-Aldrich of St.Louis MO购得。用于由在化学方法中应用的细胞溶解产物充分纯化AAM多肽的适合方法在以下参考文献中揭露Chirpich，T.P.等人，J.Biol.Chem.，1970，245，1778-1789；和Petrovich，R.M.等人，J.Biol.Chem.，1991，266，7656-7660，两篇文献均以引用的方式并入本文中。
筛检在执行若干轮的有指导的进化之后，筛检所得示范性AAM多肽文库。表达具有AAM活性的多肽的经转化细胞的筛检一般为两步法。首先，将细胞用物理方法分离且接着确定哪些细胞具有所要特性和不具所要特性。选择为一种筛检形式，其中通过表达选择标记物同时实现鉴别和物理分离，所述选择标记物在某些基因环境中允许表达标记物的细胞存活，而其他细胞死亡(或反之亦然)。示范性筛检标记物包括荧光素酶、β-半乳糖酶和绿色荧光蛋白质。选择标记物包括药物和毒素抗性基因，诸如对氯霉素、氨苄青霉素和其类似物有抗性。虽然自发选择可以且确实发生在自然进化过程中，但在本发明方法中选择是人工来执行的。
根据实例8中所述的方案来筛检通过突变发生或有指导的进化方法所产生的AAM多核苷酸以鉴别那些具有增强的活性、适用于作为本发明的改良AAM多肽而包含在内的AAM多核苷酸。在实例8的方法中，使用液体色谱法和质谱分析，通过测量α-丙氨酸向β-丙氨酸的转化来执行对于增强的AAM活性筛检来自表达文库的克隆。基于筛检结果，将本发明的AAM多肽和其相对于一亲本AAM多肽(意即SEQ ID NO59的多肽)的残基变化与增强的AAM活性列于以下表2中。
表2

在以上表2中，可见本发明的AAM多肽与其亲本SEQ ID NO59多肽相比具有2至11个残基差异，且如通过实例8的检定中β-丙氨酸的产生所证明，具有极其显著的AAM活性。相比而言，在用于测试AAM变异体的检定条件下未检测到SEQ ID NO59的β-丙氨酸。然而，在基于定性生长的互补检定中，检测到的亲本SEQ ID NO59的一些β-丙氨酸产生。
参看以上表2，相对于SEQ ID NO59的亲本序列，本发明AAM多肽的两个优选残基变化为G308R和F416S。相对于SEQ ID NO59的亲本序列，在长度为至少447个残基的本发明的那些AAM多肽中，另一优选的残基变化为D447G。另外适合的残基变化为G308K、F416M和D447L、A、I或V。因此，在一方面中，本发明涉及相对于SEQ IDNO59具有至少5个氨基酸残基变化，通常5-11个残基变化的AAM多肽或如本文所教示的其截短片段，所述残基变化包括1至3个选自由G308R、G308K、F416S、F416M、D447G、D447L、D447A、D447I和D447V组成的群组的残基变化。
基于表2中的AAM活性，本发明的尤其优选的AAM多肽为具有与SEQ ID NO34的多肽95％序列同源性的多肽，更优选地98％同源性，最优选地99％同源性。
SEQ ID NOs53、55和57的亲本多肽表明N末端的残基1-8和C末端的残基434-473不是KAM或AAM活性所必需的。同样，为399个残基表达产物的SEQ ID NO51的多肽片段揭露相对于SEQ ID NO59的亲本克隆，N末端的最前72个氨基酸不是AAM活性所必需的(参见表2)。因此，以下描述也在本发明的范围内本文所述的多肽包括相对于SEQ ID NO59的亲本序列缺少自其N末端的1至72个残基，通常1至40个残基，更通常1至20个残基，最通常1至11个残基的其片段。上述N末端截短与如本文于他处所述的C末端截短组合使用也在本发明的范围内。
发现亲本枯草杆菌KAM(SEQ ID NO59)主结构的仅极少(≤0.5％)突变是有益的。特定来说，对于所筛检的每1000个克隆，仅发生3-5个有益的单点或双点突变。实际上，发现一些突变是有害的。
以下两套序列的第一套提供现有技术的野生型枯草杆菌赖氨酸2，3-氨基变位酶(KAM)多肽，如所寄存的(GI_2529467_GB_AAB81159.1_)。此序列(SEQ ID NO60)不是用作亲本序列，而仅出于比较目的提供。
M K N K W Y K P K R H W K E I E L W K D V P E E K W N D W L W Q L T H TV R T L D D L K K V I N L T E D E E E G V R I S T K T I P L N I T P Y Y A S LM D P D N P R C P V R M Q S V P L S E E M H K T K Y D L E D P L H E D E DS R V P G L T H R Y P D R V L F L V T N Q C S M Y C R Y C T R R R F S G Q IG M G V P K K Q L D A A I A Y I R E T P E I R D C L I S G G D G L L I N D Q IL E Y I L K E L R S I P H L E V I R I G T R A P V V F P Q R I T D H L C E I L KK Y H P V W L N T H F N T S I E M T E E S V E A C E K L V N A G V P V G NQ A V V L A G I N D S V P I M K K L M H D L V K I R V R P Y Y I Y Q C D L SE G I G H F R A P V S K G L E I I E G L R G H T S G Y A V P T F V V D A P G GG G K I A L Q P N Y V L S Q S P D K V I L R N F E G V I T S Y P E P E N Y I PN Q A D A Y F E S V F P E T A D K K E P I G L S A I F A D K E V S F T P E N VD R I K R R E A Y I A N P E H E T L K D R R E R R D Q L K E K K F L A Q Q KK Q K E T E C G G D S S第二套序列通过将那些在与野生型枯草杆菌KAM序列的残基不同的申请者AAM多肽中的残基上标明字母“X”，其后标明残基数目，来显示本发明的AAM多肽相对于已知野生型枯草杆菌KAM序列的多样性M X2N K W Y K P K R H W X13E I E X17W X19D V P X23X24K W N D W L WX32L T X35T V X38T L D D X43K K V I N L T E D E E E G V R I S T K T I P LX67I T P X71X72X73X74L M D P X79X80P R C P V R M Q S V P L X93E E X96HX98X99K Y D L E D P L X108X109D E D S X114V P G X118T H R Y P X124R V L FL V T X132Q X134X135X136X137C R X140X141T R R X145F S G Q I G M G V PX156K Q L D A A I A Y I R E T P E I R D C L I S G G D G L L I N X187Q I L E Y IL K E X197R S X200P H X203X204V I R I G T R A P V V F P Q R I T D H X224C E IL K X230X231H P V X235L X237T H X240N T S I E M T E E X250V E A X254E K LV N A G V P V G N Q A V V L A G I N X276S V P X280X281K K L M H D L V K IR V R P Y Y I Y Q C D L S E G X307X308H X310X311A P V S K G L X319I I E G LR G H T X329G X331A V P T F V V X339A P G G G G K I A L X350P N Y V L S QS P X360K V I L R N F E G V I T S Y P E P E N X380X381P N Q A D A Y F E S VX393P X395T A D K K E P I G L S A X408F A X411K E V S X416T P E N V X422R IK R R E A Y I A N P E H E T L X440D R R E X445R X447Q L K E K K X454X455AQ Q K K Q K E T E C G G D S S本发明的AAM多肽的各种残基位置处的多样性变化在以下表2中显示在箭头的右边且相对的枯草杆菌的野生型KAM的氨基酸残基(GI_2529467_GB_AAB81159.1_)(SEQ ID NO60)显示在箭头的左边表3


在第四方面中，本发明涉及制造本发明的AAM核酸多肽的方法，其包含(a)在适于产生所述多肽的条件下培养经编码本发明的AAM多肽的核酸序列转化的宿主细胞；和(b)在适于产生β-丙氨酸的条件下向所培养的宿主细胞提供葡萄糖。β-丙氨酸可视情况由细胞回收。
实例1aam文库/ΔpanD菌株的转化方案将由Datsenko，K.A.和Wanner，B.L.，Proc.Natl.Acad.Sci.USA 976640-6645(2000)中所述的BW25113衍生而来的突变大肠杆菌菌株-ΔpanD用作宿主菌株以用于筛检aam基因文库。用于制造缺失的方案详细描述于Cargill专利申请案WO 03/062173的实例4中。
由-80℃的冷冻储存环境中移除化学胜任的大肠杆菌ΔpanD且将其解冻。此后，将其保持于冰上直至使用。将等分试样(每次转化100μl)转移至无菌1.5ml的离心管中。添加KCM(5×)盐溶液直至等分试样中的浓度为1×。KCM是由700mM KCl；10mMpH值调节至5.8的吗啉基丙烷磺酸(MOPS)组成。将1-5μl连接混合物添加至细胞中。首先将含有连接混合物的细胞在冰上培育30分钟。在42℃下将细胞热震动1分钟，且随后于冰上培育2分钟。将500μl SOC(Maniatis，T.，Fritsch，E.F.，and Sambrook，J.(1982)Molecular CloningA Laboratory Manual，第一版，第A.2和A.3页，Cold Spring HarborLaboratory，Cold Spring Harbor，NY)添加至细胞中，且伴随搅拌将细胞在37℃下培育1小时。接着将细胞以5000rpm的速率离心3分钟，且移除SOC。将细胞沉淀物再悬浮于500μl M9选择培养基中(Maniatis，T.，Fritsch，E.F.，and Sambrook，J.(1982)MolecularCloningA Laboratory Manual，第一版，第A.2和A.3页，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY)且伴随搅拌在30℃下培育2-4小时。接着将细胞涂于补充有1％甘露糖、20μM柠檬酸铁、5.0g/lα-丙氨酸、0.1mM异丙基-β-D-硫代半乳糖苷(IPTG)(Sigma Chemical Corp.，St.Louis，MO)、50mM MOPS、25mM碳酸氢盐和30μg/ml氯霉素的M9最小琼脂培养基上。将经涂盘的细胞在30℃下培育3天或直至菌落足够大以使得可以使用Q-BOTTM自动菌落采集器(Genetix USA，Inc，Boston MA)来采集。
在第二轮转化中，紧接着进行上述程序，除了将程序中的最后两次培育的培育温度增加至37℃且M9最小选择培养基不用α-丙氨酸(0g/L α-丙氨酸)补充之外。
A.aam文库/ΔpanD KIfldA菌株的预备转化方案将由Datsenko，K.A.and Wanner，B.L.，Proc.Natl.Acad.Sci.USA 976640-6645(2000)中所述的BW25113衍生而来的突变大肠杆菌菌株ΔpanD用作宿主菌株以用于筛检aam基因文库。用于制造缺失的方案详细描述于国际专利申请案WO 03/062173的实例4中。视情况，将另外具有黄素氧化还原蛋白(fldA)基因表达增强的菌株用作宿主菌株以用于筛检aam基因文库，这是由于增加的黄素氧化还原蛋白产生于大肠杆菌中时，其增强氨基变位酶的活性。参见USSN______，其是由Cargill，Inc.(Liao，等人)于2005年10月14日申请且标题为“Increasing the Activity of Radical S-Adenosyl Methionine(SAM)Enzymes”，所述文献于实例1-4中描述β-丙氨酸由表达AAM和过度表达黄素氧化还原蛋白的细胞的产生，且这些实例以引用的方式并入本文中。由Cargill，Inc.(Liao，等人)于2005年10月14日申请的同一申请案USSN_____，于实例4(并入本文中)中描述大肠杆菌菌株的构建，其中人造Plac/ara杂交启动子位于fldA基因的紧邻上游。将在fldA基因前带有人造启动子的菌株命名为KifldA，其中KI是指“敲入”)。
使用标准方案用化学或电化学方法来制备大肠杆菌ΔpanD KIfldA的胜任细胞。由-80℃的冷冻储存环境中移除胜任的大肠杆菌ΔpanD KifldA且将其解冻。此后，将其保持于冰上直至使用。将等分试样(每次转化100μl)转移至无菌1.5ml的离心管中。添加KCM(5×)盐溶液直至等分试样中的浓度为1×。KCM是由700mM KCl；10mM pH值调节至5.8的吗啉基丙烷磺酸(MOPS)组成。将1-5μl连接混合物添加至细胞中。首先将含有连接混合物的细胞在冰上培育30分钟。在42℃下将细胞热震动1分钟，且随后于冰上培育2分钟。将500μl SOC(Maniatis，T.，Fritsch，E.F.，and Sambrook，J.(1982)Molecular CloningA Laboratory Manual，第一版，第A.2和A.3页，Cold Spring HarborLaboratory，Cold Spring Harbor，NY)添加至细胞中，且伴随搅拌将细胞在37℃下培育1小时。接着将细胞以5000rpm的速率离心3分钟，且移除SOC。随后将沉淀物再悬浮于适用于互补检定(实例3)或生物转化检定(实例4)任一者的培养基中。
实例2aam基因向pCK110900系列载体中的克隆用于将丙氨酸氨基变位酶基因克隆至可诱导表达系统中的策略涉及通过PCR分离aam基因且将PCR片段克隆至突变lac启动子/操纵子系统下游的SfiI限制位点。最初，设计PCR引物以含有对aam基因的5′和3′末端有特异性的核苷酸序列，以及核糖体结合位点的Shine-Delgarno序列，和独特的SfiI限制位点。接着将基因由模板扩增，纯化且用限制性核酸内切酶SfiI来消化。将经限制的PCR片段使用QIAquick PCR纯化试剂盒(Qiagen)来纯化，且在lac启动子和lacI阻遏物基因的控制下将其克隆至图3的表达载体pCK110900-I Bla的SfiI位点。所述表达载体也含有P15a复制起点和氯霉素抗性基因。通过相同的方法来克隆改组aam基因文库。发现若干克隆表达活性丙氨酸2，3-氨基变位酶(按照实例8的方法)且对合成基因测序。将名为BSAAM的多核苷酸序列(SEQ ID NO58)用作所有更多的突变和改组的起始材料。BSAAM(SEQ ID NO58)具有与野生型枯草杆菌赖氨酸氨基变位酶(GenBank寄存编号H10329)大约99.2％的核苷酸一致性。
实例3经由Tier 2a生长检定进行的筛检Tier 2a生长检定生长检定鉴别能够在大肠杆菌ΔpanD宿主菌株中经由AAM变异体所产生的β-丙氨酸产生基本代谢物乙酰基CoA的变异体。因此，生长为CoA产生的函数，且与AAM活性间接相关。
A.程序将来自tier 1互补检定的AAM活性克隆用QBOTTM自动菌落采集器(Genetix USA，Inc.，Boston MA)采集且接种于96-孔主培养皿(master plate)中。使接种物在96孔主培养皿中于其中添加有0.1μM β-丙氨酸和0.5g/L α-丙氨酸的经缓冲的最小选择培养基(Na2HPO.7H2O 12.8g/L；KH2PO43g/L；NaCl 0.5g/L；NH4Cl 1g/L；MgSO42mM；CaCl20.04mM；甘露糖2％；PTG 1mM；柠檬酸铁20μM；氯霉素30μg/ml；MOPS pH 7，50mM；和碳酸氢钠pH 9，25mM)(在下文中称作“MSM”)上生长。将培养皿使用AirPoreTM多微孔带(Qiagen，Inc.)覆盖且于25℃，250rpm，85％湿度下培育直至培养物达到饱和，在此时将甘油添加至培养物中直至最终浓度为20-30％，且将培养皿于-80℃下储存。
将来自冷冻主培养皿的样品排列于含有如上所述的经缓冲最小选择培养基(MSM)(其另外含有0.5g/L α-丙氨酸)的“接种物”培养皿中。将接种物培养皿用AirPoreTM多微孔带(Qiagen，Inc.)覆盖且于25℃，250rpm，85％湿度下培育直至培养物达到饱和。
将来自接种物培养皿的15μl物质接种于含有185μl包含0.5g/L α-丙氨酸的新鲜MSM的96-孔“检定”培养皿中。将检定培养皿用AirPoreTM多微孔带(Qiagen.，Inc.)和盖子覆盖，且于25℃，85％湿度，250rpm下培育。在大约(～)40小时时期的离散时间下进行于600nm处的OD的测量。
B.数据分析由于文库变异体呈现独特生长概况，因此优选在三(3)个不同生长阶段(早期、中期和晚期)计算和比较生长率(斜率)以鉴别所有可能经改良的变异体。将在三(3)个阶段的任一者中呈现高于培养皿一般水平三(3)个标准差的克隆指定为可能经改良的变异体且在tier 2b中再测试以进行比较评定。
实例4经由Tier 2b生长检定进行的筛检通过将α-丙氨酸(AAM的底物)排除在培养基之外使得在Tier 2b中生长筛检的严格性增加。在这些条件下，细胞依靠α-丙氨酸的内部/细胞池，α-丙氨酸充当AAM底物且随后用于细胞生长。能够利用α-丙氨酸的低细胞内池的AAM变异体可能表示低KM变异体。
A.程序将来自冷冻主培养皿的样品排列于含有如上所述的经缓冲最小选择培养基(MSM)(其另外含有0.5g/L α-丙氨酸)的“接种物”培养皿中。将接种物培养皿用AirPoreTM多微孔带(Qiagen，Inc.)覆盖且于25℃，250rpm，85％湿度下培育直至培养物达到生长饱和。
使用TECANTM自动样品处理器(Columbus，Ohio)移除10μl来自接种物培养皿的每个Tier 2a变异体的接种物且将所述接种物重复8次播种于以下各物的每一者中含有190μl新鲜MSM、0.5g/L α-丙氨酸的96-孔检定培养皿。
含有190μl新鲜MSM但不含α-丙氨酸的96-孔检定培养皿。
将检定培养皿用AirPoreTM多微孔带和盖子覆盖且于25℃，85％湿度，250rpm下生长。在大约3-4天的时间点收集样品且测量每一样品的OD600nm。
