CN109468304A - 乙内酰脲酶的突变体 - Google Patents

乙内酰脲酶的突变体 Download PDF

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CN109468304A
CN109468304A CN201811207287.XA CN201811207287A CN109468304A CN 109468304 A CN109468304 A CN 109468304A CN 201811207287 A CN201811207287 A CN 201811207287A CN 109468304 A CN109468304 A CN 109468304A
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S·奥斯瓦尔德
H·舒斯特
J·罗斯
A·卡劳
U·施瓦内贝格
R·马丁内斯
H·蒙德哈达
U·霍尔特
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Abstract

本发明涉及具有选自(i)或(ii)的氨基酸序列的乙内酰脲酶：(i)选自SEQ ID NO:6‑20和SEQ ID NO:73‑119的氨基酸序列；(ii)氨基酸序列，其中在SEQ ID NO:6‑20和SEQ ID NO:73‑119的氨基酸序列中，1至75个氨基酸残基已经被取代、删除、插入和/或添加，以及其中进一步，乙内酰脲酶的催化活性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性高至少1.2倍。本发明进一步涉及制备氨基酸的方法，其中使用所述乙内酰脲酶。

Description

乙内酰脲酶的突变体
本申请是申请日为2012年11月16日，申请号为201280067105.8，标题为“乙内酰脲酶的突变体”的专利申请的分案申请。
本发明总体上涉及乙内酰脲酶(hydantoinase)。更具体地，本发明涉及一系列修饰的乙内酰脲酶及其在制备氨基酸的方法中的用途，特别是在全细胞催化剂中的用途，所述修饰的乙内酰脲酶相对于先前分离的乙内酰脲酶呈现出提高的酶活性。
背景技术
使用酶产生氨基酸的方法是已知的。此外，有这样的方法，其包括将作为起始材料的低成本化学合成的5-取代的乙内酰脲化合物不对称分解为光学活性氨基酸。这些从5-取代的乙内酰脲化合物中生产光学活性氨基酸的方法对于药物制备物的生产、化学工业产品、食品添加剂等是重要的。
在这种从5-取代的乙内酰脲化合物生产氨基酸的方法中，需要两种酶：(1)称作乙内酰脲酶(乙内酰脲水解酶)的酶，其催化通过水解将5-取代的乙内酰脲化合物转化为相应的N-氨基甲酰氨基酸形成N-氨基甲酰氨基酸的反应；(2)称作N-氨基甲酰氨基酸水解酶的酶，其催化通过水解将上述N-氨基甲酰氨基酸转化为相应的氨基酸形成光学活性的氨基酸的反应。
乙内酰脲水解酶，在本文称作“乙内酰脲酶”，包含具有广泛特异性和生物学功能的各种类别的酶。乙内酰脲酶的重要性质是其对映体选择性(enantioselectivity)，这使其对于生产光学纯的D-或L-氨基酸是有价值的。为了从5-取代的乙内酰脲化合物产生光学活性氨基酸，乙内酰脲酶和/或N-氨基甲酰氨基酸水解酶必须是对映体选择性酶。已经描述了通过生产L-氨基酸的微生物或者通过由所述微生物产生的含有酶的材料生产L-氨基酸的方法，其中采用了各种属的微生物如黄杆菌属(Flavobacterium)、芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和节杆菌属(Arthrobacter)(J.Biotechnol.46,63,1996)。
已经从节杆菌属微生物分离了乙内酰脲酶基因和N-氨基甲酰氨基酸水解酶基因，由这些基因编码的重组蛋白质用于生产L-氨基酸。
鉴于乙内酰脲酶在产生光学纯的氨基酸中的重要性，已经致力于开发具有改良的氨基酸生产性质的修饰的酶。作为这种努力的结果，已经分离和鉴定了许多微生物，其产生具有令人满意的酶性质的乙内酰脲酶。美国专利No.5,516,660公开了节杆菌(Arthrobacter sp.)菌株，其产生能从D-、L-和/或D,L-5-单取代的乙内酰脲产生L-α-氨基酸的乙内酰脲酶。
较新的的进展是利用全细胞催化剂技术(WO 2002/077212、WO 2004/042047、WO2000/058449、WO 2003/042412)在工业规模从相应的乙内酰脲中产生D-和L-氨基酸。所述催化剂含有在微生物如大肠杆菌(E.coli)中共表达的D-乙内酰脲酶、乙内酰脲消旋酶和D-氨甲酰水解酶。
国际专利申请WO 00/58449公开了源自节杆菌属的乙内酰脲酶的突变体，使得活性提高大约40％。
对源自可利用的节杆菌的这些标准催化剂的检测显示从相应的色氨酸乙内酰脲产生D-色氨酸是可能的，但是转化率很低。低转化率是由于可利用的野生型节杆菌乙内酰脲酶接受色氨酸乙内酰脲作为底物的性能不足所致。
本发明的目的是提供具有关于5-取代的乙内酰脲、优选色氨酸乙内酰脲提高的转化率的乙内酰脲酶，及使用乙内酰脲消旋酶、乙内酰脲酶和氨甲酰水解酶生产氨基酸的方法，优选地，所述氨基酸是D-色氨酸；特别地，所述方法从L-色氨酸乙内酰脲起始；优选地，所述乙内酰脲消旋酶、乙内酰脲酶和氨甲酰水解酶在大肠杆菌宿主中共表达。
因此，本发明的目的是提供改良的乙内酰脲酶，其具有增强的转化率，特别是具有关于色氨酸乙内酰脲的增强的转化率。此外，可有益于提高乙内酰脲酶的对映体选择性。
发明内容
本发明的目的通过具有选自(i)或(ii)的氨基酸序列的乙内酰脲酶得以解决：
(i)氨基酸序列，其选自：SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ IDNO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ IDNO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ IDNO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ IDNO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ IDNO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118或SEQ ID NO:119；
(ii)氨基酸序列，其中在SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ IDNO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ IDNO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ IDNO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ IDNO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ IDNO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118或SEQ ID NO:119的氨基酸序列中，1至个氨基酸残基被取代、缺失、插入和/或添加，
其中定义为
以及其中进一步地，所述乙内酰脲酶的催化活性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性高至少ζ倍，
以及其中ζ定义为ζ＝1.2。
在本发明中，如果未另外定义，则乙内酰脲酶的催化活性应理解为是这种酶关于将5-取代的D-乙内酰脲化合物转化为相应的D-氨基甲酰氨基酸的催化活性。
在本发明中，乙内酰脲酶的对映体选择性定义为Dact/Lact比率，Dact＝这种酶将5-取代的D-乙内酰脲化合物转化为相应的D-氨基甲酰氨基酸的催化活性，Lact＝这种酶将5-取代的D-乙内酰脲化合物的对映异构体转化为相应的D-氨基甲酰氨基酸的对映异构体的催化活性，换句话说，Lact＝这种酶将相应5-取代的L-乙内酰脲化合物转化为相应的L-氨基甲酰氨基酸的催化活性。
在本发明中，术语“添加氨基酸”是指在多肽的N-末端或C-末端添加氨基酸残基。
在本发明优选的实施方案中，选自70、60、50、40、30、20、10、9、8、7、6、5、4、3、2或1。
在本发明进一步优选的实施方案中，ζ选自1.5、2、3、4、5、10、15、20、25或30。
在本发明进一步优选的实施方案中，如下所述选择和ζ：
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30。
根据本发明，提供了修饰的乙内酰脲酶(乙内酰脲水解酶)，其与由微生物节杆菌产生的野生型乙内酰脲酶相比具有增强的催化活性。进一步，本发明的乙内酰脲酶可具有与由微生物节杆菌产生的野生型乙内酰脲酶相比提高的对映体选择性。本发明的乙内酰脲酶呈现关于5-取代的乙内酰脲化合物，特别是D-色氨酸乙内酰脲的增强的转化率，因此适合于用于产生D-氨基酸的方法。优选地，这种方法适于从相应的色氨酸乙内酰脲中起始产生D-色氨酸，特别是使用D-乙内酰脲酶、D-氨甲酰水解酶及任选地，乙内酰脲消旋酶。优选地，这些酶在微生物如大肠杆菌中共表达。在从5-取代的乙内酰脲化合物产生氨基酸的这种方法中，所述乙内酰脲酶催化形成N-氨基甲酰氨基酸的反应，所述反应通过水解5-取代的乙内酰脲化合物，优选(D-色氨酸乙内酰脲)以产生相应的N-氨基甲酰氨基酸(优选N-氨基甲酰D-色氨酸)。这种N-氨基甲酰氨基酸由N-氨基甲酰氨基酸水解酶水解形成相应的氨基酸(优选D-色氨酸)。
对于色氨酸的制备，也可以使用D,L-色氨酸乙内酰脲或L-色氨酸乙内酰脲作为起始材料，其中特别地，存在乙内酰脲消旋酶有利于为本发明的乙内酰脲酶提供D-色氨酸乙内酰脲底物。优选地，这种方法适于从相应的D,L-或L-色氨酸乙内酰脲起始产生D-色氨酸，特别是使用乙内酰脲消旋酶、D-乙内酰脲酶和D-氨甲酰水解酶。优选地，这三种酶在微生物如大肠杆菌中共表达。
本领域技术人员理解关于特定参考的序列的特定氨基酸位置(例如152位置)给出的信息可以移至变体乙内酰脲酶和同源氨基酸序列：本领域技术人员能比对所述参考氨基酸序列与变体序列，并确定变体序列中的相应位置(例如氨基酸位置152)。关于这个例子，本领域技术人员理解在变体序列中，对应于参考序列的氨基酸位置152的氨基酸位置可以不必须是变体序列的第152位氨基酸残基，然而在变体酶的三维结构中仍具有相同位置。排列对比氨基酸序列的方法在下文进一步描述。也可采用其它方法以确定与参考序列相比在变体序列中的相应位置。
附图说明
图1显示质粒pAS6-HyuH，其含有表达具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的野生型基因hyuH及表达具有SEQ ID NO:4所示氨基酸序列的D-氨甲酰水解酶的基因。在质粒图谱中标示的缩写代表如下元件：D-Carb.＝D-氨甲酰水解酶基因NRRL，农杆菌(Agrobacterium sp.)；rhaP＝鼠李糖启动子；hyuH＝乙内酰脲酶DSM9771；rrnB＝转录终止子；bla＝β-内酰胺酶；ori＝复制起点ColEI。
序列描述
在本发明中，多肽序列的变体有时通过表示与AA1XAA2形式的原始序列相比在变体序列中存在的每个氨基酸取代进行描述，其中AA1表示在原始序列中X位置存在的氨基酸残基，AA2表示在变体序列中X位置存在的氨基酸残基。
SEQ ID NO:1显示来自节杆菌(Arthrobacter sp.)的野生型乙内酰脲酶的氨基酸序列。
SEQ ID NO:2显示编码来自节杆菌的野生型乙内酰脲酶的核苷酸序列。
SEQ ID NO:3显示来自节杆菌的野生型乙内酰脲酶消旋酶的氨基酸序列。
SEQ ID NO:4显示来自节杆菌的野生型D-氨甲酰水解酶的氨基酸序列。
SEQ ID NO:5显示作为参考的氨基酸序列，其中在位置152、64、71、72、95、154、181、284、285、398和452的氨基酸残基X根据权利要求书定义，在位置152的氨基酸残基代表除了丙氨酸之外的任何氨基酸残基。
SEQ ID NO:6显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由甘氨酸置换。
SEQ ID NO:7显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由半胱氨酸置换。
SEQ ID NO:8显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:9显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由苏氨酸置换。
SEQ ID NO:10显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由缬氨酸置换。
SEQ ID NO:11显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由丝氨酸置换，及在154位的缬氨酸已经由异亮氨酸置换。
SEQ ID NO:12显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由缬氨酸置换，及在154位的缬氨酸已经由半胱氨酸置换。
SEQ ID NO:13显示变体乙内酰脲酶的氨基酸序列，其中在152位的丙氨酸已经由半胱氨酸置换，及在154位的缬氨酸已经由丝氨酸置换。
SEQ ID NO:14显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由天冬酰胺置换，在72位的酪氨酸已经由组氨酸置换，及在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:15显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由天冬酰胺置换，在72位的酪氨酸已经由精氨酸置换，及在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:16显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由缬氨酸置换，在72位的酪氨酸已经由精氨酸置换，及在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:17显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由丝氨酸置换，在72位的酪氨酸已经由精氨酸置换，及在152位的丙氨酸已经由半胱氨酸置换，在154位的缬氨酸已经由丝氨酸置换，在181位的缬氨酸已经由丙氨酸置换。
SEQ ID NO:18显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由酪氨酸置换，在72位的酪氨酸已经由精氨酸置换，及在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:19显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由亮氨酸置换，及在152位的丙氨酸已经由半胱氨酸置换，在154位的缬氨酸已经由丝氨酸置换。
SEQ ID NO:20显示变体乙内酰脲酶的氨基酸序列，其中在71位的精氨酸已经由酪氨酸置换，在72位的酪氨酸已经由组氨酸置换，及在152位的丙氨酸已经由丝氨酸置换。
SEQ ID NO:21-72显示如表1所示的用于诱变乙内酰脲酶的各种氨基酸位置的引物序列。
SEQ ID NO:73显示与SEQ ID NO:1相比携带取代I95H的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:74显示与SEQ ID NO:1相比携带取代V154G的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:75显示与SEQ ID NO:1相比携带取代V154N的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:76显示与SEQ ID NO:1相比携带取代R71D的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:77显示与SEQ ID NO:1相比携带取代R71E的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:78显示与SEQ ID NO:1相比携带取代V284F的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:79显示与SEQ ID NO:1相比携带取代V154A的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:80显示与SEQ ID NO:1相比携带取代I64T的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:81显示与SEQ ID NO:1相比携带取代I64V的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:82显示与SEQ ID NO:1相比携带取代V154S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:83显示与SEQ ID NO:1相比携带取代V154A、T398A、H452L的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:84显示与SEQ ID NO:1相比携带取代A152S、V154I的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:85显示与SEQ ID NO:1相比携带取代R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:86显示与SEQ ID NO:1相比携带取代R71V、Y72N、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:87显示与SEQ ID NO:1相比携带取代R71S、Y72N、A152C、V154S、V181A的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:88显示与SEQ ID NO:1相比携带取代R71D、Y72N、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:89显示与SEQ ID NO:1相比携带取代R71Y、Y72N、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:90显示与SEQ ID NO:1相比携带取代I64V、R71I、Y72N、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:91显示与SEQ ID NO:1相比携带取代I64C、R71Y、Y72N、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:92显示与SEQ ID NO:1相比携带取代I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:93显示与SEQ ID NO:1相比携带取代I64C、R71D、Y72H、A152S、F448L的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:94显示与SEQ ID NO:1相比携带取代I64C、R71D、Y72H、A152S、M417V的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:95显示与SEQ ID NO:1相比携带取代S14G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:96显示与SEQ ID NO:1相比携带取代S37R、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:97显示与SEQ ID NO:1相比携带取代S14G、I64C、R71D、Y72H、A152S、E358K的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:98显示与SEQ ID NO:1相比携带取代S37R、I64C、R71D、Y72H、A152S、V318A的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:99显示与SEQ ID NO:1相比携带取代S37R、I64C、R71D、Y72H、A152S、N303S、Q404R的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:100显示与SEQ ID NO:1相比携带取代S14G、I64C、R71D、Y72H、A152S、N70D的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:101显示与SEQ ID NO:1相比携带取代S14G、I64C、R71D、Y72H、A152S、V154A的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:102显示与SEQ ID NO:1相比携带取代S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:103显示与SEQ ID NO:1相比携带取代S14A、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:104显示与SEQ ID NO:1相比携带取代S14V、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:105显示与SEQ ID NO:1相比携带取代S14G、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:106显示与SEQ ID NO:1相比携带取代S14A、S37W、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:107显示与SEQ ID NO:1相比携带取代S14A、S37V、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:108显示与SEQ ID NO:1相比携带取代D15N、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:109显示与SEQ ID NO:1相比携带取代S14V、S37R、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:110显示与SEQ ID NO:1相比携带取代D15N、I64C、R71D、Y72H、A152S、V154C的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:111显示与SEQ ID NO:1相比携带取代D15N、I64C、R71D、Y72H、A152S、V154I的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:112显示与SEQ ID NO:1相比携带取代S14P、D15G、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:113显示与SEQ ID NO:1相比携带取代S14G、D15R、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:114显示与SEQ ID NO:1相比携带取代S14G、D15Q、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:115显示与SEQ ID NO:1相比携带取代S14F、D15A、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:116显示与SEQ ID NO:1相比携带取代D15S、S37G、I64C、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:117显示与SEQ ID NO:1相比携带取代S14P、D15G、S37G、R71D、Y72H、A152S的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:118显示与SEQ ID NO:1相比携带取代S14P、D15G、S37G、I64C、R71D、Y72H的变体乙内酰脲酶氨基酸序列。
SEQ ID NO:119显示与SEQ ID NO:1相比携带取代D15S、S37G、I64C、R71D、Y72H的变体乙内酰脲酶氨基酸序列。
发明详述
如上所述，本发明提供衍生自来自节杆菌的具有SEQ ID NO:1所示氨基酸序列的野生型乙内酰脲酶的新的乙内酰脲酶，其具有对于D-5-取代的乙内酰脲底物，特别是D-色氨酸乙内酰脲提高的催化活性及任选提高的对映体选择性。
本发明的乙内酰脲酶具有选自(i)或(ii)的氨基酸序列：
(i)氨基酸序列，其选自：SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ IDNO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ IDNO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ IDNO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ IDNO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ IDNO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118或SEQ ID NO:119；
(ii)氨基酸序列，其中在SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ IDNO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ IDNO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ IDNO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ IDNO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ IDNO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、或SEQ IDNO:119的氨基酸序列中，1至个氨基酸残基已经被取代、缺失、插入和/或添加，
其中定义为
及其中进一步地，所述乙内酰脲酶的催化活性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性高至少ζ倍，
以及其中ζ定义为ζ＝1.2。
在本发明中，如果未另外定义，乙内酰脲酶的催化活性定义为这种酶将5-取代的D-乙内酰脲化合物转化为相应的D-氨基甲酰氨基酸的催化活性。
在本发明中，乙内酰脲酶的对映体选择性定义为Dact/Lact比率，Dact＝这种酶将5-取代的D-乙内酰脲化合物转化为相应的D-氨基甲酰氨基酸的催化活性，Lact＝这种酶将5-取代的D-乙内酰脲化合物的对映异构体转化为相应的D-氨基甲酰氨基酸的对映异构体的催化活性，换言之，Lact＝这种酶将相应5-取代的L-乙内酰脲化合物转化为相应L-氨基甲酰氨基酸的催化活性。
在本发明优选的实施方案中，选自70、60、50、40、30、20、10、9、8、7、6、5、4、3、2或1。
在本发明进一步优选的实施方案中，ζ选自1.5、2、3、4、5、10、15、20、25或30。
在本发明进一步优选的实施方案中，如下所述选择和ζ：
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4， 且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25，且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30，
且ζ＝1.5，且ζ＝2，且ζ＝3，且ζ＝4，且ζ＝5，且ζ＝10，且ζ＝15，且ζ＝20，且ζ＝25， 且ζ＝30。
在本发明的另一实施方案中，本发明的乙内酰脲酶与具有氨基酸序列SEQ ID NO:5的乙内酰脲酶相比具有至少80％相同性或者具有包含最多90个氨基酸残基的删除、取代、插入和/或添加的氨基酸序列；
其中将在152、64、71、72、95、154、181、284、285、398和452位置的氨基酸残基定义为X152、X64、X71、X72、X95、X154、X181、X284、X285、X398和X452，并且其中这些氨基酸残基进行选择如下：
X152＝除了丙氨酸之外的任何氨基酸残基；
X64＝任何氨基酸残基；
X71＝任何氨基酸残基；
X72＝任何氨基酸残基；
X95＝任何氨基酸残基；
X154＝任何氨基酸残基；
X181＝任何氨基酸残基；
X284＝任何氨基酸残基；
X285＝任何氨基酸残基；
X398＝任何氨基酸残基；
X452＝任何氨基酸残基；
并且其中进一步地，所述乙内酰脲酶的催化活性与具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性相比高至少1.2倍。
在一优选的实施方案中，所述催化活性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性高至少1.5、2、3、4、5、10、15、20、25或30倍。
在另一优选的实施方案中，对于D-乙内酰脲的对映体选择性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的对映体选择性高至少1.2、1.5、2、3、4、5、10、15、20、25、30、50、100或150倍。在更优选的实施方案中，针对D-5-吲哚基甲基乙内酰脲(D-色氨酸乙内酰脲)的乙内酰脲酶的对映体选择性与具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶相比增加至少1.2倍，优选至少1.5、2、3、4、5、10、15、20、25、30、50、100或150倍。
本领域技术人员理解如本文所述的乙内酰脲酶的变体也包括在本发明范围内。这些变体乙内酰脲酶与具有SEQ ID NO:1和SEQ ID NO:5所示氨基酸序列的乙内酰脲酶相比分别具有至少80％相同性或者具有包含最多90个氨基酸残基的删除、取代、插入和/或添加的氨基酸序列。在这些变体中，保留根据本发明的关于参考氨基酸序列(分别为SEQ ID NO:1和SEQ ID NO:5)的特定氨基酸取代。
此外，提供了具有乙内酰脲酶活性的上述蛋白质的衍生物，该衍生物与SEQ IDNO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20分别具有至少80％相同性，优选至少85％相同性，进一步优选至少90％相同性，再进一步优选至少95％相同性，进一步优选至少96％相同性，进一步优选至少97％相同性，进一步优选至少98％相同性，最优选至少99％相同性。如上所述，在这些变体中，保留了根据本发明的相对于参考序列(分别为SEQ ID NO:1和SEQ ID NO:5)的氨基酸取代。
此外，提供了具有乙内酰脲酶活性的上述蛋白质的衍生物，该衍生物由具有与SEQID NO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示氨基酸序列相比包含删除、取代、插入和/或添加最多90个氨基酸残基的氨基酸序列的蛋白质表示。如上所述，在这些变体中，保留了相对于参考序列(分别为SEQ ID NO:1和SEQ ID NO:5)的根据本发明的氨基酸取代。
本发明还涉及变体乙内酰脲酶，其具有包含删除、取代、插入和/或添加最多90个氨基酸残基的氨基酸序列。在本文使用的短语“包含最多90个氨基酸残基的删除、取代、插入和/或添加”中“最多90个氨基酸残基”的范围可限于最多70个、优选最多45个、进一步优选最多40个、更优选最多35个、进一步优选最多30个、进一步优选最多25个、进一步优选最多20个、更优选最多15个、进一步优选最多10个及最优选最多5个氨基酸残基。在本文使用的短语“包含最多90个氨基酸残基的删除、取代、插入和/或添加”中术语“最多90个氨基酸残基”优选是指1-70个、优选1-45个、进一步优选1-40个、更优选1-35个、进一步优选1-30个、进一步优选1-25个、进一步优选1-20个、更优选1-15个、进一步优选1-10个，及最优选1-5个氨基酸残基。术语“添加”是指相对于上述序列在N-末端或C-末端添加氨基酸残基。
此外，本发明提供了分别具有SEQ ID NO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示氨基酸序列的具有乙内酰脲酶活性的上述蛋白质的片段，其中优选所述片段含有SEQ ID NO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示氨基酸序列的至少300个、进一步优选至少350个及特别优选至少400个及最优选至少450个连续氨基酸，或者如上定义的其衍生物。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys、Gly、Ser、Thr或Val；
X64＝Ile、Thr或Val；
X71＝Arg、Gln、Asp、Glu、Val、Ser、Tyr或Leu；
X72＝Tyr、His或Asn；
X95＝Ile或His；
X154＝Val、Ala、Cys、Gly、Ile、Asn或Ser；
X181＝Val或Ala；
X284＝Val或Phe；
X285＝Ala或Asp；
X398＝Thr或Ala；
X452＝His或Leu。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys、Ser或Val；
X64＝Ile、Thr或Val
X71＝Arg、Gln、Asp、Glu、Val、Ser、Tyr或Leu
X72＝Tyr、His或Asn
X95＝Ile或His；
X154＝Cys、Ile或Ser；
X181＝Val或Ala；
X284＝Val或Phe；
X285＝Ala或Asp；
X398＝Thr或Ala；
X452＝His或Leu。
在本发明的另一实施方案中，X152、X154进一步限定如下：
X152＝Cys、Gly、Ser、Thr或Val；
X154＝Ala、Cys、Gly、Ile、Asn或Ser。
在本发明的另一实施方案中，X152、X154进一步限定如下：
X152＝Cys、Ser或Val；
X154＝Cys、Ile或Ser。
在本发明的另一实施方案中，X152进一步限定如下：
X152＝Ser；X152＝Cys；X152＝Val；X152＝Gly或者X152＝Thr。
在本发明的另一实施方案中，X152和X154进一步限定如下：
X152＝Ser；X154＝Ile。
在本发明的另一实施方案中，X152和X154进一步限定如下：
X152＝Val；X154＝Cys。
在本发明的另一实施方案中，X152和X154进一步限定如下：
X152＝Cys；X154＝Ser。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Ser；X71＝Asp；X72＝His；X154＝Val；X181＝Val。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Ser；X71＝Asp；X72＝Asn；X154＝Val；X181＝Val。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Ser；X71＝Val；X72＝Asn；X154＝Val；X181＝Val。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Cys；X71＝Ser；X72＝Asn；X154＝Ser；X181＝Ala。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Ser；X71＝Tyr；X72＝Asn；X154＝Val；X181＝Val。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Cys；X71＝Leu；X72＝Tyr；X154＝Ser；X181＝Val。
在本发明的另一实施方案中，X152、X71、X72、X154、X181进一步限定如下：
X152＝Ser；X71＝Tyr；X72＝His；X154＝Val；X181＝Val。
在本发明的另一实施方案中，提供了乙内酰脲酶，其中X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Val；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Gly；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Thr；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Ile；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Val；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Cys；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys；X64＝Ile；X71＝Arg；X72＝Tyr；X95＝Ile；X154＝Ser；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Asp；X72＝His；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Asp；X72＝Asn；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Val；X72＝Asn；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys；X64＝Ile；X71＝Ser；X72＝Asn；X95＝Ile；X154＝Ser；X181＝Ala；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Tyr；X72＝Asn；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Cys；X64＝Ile；X71＝Leu；X72＝Tyr；X95＝Ile；X154＝Ser；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，X152、X64、X71、X72、X95、X154、X181、X284、X285、X398、X452进一步限定如下：
X152＝Ser；X64＝Ile；X71＝Tyr；X72＝His；X95＝Ile；X154＝Val；X181＝Val；X284＝Val；X285＝Ala；X398＝Thr；X452＝His。
在本发明的另一实施方案中，本发明的乙内酰脲酶将下列底物的至少一种转化为相应的氨基甲酰氨基酸，所述底物包括乙内酰脲、5-甲基乙内酰脲、5-苯甲基乙内酰脲、5-(4-羟基苯甲基)乙内酰脲、5-吲哚基甲基乙内酰脲、5-(3,4-二羟基苯甲基)乙内酰脲、5-甲基硫代乙基乙内酰脲、5-异丙基乙内酰脲、5-异丁基乙内酰脲、5-仲-丁基乙内酰脲、5-(4-氨基丁基)乙内酰脲、5-羟基甲基乙内酰脲或者对应于非天然氨基酸的5-取代的乙内酰脲化合物、其相应的衍生物，其中所述乙内酰脲酶转化所述底物的催化活性至少与具有SEQID NO:1所示氨基酸序列的乙内酰脲酶的转化D-5-吲哚基甲基乙内酰脲的催化活性一样高。
如上所述，本发明的乙内酰脲酶的底物可以是任何D-5-取代的乙内酰脲化合物，其能够作为这种酶的特异性底物而被水解。也包括对应于非天然氨基酸或其衍生物的5-取代的乙内酰脲化合物。非天然氨基酸或其衍生物是指不是由标准遗传密码编码的氨基酸。非天然氨基酸的相应的乙内酰脲或其衍生物包括5-苯基乙内酰脲、5-(4-羟基苯基)乙内酰脲、5-甲氧基甲基乙内酰脲、5-苄氧基甲基乙内酰脲、5-(3,4-甲二氧基苯甲基)乙内酰脲、二氢尿嘧啶。优选地，本发明的乙内酰脲酶的底物是D,L-5-吲哚基甲基乙内酰脲，更优选D-5-吲哚基甲基乙内酰脲。
本发明还提供编码本发明的乙内酰脲酶的多核苷酸。
因此，本发明提供了分离的或重组的多核苷酸，其在严格条件下与具有如上述本发明核苷酸序列的多核苷酸的全长互补序列杂交，所述严格条件包括在1×氯化钠/柠檬酸钠(SSC)和0.1％十二烷基硫酸钠(SDS)溶液中在65℃洗涤；
其中所述乙内酰脲酶的催化活性比具有SEQ ID NO:1所示氨基酸序列的乙内酰脲酶的催化活性高至少ζ倍，
及其中ζ定义为ζ＝1.2。
本发明还提供包含如上述编码本发明乙内酰脲酶的多核苷酸的载体。优选地，所述载体进一步包含与所述多核苷酸可操纵地连接并且适于在宿主细胞中表达的调节序列。
本发明进一步提供分离的或转化的宿主细胞，其包含如上述编码本发明乙内酰脲酶的多核苷酸或者包含所述多核苷酸的载体。优选地，所述宿主选自大肠杆菌(Escherichia coli)、棒状细菌、芽孢杆菌(Bacillus sp.)。所述宿主细胞表达本发明的乙内酰脲酶。在一优选的实施方案中，宿主细胞表达至少选自以下一组的酶活性：乙内酰脲酶；乙内酰脲酶和乙内酰脲消旋酶；乙内酰脲酶和氨甲酰水解酶；或者乙内酰脲酶和乙内酰脲消旋酶及氨甲酰水解酶。
此外，本发明提供了一种制备氨基酸的方法，包括如下步骤：
(a)向反应介质提供本发明的乙内酰脲酶的乙内酰脲酶活性和氨甲酰水解酶活性及至少一种5-取代的乙内酰脲，任选向所述反应介质提供乙内酰脲消旋酶活性；
(b)温育所述反应介质以使得通过在(a)中提供的酶活性将5-取代的乙内酰脲转化为相应的氨基酸；及
(c)从反应介质中回收得自相应的5-取代的乙内酰脲的酶促转化的氨基酸。
在优选的方法中，所述5-取代的乙内酰脲选自D,L-5-取代的乙内酰脲、D,L-5-吲哚基甲基乙内酰脲(＝D,L-色氨酸乙内酰脲)、D-5-取代的乙内酰脲、D-5-吲哚基甲基乙内酰脲(＝D-色氨酸乙内酰脲)。
在进一步优选的方法中，提供给反应介质的所述5-取代的乙内酰脲是L-5-取代的乙内酰脲或者D,L-5-取代的乙内酰脲，此外，在步骤(a)中给反应介质提供乙内酰脲消旋酶活性以将L-5-取代的乙内酰脲或者D,L-5-取代的乙内酰脲转化为D-5-取代的乙内酰脲。
在另一优选的方法中，要制备的氨基酸是D-色氨酸，及5-取代的乙内酰脲选自D-5-吲哚基甲基乙内酰脲、L-5-吲哚基甲基乙内酰脲或者D,L-5-吲哚基甲基乙内酰脲。
在所述方法中，5-取代的乙内酰脲由本发明的乙内酰脲酶转化为相应的氨基甲酰氨基酸，其通过所述氨甲酰水解酶活性被转化为相应的氨基酸。在特别优选的实施方案中，所述氨基甲酰氨基酸选自N-(氨基羰基)-DL-色氨酸(＝N-氨基甲酰-DL-色氨酸；＝CA索引名:N-(氨基羰基)-色氨酸；＝登记号:98299-50-4；化学式:C12H13N3O3)或者N-氨基甲酰-L-色氨酸(＝CA索引名:N-(氨基羰基)-L-色氨酸＝登记号:89595-64-2；化学式:C12H13N3O3)。
在另一优选的方法中，反应介质是部分或全部由细胞培养基组成的介质，所述乙内酰脲酶活性由本发明的宿主细胞提供，所述宿主细胞在所述细胞反应介质中培养。
在特别优选的方法中，所述反应介质是部分或全部由细胞培养基组成的介质，及所述氨甲酰水解酶活性由本发明的宿主细胞或者由第二种宿主细胞提供，所述宿主细胞在所述反应介质中培养。
在一优选的实施方案中，将在不同的细胞或者在相同细胞中表达乙内酰脲酶活性和氨甲酰水解酶活性及任选地，乙内酰脲消旋酶活性的宿主细胞在细胞培养基中培养，以产生生物量，然后分离细胞培养基与生物量，使用缓冲液或水使生物量重悬浮。在加入所述生物量之前、期间或之后在所述缓冲液或水中加入底物，以使底物的转化开始。
本发明的乙内酰脲酶可以如，例如，在Biocatalytic Production of AminoAcids and Derivatives(Rozzell,J.D.and Wagner,F.eds.,Hanser Publisher,NY,atpages 75-176(1992))中所述使用，用于从D,L-5-单取代的乙内酰脲生产光学纯的氨基酸。乙内酰脲酶的一般应用也在“Enzyme catalysis in organic synthesis”(Dranz,K.andWaldmann,H.eds.,VCH-Verlag,Weinheim,at pages409-431(1995))和Wagner,T.et al.(Production of 1-methionine from D,L-5-(2-methylthioethyl)hydantoin byresting cells of a new mutant strain of Arthrobacter species DSM 7330,Journalof Biotechnology 46:63-68(1996))中描述。
对于将D,L-或L-5-取代的乙内酰脲或者D,L-或L-氨基甲酰氨基酸经酶转化为相应的D-氨基酸，优选应用全细胞催化技术，其包括表达本发明的乙内酰脲酶及表达氨甲酰水解酶，进一步优选也表达乙内酰脲消旋酶。
本发明的乙内酰脲酶可以其游离或固定化形式用于这种方法中。氨甲酰水解酶和乙内酰脲消旋酶也可以被固定化。固定化酶的技术为本领域技术人员熟知。优选的方法在Bhavender P.Sharma,Lorraine F.Bailey and Ralph A.Messing(ImmobilisierteBiomaterialien Techniken und Anwendungen,Angew.Chem.,94,pp.836-852(1992))；Dordick et al.(J.Am.Chem.Soc.,116,pp.5009-5010(1994))；Okahata et al.,Tetrahedron Lett.,38,pp.1971-1974(1997))；Adlercreutz et al.(Biocatalysis,6,291-305(1992))；Goto et al.(Biotechnol.Prog.10,pp.263-268(1994))；Kamiya et al.(Biotechnol.Prog.,11,pp.270-275(1995))；Okahata et al.(Tibtech,February 1997,15,pp.50-54)；Fishman et al.(Biotechnol.Lett.,20,pp.535-538(1998))中提及。
将5-取代的乙内酰脲或氨基甲酰氨基酸转化为相应的氨基酸可以分批或者连续方式进行。有利地，使用酶-膜-反应器作为反应容器(Wandrey et al.in Jahrbuch 1998,Verfahrenstechnik und Chemieingenieurwesen,VDI S.151ff.；Wandrey et al.inApplied Homogeneous Catalysis with Organometallic Compounds,Vol.2,VCH 1996,pp.832ff.；Kragl et al.,Angew.Chem.,6,pp.684f.(1996))。
关于使用全细胞催化方法生产氨基酸，有利地，由于高再生率和应用的生长条件易于达到而使用细菌细胞。本领域技术人员已知有一些细菌可用于这方面。优选地，在这方面，可以使用大肠杆菌作为所述细胞和表达系统(Yanisch-Perron et al.,Gene,33,103-109(1985))。
为了生产氨基酸，表达本发明的乙内酰脲酶及优选另外表达氨甲酰水解酶及进一步优选另外表达乙内酰脲消旋酶的细胞可用于培养基中。或者，相应的分离的细胞、洗涤的细胞、细胞处理产物、得自细胞处理产物的粗制酶溶液或者纯化的酶溶液可用于反应混合物中。为了进行反应，将分别含有5-取代的乙内酰脲化合物作为底物的所述培养基或反应混合物保持在温度15-45℃，优选25-40℃，pH 5-9，优选pH 6-8，同时静置或搅动8小时至5天。所述培养基或反应混合物分别可进一步含有过渡金属离子，优选地，所述过度金属离子选自Zn2+、Mn2+和Co2+，浓度为0.02mM-10mM，优选0.1mM-5mM。当使用完整细胞时，所述反应混合物可进一步含有为转化的细胞生长必需的营养成分，如碳源、氮源和无机离子。此外，可以在培养基或反应混合物中按份数加入底物(5-取代的乙内酰脲或氨基甲酰氨基酸化合物)。
由于所述方法中采用的酶也能催化相应的逆反应，因此本发明另外还提供制备如上述的D-氨基酸的方法，其中使用N-氨基甲酰-D,L-氨基酸或N-氨基甲酰-L-氨基酸代替5-取代的乙内酰脲作为底物：
所述N-氨基甲酰-D,L-氨基酸或N-氨基甲酰-L-氨基酸通过本发明的乙内酰脲酶被转化为相应的D,L-5-取代的乙内酰脲或L-5-取代的乙内酰脲化合物。此外，通过乙内酰脲消旋酶将所述D,L-5-取代的乙内酰脲或L-5-取代的乙内酰脲化合物转化为D-对映异构体，即相应的D-5-取代的乙内酰脲化合物。然后，D-5-取代的乙内酰脲化合物通过本发明的乙内酰脲酶被转化为相应的N-氨基甲酰-D-氨基酸。随后，氨甲酰水解酶将N-氨基甲酰-D-氨基酸转化为相应的D-氨基酸。在这个方法的特别优选的实施方案中，用作底物的N-氨基甲酰氨基酸是N-(氨基羰基)-DL-色氨酸(＝N-氨基甲酰-DL-色氨酸；＝CA索引名:N-(氨基羰基)-色氨酸；＝登记号:98299-50-4；化学式:C12 H13 N3 O3)或者N-氨基甲酰-L-色氨酸(＝CA索引名:N-(氨基羰基)-L-色氨酸＝登记号:89595-64-2；化学式:C12H13N3O3)。
在培养基或反应混合物中产生的氨基酸可以通过已知方法快速定量。为此，可以使用高压液相层析(HPLC)，采用C-18柱及例如含有1％乙腈的20mM磷酸钾(pH2.3)及含有10％H2O的乙腈的混合物作为洗脱液。
在培养基或反应混合物中积聚的氨基酸可以通过标准方法从培养基或反应混合物中收集。例如，可以采用如过滤、离心、真空浓缩、离子交换或吸附层析、结晶化等方法，如果需要可以彼此组合。
如下反应方案显示催化的转化(以转化D-乙内酰脲为例)中包含的反应步骤和酶。通过D-乙内酰脲酶的催化活性将D-乙内酰脲转化为N-氨基甲酰-D-氨基酸，所述D-乙内酰脲酶催化水解D-5-取代的乙内酰脲化合物的反应，产生N-氨基甲酰-D-氨基酸。在进一步的反应步骤中，称作N-氨基甲酰氨基酸水解酶的一种酶催化水解N-氨基甲酰-D-氨基酸的反应，产生光学活性D-氨基酸。进一步地，称作乙内酰脲消旋酶的一种酶催化光学活性的L-乙内酰脲化合物转化为光学活性的D-乙内酰脲化合物，反之亦然。
反应方案
定义
乙内酰脲酶：术语“乙内酰脲酶(hydantoinase)”或“乙内酰脲酶活性”在本文是指水解乙内酰脲，特别是5-取代的乙内酰脲，的酶或者酶活性。乙内酰脲酶催化水解5-取代的乙内酰脲形成相应的N-氨基甲酰氨基酸的反应。特别地，本发明的乙内酰脲酶催化水解D-5-吲哚基甲基乙内酰脲形成N-氨基甲酰-D-色氨酸的反应，或者水解L-5-吲哚基甲基乙内酰脲形成N-氨基甲酰-L-色氨酸的反应。乙内酰脲酶的一个实例是具有SEQ ID NO:1所示氨基酸序列的蛋白质。乙内酰脲酶活性的一个实例是具有SEQ ID NO:1所示氨基酸序列的蛋白质对于化合物如L-5-甲基硫乙基乙内酰脲、L-5-(4-羟基苯甲基)乙内酰脲或L-5-苯甲基乙内酰脲的催化活性，通过所述乙内酰脲酶的活性将所述化合物转化为相应的N-氨基甲酰氨基酸。
乙内酰脲消旋酶：术语“乙内酰脲消旋酶”或“乙内酰脲消旋酶活性”在本文是指将L-乙内酰脲转化为D-乙内酰脲的酶或酶活性，反之亦然。特别地，N-乙内酰脲消旋酶催化转化L-5-吲哚基甲基乙内酰脲形成D-5-吲哚基甲基乙内酰脲的反应，或者催化转化D-5-吲哚基甲基乙内酰脲形成L-5-吲哚基甲基乙内酰脲的反应。乙内酰脲消旋酶的一个实例是具有SEQ ID NO:3所示氨基酸序列的蛋白质。乙内酰脲消旋酶活性的一个实例是具有SEQ IDNO:3所示氨基酸序列的蛋白质对于化合物如L-5-甲基硫乙基乙内酰脲、L-5-(4-羟基苯甲基)乙内酰脲、L-5-苯甲基乙内酰脲或L-5-吲哚基甲基乙内酰脲的催化活性，通过所述乙内酰脲消旋酶的活性将所述化合物转化为相应的D-对映异构体。
N-氨甲酰水解酶：术语“N-氨甲酰水解酶”或者“N-氨甲酰水解酶活性”在本文是指水解N-氨基甲酰氨基酸，特别是N-氨基甲酰-D-氨基酸，的酶或酶活性。N-氨甲酰水解酶或N-氨基甲酰氨基酸水解酶分别催化水解所述N-氨基甲酰氨基酸，特别是N-氨基甲酰-D-氨基酸，产生相应的氨基酸的反应，特别是产生光学活性D-氨基酸。特别地，N-氨甲酰水解酶催化水解N-氨基甲酰-D-色氨酸形成D-色氨酸的反应，或者催化水解N-氨基甲酰-L-色氨酸形成L-色氨酸的反应。N-氨甲酰水解酶的一个实例是具有SEQ ID NO:4所示氨基酸序列的蛋白质。N-氨甲酰水解酶活性的一个实例是具有SEQ ID NO:4所示氨基酸序列的蛋白质对于化合物如N-氨基甲酰甲硫氨酸、N-氨基甲酰酪氨酸、N-氨基甲酰苯丙氨酸或N-氨基甲酰色氨酸的催化活性，通过所述N-氨甲酰水解酶将所述化合物转化为相应的氨基酸。
5-取代的乙内酰脲：术语“5-取代的乙内酰脲”是指根据如下化学式(I)的化合物，其中R代表氢、-CH3、-CH(CH3)2、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、苯甲基、4-羟基苯甲基、吲哚基甲基、-CH2-COOH、-CH2-CONH2、-CH2-CH2-COOH、-CH2-CH2-CONH2、-CH2-OH、-CH(OH)-CH3、-CH2-SH、-CH2-CH2-S-CH3、咪唑基甲基、-CH2-CH2-CH2-NH-C(NH)NH2、-CH2-CH2-CH2-CH2-NH2，或者术语“5-取代的乙内酰脲”是指脯氨酸乙内酰脲。所述残基对应于天然存在的氨基酸，所述氨基酸经由乙内酰脲酶水解相应的乙内酰脲化合物及随后N-氨基甲酰水解酶水解相应的N-氨基甲酰氨基酸而获得。此外，5-取代的乙内酰脲化合物可对应于非天然氨基酸或其衍生物，如5-苯基乙内酰脲、5-(4-羟基苯基)乙内酰脲、5-甲氧基甲基乙内酰脲、5-苄氧基甲基乙内酰脲、5-(3,4-甲二氧基苯甲基)乙内酰脲、二氢尿嘧啶。应注意化学式(I)显示5-取代的乙内酰脲的D-对映异构体。
化学式(I):
核酸构建体：如本文所用，术语“核酸构建体”是指单链或双链的核酸分子，其分离自天然存在的基因或者被修饰为含有天然不存在的核酸区段。当所述核酸构建体含有表达本发明编码序列所需的控制序列时，术语核酸构建体与术语“表达盒”同义。
控制序列：术语“控制序列”在本文是指包括表达编码本发明多肽的多核苷酸所需或有利的所有元件。各控制序列对于编码多肽的核苷酸序列可以是天然的或者是外来的，或者彼此是天然或者外来的。这种控制序列包括但不限于前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列及转录终止子。最低限度，控制序列包括启动子和转录及翻译终止信号。可以为所述控制序列提供接头以导入特定限制位点，帮助控制序列与编码多肽的核苷酸序列的编码区连接。
可操纵地连接：术语“可操纵地连接”在本文是指这样的构型，其中控制序列置于相对于多核苷酸序列的编码序列的适当位置，由此所述控制序列指导多肽编码序列的表达。
编码序列：当在本文使用时，术语“编码序列”是指这样的核苷酸序列，其直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
表达：术语“表达”包括多肽产生中包含的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。
表达载体：术语“表达载体”在本文是指线性或环形DNA分子，其包含编码本发明多肽的多核苷酸，所述多核苷酸与为所述多核苷酸表达而提供的另外的核苷酸可操纵地连接。
宿主细胞：如本文所用，术语“宿主细胞”包括对用包含本发明多核苷酸的核酸构建体或表达载体进行的转化、转染、转导等易感的任何细胞类型。
细胞培养基：术语“细胞培养基”一般是指用于培养微生物包括细菌、酵母和真菌的培养基，也可称作“发酵培养基”。
非天然氨基酸：术语“非天然氨基酸”是指具有化学式NH2-CH(R)-COOH的α氨基酸，其中R表示蛋白质氨基酸残基的修饰的残基，所述蛋白质氨基酸残基选自氢、-CH3、-CH(CH3)2、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、苯甲基、4-羟基苯甲基、吲哚基甲基、-CH2-COOH、-CH2-CONH2、-CH2-CH2-COOH、-CH2-CH2-CONH2、-CH2-OH、-CH(OH)-CH3、-CH2-SH、-CH2-CH2-S-CH3、咪唑基甲基、-CH2-CH2-CH2-NH-C(NH)NH2、-CH2-CH2-CH2-CH2-NH2，或者术语“非天然氨基酸”是指脯氨酸的衍生物，其可以在其环形结构的-CH2-CH2-CH2-基团进行修饰。上述修饰可以是氢原子由脂肪族基团、芳基、杂环基团、酸性基团、碱性基团、羟基基团和/或卤素如F取代。
修饰：术语“修饰”在本文是指对由本发明的多肽或其同源序列组成的多肽的任何化学修饰；以及对编码这种多肽的DNA的遗传操作。所述修饰可以是取代、删除、插入和/或添加一或多个氨基酸。
变体：如本文所用，术语“变体”是指具有由表达本发明多肽的修饰的核苷酸序列或其同源序列的生物体产生的乙内酰脲酶活性的多肽。所述修饰的核苷酸序列通过修饰所述核苷酸序列的人工干预获得。变体多肽序列也可以是天然存在的。例如，已经描述了分离自液化微杆菌(Microbacterium liquefaciens)AJ3912的乙内酰脲酶，其与具有SEQ IDNO:1所示氨基酸序列的乙内酰脲酶具有大约83％相同性。这种液化微杆菌乙内酰脲酶可以被认为是氨基酸序列SEQ ID NO:1的(天然存在的)变体。
本发明的蛋白质可以例如是合成的、从纯化的蛋白质中制备的或者使用本领域已知的重组方法和技术产生的。尽管本文描述了特定的制备技术，但是应理解适于产生这些肽的所有合适的技术均包含在本发明范围内。通常地，这些技术包括DNA和蛋白质测序、克隆、表达及允许构建编码和表达每种本发明蛋白质的原核和真核载体的其它重组工程技术。
术语“肽”和“寡肽”被认为是同义的(通常认可的)，每个术语均可以根据上下文要求互换使用，表示通过肽键偶联的至少两个氨基酸的链。术语“多肽”在本文是指含有十个以上氨基酸残基的链。本文中的所有寡肽和多肽化学式或序列均是从左至右书写的，以从氨基末端至羧基末端的方向。本文使用的一个字母的氨基酸代码为本领域熟知，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold SpringHarbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