B.Tier 2b数据分析通过以下三个标准对变异体分级i)等于在不含α-丙氨酸的培养基上的最终培养物OD600/在含有α-丙氨酸的培养基上的最终培养物OD600nm的生长率；ii)最终培养物OD600；和iii)初始生长率(在第一阶段中，来自大约0-20小时)保留最终培养物OD600nm＞0.7的克隆。
接着基于标准(i)的生长率对克隆分级。保留OD600nm＞0.7的任何克隆。然而，不满足上述两个标准但具有极佳初始生长率(iii)的克隆也选出用于进一步评估。
实例5经由Tier 2c-PCR分析进行的筛检PCR筛检鉴别在进一步筛检功能前于表达载体中含有正确尺寸基因的变异体。其排除在筛检过程中可能经历缺失/截短的不稳定基因变异体。
A.程序将来自冷冻主培养皿的可能经改良变异体接种于含有具有1％葡萄糖和30μg/mL氯霉素的LB培养基的96-微孔培养皿中。在25℃，250rpm，85％湿度下使培养物于用AirPoreTM多微孔带(Qiagen，Inc.)覆盖的培养皿中生长直至培养物达到饱和历时大约2天。将10μL的培养物转移至PCR培养皿中且于99℃下煮沸历时10分钟以使细胞破裂。此后，将90μL以下PCR预混液(master mix)添加至破裂的细胞中PCR预混液10μL 10×Taq聚合酶缓冲液(QIAGEN，Valencia CA)4μL 25mM MgCl22μL 10mM dNTPs1.25μL 20μL引物-B正向(特异于BsAAM基因)1.25μL 20μM引物-B反向(特异于BsAAM基因)1μL 5U/μLTaq聚合酶(QIAGEN)70.5μL 无菌水90μL 总体积如下为用于PCR反应中的芽孢杆菌特异性引物B-正向5′ccagcctggccataaggagatatacatatgaaaaacaaatggtataaac 3′SEQ ID NO63
B-反向5′atggtgatggtgatggtggccagtttggccttatgaagaatcccctccgc 3′SEQ ID NO64使扩增反应进行30个循环。第一循环是在94℃下进行1分钟。此后，使延伸程序进行29个循环在94.0℃下历时1分钟；在55.0℃下历时30秒；且在72.0℃下历时1分钟。最终的延伸是在72.0℃下进行5分钟。通过于0.8％琼脂糖凝胶上的凝胶电泳来分析PCR反应产物。
实例6产生β-丙氨酸的AAM变异体的生长(50ml规模)鉴别AAM活性的细胞选择方法。
为了鉴别编码可行使丙氨酸2，3-氨基变位酶反应的多肽的基因，需要对于所要活性的有效筛检或选择。因此，通过认识到大肠杆菌使用β-丙氨酸来合成泛酸而开发出选择方法，泛酸又是辅酶A(CoA)和酰基载体蛋白质(ACP)的组份。CoA和ACP为活有机体中的主要酰基载体且为生长所必需。
在大肠杆菌中，产生β-丙氨酸的主要路径为来自由天冬氨酸脱羧酶(E.C.4.1.1.11)催化的反应中的天冬氨酸，天冬氨酸脱羧酶是由panD基因编码。panD的功能性缺失突变(显示为ΔpanD)导致β-丙氨酸营养缺陷型和生长抑制，其可通过泛酸盐或β-丙氨酸的外源加入来减轻或通过来自另一来源的β-丙氨酸的产生来减轻。
菌株描述大肠杆菌ΔpanD宿主(衍生自BW25113，描述于Datsenko，K.A.and Wanner，B.L.，Proc.Natl.Acad.Sci.USA 976640-6645(2000)中)，经pCK110900-I Bla载体(由突变的lac启动子序列造成的低启动子强度)转化。使接种培养物在如下经缓冲的最小选择培养基(MSM)中生长M9盐，pH 7.0-7.4；50mM MOPs，pH 7.0；25mM碳酸氢钠，pH 9.0；1mM异丙基-β-D-硫代半乳糖苷(IPTG)；30μg/ml氯霉素；0.1g/L丙氨酸；5μM吡哆醇HCl和20μM柠檬酸铁。将接种物的1∶20稀释液用于接种50ml如上所述的MSM培养基。将培养物在25℃，250rpm下培育大约3天或直至培养物达到OD600nm～1。接着将α-丙氨酸添加至培养基中直至最终浓度为300mM，且将泛酸盐添加至约300μM。在25℃，250rpm下继续进行经补充的培养基的培育。在添加α-丙氨酸之后t＝0至t＝5时间点将样品由培养基中移出用于分析。
实例7用于β-丙氨酸检测的提取细胞的方法通过离心培养物收集来自实例6的培养物的细胞。将上清液(用过的培养基)倾析且贮存用于进一步分析(如下)。将细胞沉淀物用水洗涤。将沉淀物于-80℃下储存用于以后的提取。于测试管中将50ml细胞沉淀物(OD～4.0)完全再悬浮于0.9ml水中。将每一样品的提取体积调节为根据收集OD600的此比例。添加等体积的甲醇(-20℃)和200μL的微玻璃珠且使混合物强烈地涡流。将含有混合物的试管置于干冰/EtOH上或于-80℃冷却器中历时约30分钟。于室温下将试管中的冷冻内容物解冻且再次强烈地涡流且于最大速度下离心约10分钟。使用0.2-0.45微米过滤板或针筒式过滤器过滤上清液。
使用0.2-0.45微米过滤板或针筒式过滤器过滤用过的培养基。于-20℃甲醇/水(最终甲醇浓度50％)将经过滤的用过的培养基稀释至1∶10。
通过LC/MS/MS(实例8)来分析细胞提取物和用过的培养基的β-丙氨酸含量。
对于用过的培养基样品，将第一分钟的转为废料。β-丙氨酸峰值在大约2分钟时达到。
如果仅分析用过的培养基的话，那么可将检定规模设为2ml。
实例8β-丙氨酸的检定(LC/MS/MS)使用液体色谱法与质谱分析的组合来测定β-丙氨酸。适合的分析物为如实例7中所制备的细胞提取物和用过的培养基。
使用ASTEC CHIROBIOTICTMT4.6cm×50mm手性LC管柱(Advanced SeparationTechnologies，Inc.，Whippany，N.J.USA)执行液体色谱(LC)相。流动相由以下两种溶液组成A0.25％含水乙酸；和B0.25％(v/v)于甲醇中的乙酸。洗提在0.6ml/分钟下为等度的。
使用以下调准参数于Micromass Ultima Triple Quad质谱仪上执行质谱(MS)分析毛细管3.5kV；锥孔20V；hex 115V；孔隙1.0V；源温度100℃；去溶剂化温度350℃；锥孔气体40L/hr；去溶剂化气体500L/h；低质量分辨(Q1)12；高质量分辨(Q1)12；离子能量(Q1)0.1；碰撞室入口-5；碰撞能量14；出口1；低质量分辨(Q2)15；高质量分辨(Q2)15；离子能量(Q2)3.0；倍增器650V。
MS方法丙氨酸转变

内部通道滞留为0.1秒。
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ccaggcggag ggggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcccgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattgt1140acccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acccgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattagaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>2<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>2Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125
Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Arg Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Pro Val Glu Ala Arg Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Gly Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365
Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Cys Thr Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Glu Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Lau Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>3<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>3atggaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtgcccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcagt gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600
ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcgtt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgga cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctggc1080agagtgatct taagaaattt tgaaggtgtg attacgtcat acccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>4<211>471<212>PRT<213>人造序列<220>
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Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Val Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asp Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300
Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Gly Arg Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>5<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>5atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag ggaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180
accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaataca caaaacaaaa300tacgatatgg aagacccgct tcatggggat gaagactcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttctgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgcccccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>6<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>6Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15
Leu Trp Lys Asp Val Pro Glu Gly Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Ile85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Gly Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255
Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470
<210>7<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>7atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacca tactatgcga gcttaatgga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgtg ccgctttccg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc cgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tctccaaagg tttggagatc960attgaagggc tgagaggtca taccccaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgtttccc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcctac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat tttcggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416
<210>8<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>8Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190
Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Pro210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Pro Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Ser Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430
Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Ser Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>9<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>9atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtgcgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tgtttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080
aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>10<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>10Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140
Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Val Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365
Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>11<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>11atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gtcccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aggaacgtta120gatgatttaa agaaagtcat caatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaag gttccgtgta ctgccgccac420cgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatccgg aagtcatccg catcggaaca cgtgctcccg tcgtcttccc gcagcgcatt660
accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaact840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggcca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>12<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>12Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Gly Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80
Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Gly Ser Val Tyr Cys Arg His Arg Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Pro Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Thr Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320
Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>13<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>13atgaaaaaca aatggtataa accgaaacgg cattgggagg agatcgagcg atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ccttaatgga ccccgacaat240
ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc gggatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagcc gcgcagcact600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840gtgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcattcc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>14<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>14Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Glu Glu Ile Glu1 5 10 15Arg Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30
Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Pro Arg Ser Thr Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255
Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Val Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Ser Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>15<211>1416
<212>DNA<213>人造序列<220>
<223>合成构筑体<400>15atgaaaaaca aatggtataa accgaaacgg cattgggagg agatcgagcg atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ccttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc gggatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagcc gcgcagcact600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840gtgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcattcc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>16<211>471
<212>PRT<213>人造序列<220>
<223>合成构筑体<400>16Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Glu Glu Ile Glu1 5 10 15Arg Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Pro Arg Ser Thr Pro His Leu Glu Val Ile Arg Ile195 200 205
Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Val Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Ser Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430
Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>17<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>17atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacggctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctc ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttccg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagatacac cggtacccgg tccgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gctccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140
atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>18<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>18Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Arg20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Pro Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Thr Pro Val Pro Gly Pro Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140
Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg SerIle Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380
Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>19<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>19atggaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720
aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt ctgagggctt ggggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtcaca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtttac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagaga tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>20<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>20Met Glu Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95
His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Leu Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320
Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Asp Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>21<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>21atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gtcccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgagg aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct taccatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300
tacgacatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tccgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggctcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcgtt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatctt720aacacaagca tcgaaatgac agaagaaccc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcgggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc tgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggagcc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>22<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>22Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30
Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr His Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Pro Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Leu Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Val Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Leu225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Pro Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270
Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Cys Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>23<211>1416<212>DNA<213>人造序列
<220>
<223>合成构筑体<400>23atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaagac60gttccggacg aaaagtggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgattcaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccactttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg gccgtgtgct gtttcttgtc acgaatcaat gttccgtgca ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccgaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct ggcaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>24<211>471<212>PRT<213>人造序列
<220>
<223>合成构筑体<400>24Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Asp Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Ser Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Gly Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val His Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Glu Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205
Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Gly Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445
Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>25<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>25atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgttg120gatgatttaa agaaagtcat taacctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaaa240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgacctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggggatc960attgaagggc tgggaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcgg ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaagaag1200
gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>26<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>26Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Lys65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160
Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Gly Ile305 310 315 320Ile Glu Gly Leu Gly Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Arg Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380
Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>27<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>27atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccgggag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tgctatgctc ctttaatgga ccccgacaac240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcgtgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacgg ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagcg tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780
ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag ggggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtaatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctggaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ctttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>28<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>28Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Gly Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Cys Tyr Ala Pro Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95
His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu Arg Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Gly Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Val Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335
Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Gly Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Ser Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>29<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>29atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacggctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaag tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aggaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360
cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccgc420tgcacacgcc ggcgcttttc cggacagatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cctggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat acggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>30<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>30Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Lau Trp Arg20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45
Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Ser Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg Arg Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270
Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>31<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>31atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacgcactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgcga gcttaatgga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca cacaagcaaa300tatgacatgg aagatccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgagtcaat gtcccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg gagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaga tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>32<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>32Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr Arg Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Thr Ser Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Ser Gln Cys Pro Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Gly Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220
Cys Glu Ile Leu Lys Arg Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445
Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>33<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>33atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg actgtctgtt gtctggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcacc tgtgcgagat gttaaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc gctgtttgct gacaaagaag tttcgtctac acctgaaaat1260
gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>34<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>34Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160
Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Leu Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Met Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400
Glu Pro Ile Gly Leu Ser Ala Leu Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>35<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>35atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tatcacacct tactatgcga gcttaatgga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgtg ccgcttcctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acggatcaat gttccgtgta ctgccgccac420cgcacacgcc ggcgcttctc cggacaaatc ggaatgggcg tccccgaaaa acagcttgat480gctgcaattg cttacatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa catcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat atgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgttgta ttagcaggta ttaatgattc ggttccaatt840
ataaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgacctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>36<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>36Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Pro Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110
Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asp Gln Cys Ser Val Tyr Cys Arg His Arg Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Glu Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys His His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Tyr Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Ile Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335
Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>37<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>37atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatccca accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420
tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg actgtctgtt gtctggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcattcg tatcggttct cgtgcgccag tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat artgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat agggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtcaca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtttac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagaga tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>38<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>38Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45
Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asn Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Leu Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Ser Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285
Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Asp Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>39<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体
<400>39atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccgctttctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgca ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcacctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gtggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct ggcaaagaag tttcgtctac acctgaaaat1260gtagtcagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>40<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体
<400>40Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val His Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220
Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Gly Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Val Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460
Glu Cys Gly Gly Asp Ser Ser465 470<210>41<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>41atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggagggac60gtcccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgctt ctttaatgga ccccgacaat240ccgaggtgcc cggtacgcat gcagtctgtg ccactgtctg aggaaatgca caaaagcaaa300tatgacatgg aagatccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccgggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcgtac atcgcaaatc cggagcatga aacattaaaa1320
gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>42<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>42Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Arg Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Ser Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175
Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400
Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Lau Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>43<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>43atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacca tactatgcga gcttaatgga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgtg ccgctttccg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc cgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900
tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tctccaaagg tttggagatc960attgaagggc tgagaggtca taccccaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgtttccc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcctac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat tttcggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>44<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>44Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110
Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Pro210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Pro Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350
Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Ser Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Ser Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>45<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>45atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt acggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cgcacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgcga gcttaattga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgcg ccgctgtctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaacgggcg tccccaaaaa acagcttgat480