“分离的”多肽或蛋白质是指从其天然环境中分离的多肽或蛋白质。例如，就本发明的目的而言，在宿主细胞中表达的重组产生的多肽及蛋白质认为是分离的，这与已经通过任何合适技术基本上纯化的天然或重组的多肽一样，所述技术例如在Smith andJohnson,Gene 67:31-40(1988)中揭示的单步骤纯化方法。
本发明的乙内酰脲酶可以通过本领域熟知的方法自重组细胞培养物中回收及纯化，所述方法包括硫酸铵或乙醇沉淀、酸提取、阴离子或阳离子交换层析、磷酸纤维素层析、疏水性相互作用层析、亲和性层析、羟磷灰石层析及凝集素层析。最优选地，采用高效液相层析(HPLC)进行纯化。
本发明的多肽包括天然纯化的产物、化学合成的产物及通过重组技术从原核或真核宿主中产生的产物，所述宿主包括例如细菌、酵母、真菌、高等植物、昆虫和哺乳动物细胞。根据在重组产生方法中使用的宿主，本发明的多肽可以是糖基化或非糖基化的。此外，本发明的多肽也可以包括修饰的起始甲硫氨酸残基，在一些情况中是宿主介导的处理的结果。
本发明的蛋白质可以与非-乙内酰脲酶多肽(例如异源氨基酸序列)可操纵地连接，形成融合蛋白。如本文所用，乙内酰脲酶“嵌合蛋白”或“融合蛋白”包含与非-乙内酰脲酶多肽可操纵地连接的乙内酰脲酶多肽。
例如，在一个实施方案中，所述融合蛋白是GST-乙内酰脲酶融合蛋白，其中乙内酰脲酶序列与GST序列的C-末端融合。这种融合蛋白可帮助重组乙内酰脲酶的纯化。在另一实施方案中，所述融合蛋白是在其N末端含有异源信号序列的乙内酰脲酶蛋白。在某些宿主细胞中(例如哺乳动物和酵母宿主细胞)，可以通过使用异源信号序列增加乙内酰脲酶的表达和/或分泌。
信号序列可用于帮助本发明蛋白质或多肽的分泌和分离。信号序列典型特征在于疏水性氨基酸核心，其通常在分泌期间在一或多个裂解事件中自成熟蛋白质中裂解。这种信号肽含有使得信号序列随着其经过分泌途径从成熟蛋白质裂解的加工位点。信号序列指导蛋白质分泌，如从转化了表达载体的真核宿主中分泌，所述信号序列随后或同时被裂解。然后，蛋白质可以容易地通过本领域公认的方法从胞外介质中纯化。或者，信号序列可以通过使用帮助纯化的序列如GST结构域而与感兴趣的蛋白质连接。因此，例如，编码多肽的序列可以与标记序列融合，所述标记序列如编码肽的序列，其帮助所述融合多肽的纯化。在本发明这方面的某些实施方案中，标记序列是六组氨酸肽，如在pQE载体中提供的标签(Qiagen,Inc.)，其中许多是可以商购的。例如，如在Gentz et al,Proc.Natl.Acad.Sci.USA 86:821-824(1989)中所述，提供六组氨酸以方便融合蛋白的纯化。HA标签是可用于纯化的另一种肽，其对应于流感血凝素蛋白衍生的表位，如Wilson etal.,Cell 37:767(1984)所述。
优选地，本发明的嵌合或融合蛋白是通过标准重组DNA技术产生的。例如，编码不同多肽序列的DNA片段根据常规技术符合读框地连接在一起，例如通过采用平末端或交错末端(stagger-ended)末端进行连接，采用限制酶消化以提供合适末端，适当填充粘端，碱性磷酸酶处理以避免不希望的连接，以及酶连接。在另一实施方案中，所述融合基因可以通过常规技术，包括自动化DNA合成仪，合成。或者，可以使用锚定引物进行PCR扩增基因片段，，所述锚定引物在两个连续的基因片段之间产生互补突出部分，随后，所述突出部分可退火并再扩增，产生嵌合基因序列(见例如Current Protocols in Molecular Biology,eds.Ausubel et al.John Wiley&Sons:1992)。此外，许多可商购的表达载体已经编码融合部分(例如GST多肽)。本发明的核酸可以克隆进这种表达载体中，由此所述融合部分与融合组分符合读框地连接，以表达包含本发明蛋白质的融合蛋白。
术语“保守取代”或者由“同源”氨基酸残基取代是指其中氨基酸残基由具有相似侧链的氨基酸残基置换的取代。这些家族为本领域已知，包括碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸；负电荷的谷氨酸、天冬氨酸；疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸；极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺；芳香性的苯丙氨酸、酪氨酸、色氨酸；化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。
可以取代蛋白质中原始氨基酸并且被认为是保守取代的氨基酸的例子包括：Ser取代Ala；Lys取代Arg；Gln或His取代Asn；Glu取代Asp；Ser取代Cys；Asn取代Gln；Asp取代Glu；Pro取代Gly；Asn或Gln取代His；Leu或Val取代Ile；Ile或Val取代Leu；Arg或Gln取代Lys；Leu或Ile取代Met；Met、Leu或Tyr取代Phe；Thr取代Ser；Ser取代Thr；Tyr取代Trp；Trp或Phe取代Tyr；及Ile或Leu取代Val。
例如，在Bowie,J.U.et al.,Science 247:1306-1310(1990)中提供了关于怎样产生表型沉默氨基酸取代的指导，其中作者指出有两种主要方法研究氨基酸序列对改变的耐受性。第一种方法依赖于评估方法，其中自然选择接受或排斥突变。第二种方法使用遗传工程以在克隆的基因的特定部位导入氨基酸改变，并进行选择或筛选以鉴定维持功能性的序列。正如作者说明的，这些研究表明蛋白质令人惊奇地耐受氨基酸取代。作者进一步指出这种改变在所述蛋白质的某一部位可能是允许的。例如，大多数埋藏(buried)的氨基酸残基需要非极性侧链，而很少的表面侧链特征通常是保守的。其它这种表型沉默取代在如前文Bowie等所述，在此引入作参考。
如本文所定义，术语“基本同源的”是指第一个氨基酸序列含有与第二个氨基酸序列足够的或最小数目的相同或等价(例如具有相似侧链)氨基酸，由此第一个和第二个氨基酸序列具有一个共同的结构域。例如，含有具有大约80％、85％、90％、95％、96％、97％、98％或99％或更高相同性的共同结构域的氨基酸序列在本文被定义为足够相同的，条件是保留如本文定义的本发明的氨基酸位置的取代。
因此具有与本发明的核苷酸序列不同的核苷酸序列的编码其它乙内酰脲酶家族成员的核酸也在本发明范围内，条件是保留如本文定义的根据本发明的编码的氨基酸序列中的氨基酸位置的取代。对应于本发明DNA的变体(例如天然等位基因变体)和同源物的这种核酸分子可以基于其与本文公开的核酸的同源性，使用本文公开的cDNA或其适当片段作为杂交探针根据标准杂交技术而分离，优选地，在高度严格杂交条件下进行分离。
除了本文提供的所述序列的天然存在的等位基因变体之外，本领域技术人员会认可可以通过突变在本发明核苷酸序列中导入进一步的改变，从而导致乙内酰脲酶蛋白质的氨基酸序列改变而不实质上改变该蛋白质的功能，条件是保留在如本文定义的本发明的编码的氨基酸序列中的氨基酸位置的取代。
根据本发明，提供了改良的乙内酰脲酶。改良的乙内酰脲酶是催化活性和/或对映体选择性被改良的蛋白质。这种蛋白质可以通过在全部或部分编码序列中随机导入的突变(例如通过饱和诱变)获得，所得突变体可以重组表达并筛选相应的生物学活性。例如，本领域提供了测量乙内酰脲酶的催化活性的标准试验，因此可易于选择改良的蛋白质。
如本文所用，术语“基因”和“重组基因”是指可自染色体DNA分离的核酸分子，其包括编码蛋白质如乙内酰脲酶的一个开放读框。基因可包括编码序列、非编码序列、内含子和调节序列。此外，基因是指如本文定义的分离的核酸分子。
本发明的核酸分子或其功能等价物可以使用标准分子生物学技术和本文提供的序列信息分离。例如，使用如本文所述的全部或部分核酸序列作为杂交探针，可以使用标准杂交技术和克隆技术分离本发明的核酸分子(例如在Sambrook,J.,Fritsh,E.F.,andManiatis,T.Molecular Cloning:A Laboratory Manual 2nd,ed.,Cold Spring HarborLaboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989中描述)。
此外，涵盖本发明的全部或部分核酸序列的核酸分子可以通过聚合酶链反应(PCR)分离，所述PCR使用基于所述序列中包含的序列信息设计合成的寡核苷酸引物。
本发明的核酸可以使用cDNA、mRNA或者基因组DNA作为模板及合适的寡核苷酸引物根据标准PCR扩增技术进行扩增。如此扩增的核酸可以克隆进合适的载体中，并通过DNA序列分析进行表征。
此外，对应于或者可以与本发明的核苷酸序列杂交的寡核苷酸可以通过标准的合成技术制备，例如使用自动化DNA合成仪制备。
与其它核苷酸序列互补的核酸分子是与该核苷酸序列充分互补的分子，使得其可以与其他核苷酸序列杂交，从而形成稳定双链体。
“分离的多核苷酸”或者“分离的核酸”是DNA或RNA，其源自天然存在的生物体基因组，其与在所述基因组中与其直接相邻的(一个在5’末端及一个在3’末端)编码序列均不直接相邻。因此，在一个实施方案中，分离的核酸包括一些或所有5’非编码(例如启动子)序列，其与编码序列直接相邻。因此该术语包括例如重组DNA，其掺入载体中、掺入自主复制质粒或病毒中，或者掺入原核细胞或真核细胞基因组DNA中，或者不依赖于其它序列作为单独的分子存在(例如通过PCR或限制性核酸内切酶处理产生的cDNA或基因组DNA片段)。也包括重组DNA，其是编码另外的多肽的杂合基因的一部分，所述杂合基因基本上没有细胞材料、病毒材料或者培养基(当通过重组DNA技术产生时)，或者化学前体或其它化合物(当化学合成时)。此外，“分离的核酸片段”是非天然存在的片段，在天然状态中未发现。
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，但是优选是双链DNA。所述核酸可以使用寡核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)合成。这种寡核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。
本发明还包括本文描述的核酸分子的互补链。
本发明提供的序列信息不应狭义地解释为需要包含错误鉴定的碱基。本文公开的特定序列可以容易地用于从节杆菌(Arthrobacter sp.)中分离完整基因，其随后可易于进行进一步序列分析，从而鉴定测序错误。
除非另外指出，通过对本文所述DNA分子测序确定的所有核苷酸序列均使用自动DNA测序仪确定，由本文确定的DNA分子编码的多肽的所有氨基酸序列通过如上述确定的DNA分子的翻译而预测。因此，正如本领域所已知，对于通过这种自动化方法确定的任何DNA序列，本文确定的任何核苷酸序列均可含有一些误差。通过自动化确定的核苷酸序列与所测序的DNA分子的实际核苷酸序列通常至少大约95％到至少大约99.9％相同的。所述实际序列可以通过其它方法，包括本领域熟知的人工DNA测序方法，更精确地确定。正如本领域所已知，与实际序列相比，在确定的核苷酸序列中的一个单一插入或删除将导致在核苷酸序列翻译中的框移，以致由所确定的核苷酸序列编码的预测的氨基酸序列自这种插入或删除的点开始与由所测序的DNA分子实际编码的氨基酸序列完全不同。
本领域技术人员能鉴定这种错误鉴定的碱基并知道怎样校正这些误差。
术语“同源性”或“相同性百分比”在本文可互换使用。对于本发明，在此定义，为确定两个氨基酸序列或两个核酸序列的相同性百分比，以最佳比较为目的比对序列(例如在第一个氨基酸或核酸序列中可导入缺口，以与第二个氨基酸或核酸序列进行最佳比对)。然后比较在相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一个序列中的位置在第二个序列中相应位置由相同氨基酸残基或核苷酸占据时，则这些分子在这个位置是相同的。两个序列之间的相同性百分比是所述序列共有的相同位置的数量的函数(即％相同性＝相同位置的数量/位置(即重叠位置)的总数量×100)。优选地，这两个序列是相同长度的。
本领域技术人员知道这样的事实，一些不同的计算机程序可用于确定两个序列之间的同源性。例如，序列比较及确定两个序列之间的相同性百分比可以使用数学算法实现。在一优选的实施方案中，两个氨基酸序列之间的相同性百分比是使用Needleman andWunsch(J.Mol.Biol.(48):444-453(1970))算法，使用Blossom 62矩阵或PAM250矩阵，gap权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6确定，所述Needleman and Wunsch算法已经并入GCG软件包的GAP程序中(可在http://www.gcg.com获得)。本领域技术人员会理解所有这些不同参数将产生略有不同的结果，但是当使用不同算法时两个序列的整体相同性百分比不显著改变。
在另一实施方案中，两个核苷酸序列之间的相同性百分比是使用GCG软件包中的GAP程序(可在http://www.gcg.com获得)，使用NWSgapdna.CMP矩阵及gap权重为40、50、60、70或80，长度权重为1、2、3、4、5或6确定的。在另一实施方案中，两个氨基酸或核苷酸序列的相同性百分比是使用E.Meyers and W.Miller(CABIOS,4:11-17(1989)算法，使用PAM120权重残基表，gap长度罚分为12，gap罚分为4确定的，所述E.Meyers and W.Miller算法已经并入ALIGN程序(version 2.0)中(在http://vega.igh.cnrs.fr/bin/align-guess.cgi可获得)。
本发明的核酸和蛋白质序列可进一步用作“查询序列”以搜索公众数据库，例如，以鉴定其它家族成员或相关序列。这种搜索可使用Altschul,et al.(1990)J.Mol.Biol.215:403-10的BLASTN和BLASTX程序(2.0版)进行。可以使用BLASTN程序，分值＝100，字长＝12，进行BLAST核苷酸搜索以获得与本发明的PLP03核酸分子同源的核苷酸序列。可以使用BLASTX程序，分值＝50，字长＝3，进行BLAST蛋白质搜索以获得与本发明的PLP03蛋白质分子同源的氨基酸序列。为了获得以比较为目的的缺口比对，可使用如Altschul et al.,(1997)Nucleic Acids Res.25(17):3389-3402所述的Gapped BLAST。当使用BLAST和Gapped BLAST程序时，可以使用各个程序的默认参数(例如BLASTX和BLASTN)。见http://www.ncbi.nlm.nih.gov。
“氨基酸相同性百分比”或者“氨基酸序列相同性百分比”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有大约指定的相同氨基酸百分比。例如，“95％氨基酸相同性”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有95％氨基酸相同性。优选地，不相同的残基位置是由于保守取代而不同。例如，具有相似化学性质如电荷或极性的氨基酸取代不太可能影响蛋白质的性质。这样的例子包括谷氨酰胺取代天冬酰胺或者谷氨酸取代天冬氨酸。
如本文所用，术语“杂交”是指描述杂交和洗涤条件，在此条件下核苷酸序列彼此至少大约90％、优选至少大约95％、更优选至少大约96％、更优选至少98％同源的核苷酸序列通常保持彼此杂交。本领域技术人员理解进一步的条件是，保留所编码的如本文定义的本发明的氨基酸序列中的氨基酸位置取代。
这种杂交条件的一个优选的非限制性实例是在6×氯化钠/柠檬酸钠(SSC)中在大约45℃杂交，随后在1×SSC、0.1％SDS中在50℃、优选在55℃、进一步优选在60℃及更优选在65℃洗涤一或多次。
高度严格条件包括，例如，在5×SSC/5×Denhardt's溶液/1.0％SDS中在68℃杂交及在0.2×SSC/0.1％SDS中在室温洗涤。或者，洗涤可以在42℃进行。
本领域技术人员知道何种条件用于严格和高度严格杂交条件。关于这种条件的其它指导在本领域容易获得，例如，在Sambrook et al.,1989,Molecular Cloning,ALaboratory Manual,Cold Spring Harbor Press,N.Y.；和Ausubel et al.(eds.),1995,Current Protocols in Molecular Biology,(John Wiley&Sons,N.Y.)中。
当然，仅与poly A序列(如mRNA的3’末端poly(A)序列段(tract))或者与互补的一段T(或U)残基杂交的多核苷酸不包括在用于与本发明的核酸的一部分特异性杂交的本发明的多核苷酸范围内，因为这种多核苷酸将与含有一段poly(A)或其互补序列(例如实际上任何双链cDNA克隆)的任何核酸分子杂交。
本发明还涉及包含与一或多个控制序列可操纵地连接的本发明的分离的多核苷酸的核酸构建体，所述控制序列指导编码序列在合适的宿主细胞中在与所述控制序列相容的条件下表达。
编码本发明多肽的分离的多核苷酸可以进行各种操作，以供多肽的表达。在将其插入载体之前对多核苷酸的操作根据表达载体可以是可取的或必需的。利用重组DNA方法修饰多核苷酸序列的技术为本领域熟知。
所述控制序列可以是合适的启动子序列，一种由宿主细胞识别以表达编码本发明多肽的多核苷酸的核苷酸序列。所述启动子序列含有介导所述多肽的表达的转录控制序列。所述启动子可以是在所选择的宿主细胞中表现出转录活性的任何核苷酸序列，包括突变体、截短的和杂合的启动子，并且可以得自与宿主细胞同源或异源的编码胞外或胞内多肽的基因。
指导本发明的核酸构建体转录(尤其是在细菌宿主细胞中)的合适启动子的实例是得自如下来源的启动子：大肠杆菌lac操纵子，天蓝色链霉菌(Streptomycescoelicolor)琼脂糖酶基因(dagA)，枯草芽孢杆菌(Bacillus sutilis)果聚糖蔗糖酶(levansucrase)基因(sacB)，地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)，嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽糖淀粉酶基因(amyM)，解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)，地衣芽孢杆菌青霉素酶基因(penP)，枯草芽孢杆菌xylA和xylB基因，及原核β-内酰胺酶基因(ViIIa-Kamaroffet al.,1978,Proceedings of the National Academy of Sciences USA 75:3727-3731)，以及tac启动子(DeBoer et al.,1983,Proceedings of the National Academy ofSciences USA 80:21-25)。另外的启动子在"Useful proteins from recombinantbacteria"in Scientific American,1980,242:74-94及在如前述Sambrook et al.,1989中描述。
作为核糖体结合序列，任何序列均可使用，只要其可以在宿主细胞中表达即可。然而，优选使用其中Shine-Dalgarno序列与起始密码子之间被调节为具有适当距离(例如6-18个碱基)的质粒。
为了有效地进行转录和翻译，可以表达这样的蛋白质，其中具有所述蛋白质活性的蛋白质或者通过缺失一部分而衍生自这种蛋白质的蛋白质的N-末端可以与由表达载体编码的蛋白质的N-末端融合。
控制序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。尽管转录终止序列不总是感兴趣的蛋白质表达所必需的，但是其通常是可商购的载体部分。终止子序列与编码所述多肽的多核苷酸序列的3’末端可操纵地连接。优选将转录终止序列直接置于结构基因之下。在所选择的宿主细胞中起作用的任何终止子均可以用于本发明中。
控制序列也可以是合适的前导序列，一种对于由宿主细胞翻译重要的mRNA非翻译区。所述前导序列与编码多肽的核苷酸序列的5’末端可操纵地连接。在所选择的宿主细胞中起作用的任何前导序列均可以用于本发明中。
控制序列也可以是聚腺苷酸化序列，一种与核苷酸序列的3’末端可操纵地连接，并且当转录时由宿主细胞识别为信号以将聚腺苷残基加入转录的mRNA的序列。在所选择的宿主细胞中起作用的任何聚腺苷酸化序列均可用于本发明中。
控制序列也可以是信号肽编码区，其编码与多肽的氨基末端连接的氨基酸序列，及指导编码的多肽进入细胞分泌途径。核苷酸序列的编码序列的5’末端可固有地含有信号肽编码区，其与编码区区段的按照翻译读框天然连接，所述编码区编码分泌的多肽。或者，编码序列的5’末端可含有与所述编码序列是外来的信号肽编码区。所述外来的信号肽编码区在所述编码序列不天然含有信号肽编码区之处是必要的。或者，外来的信号肽编码区可以简单地置换天然信号肽编码区，以增强多肽的分泌。然而，指导表达的多肽进入选择的所宿主细胞的分泌途径(即分泌进培养基中)的任何信号肽编码区均可以用于本发明中。
控制序列也可以是前肽编码区，其编码位于多肽氨基末端的氨基酸序列。所得多肽已知是酶原或多肽原(或者在一些情况中的酵素原)。多肽原通常是失活的，通过前肽的催化或自催化裂解可以从多肽原转化为成熟活性多肽。前肽编码区可以得自如下基因：枯草芽孢杆菌碱性蛋白酶(aprE)，枯草芽孢杆菌中性蛋白酶(nprT)，酿酒酵母(Saccharomyces cerevisiae)α-因子，米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶及嗜热毁丝霉(Myceliophthora thermophila)漆酶。
在信号肽与前肽区均存在于多肽的氨基末端的情况中，前肽区紧挨着多肽的氨基末端，信号肽区紧挨着前肽区的氨基末端。也可以加入调节序列，其使得多肽的表达调节与宿主细胞的生长相关。调节系统的例子是使得基因表达应答化学或物理刺激而被打开或关闭的那些系统，包括调节化合物的存在。原核细胞系统中的调节系统包括lac、tac和trp操纵子系统。在酵母中，可以使用ADH2系统或GAL1系统。在丝状真菌中，可以使用TAKAα-淀粉酶启动子、黑曲霉(Aspergillus niger)葡萄糖淀粉酶启动子和米曲霉(Aspergillusoryzae)葡萄糖淀粉酶启动子作为调节序列。调节序列的其它实例是使基因可以扩增的那些序列。在真核细胞系统中，这些包括二氢叶酸还原酶基因，其在存在氨甲喋呤的条件下扩增，及金属硫蛋白基因，其在存在重金属的条件下扩增。在这些情况中，编码多肽的核苷酸序列与调节序列可操纵地连接。
本发明还涉及重组表达载体，其包含本发明的多核苷酸、启动子和转录及翻译终止信号。本文描述的各种核酸和控制序列可以结合在一起以产生重组表达载体，其可包括一或多个方便的限制位点以允许编码多肽的核苷酸序列在这种位点的插入或取代。或者，本发明的核苷酸序列可以通过在合适的表达载体中插入核苷酸序列或者包含该序列的核酸构建体进行表达。在产生表达载体中，所述编码序列位于所述载体，以便所述编码序列与合适的控制序列可操纵地连接以表达。
重组表达载体可以是任何载体(例如质粒或病毒)，可以方便地对其实施重组DNA方法，且其可引起所述核苷酸序列的表达。载体的选择通常依赖于载体与该载体所导入的宿主细胞的相容性。载体可以是线性的或闭环质粒。
载体可以是自主复制载体，即作为染色体外实体存在的载体，其复制不依赖于染色体复制，例如质粒，染色体外元件，微型染色体或者人工染色体。载体可含有任何保证自主复制的方法。或者，当导入宿主细胞中时，载体可以整合入基因组中并与其所整合的染色体一起复制。此外，可以使用含有要导入宿主细胞基因组中的全部DNA的单一载体或质粒或者两或多个载体或质粒，或者转座子。
为了鉴定和选择这些转化体，编码可选择标记(例如抗生素抗性)的基因通常与感兴趣的基因一起被导入宿主细胞中。本发明的载体优选含有一或多个可选择标记，其使得可以对转化、转染、转导等的细胞进行简单的选择。可选择标记是一种基因，其产物提供生物杀灭剂或病毒抗性、重金属抗性、原养型至营养缺陷型等。细菌可选择标记是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或者赋予抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记。用导入的核酸转化的细胞可以通过药物选择鉴定(已经整合入可选择标记基因的细胞存活，而其它细胞死亡)。对于酵母宿主细胞合适的标记是ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。用于丝状真菌宿主细胞中的选择标记包括但不限于amdS(乙酰胺酶)、argB(鸟氨酸氨基甲酰转移酶)、bar(草丁膦乙酰转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5'-磷酸脱羧酶)、sC(硫酸腺嘌呤转移酶)及trpC(邻氨基苯甲酸合成酶)，以及其等价物。
本发明的载体可含有使得载体整合进宿主细胞基因组中或者载体在细胞中不依赖于基因组而自主复制的元件。
为了整合进宿主细胞基因组中，载体可依赖于编码多肽的多核苷酸序列或者载体的任何其它元件以通过同源或非同源重组整合进基因组中。或者，载体可含有另外的核苷酸序列以指导通过同源重组在染色体的精确位置整合进宿主细胞基因组中。为了提高在精确位置整合的可能性，整合元件应优选含有足够数目的核酸。所述整合元件可以是与宿主细胞基因组中靶序列同源的任何序列。此外，整合元件可以是非编码或编码核苷酸序列。另一方面，载体可以通过非同源重组整合进宿主细胞基因组中。
对于自主复制，载体可进一步包含复制起点，使得载体能在所述宿主细胞中自主复制。所述复制起点可以是在细胞中起作用的介导自主复制的任何质粒复制基因。术语“复制起点”或者“质粒复制基因”在本文定义为使得质粒或载体在体内复制的核苷酸序列。
细菌复制起点的实例是允许在大肠杆菌中复制的质粒pBR322、pUC19、pACYC177和pACYC184的复制起点，以及允许在芽孢杆菌中复制的pUB110、pE194、pTA1060和pAMR1的复制起点。
用于酵母宿主细胞中的复制起点的实例是2微米复制起点ARS1、ARS4，ARS1与CEN3的组合及ARS4与CEN6的组合。
可以将本发明多核苷酸的一个以上拷贝插入宿主细胞中，以增加基因产物的产生。多核苷酸的拷贝数的增加可以通过将所述序列的至少一个额外拷贝整合进宿主细胞基因组中或者通过在多核苷酸中包括可扩增的可选择标记基因而获得，这样可以通过将细胞在存在合适选择剂的条件下培养而选择含有可选择标记基因的扩增的拷贝，从而含有多核苷酸的额外的拷贝的细胞。
用于连接上述元件以构建本发明的重组表达载体的方法为本领域技术人员熟知(见例如前述Sambrook et al.,1989)。
本发明的重组表达载体包含本发明的核酸，其处于适于所述核酸在宿主细胞中表达的形式，这意味着重组表达载体包括一或多个基于用于表达的宿主细胞而选择的调节序列，其与要表达的核酸序列可操纵地连接。在重组表达载体中，“可操纵地连接”是指感兴趣的核苷酸序列与调节序列以使得所述核苷酸序列表达方式(例如在体外转录/翻译系统中或者当载体导入宿主细胞中时在宿主细胞中)连接。术语“调节序列”是指包括启动子、增强子及其它表达控制元件。这种调节序列在，例如，Goeddel；Gene Expression Technology:Methods in Enzymology 185,Academic Press,San Diego,Calif.(1990)中描述。调节序列包括指导核苷酸序列在许多类型宿主细胞中组成型或可诱导表达的那些序列。
DNA插入体应与合适启动子可操纵地连接，如噬菌体λPL启动子、大肠杆菌lacI和lacZ启动子、T3和T7启动子、gpt启动子、λPR、PL启动子及trp启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、逆转录病毒LTR(如Rous肉瘤病毒(RSV)、CMV(巨细胞病毒)的LTR)启动子和金属硫蛋白启动子(如小鼠金属硫蛋白-I启动子)。本领域技术人员知道其它合适的启动子。蛋白质在原核细胞中的表达通常在大肠杆菌中，用含有指导蛋白质的表达的组成型或可诱导的启动子的载体进行。在一特定实施方案中，启动子优选能指导乙内酰脲酶在微生物中高水平表达的，所述微生物优选大肠杆菌、枯草芽孢杆菌、棒杆菌。这种启动子为本领域已知。表达构建体可含有转录起始、终止位点，及在转录区中含有核糖体结合位点以进行翻译。由所述构建体表达的成熟转录物的编码部分包括翻译起始AUG和位于要翻译的多肽末端合适位置的终止密码子。
载体DNA可以通过常规转化或转染技术导入原核或真核细胞中。如本文所用，术语“转化”和“转染”是指将外源核酸(例如DNA)导入宿主细胞中的各种本领域公认的技术，包括磷酸钙或氯化钙共沉淀、DEAE-葡聚糖介导的转染、转导、感染、脂质转染、阳离子脂质介导的转染或者电穿孔。转化或转染宿主细胞的合适方法可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)、Davis etal.,Basic Methods in Molecular Biology(1986)及其它实验室手册。
优选用于细菌中的载体是pQE70、pQE60和PQE-9，可得自Qiagen；pBS载体，Phagescript载体，Bluescript载体，pNH8A，pNH16A，pNH18A，pNH46A，可得自Stratagene；及ptrc99a，pKK223-3，pKK233-3，pDR540，pRIT5，可得自Pharmacia。优选的真核载体是PWLNEO，pSV2CAT，pOG44，pZT1和pSG，可得自Stratagene；及pSVK3，pBPV，pMSG和pSVL，可得自Pharmacia。其它合适的载体将为本领域技术人员显然的。
本发明还涉及重组宿主细胞，其包含本发明的多核苷酸，所述多核苷酸有利地用于乙内酰脲酶多肽的重组产生中。包含本发明多核苷酸的载体被导入宿主细胞中，由此所述载体作为染色体整合体或作为自身复制染色体外载体被保留。术语“宿主细胞”还涵盖了亲代细胞的任何后代，其由于在复制期间发生的突变而与亲代细胞不相同。宿主细胞的选择很大程度依赖于编码多肽的基因及其来源。
还涉及“转化的细胞”或“重组细胞”，其是通过重组DNA技术已经向其中(或其祖细胞中)导入了本发明核酸的(宿主)细胞。
包括原核细胞和真核细胞，例如细菌、真菌、酵母等。特别地，可用作宿主细胞的微生物是细菌细胞(如革兰氏阳性或革兰氏阴性细菌，包括但不限于芽孢杆菌如枯草芽孢杆菌，链霉菌(Streptomyces sp.)，大肠杆菌，假单胞菌(Pseudomonas sp.)，鼠伤寒沙门氏菌(Salmonella typhimurium)，棒状杆菌)，以及放线菌，真菌细胞如酵母，动物细胞和植物细胞。
将载体导入细菌宿主细胞中可，例如，通过如下方式实现：原生质体转化(见例如Chang and Cohen,1979,Molecular General Genetics 168:111-115)，使用感受态细胞(见例如Young and Spizizen,1961,Journal of Bacteriology 81:823-829或者Dubnauand Davidoff-Abelson,1971,Journal of Molecular Biology 56:209-221)，电穿孔(见例如Shigekawa and Dower,1988,Biotechniques 6:742-751)，或者缀合(见例如Koehlerand Thorne,1987,Journal of Bacteriology 169:5771-5278)。
下列实施例提供了关于用于鉴定和分离本发明修饰的乙内酰脲酶的方法的其它细节。本发明不限于所述实施例的描述。
实施例
实施例1:乙内酰脲酶的克隆和表达
将具有SEQ ID NO:1所示氨基酸序列的节杆菌(Arthrobacter sp.)乙内酰脲酶(HyuH)提供给pOM18载体。为了进一步处理，需要具有正确方向的HyuH基因亚克隆。NdeI和HindIII用于双消化pOM18载体。对载体骨架、来自pAS6的carbA基因(氨甲酰水解酶)和来自pOM18的HyuH基因进行凝胶提取PCR(gel extracted PCR)。所有这三种凝胶提取的产物均经柱纯化(Machery Nagel)、连接及随后转化大肠杆菌DH5α。将克隆在LB-amp上生长过夜，使用来自carbA的基因特异性正向引物及反向引物对培养物进行PCR以检查正确方向。获得的质粒pAS6-HyuH含有表达具有SEQ ID NO:1所示氨基酸序列的蛋白质的野生型乙内酰脲酶基因，及表达具有SEQ ID NO:4所示氨基酸序列的蛋白质的氨甲酰水解酶基因。制备想要的克隆的质粒，并在含有pOM21c(表达乙内酰脲消旋酶的质粒)的JM109细胞中转化，以进行表达及活性检查。对于表达分析，将预培养物在LB培养基(具有合适抗生素)中在37℃生长过夜。将10μl预培养物加入3ml诱导培养基(LB培养基+2mg/ml鼠李糖)中。将培养物在30℃生长20小时。对5μl的细胞进行SDS PAGE分析。使用基于荧光的筛选测定检查酶活性。
实施例2：乙内酰脲酶的突变体
确定野生型节杆菌乙内酰脲酶(SEQ ID NO:1)内的一些位点以进行诱变。突变体文库通过在选择的氨基酸位置进行饱和诱变而制备。然后使用高通量筛选试验针对提高的活性筛选合理的突变体文库。选择用于位点饱和的5个位点(Y72、I95、V154、P155、H316)，及用于诱变的2个位点(A152G、S153G)。对于确定的氨基酸位置，进行5个位点饱和诱变和1个定点诱变。
实施例2.1：构建聚焦(focused)突变体文库(SSM&SDM)
Phusion聚合酶用于位点饱和诱变(ssm)和定点诱变(sdm)，所述ssm和sdm使用在实施例1中获得的表达野生型乙内酰脲酶的质粒(pAS6-HyuH；图1)或者在随后的步骤中获得的携带所述乙内酰脲酶突变体的质粒。
对于位点饱和/定点诱变，应用一种两步PCR方法以产生聚焦的突变体文库。将PCR产物经柱纯化并进行DpnI消化过夜以除去亲代质粒。然后将代表具有pAS6-HyuH所有元件的全长质粒的产物在含有pOM21c的JM109细胞中转化。
详细地，根据两步PCR方案进行位点饱和/定点诱变(SSM)。50μl PCR反应混合物含有1ng/μl质粒模板、1×Phusion缓冲液(New England Biolabs,Frankfurt,Germany)、1U的Phusion聚合酶及300μM dNTP。将反应混合物分成两份等体积，将进行各自氨基酸位置位点饱和的400nM正向引物和反向引物(见表1)加入分开的反应混合物中。PCR程序是98℃进行45秒(1个循环)；98℃进行15秒、65℃进行30秒、72℃进行3分30秒(5个循环)；72℃进行5分钟(1个循环)。在第一个步骤之后，集合这两个反应物，进行相同程序，再重复18次。
表1概括了用于所述PCR方法的引物(F＝正向引物；R＝反向引物)，也示出了被修饰的SEQ ID NO:1的氨基酸位置。
实施例2.2：构建节杆菌乙内酰脲酶(HyuH)的随机突变体文库
为了产生epPCR突变体文库，使用MnCl2产生易错条件。PCR最初是使用各种MnCl2浓度进行(0.05mM、0.1mM、0.2mM、0.5mM)。反应混合物含有0.3ng/μl质粒模板(使用在实施例1中获得的表达野生型乙内酰脲酶质粒[pAS6-HyuH；图1]或者在随后研究阶段获得的携带获得的所述乙内酰脲酶突变体的质粒)、 Taq缓冲液(SeSaM-Biotech GmbH,Bremen,Germany)、1U的 Taq聚合酶、200μM dNTP和250nM如表1所示的HyuH_GS_F及HyuH_R引物，各连同各种MnCl2浓度。PCR程序是94℃进行2分钟(1个循环)；94℃进行30秒、56℃进行30秒、72℃进行50秒(25个循环)；72℃进行5分钟(1个循环)。方案(Mundhada et al.“SeSaM-Tv-II generates a protein sequence space that isunobtainable by epPCR”Chembiochem.2011Jul 4；12(10):1595-601)用于构建基于颠换的突变体文库。将PCR产物经柱纯化，并进行Megawhop以克隆。50μl PCR反应混合物含有1ng/μl质粒模板、1×Phusion缓冲液(New England Biolabs,Frankfurt,Germany)、1U的Phusion聚合酶、300μM dNTP和5ng/μl来自epPCR或的突变的模板。Megawhop的PCR程序是72℃进行3分钟(1个循环)；98℃进行45秒(1个循环)；98℃进行15秒、62℃进行30秒、72℃进行5分钟(25个循环)；72℃进行5分钟(1个循环)。然后将PCR混合物经柱纯化，并进行DpnI(10U)消化过夜。代表具有pAS6-HyuH所有元件的全长质粒的PCR产物在化学感受态JM109细胞(Promega,Mannheim,Germany)中转化以进行蛋白质表达及随后的分析。
实施例2.3：构建聚焦的Omnichange突变体文库
Omnichange方案使得可以在序列中的远程位置进行多位点饱和诱变。这种方案进行四个步骤，从使用含有NNK-密码子和硫代磷酸二酯键的引物通过PCR产生片段开始，然后使用碘元素进行DNA裂解反应以产生互补的5’突出端，第三步是DNA-杂交，最后是转化和缺口(nick)修复。
详细地，50μl PCR反应混合物含有0.5ng/μl质粒模板、1×Phusion缓冲液(NewEngland Biolabs.Frankfurt.Germany)、5U的Phusion聚合酶、200μM dNTP’s及400μM片段特异性正向和反向引物。针对短片段的PCR程序是94℃进行3分钟(1个循环)；94℃进行30秒、55℃进行30秒、72℃进行30秒(25个循环)；72℃进行1分钟(1个循环)。对于载体片段，PCR程序改变为在94℃进行3分钟(1个循环)；94℃进行30秒、55℃进行30秒、72℃进行1.5分钟(25个循环)；72℃进行3分钟(1个循环)。然后将此PCR混合物进行柱纯化，经DpnI(10U)消化过夜、柱纯化并稀释至0.05pmol/μl。根据原始Omnichange方案(Dennig et al.“OmniChange:The Sequence Independent Method for Simultaneous Site-Saturationof Five Codons”PLoS ONE,2011,6(10):e26222)进行碘裂解和DNA-片段杂交。然后将杂交产物在化学感受态JM109细胞(Promega,Mannheim,Germany)中转化以进行蛋白质表达及随后的分析。
实施例3：针对微滴定平板(MTP)的基于荧光的筛选测定
预培养和表达：使用牙签将阳性和阴性对照以及获得的克隆接种在96孔微滴定平板(MTP)中的含有合适抗生素的100μl的LB培养基。将平板在37℃(900rpm)温育过夜。为了制备筛选平板，使用96-孔复制器，将克隆移至具有200μl/孔具有抗生素和2mg/ml鼠李糖的培养基的新的MTP中。为了保持平板的材料，加入100μL的50％甘油(w/w)，简单混合，将平板在-80℃贮存。
生物转化：在30℃(900rpm)温育过夜后，向筛选平板加入100μL/孔均质的L-或D-色氨酸乙内酰脲悬浮液(5mg/mL于PBS缓冲液中，pH 7.4)。将该平板在30℃和900rpm温育，在不同时间点使用合适的平板分析仪在300/350nm测量荧光。