gctgcaactg cttatatccg ggaaacaccc gaaatccgcg attgtttaat tccaggcggt540gatgggctgc tcatcaacga ccaaatttta ggatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgcccccg tcggctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tatgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg ccctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>46<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>46Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Arg Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60
Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Ile Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Ala Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Thr Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Thr Ala Tyr Ile Arg Glu Thr Pro GluIle Arg Asp Cys Leu165 170 175Ile Pro Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Gly Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Gly Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285
Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Ala Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>47<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>47atggaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60
gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactatgcga gcttaattga tccagaaaac240ccacgttgtc cggtacgcat gcagtctgtg ccgctttccg aagaaatgca caaaacaaaa300tacgatatgg aagatccgct tcatgaggat gaagattcac cggtacccgg cctgacacac360cgctatcccg accgtgtgct gtttcttgtc gcgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatccgg aagtcatccg catcggaaca cgtgcccccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgcccctg tttccaaagg tttggagatc960attgaagggc tgagaggtca tacctcaggc tgtgcggttc ctacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaacc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagggg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>48<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>48
Met Glu Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Ile Asp Pro Glu Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Ala Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Pro Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240
Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Cys Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460
Glu Cys Gly Gly Asp Ser Ser465 470<210>49<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<400>49atgaaaaaca aatggtataa accgaaacgg cattggaagg agatcgagtt atggaaggac60gttccggaag agaaatggaa cgattggctt tgacagctga cacacactgt aagaacgtta120gatgatttaa agaaagtcat taatctgacc gaggatgaag aggaaggcgt ccgtatttct180accaaaacga tccccttaaa tattacacct tactaggttt ctttaatgga ccccgacaat240ccgagatgcc cggtacgcat gcagtctgtg ccactgtctg aagaaatgca caaaacaaaa300tacgatatgg aagacccgct tcatgaggat gaagattcac cggtacccgg tctgacacac360cgctatcccg accgtgtgct gtttcttgtc acgaatcaat gttccgtgta ctgccgccac420tgcacacgcc ggcgcttttc cggacaaatc ggaatgggcg tccccaaaaa acagcttgat480gctgcaattg cttatatccg ggaaacaccc gaaatccgcg attgtttaat ttcaggcggt540gatgggctgc tcatcaacga ccaaatttta gaatatattt taaaagagct gcgcagcatt600ccgcatctgg aagtcatccg catcggaaca cgtgctcccg tcgtctttcc gcagcgcatt660accgatcatc tgtgcgagat attgaaaaaa tatcatccgg tctggctgaa cacccatttt720aacacaagca tcgaaatgac agaagaatcc gttgaggcat gtgaaaagct ggtgaacgcg780ggagtgccgg tcggaaatca ggctgtcgta ttagcaggta ttaatgattc ggttccaatt840atgaaaaagc tcatgcatga cttggtaaaa atcagagtcc gtccttatta tatttaccaa900tgtgatctgt cagaaggaat aaggcatttc cgtgctcctg tttccaaagg tttggagatc960attgaagggc tgagaggtca cacctcaggc aatgcggttc ccacctttgt cgttcacgca1020ccaggcggag gaggtaaaat cgccctgcag ccgaactatg tcctgtctca aagtcctgac1080aaagtgatct taagaaattt tgaaggtgtg attacgtcat atccggaacc agagaattat1140atccccaatc aggcagacgc ctattttgag tccgttttcc ctgaaaccgc tgacaaaaag1200gagccgatcg ggctgagtgc catttttgct gacaaagaag tttcgtctac acctgaaaat1260gtagacagaa tcaaacggcg tgaggcatac atcgcaaatc cggagcatga aacattaaaa1320gatcggcgtg agaaaagagg tcagctcaaa gaaaagaaat ttttggcgca gcagaaaaaa1380
cagaaagaga ctgaatgcgg aggggattct tcataa 1416<210>50<211>71<212>PRT<213>人造序列<220>
<223>合成构筑体<400>50Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr65 70<210>51<211>399<212>PRT<213>人造序列<220>
<223>合成构筑体<400>51Val Ser Leu Met Asp Pro Asp Asn Pro Arg Cys Pro Val Arg Met Gln1 5 10 15Ser Val Pro Leu Ser Glu Glu Met His Lys Thr Lys Tyr Asp Met Glu20 25 30Asp Pro Leu His Glu Asp Glu Asp Ser Pro Val Pro Gly Leu Thr His35 40 45Arg Tyr Pro Asp Arg Val Leu Phe Leu Val Thr Asn Gln Cys Ser Val50 55 60
Tyr Cys Arg His Cys Thr Arg Arg Arg Phe Ser Gly Gln Ile Gly Met65 70 75 80Gly Val Pro Lys Lys Gln Leu Asp Ala Ala Ile Ala Tyr Ile Arg Glu85 90 95Thr Pro Glu Ile Arg Asp Cys Leu Ile Ser Gly Gly Asp Gly Leu Leu100 105 110Ile Asn Asp Gln Ile Leu Glu Tyr Ile Leu Lys Glu Leu Arg Ser Ile115 120 125Pro His Leu Glu Val Ile Arg Ile Gly Thr Arg Ala Pro Val Val Phe130 135 140Pro Gln Arg Ile Thr Asp His Leu Cys Glu Ile Leu Lys Lys Tyr His145 150 155 160Pro Val Trp Leu Asn Thr His Phe Asn Thr Ser Ile Glu Met Thr Glu165 170 175Glu Ser Val Glu Ala Cys Glu Lys Leu Val Asn Ala Gly Val Pro Val180 185 190Gly Asn Gln Ala Val Val Leu Ala Gly Ile Asn Asp Ser Val Pro Ile195 200 205Met Lys Lys Leu Met His Asp Leu Val Lys Ile Arg Val Arg Pro Tyr210 215 220Tyr Ile Tyr Gln Cys Asp Leu Ser Glu Gly Ile Arg His Phe Arg Ala225 230 235 240Pro Val Ser Lys Gly Leu Glu Ile Ile Glu Gly Leu Arg Gly His Thr245 250 255Ser Gly Asn Ala Val Pro Thr Phe Val Val His Ala Pro Gly Gly Gly260 265 270Gly Lys Ile Ala Leu Gln Pro Asn Tyr Val Leu Ser Gln Ser Pro Asp275 280 285
Lys Val Ile Leu Arg Asn Phe Glu Gly Val Ile Thr Ser Tyr Pro Glu290 295 300Pro Glu Asn Tyr Ile Pro Asn Gln Ala Asp Ala Tyr Phe Glu Ser Val305 310 315 320Phe Pro Glu Thr Ala Asp Lys Lys Glu Pro Ile Gly Leu Ser Ala Ile325 330 335Phe Ala Asp Lys Glu Val Ser Ser Thr Pro Glu Asn Val Asp Arg Ile340 345 350Lys Arg Arg Glu Ala Tyr Ile Ala Asn Pro Glu His Glu Thr Leu Lys355 360 365Asp Arg Arg Glu Lys Arg Gly Gln Leu Lys Glu Lys Lys Phe Leu Ala370 375 380Gln Gln Lys Lys Gln Lys Glu Thr Glu Cys Gly Gly Asp Ser Ser385 390 395<210>52<211>1245<212>DNA<213>人造序列<220>
<223>合成构筑体<220>
<221>misc_feature<223>此亲本序列是斯氏梭菌(Clostridium stricklandii)的野生型KAM的修饰<220>
<221>CDS<222>(1)..(1245)<400>52atg agt tta aag gat aag ttt ttt aca cat gta agc caa gaa gat tgg48Met Ser Leu Lys Asp Lys Phe Phe Thr His Val Ser Gln Glu Asp Trp1 5 10 15aat gat tgg aaa tgg caa gta aga aat cgt ata aag act gtt gaa gaa96Asn Asp Trp Lys Trp Gln Val Arg Asn Arg Ile Lys Thr Val Glu Glu20 25 30ctt aaa aaa tat att cca ctt act cca gaa gaa gaa gaa ggg gta aaa144Leu Lys Lys Tyr Ile Pro Leu Thr Pro Glu Glu Glu Glu Gly Val Lys35 40 45
cgc tgt ctt gat aca tta cgt atg gct att act cca tac tat cta tcg192Arg Cys Leu Asp Thr Leu Arg Met Ala Ile Thr Pro Tyr Tyr Leu Ser50 55 60cta att gat gta gaa aat cca aat gac cct gta aga aag caa gct gta240Leu Ile Asp Val Glu Asn Pro Asn Asp Pro Val Arg Lys Gln Ala Val65 70 75 80cct ctt tct tta gag ctg cat cgc gca gcg tct gat atg gaa gac cca288Pro Leu Ser Leu Glu Leu His Arg Ala Ala Ser Asp Met Glu Asp Pro85 90 95ctt cat gaa gat gga gat tct cca gtt cca gga ctt aca cat cgc tat336Leu His Glu Asp Gly Asp Ser Pro Val Pro Gly Leu Thr His Arg Tyr100 105 110cct gat cgc gtt ctt ctt tta atg act gat caa tgt tca gta tac tgc384Pro Asp Arg Val Leu Leu Leu Met Thr Asp Gln Cys Ser Val Tyr Cys115 120 125cgc cac tgt act cgt aga cgc ttc gct ggt cga aca gat tct gct gtt432Arg His Cys Thr Arg Arg Arg Phe Ala Gly Arg Thr Asp Ser Ala Val130 135 140gat acg aag caa ata gat gct gcg att gaa tat atc aaa aat act cca480Asp Thr Lys Gln Ile Asp Ala Ala Ile Glu Tyr Ile Lys Asn Thr Pro145 150 155 160caa gta aga gac gtt cta ctt tca gga gga gat gct cta tta atc tca528Gln Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu Leu Ile Ser165 170 175gat gaa aag ctt gag tac aca atc aga aga ctt cgt gaa ata cca cac576Asp Glu Lys Leu Glu Tyr Thr Ile Arg Arg Leu Arg Glu Ile Pro His180 185 190gtt gag gtt att cgt att gga tca cgt gta cca gtt gta atg cca caa624Val Glu Val Ile Arg Ile Gly Ser Arg Val Pro Val Val Met Pro Gln195 200 205cgt att aca cca gaa cta gtt tct atg ctt aaa aag tat cat cca gta672Arg Ile Thr Pro Glu Lau Val Ser Met Leu Lys Lys Tyr His Pro Val210 215 220tgg tta aat aca cac ttc aac cat cct aat gaa att act gaa gag tct720Trp Leu Asn Thr His Phe Asn His Pro Asn Glu Ile Thr Glu Glu Ser225 230 235 240aaa cgt gca tgt gag tta ctt gct gat gca ggt att cct ctt gga aat768Lys Arg Ala Cys Glu Leu Leu Ala Asp Ala Gly Ile Pro Leu Gly Asn245 250 255caa agt gtg ctt ctt gca ggt gta aat gat tgc atg cac gtt atg aaa816Gln Ser Val Leu Leu Ala Gly Val Asn Asp Cys Met His Val Met Lys260 265 270aaa cta gta aat gac tta gtt aaa ata cgc gta cgt cct tac tat att864
Lys Leu Val Asn Asp Leu Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile275 280 285tat caa tgt gac ctt tca gtt gga att gag cac ttt cgc act cca gtt912Tyr Gln Cys Asp Leu Ser Val Gly Ile Glu His Phe Arg Thr Pro Val290 295 300gca aag gga ata gaa ata att gaa ggc tta aga gga cat act tca gga960Ala Lys Gly Ile Glu Ile Ile Glu Gly Leu Arg Gly His Thr Ser Gly305 310 315 320tac tgc gtt cct aca ttt gtt gtg cat gca cct ggt ggt gga gga aaa1008Tyr Cys Val Pro Thr Phe Val Val His Ala Pro Gly Gly Gly Gly Lys325 330 335act cca gtt atg cca aac tat gtt att tca caa aat cac aat aaa gtt1056Thr Pro Val Met Pro Asn Tyr Val Ile Ser Gln Asn His Asn Lys Val340 345 350att tta cgt aac ttt gaa ggt gta att aca act tac gat gag cct gat1104Ile Leu Arg Asn Phe Glu Gly Val Ile Thr Thr Tyr Asp Glu Pro Asp355 360 365cat tat act ttc cac tgt gac tgt gat gta tgc act gga aaa aca aat1152His Tyr Thr Phe His Cys Asp Cys Asp Val Cys Thr Gly Lys Thr Asn370 375 380gtt cat aag gtt gga gta gct gga ctt cta aat gga gag aca gcg aca1200Val His Lys Val Gly Val Ala Gly Leu Leu Asn Gly Glu Thr Ala Thr385 390 395 400ctt gaa cct gag ggt ttg gaa aga aaa caa aga gga cat cac taa1245Leu Glu Pro Glu Gly Leu Glu Arg Lys Gln Arg Gly His His405 410<210>53<211>414<212>PRT<213>人造序列<220>
<223>合成构筑体<400>53Met Ser Leu Lys Asp Lys Phe Phe Thr His Val Ser Gln Glu Asp Trp1 5 10 15Asn Asp Trp Lys Trp Gln Val Arg Asn Arg Ile Lys Thr Val Glu Glu20 25 30Leu Lys Lys Tyr Ile Pro Leu Thr Pro Glu Glu Glu Glu Gly Val Lys35 40 45
Arg Cys Leu Asp Thr Leu Arg Met Ala Ile Thr Pro Tyr Tyr Leu Ser50 55 60Leu Ile Asp Val Glu Asn Pro Asn Asp Pro Val Arg Lys Gln Ala Val65 70 75 80Pro Leu Ser Leu Glu Leu His Arg Ala Ala Ser Asp Met Glu Asp Pro85 90 95Leu His Glu Asp Gly Asp Ser Pro Val Pro Gly Leu Thr His Arg Tyr100 105 110Pro Asp Arg Val Leu Leu Leu Met Thr Asp Gln Cys Ser Val Tyr Cys115 120 125Arg His Cys Thr Arg Arg Arg Phe Ala Gly Arg Thr Asp Ser Ala Val130 135 140Asp Thr Lys Gln Ile Asp Ala Ala Ile Glu Tyr Ile Lys Asn Thr Pro145 150 155 160Gln Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu Leu Ile Ser165 170 175Asp Glu Lys Leu Glu Tyr Thr Ile Arg Arg Leu Arg Glu Ile Pro His180 185 190Val Glu Val Ile Arg Ile Gly Ser Arg Val Pro Val Val Met Pro Gln195 200 205Arg Ile Thr Pro Glu Leu Val Ser Met Leu Lys Lys Tyr His Pro Val210 215 220Trp Leu Asn Thr His Phe Asn His Pro Asn Glu Ile Thr Glu Glu Ser225 230 235 240Lys Arg Ala Cys Glu Leu Leu Ala Asp Ala Gly Ile Pro Leu Gly Asn245 250 255Gln Ser Val Leu Leu Ala Gly Val Asn Asp Cys Met His Val Met Lys260 265 270Lys Leu Val Asn Asp Leu Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile275 280 285
Tyr Gln Cys Asp Leu Ser Val Gly Ile Glu His Phe Arg Thr Pro Val290 295 300Ala Lys Gly Ile Glu Ile Ile Glu Gly Leu Arg Gly His Thr Ser Gly305 310 315 320Tyr Cys Val Pro Thr Phe Val Val His Ala Pro Gly Gly Gly Gly Lys325 330 335Thr Pro Val Met Pro Asn Tyr Val Ile Ser Gln Asn His Asn Lys Val340 345 350Ile Leu Arg Asn Phe Glu Gly Val Ile Thr Thr Tyr Asp Glu Pro Asp355 360 365His Tyr Thr Phe His Cys Asp Cys Asp Val Cys Thr Gly Lys Thr Asn370 375 380Val His Lys Val Gly Val Ala Gly Leu Leu Asn Gly Glu Thr Ala Thr385 390 395 400Leu Glu Pro Glu Gly Leu Glu Arg Lys Gln Arg Gly His His405 410<210>54<211>1251<212>DNA<213>人造序列<220>
<223>合成构筑体<220>
<221>CDS<222>(1)..(1251)<400>54atg gca gaa agt cgt aga aag tat tat ttc cct gat gtc acc gat gag48Met Ala Glu Ser Arg Arg Lys Tyr Tyr Phe Pro Asp Val Thr Asp Glu1 5 10 15caa tgg tac gac tgg cat tgg cag gtc ctc aat cga att aag acg ctc96Gln Trp Tyr Asp Trp His Trp Gln Val Leu Asn Arg Ile Lys Thr Leu20 25 30gac cag ctg aaa aag tac gtt aca ctc acc gct gaa gaa gaa gag gga144Asp Gln Leu Lys Lys Tyr Val Thr Leu Thr Ala Glu Glu Glu Glu Gly
35 40 45gta aaa gaa tcg ccc aaa gta ctc cga atg gct atc aca cct tat tat192Val Lys Glu Ser Pro Lys Val Leu Arg Met Ala Ile Thr Pro Tyr Tyr50 55 60ttg agt ttg ata gac ccc gag aat cct aat tgt ccg att cgt aaa caa240Leu Ser Leu Ile Asp Pro Glu Asn Pro Asn Cys Pro Ile Arg Lys Gln65 70 75 80gcc att cct act caa cag gaa ctg gta cgt gct cct gaa gat cag gta288Ala Ile Pro Thr Gln Gln Glu Leu Val Arg Ala Pro Glu Asp Gln Val85 90 95gac cca ctt agt gaa gat gaa gat tcg ccc gta ccc gga ctg act cat336Asp Pro Leu Ser Glu Asp Glu Asp Ser Pro Val Pro Gly Leu Thr His100 105 110cgt tat ccg gat cgt gta ttg ttc ctt atc acg gac aaa tgt tcg atg384Arg Tyr Pro Asp Arg Val Leu Phe Leu Ile Thr Asp Lys Cys Ser Met115 120 125tac tgt cgt cat tgt act cgc cgt cgc ttc gca gga cag aaa gat gct432Tyr Cys Arg His Cys Thr Arg Arg Arg Phe Ala Gly Gln Lys Asp Ala130 135 140tct tct cct tct gag cgc atc gat cga tgc att gac tat ata gcc aat480Ser Ser Pro Ser Glu Arg Ile Asp Arg Cys Ile Asp Tyr Ile Ala Asn145 150 155 160aca ccg aca gtc cgc gat gtt ttg cta tcg gga ggc gat gcc ctc ctt528Thr Pro Thr Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu Leu165 170 175gtc agc gac gaa cgc ttg gaa tac ata ttg aag cgt ctg cgc gaa gta576Val Ser Asp Glu Arg Leu Glu Tyr Ile Leu Lys Arg Leu Arg Glu Val180 185 190cct cat gtg gag att gtt cgt ata gga agc cgt acg ccg gta gtc ctc624Pro His Val Glu Ile Val Arg Ile Gly Ser Arg Thr Pro Val Val Leu195 200 205cct cag cgt ata acg cct caa ttg gtg gat atg ctc aaa aaa tat cat672Pro Gln Arg Ile Thr Pro Gln Leu Val Asp Met Leu Lys Lys Tyr His210 215 220ccg gtg tgg ctg aac act cac ttc aac cac ccg aat gaa gtt acc gaa720Pro Val Trp Leu Asn Thr His Phe Asn His Pro Asn Glu Val Thr Glu225 230 235 240gaa gca gtg gag gct tgt gaa aga atg gcc aat gcc ggt att ccg ttg768Glu Ala Val Glu Ala Cys Glu Arg Met Ala Asn Ala Gly Ile Pro Leu245 250 255ggt aac caa acg gtt tta ttg cgt gga atc aat gat tgt aca cat gtg816Gly Asn Gln Thr Val Leu Leu Arg Gly Ile Asn Asp Cys Thr His Val260 265 270
atg aag aga ttg gta cat ttg ctg gta aag atg cgt gtg cgt cct tac864Met Lys Arg Leu Val His Leu Leu Val Lys Met Arg Val Arg Pro Tyr275 280 285tat ata tat gta tgc gat ctt tcg ctt gga ata ggt cat ttc cgc acg912Tyr Ile Tyr Val Cys Asp Leu Ser Leu Gly Ile Gly His Phe Arg Thr290 295 300ccg gta tct aaa gga atc gaa att atc gaa aat ttg cgc gga cac acc960Pro Val Ser Lys Gly Ile Glu Ile Ile Glu Asn Leu Arg Gly His Thr305 310 315 320tcg ggc tat gca gtt cct acc ttt gtg gta ggt gct ccg ggg ggt ggt1008Ser Gly Tyr Ala Val Pro Thr Phe Val Val Gly Ala Pro Gly Gly Gly325 330 335ggt aag ata cct gta acg ccg aac tat gtt gta tct cag tcc cca cga1056Gly Lys Ile Pro Val Thr Pro Asn Tyr Val Val Ser Gln Ser Pro Arg340 345 350cat gtg gtt ctt cgc aat tat gaa ggt gtt atc aca acc tat acg gag1104His Val Val Leu Arg Asn Tyr Glu Gly Val Ile Thr Thr Tyr Thr Glu355 360 365ccg gag aat tat cat gag gag tgc gat tgt gag gac tgt cga gcc ggt1152Pro Glu Asn Tyr His Glu Glu Cys Asp Cys Glu Asp Cys Arg Ala Gly370 375 380aag cat aaa gag ggt gta gct gca ctt tcc gga ggt cag cag ttg gct1200Lys His Lys Glu Gly Val Ala Ala Leu Ser Gly Gly Gln Gln Leu Ala385 390 395 400atc gag cct tcc gac tta gct cgc aaa aaa cgc aag ttt gat aag aac1248Ile Glu Pro Ser Asp Leu Ala Arg Lys Lys Arg Lys Phe Asp Lys Asn405 410 415taa1251<210>55<211>416<212>PRT<213>人造序列<220>
<223>合成构筑体<400>55Met Ala Glu Ser Arg Arg Lys Tyr Tyr Phe Pro Asp Val Thr Asp Glu1 5 10 15Gln Trp Tyr Asp Trp His Trp Gln Val Leu Asn Arg Ile Lys Thr Leu20 25 30Asp Gln Leu Lys Lys Tyr Val Thr Leu Thr Ala Glu Glu Glu Glu Gly
35 40 45Val Lys Glu Ser Pro Lys Val Leu Arg Met Ala Ile Thr Pro Tyr Tyr50 55 60Leu Ser Leu Ile Asp Pro Glu Asn Pro Asn Cys Pro Ile Arg Lys Gln65 70 75 80Ala Ile Pro Thr Gln Gln Glu Leu Val Arg Ala Pro Glu Asp Gln Val85 90 95Asp Pro Leu Ser Glu Asp Glu Asp Ser Pro Val Pro Gly Leu Thr His100 105 110Arg Tyr Pro Asp Arg Val Leu Phe Leu Ile Thr Asp Lys Cys Ser Met115 120 125Tyr Cys Arg His Cys Thr Arg Arg Arg Phe Ala Gly Gln Lys Asp Ala130 135 140Ser Ser Pro Ser Glu Arg Ile Asp Arg Cys Ile Asp Tyr Ile Ala Asn145 150 155 160Thr Pro Thr Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu Leu165 170 175Val Ser Asp Glu Arg Leu Glu Tyr Ile Leu Lys Arg Leu Arg Glu Val180 185 190Pro His Val Glu Ile Val Arg Ile Gly Ser Arg Thr Pro Val Val Leu195 200 205Pro Gln Arg Ile Thr Pro Gln Leu Val Asp Met Leu Lys Lys Tyr His210 215 220Pro Val Trp Leu Asn Thr His Phe Asn His Pro Asn Glu Val Thr Glu225 230 235 240Glu Ala Val Glu Ala Cys Glu Arg Met Ala Asn Ala Gly Ile Pro Leu245 250 255Gly Asn Gln Thr Val Leu Leu Arg Gly Ile Asn Asp Cys Thr His Val260 265 270