实施例4：结果
使用实施例3所述的荧光测定，筛选得自实施例2的位点饱和诱变实验和随机诱变文库的突变体乙内酰脲酶的活性。读出数涉及在40分钟与60分钟之间的不同测量。每个测量均一式三份进行。如上所述，分别使用L-5-吲哚基甲基乙内酰脲(＝L-色氨酸乙内酰脲)和D-5-吲哚基甲基乙内酰脲(＝D-色氨酸乙内酰脲)作为底物，测定突变体乙内酰脲酶的活性，并将其活性与具有SEQ ID NO:1所示氨基酸序列的野生型乙内酰脲酶的活性相比较。
在表2中，对获得结果进行了概括。如表中示出，获得一系列改良的突变体，它们与由微生物节杆菌产生的具有SEQ ID NO:1所示氨基酸序列的野生型乙内酰脲酶相比表现更高的催化活性和/或更高的对映体选择性。
在表2中，“诱变循环”一栏表示突变体从何种类型诱变产生(SSM＝位点饱和诱变；epPCR＝易错PCR；combi SSM＝组合的位点饱和诱变，在一个以上氨基酸位置的诱变)。在称作“取代”的一栏中，指出在SEQ ID NO:1氨基酸序列中在指定位置的氨基酸残基的取代。
称作“D-Trp-hyd与HyuH wt的活性比率”的一栏表示使用D-5-吲哚基甲基乙内酰脲(＝D-色氨酸乙内酰脲)作为底物，相对于野生型乙内酰脲酶的催化活性的提高倍数。称作“L-Trp-hyd与HyuH wt活性比率”的一栏表示使用L-5-吲哚基甲基乙内酰脲(＝L-色氨酸乙内酰脲)作为底物，相对于野生型乙内酰脲酶的活性的提高倍数。
“D-Trp-hyd与L-Trp-hyd的活性比率”一栏表示突变体乙内酰脲酶转化D-5-吲哚基甲基乙内酰脲的催化活性与突变体乙内酰脲酶转化L-5-吲哚基甲基乙内酰脲的催化活性的比率，说明对于任一对映异构体的过度偏爱倍数(fold excess preference)。换句话说，“D-Trp-hyd与L-Trp-hyd的活性比率”一栏表示各个乙内酰脲酶的对映体选择性。
本发明的乙内酰脲酶显示对于5-取代的乙内酰脲化合物，特别是D-色氨酸乙内酰脲的增强的转化率。因此，本文描述的本发明的乙内酰脲酶突变体被认为适用于生产D-氨基酸，特别是D-色氨酸的方法中，所述方法从D-色氨酸乙内酰脲开始产生，使用本发明的乙内酰脲酶和D-氨甲酰水解酶进行。
表1：引物及其序列表。引物名称表示针对聚焦的突变体文库的靶位点
表2:所有突变体的活性和选择性概括
序列表
<110> 赢创工业化学有限公司
<120> 乙内酰脲酶的突变体
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Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
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Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
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Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
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Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
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Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
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Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
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Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
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Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
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His Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His
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Leu Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr
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Asp Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg
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Trp Pro Glu Thr Trp Arg Val Met Gly Leu Lys Gly Ala Glu Ile Ile
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Cys Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His
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Asp His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser
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Glu Gly Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly
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Glu Ile Val Ala Leu Thr Thr Thr Leu Glu Asp Glu Val Ile Thr Ala
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Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
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Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
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Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
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Asp Met Asp Leu Lys Asn Xaa Xaa Gly Arg Phe Glu Leu Asp Ser Glu
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Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Xaa Ser Xaa Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Xaa Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
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195 200 205
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210 215 220
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225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Xaa Xaa Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Xaa Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg Xaa Asp Tyr Glu Ala Ser Lys
450 455
<210> 6
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by G
<400> 6
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Gly Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 7
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by C
<400> 7
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Cys Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 8
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 8
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 9
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by T
<400> 9
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Thr Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 10
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by V
<400> 10
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Val Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 11
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<220>
<221> VARIANT
<222> (154)..(154)
<223> V replaced by I
<400> 11
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Ile Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 12
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by V
<220>
<221> VARIANT
<222> (154)..(154)
<223> V replaced by C
<400> 12
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Val Ser Cys Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 13
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by C
<220>
<221> VARIANT
<222> (154)..(154)
<223> V replaced by S
<400> 13
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Cys Ser Ser Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 14
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by D
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by H
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 14
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 15
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by D
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by N
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 15
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 16
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by V
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by N
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 16
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Val Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 17
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by S
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by N
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by C
<220>
<221> VARIANT
<222> (154)..(154)
<223> V replaced by S
<220>
<221> VARIANT
<222> (181)..(181)
<223> V replaced by A
<400> 17
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Ser Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Cys Ser Ser Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Ala Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 18
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by Y
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by N
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 18
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Tyr Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 19
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by L
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by C
<220>
<221> VARIANT
<222> (154)..(154)
<223> V replaced by S
<400> 19
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Leu Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Cys Ser Ser Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 20
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<220>
<221> VARIANT
<222> (71)..(71)
<223> R replaced by Y
<220>
<221> VARIANT
<222> (72)..(72)
<223> Y replaced by H
<220>
<221> VARIANT
<222> (152)..(152)
<223> A replaced by S
<400> 20
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Tyr His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 21
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic peptide
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 21
atctgaagaa ccggnnkggc cgcttcgaac t 31
<210> 22
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 22
agttcgaagc ggccmnnccg gttcttcaga t 31
<210> 23
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 23
catcgagatg ccgnnkacct tcccgcccac 30
<210> 24
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 24
gtgggcggga aggtmnncgg catctcgatg 30
<210> 25
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 25
tgatggcagc ctcannkccg ggcatgttcg 30
<210> 26
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 26
cgaacatgcc cggmnntgag gctgccatca 30
<210> 27
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 27
tggcagcctc agttnnkggc atgttcgacg 30
<210> 28
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 28
gcgtcgaaca tgccmnnaac tgaggctgcc a 31
<210> 29
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 29