Met Lys Arg Leu Val His Leu Leu Val Lys Met Arg Val Arg Pro Tyr275 280 285Tyr Ile Tyr Val Cys Asp Leu Ser Leu Gly Ile Gly His Phe Arg Thr290 295 300Pro Val Ser Lys Gly Ile Glu Ile Ile Glu Asn Leu Arg Gly His Thr305 310 315 320Ser Gly Tyr Ala Val Pro Thr Phe Val Val Gly Ala Pro Gly Gly Gly325 330 335Gly Lys Ile Pro Val Thr Pro Asn Tyr Val Val Ser Gln Ser Pro Arg340 345 350His Val Val Leu Arg Asn Tyr Glu Gly Val Ile Thr Thr Tyr Thr Glu355 360 365Pro Glu Asn Tyr His Glu Glu Cys Asp Cys Glu Asp Cys Arg Ala Gly370 375 380Lys His Lys Glu Gly Val Ala Ala Leu Ser Gly Gly Gln Gln Leu Ala385 390 395 400Ile Glu Pro Ser Asp Leu Ala Arg Lys Lys Arg Lys Phe Asp Lys Asn405 410 415<210>56<211>1278<212>DNA<213>人造序列<220>
<223>合成构筑体<220>
<221>CDS<222>(1)..(1278)<400>56atg aat aca gtt aat act cgt aaa aaa ttt ttc cca aat gta act gat48Met Asn Thr Val Asn Thr Arg Lys Lys Phe Phe Pro Asn Val Thr Asp1 5 10 15gaa gaa tgg aat gat tgg aca tgg caa gta aaa aac cgc ctt aaa agt96Glu Glu Trp Asn Asp Trp Thr Trp Gln Val Lys Asn Arg Leu Lys Ser20 25 30
gtt gaa gat tta gaa aaa tat gtt gat tta agt gaa gaa gaa aca gaa144Val Glu Asp Leu Glu Lys Tyr Val Asp Leu Ser Glu Glu Glu Thr Glu35 40 45ggg gtt gta cgc act ctt gaa act tta cgt atg gca atc act cca ttt192Gly Val Val Arg Thr Leu Glu Thr Leu Arg Met Ala Ile Thr Pro Phe50 55 60tac ttc tca ttg ata gat ttg aat agt gat cgc tgc cca ata cgt aag240Tyr Phe Ser Leu Ile Asp Leu Asn Ser Asp Arg Cys Pro Ile Arg Lys65 70 75 80caa gct ata cct act ata cga gaa ata cat caa tct gat gct gat atg288Gln Ala Ile Pro Thr Ile Arg Glu Ile His Gln Ser Asp Ala Asp Met85 90 95ttg gat cct cta cat gaa gat gaa gac tct cca gta cca gga tta act336Leu Asp Pro Leu His Glu Asp Glu Asp Ser Pro Val Pro Gly Leu Thr100 105 110cat cgc tat cca gat cgt gtt tta ctt cta ata aca gac atg tgt tct384His Arg Tyr Pro Asp Arg Val Leu Leu Leu Ile Thr Asp Met Cys Ser115 120 125gta tac tgt cgc cac tgc act cgt cgc aga ttt gct ggg tca agt gat432Val Tyr Cys Arg His Cys Thr Arg Arg Arg Phe Ala Gly Ser Ser Asp130 135 140ggt gct atg cct atg gat aga att gac aaa gca ata gaa tat att gca480Gly Ala Met Pro Met Asp Arg Ile Asp Lys Ala Ile Glu Tyr Ile Ala145 150 155 160aaa act cca caa gta agg gat gta ttg tta tca gga gga gat gca ctt528Lys Thr Pro Gln Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu165 170 175cta gtt tct aat aaa aaa tta gaa agc ata atc caa aaa cta cgc gca576Leu Val Ser Asn Lys Lys Leu Glu Ser Ile Ile Gln Lys Leu Arg Ala180 185 190ata cct cat gtt gaa ata atc aga ata gga agt cgt aca cca gtt gtt624Ile Pro His Val Glu Ile Ile Arg Ile Gly Ser Arg Thr Pro Val Val195 200 205tta cct caa aga att act cct gaa tta tgt aat atg tta aag aaa tat672Leu Pro Gln Arg Ile Thr Pro Glu Leu Cys Asn Met Leu Lys Lys Tyr210 215 220cat cca att tgg atg aat act cat ttt aac cac cct caa gaa gta acg720His Pro Ile Trp Met Asn Thr His Phe Asn His Pro Gln Glu Val Thr225 230 235 240cca gaa gct aaa aaa gct tgt gaa atg ttg gca gat gca gga gtt cca768Pro Glu Ala Lys Lys Ala Cys Glu Met Leu Ala Asp Ala Gly Val Pro245 250 255tta gga aat caa act gta cta tta aga gga ata aat gac agt gta cct816
Leu Gly Asn Gln Thr Val Leu Leu Arg Gly Ile Asn Asp Ser Val Pro260 265 270gta atg aaa agg tta gta cat gat tta gta atg atg cgt gta cgc cct864Val Met Lys Arg Leu Val His Asp Leu Val Met Met Arg Val Arg Pro275 280 285tat tat att tac caa tgt gac tta tct atg gga ctc gaa cac ttc cgc912Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser Met Gly Leu Glu His Phe Arg290 295 300aca cca gtt tct aaa ggt ata gaa att att gaa gga tta cgt gga cat960Thr Pro Val Ser Lys Gly Ile Glu Ile Ile Glu Gly Leu Arg Gly His305 310 315 320aca tct gga tat gca gta cca aca ttt gtt gtg cat gca cct ggt ggt1008Thr Ser Gly Tyr Ala Val Pro Thr Phe Val Val His Ala Pro Gly Gly325 330 335gga gga aaa act cca gta atg cct caa tat gta att tct caa tct cct1056Gly Gly Lys Thr Pro Val Met Pro Gln Tyr Val Ile Ser Gln Ser Pro340 345 350cat cgt gta gtt tta cgc aac ttt gaa gga gtt ata aca act tat aca1104His Arg Val Val Leu Arg Asn Phe Glu Gly Val Ile Thr Thr Tyr Thr355 360 365gaa cca gaa aat tat aca cat gaa cct tgt tat gat gaa gaa aaa ttt1152Glu Pro Glu Asn Tyr Thr His Glu Pro Cys Tyr Asp Glu Glu Lys Phe370 375 380gaa aaa atg tat gaa ata agt gga gtt tat atg cta gat gaa gga tta1200Glu Lys Met Tyr Glu Ile Ser Gly Val Tyr Met Leu Asp Glu Gly Leu385 390 395 400gaa atg tca cta gaa cct agc cac tta gca cgt cat gaa cgc aat aaa1248Glu Met Ser Leu Glu Pro Ser His Leu Ala Arg His Glu Arg Asn Lys405 410 415aag aga gca gaa gct gaa ggg aaa aaa taa1278Lys Arg Ala Glu Ala Glu Gly Lys Lys420 425<210>57<211>425<212>PRT<213>人造序列<220>
<223>合成构筑体<400>57Met Asn Thr Val Asn Thr Arg Lys Lys Phe Phe Pro Asn Val Thr Asp1 5 10 15
Glu Glu Trp Asn Asp Trp Thr Trp Gln Val Lys Asn Arg Leu Lys Ser20 25 30Val Glu Asp Leu Glu Lys Tyr Val Asp Leu Ser Glu Glu Glu Thr Glu35 40 45Gly Val Val Arg Thr Leu Glu Thr Leu Arg Met Ala Ile Thr Pro Phe50 55 60Tyr Phe Ser Leu Ile Asp Leu Asn Ser Asp Arg Cys Pro Ile Arg Lys65 70 75 80Gln Ala Ile Pro Thr Ile Arg Glu Ile His Gln Ser Asp Ala Asp Met85 90 95Leu Asp Pro Leu His Glu Asp Glu Asp Ser Pro Val Pro Gly Leu Thr100 105 110His Arg Tyr Pro Asp Arg Val Leu Leu Leu Ile Thr Asp Met Cys Ser115 120 125Val Tyr Cys Arg His Cys Thr Arg Arg Arg Phe Ala Gly Ser Ser Asp130 135 140Gly Ala Met Pro Met Asp Arg Ile Asp Lys Ala Ile Glu Tyr Ile Ala145 150 155 160Lys Thr Pro Gln Val Arg Asp Val Leu Leu Ser Gly Gly Asp Ala Leu165 170 175Leu Val Ser Asn Lys Lys Leu Glu Ser Ile Ile Gln Lys Leu Arg Ala180 185 190Ile Pro His Val Glu Ile Ile Arg Ile Gly Ser Arg Thr Pro Val Val195 200 205Leu Pro Gln Arg Ile Thr Pro Glu Leu Cys Asn Met Leu Lys Lys Tyr210 215 220His Pro Ile Trp Met Asn Thr His Phe Asn His Pro Gln Glu Val Thr225 230 235 240Pro Glu Ala Lys Lys Ala Cys Glu Met Leu Ala Asp Ala Gly Val Pro245 250 255
Leu Gly Asn Gln Thr Val Leu Leu Arg Gly Ile Asn Asp Ser Val Pro260 265 270Val Met Lys Arg Leu Val His Asp Leu Val Met Met Arg Val Arg Pro275 280 285Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser Met Gly Leu Glu His Phe Arg290 295 300Thr Pro Val Ser Lys Gly Ile Glu Ile Ile Glu Gly Leu Arg Gly His305 310 315 320Thr Ser Gly Tyr Ala Val Pro Thr Phe Val Val His Ala Pro Gly Gly325 330 335Gly Gly Lys Thr Pro Val Met Pro Gln Tyr Val Ile Ser Gln Ser Pro340 345 350His Arg Val Val Leu Arg Asn Phe Glu Gly Val Ile Thr Thr Tyr Thr355 360 365Glu Pro Glu Asn Tyr Thr His Glu Pro Cys Tyr Asp Glu Glu Lys Phe370 375 380Glu Lys Met Tyr Glu Ile Ser Gly Val Tyr Met Leu Asp Glu Gly Leu385 390 395 400Glu Met Ser Leu Glu Pro Ser His Leu Ala Arg His Glu Arg Asn Lys405 410 415Lys Arg Ala Glu Ala Glu Gly Lys Lys420 425<210>58<211>1416<212>DNA<213>人造序列<220>
<223>合成构筑体<220>
<221>CDS<222>(1)..(1416)
<400>58atg aaa aac aaa tgg tat aaa ccg aaa cgg cat tgg aag gag atc gag48Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glul 5 10 15tta tgg aag gac gtt ccg gaa gag aaa tgg aac gat tgg ctt tgg cag96Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30ctg aca cac act gta aga acg tta gat gat tta aag aaa gtc att aat144Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45ctg acc gag gat gaa gag gaa ggc gtc cgt att tct acc aaa acg atc192Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60ccc tta aat att aca cct tac tat gct tct tta atg gac ccc gac aat240Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80ccg aga tgc ccg gta cgc atg cag tct gtg ccg ctt tct gaa gaa atg288Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95cac aaa aca aaa tac gat atg gaa gac ccg ctt cat gag gat gaa gat336His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110tca ccg gta ccc ggt ctg aca cac cgc tat ccc gac cgt gtg ctg ttt384Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125ctt gtc acg aat caa tgt tcc gtg tac tgc cgc cac tgc aca cgc cgg432Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140cgc ttt tcc gga caa atc gga atg ggc gtc ccc aaa aaa cag ctt gat480Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160gct gca att gct tat atc cgg gaa aca ccc gaa atc cgc gat tgt tta528Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175att tca ggc ggt gat ggg ctg ctc atc aac gac caa att tta gaa tat576Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190att tta aaa gag ctg cgc agc att ccg cat ctg gaa gtc atc cgc atc624Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205gga aca cgt gct ccc gtc gtc ttt ccg cag cgc att acc gat cat ctg672Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220