cagcctcagt tccgnnkcag cctcagttcc g 31
<210> 30
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 30
cggaactgag gctgmnncgg aactgaggct g 31
<210> 31
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 31
cccttgggtc agacnnkggc ggacatcctg 30
<210> 32
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 32
caggatgtcc gccmnngtct gacccaaggg 30
<210> 33
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 33
cgtatatgaa ggtcnnkccg cccgtccgct ca 32
<210> 34
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 34
tgagcggacg ggcggmnnga ccttcatata cg 32
<210> 35
<211> 29
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 35
gaccgtatat gaagnnkgcg ccgcccgtc 29
<210> 36
<211> 29
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 36
gacgggcggc gcmnncttca tatacggtc 29
<210> 37
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 37
caccatcatc gagatgnnka taaccttccc g 31
<210> 38
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 38
cgggaaggtt atmnncatct cgatgatggt g 31
<210> 39
<211> 33
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 39
tgccgataac cttcnnkccc accaccactt tgg 33
<210> 40
<211> 33
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 40
ccaaagtggt ggtgggmnng aaggttatcg gca 33
<210> 41
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 41
cgataacctt cccgnnkacc accactttgg ac 32
<210> 42
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 42
gtccaaagtg gtggtmnncg ggaaggttat cg 32
<210> 43
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 43
ggatctgaag aacnnktatg gccgcttcga ac 32
<210> 44
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 44
gttcgaagcg gccatamnng ttcttcagat cc 32
<210> 45
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 45
caagtcaatg atggcannkt cagttccggg c 31
<210> 46
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 46
gcccggaact gamnntgcca tcattgactt g 31
<210> 47
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 47
cagttccggg catgnnkgac gccgtcagcg ac 32
<210> 48
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 48
gtcgctgacg gcgtcmnnca tgcccggaac tg 32
<210> 49
<211> 37
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (20)..(20)
<223> n is a, c, g, or t
<400> 49
catggatctg aagaacndtn dtggccgctt cgaactc 37
<210> 50
<211> 37
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, c, g, or t
<400> 50
gagttcgaag cggccahnah ngttcttcag atccatg 37
<210> 51
<211> 44
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, or t
<400> 51
gcttcaagtc aatgatggca ndttcandtc cgggcatgtt cgac 44
<210> 52
<211> 44
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, or t
<400> 52
gtcgaacatg cccggahntg aahntgccat cattgacttg aagc 44
<210> 53
<211> 35
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(16)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, c, g, or t
<400> 53
ggaccgtata tgaagndtnd tccgcccgtc cgctc 35
<210> 54
<211> 35
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (20)..(20)
<223> n is a, c, g, or t
<400> 54
gagcggacgg gcggahnahn cttcatatac ggtcc 35
<210> 55
<211> 33
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 55
gaacatgtgc atatcnnkga catggatctg aag 33
<210> 56
<211> 33
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 56
cttcagatcc atgtcmnnga tatgcacatg ttc 33
<210> 57
<211> 22
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 57
tcacttcgac gcctcgtagt cg 22
<210> 58
<211> 29
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 58
atgtttgacg taatagttaa gaactgccg 29
<210> 59
<211> 42
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 59
gtgtgatggc gtgaggcagc ctactgccgc caggcaaatt ct 42
<210> 60
<211> 40
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 60
cacactaccg cactccgtcg cgtcagccac agcactacgg 40
<210> 61
<211> 30
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 61
cgtatggtgt ccnnkgacgg aatcaccgag 30
<210> 62
<211> 31
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 62
ggacaccata cggcagttct taactattac g 31
<210> 63
<211> 29
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 63
agctcggaca cannkgatgt tgaggcgag 29
<210> 64
<211> 22
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 64
tgtgtccgag ctgattgcgg cg 22
<210> 65
<211> 38
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, c, g, or t
<400> 65
gtcaatgatg gcaagtndtd nyccgggcat gttcgacg 38
<210> 66
<211> 38
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22)..(22)
<223> n is a, c, g, or t
<400> 66
cgtcgaacat gcccggrnha hnacttgcca tcattgac 38
<210> 67
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, c, g, or t
<400> 67
ccgtatggtg tccbbtnnkg gaatcaccga gg 32
<210> 68
<211> 32
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 68
cctcggtgat tccmnnavvg gacaccatac gg 32
<210> 69
<211> 34
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 69
gaacatgtgc atatcatcga catggatctg aaga 34
<210> 70
<211> 34
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 70
tcttcagatc catgtcgatg atatgcacat gttc 34
<210> 71
<211> 34
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 71
caagtcaatg atggcagcct cagttccggg catg 34
<210> 72
<211> 34
<212> DNA
<213> Artificial
<220>
<223> synthetic DNA
<400> 72
catgcccgga actgaggctg ccatcattga cttg 34
<210> 73
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 73
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro His Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 74
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 74
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Gly Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 75
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 75
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Asn Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 76
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 76
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 77
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 77
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Glu Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 78
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 78
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Phe Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 79
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 79
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Ala Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 80
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 80
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Thr
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 81
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 81
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Val
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 82
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 82
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Ser Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 83
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 83
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Ala Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Ala Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg Leu Asp Tyr Glu Ala Ser Lys
450 455
<210> 84
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 84
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Arg Tyr Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Ile Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 85
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 85
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 86
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 86
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Val Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 87
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 87
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Val Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Cys Ser Ser Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Ala Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 88
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 88
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 89
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 89
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Tyr Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 90
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 90
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Ile Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 91
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 91
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Tyr Asn Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 92
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 92
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 93
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 93