tgc gag ata ttg aaa aaa tat cat ccg gtc tgg ctg aac acc cat ttt720Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240aac aca agc atc gaa atg aca gaa gaa tcc gtt gag gca tgt gaa aag768Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255ctg gtg aac gcg gga gtg ccg gtc gga aat cag gct gtc gta tta gca816Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270ggt att aat gat tcg gtt cca att atg aaa aag ctc atg cat gac ttg864Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285gta aaa atc aga gtc cgt cct tat tat att tac caa tgt gat ctg tca912Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300gaa gga ata ggg cat ttc cgt gct cct gtt tcc aaa ggt ttg gag atc960Glu Gly Ile Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320att gaa ggg ctg aga ggt cat acc tca ggc tat gcg gtt cct acc ttt1008Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335gtc gtt cac gca cca ggc gga gga ggt aaa atc gcc ctg cag ccg aac1056Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350tat gtc ctg tca caa agt cct gac aaa gtg atc tta aga aat ttt gaa1104Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365ggt gtg att acg tca tat ccg gaa cca gag aat tat atc ccc aat cag1152Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380gca gac gcc tat ttt gag tcc gtt ttc cct gaa acc gct gac aaa aag1200Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400gag ccg atc ggg ctg agt gcc att ttt gct gac aaa gaa gtt tcg ttt1248Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415aca cct gaa aat gta gac aga atc aaa cgg cgt gag gca tac atc gca1296Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430aat ccg gag cat gaa aca tta aaa gat cgg cgt gag aaa aga gat cag1344Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Asp Gln435 440 445ctc aaa gaa aag aaa ttt ttg gcg cag cag aaa aaa cag aaa gag act1392Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr
450 455 460gaa tgc gga ggg gat tct tca taa1416Glu Cys Gly Gly Asp Ser Ser465 470<210>59<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>59Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu
165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400
Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Asp Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>60<211>471<212>PRT<213>来自枯草杆菌(Bacillus subtilis)的赖氨酸2，3-氨基变位酶<400>60Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Leu Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Arg Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125
Leu Val Thr Asn Gln Cys Ser Met Tyr Cys Arg Tyr Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val Asp Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350
Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Arg Arg Asp Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>61<211>471<212>PRT<213>人造序列<220>
<223>合成构筑体<400>61Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60
Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Leu Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Met Tyr Cys Arg Tyr Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300
Glu Gly Ile Gly His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val Asp Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Phe405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Asp Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>62<211>471<212>PRT<213>人造序列<400>62Met Lys Asn Lys Trp Tyr Lys Pro Lys Arg His Trp Lys Glu Ile Glu1 5 10 15Leu Trp Lys Asp Val Pro Glu Glu Lys Trp Asn Asp Trp Leu Trp Gln
20 25 30Leu Thr His Thr Val Arg Thr Leu Asp Asp Leu Lys Lys Val Ile Asn35 40 45Leu Thr Glu Asp Glu Glu Glu Gly Val Arg Ile Ser Thr Lys Thr Ile50 55 60Pro Leu Asn Ile Thr Pro Tyr Tyr Ala Ser Leu Met Asp Pro Asp Asn65 70 75 80Pro Arg Cys Pro Val Arg Met Gln Ser Val Pro Leu Ser Glu Glu Met85 90 95His Lys Thr Lys Tyr Asp Met Glu Asp Pro Leu His Glu Asp Glu Asp100 105 110Ser Pro Val Pro Gly Leu Thr His Arg Tyr Pro Asp Arg Val Leu Phe115 120 125Leu Val Thr Asn Gln Cys Ser Val Tyr Cys Arg His Cys Thr Arg Arg130 135 140Arg Phe Ser Gly Gln Ile Gly Met Gly Val Pro Lys Lys Gln Leu Asp145 150 155 160Ala Ala Ile Ala Tyr Ile Arg Glu Thr Pro Glu Ile Arg Asp Cys Leu165 170 175Ile Ser Gly Gly Asp Gly Leu Leu Ile Asn Asp Gln Ile Leu Glu Tyr180 185 190Ile Leu Lys Glu Leu Arg Ser Ile Pro His Leu Glu Val Ile Arg Ile195 200 205Gly Thr Arg Ala Pro Val Val Phe Pro Gln Arg Ile Thr Asp His Leu210 215 220Cys Glu Ile Leu Lys Lys Tyr His Pro Val Trp Leu Asn Thr His Phe225 230 235 240Asn Thr Ser Ile Glu Met Thr Glu Glu Ser Val Glu Ala Cys Glu Lys245 250 255
Leu Val Asn Ala Gly Val Pro Val Gly Asn Gln Ala Val Val Leu Ala260 265 270Gly Ile Asn Asp Ser Val Pro Ile Met Lys Lys Leu Met His Asp Leu275 280 285Val Lys Ile Arg Val Arg Pro Tyr Tyr Ile Tyr Gln Cys Asp Leu Ser290 295 300Glu Gly Ile Arg His Phe Arg Ala Pro Val Ser Lys Gly Leu Glu Ile305 310 315 320Ile Glu Gly Leu Arg Gly His Thr Ser Gly Tyr Ala Val Pro Thr Phe325 330 335Val Val His Ala Pro Gly Gly Gly Gly Lys Ile Ala Leu Gln Pro Asn340 345 350Tyr Val Leu Ser Gln Ser Pro Asp Lys Val Ile Leu Arg Asn Phe Glu355 360 365Gly Val Ile Thr Ser Tyr Pro Glu Pro Glu Asn Tyr Ile Pro Asn Gln370 375 380Ala Asp Ala Tyr Phe Glu Ser Val Phe Pro Glu Thr Ala Asp Lys Lys385 390 395 400Glu Pro Ile Gly Leu Ser Ala Ile Phe Ala Asp Lys Glu Val Ser Ser405 410 415Thr Pro Glu Asn Val Asp Arg Ile Lys Arg Arg Glu Ala Tyr Ile Ala420 425 430Asn Pro Glu His Glu Thr Leu Lys Asp Arg Arg Glu Lys Arg Gly Gln435 440 445Leu Lys Glu Lys Lys Phe Leu Ala Gln Gln Lys Lys Gln Lys Glu Thr450 455 460Glu Cys Gly Gly Asp Ser Ser465 470<210>63
<211>49<212>DNA<213>人造序列<220>
<223>芽孢杆菌(Bacillus)特异性引物<220>
<221>misc_feature<223>正向引物<400>63ccagcctggc cataaggaga tatacatatg aaaaacaaat ggtataaac49<210>64<211>50<212>DNA<213>人造序列<220>
<223>芽孢杆菌(Bacillus)特异性引物<220>
<221>misc_feature<223>反向引物<400>64atggtgatgg tgatggtggc cagtttggcc ttatgaagaa tcccctccgc 50
权利要求
1.一种具有丙氨酸2，3-氨基变位酶活性和具有以下条件的多肽(如下称为“AAM多肽”)(a)具有选自由SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51组成的群组的氨基酸序列；(b)具有与选自由SEQ ID NO2、22、28、32和36组成的群组的氨基酸序列有至少98％的同源性的氨基酸序列；(c)具有与选自由SEQ ID NO4、6、8、12、16、24、26、30、34和40组成的群组的氨基酸序列有至少99％的同源性的氨基酸序列；(d)为由在高严格条件下与任一以下各物杂交的核酸序列编码的多肽(i)SEQID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49的核苷酸序列，(ii)至少100个核苷酸的(i)的子序列，或(iii)(i)或(ii)的互补链；或(e)为(d)的多肽的变异体，其包含由其取代、缺失和/或插入1至6个氨基酸且具有于pH7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD的AAM活性。
2.根据权利要求1所述的多肽，其具有选自由SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48和51组成的群组的氨基酸序列。
3.根据权利要求1所述的多肽，其具有与选自由SEQ ID NO2、22、28、32和36组成的群组的氨基酸序列有至少98％的同源性的氨基酸序列。
4.根据权利要求1所述的多肽，其具有与选自由SEQ ID NO4、6、8、12、16、24、26、30、34和40组成的群组的氨基酸序列有至少99％的同源性的氨基酸序列。
5.根据权利要求1所述的多肽，其为由在高严格条件下与任一以下各物杂交的核酸序列编码的多肽(i)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49的核苷酸序列，(ii)至少100个核苷酸的(i)的子序列，或(iii)(i)或(ii)的互补链。
6.根据权利要求1所述的多肽，其为(d)的多肽的变异体，其包含由其取代、缺失和/或插入1至6个氨基酸且具有于pH7.0-7.6，25℃下约1至约30μM所产生的β-丙氨酸/小时/1细胞OD的AAM活性。
7.一种AAM多肽，其具有SEQ ID NO2、6、12、16、20、24、28、30、32、34、38、44、46或48的氨基酸序列。
8.根据权利要求7所述的AAM多肽，其具有SEQ ID NO6、12、28、34、46或48的氨基酸序列。
9.根据权利要求8所述的AAM多肽，其具有SEQ ID NO28或34的氨基酸序列。
10.一种编码权利要求1所述的AAM多肽的多核苷酸。
11.一种编码具有AAM活性的多肽的多核苷酸，所述多核苷酸具有SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41、43、45、47或49。
12.一种经分离且经纯化的多核苷酸，其编码权利要求1所述的多肽。
13.一种表达载体，其包含可操作地连接至启动子的权利要求10或11所述的多核苷酸。
14.一种经转化以表达权利要求10所述的多核苷酸的宿主细胞。
15.一种制造权利要求1所述的AAM多肽的方法，其包含(a)在适于产生所述多肽的条件下培养包含核酸构筑体的宿主细胞，所述构筑体包含编码所述AAM多肽的核酸序列；和(b)回收所述AAM多肽。
16.一种呈冻干形式的权利要求1所述的AAM多肽，。
17.一种组合物，其包含于经缓冲培养基中的权利要求1所述的多肽。
18.一种相对于SEQ ID NO59具有5至11个氨基酸残基变化的AAM多肽或其片段，所述残基变化包括1至3个选自由G308R、G308K、F4-16S、F416M、D447G、D447L、D447A、D447I和D447V组成的群组的残基变化。
全文摘要
本发明涉及一种相对于野生型酶具有增强的丙氨酸2，3－氨基变位酶(AAM)活性和/或热稳定性的多肽，所述野生型酶由于与丙氨酸的交叉反应性而具有偶然的AAM活性。另外，本发明涉及编码本发明的AAM多肽的多核苷酸，包含所述多核苷酸的核酸序列，包含可操作地连接至启动子的多核苷酸的表达载体，经转化以表达AAM多肽的宿主细胞和用于制造本发明的AAM多肽的方法。
文档编号C12N15/10GK101068923SQ200580036395
公开日2007年11月7日 申请日期2005年10月25日 优先权日2004年10月25日
发明者兰吉尼·查特吉, 肯尼思·W·米切尔, 苏珊·Y·路易, 理查德·J·福克斯, 米歇尔·陈 申请人:科德克希思公司