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Leu
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 94
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 94
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Val Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 95
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 95
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 96
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 96
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Arg Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 97
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 97
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Ala Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 98
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 98
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Arg Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Lys Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 99
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 99
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Arg Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Ser Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Arg Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 100
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 100
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asp Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 101
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 101
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Ala Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 102
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 102
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 103
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 103
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ala Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 104
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 104
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Val Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 105
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 105
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 106
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 106
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ala Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Trp Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 107
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 107
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ala Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Val Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 108
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 108
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asn Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 109
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 109
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Val Asp Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Arg Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 110
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 110
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asn Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Cys Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 111
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 111
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Asn Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Ser Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Ile Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 112
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 112
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Pro Gly Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 113
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 113
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Arg Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 114
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 114
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Gly Gln Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 115
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 115
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Phe Ala Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 116
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 116
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Ser Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 117
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 117
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Pro Gly Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Ile
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ser Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 118
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 118
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Pro Gly Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455
<210> 119
<211> 458
<212> PRT
<213> Artificial
<220>
<223> mutant sequence
<400> 119
Met Phe Asp Val Ile Val Lys Asn Cys Arg Met Val Ser Ser Ser Gly
1 5 10 15
Ile Thr Glu Ala Asp Ile Leu Val Lys Asp Gly Lys Val Ala Ala Ile
20 25 30
Ser Ser Asp Thr Gly Asp Val Glu Ala Ser Arg Thr Ile Asp Ala Gly
35 40 45
Gly Lys Phe Val Met Pro Gly Val Val Asp Glu His Val His Ile Cys
50 55 60
Asp Met Asp Leu Lys Asn Asp His Gly Arg Phe Glu Leu Asp Ser Glu
65 70 75 80
Ser Ala Ala Val Gly Gly Ile Thr Thr Ile Ile Glu Met Pro Ile Thr
85 90 95
Phe Pro Pro Thr Thr Thr Leu Asp Ala Phe Leu Glu Lys Lys Lys Gln
100 105 110
Ala Gly Gln Arg Leu Lys Val Asp Phe Ala Leu Tyr Gly Gly Gly Val
115 120 125
Pro Gly Asn Leu Pro Glu Ile Arg Lys Met His Asp Ala Gly Ala Val
130 135 140
Gly Phe Lys Ser Met Met Ala Ala Ser Val Pro Gly Met Phe Asp Ala
145 150 155 160
Val Ser Asp Gly Glu Leu Phe Glu Ile Phe Gln Glu Ile Ala Ala Cys
165 170 175
Gly Ser Val Val Val Val His Ala Glu Asn Glu Thr Ile Ile Gln Ala
180 185 190
Leu Gln Lys Gln Ile Lys Ala Ala Gly Arg Lys Asp Met Ala Ala Tyr
195 200 205
Glu Ala Ser Gln Pro Val Phe Gln Glu Asn Glu Ala Ile Gln Arg Ala
210 215 220
Leu Leu Leu Gln Lys Glu Ala Gly Cys Arg Leu Ile Val Leu His Val
225 230 235 240
Ser Asn Pro Asp Gly Val Glu Leu Ile His Gln Ala Gln Ser Glu Gly
245 250 255
Gln Asp Val His Cys Glu Ser Gly Pro Gln Tyr Leu Asn Ile Thr Thr
260 265 270
Asp Asp Ala Glu Arg Ile Gly Pro Tyr Met Lys Val Ala Pro Pro Val
275 280 285
Arg Ser Ala Glu Met Asn Val Arg Leu Trp Glu Gln Leu Glu Asn Gly
290 295 300
Leu Ile Asp Thr Leu Gly Ser Asp His Gly Gly His Pro Val Glu Asp
305 310 315 320
Lys Glu Pro Gly Trp Lys Asp Val Trp Lys Ala Gly Asn Gly Ala Leu
325 330 335
Gly Leu Glu Thr Ser Leu Pro Met Met Leu Thr Asn Gly Val Asn Lys
340 345 350
Gly Arg Leu Ser Leu Glu Arg Leu Val Glu Val Met Cys Glu Lys Pro
355 360 365
Ala Lys Leu Phe Gly Ile Tyr Pro Gln Lys Gly Thr Leu Gln Val Gly
370 375 380
Ser Asp Ala Asp Leu Leu Ile Leu Asp Leu Asp Ile Asp Thr Lys Val
385 390 395 400
Asp Ala Ser Gln Phe Arg Ser Leu His Lys Tyr Ser Pro Phe Asp Gly
405 410 415
Met Pro Val Thr Gly Ala Pro Val Leu Thr Met Val Arg Gly Thr Val
420 425 430
Val Ala Glu Lys Gly Glu Val Leu Val Glu Gln Gly Phe Gly Gln Phe
435 440 445
Val Thr Arg His Asp Tyr Glu Ala Ser Lys
450 455

Claims (14)

1.由选自(i)或(ii)的氨基酸序列组成的乙内酰脲酶：
(i)氨基酸序列，其选自：SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:73、SEQID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:86、SEQID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118或SEQ ID NO:119；
(ii)氨基酸序列，其中在SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:73、SEQID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:86、SEQID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118或SEQ ID NO:119的氨基酸序列中，个氨基酸残基已经被取代、删除、插入和/或添加，
其中定义为
以及其中进一步，当使用D-5-吲哚基甲基乙内酰脲作为底物时，所述乙内酰脲酶的催化活性比由SEQ ID NO:1组成的氨基酸序列的乙内酰脲酶的催化活性高至少ζ倍，
以及其中ζ定义为ζ＝1.2。
2.权利要求1的乙内酰脲酶，其中选自20、10、9、8、7、6、5、4、3、2、1。
3.权利要求1的乙内酰脲酶，其中ζ选自1.5、2、3、4、5、10、15、20、25或30。
4.权利要求1-3任一项的乙内酰脲酶，其中所述乙内酰脲酶将如下底物中的至少一种转化为相应的氨基甲酰氨基酸：乙内酰脲、5-甲基乙内酰脲、5-苯甲基乙内酰脲、5-(4-羟基苯甲基)乙内酰脲、5-(3,4-二羟基苯甲基)乙内酰脲、5-甲基硫代乙基乙内酰脲、5-异丙基乙内酰脲、5-异丁基乙内酰脲、5-仲-丁基乙内酰脲、5-(4-氨基丁基)乙内酰脲、5-羟基甲基乙内酰脲或者对应于非天然氨基酸的5-取代的乙内酰脲化合物、其相应的衍生物，其中所述乙内酰脲酶在转化所述底物方面的催化活性至少与由SEQ ID NO:1组成的氨基酸序列的乙内酰脲酶在转化D-5-吲哚基甲基乙内酰脲方面的催化活性一样高。
5.权利要求1-4任一项的乙内酰脲酶，其中对D-5-吲哚基甲基乙内酰脲的对映体选择性比由SEQ ID NO:1组成的氨基酸序列的乙内酰脲酶的对映体选择性高至少1.2、1.5、2、3、4、5、10、15、20、25、30、50、100或150倍。
6.编码权利要求1-5任一项的乙内酰脲酶的多核苷酸。
7.包含权利要求6的多核苷酸的载体。
8.分离的或转化的宿主细胞，其包含权利要求6的多核苷酸或者权利要求7的载体。
9.权利要求8的宿主细胞，其表达至少选自以下一组的酶活性：乙内酰脲酶；乙内酰脲酶和乙内酰脲消旋酶；乙内酰脲酶和氨甲酰水解酶；或者乙内酰脲酶和乙内酰脲消旋酶及氨甲酰水解酶。
10.制备氨基酸的方法，其包括如下步骤：
(a)向反应介质提供权利要求1-5任一项的乙内酰脲酶的乙内酰脲酶活性及氨甲酰水解酶活性和至少一种5-取代的乙内酰脲；
(b)温育所述反应介质，以使得通过在(a)中提供的酶活性将5-取代的乙内酰脲转化为相应的氨基酸；及
(c)从所述反应介质中回收得自相应的5-取代的乙内酰脲的酶促转化的氨基酸，
其中所述5-取代的乙内酰脲选自D,L-5-吲哚基甲基乙内酰脲和D-5-吲哚基甲基乙内酰脲。
11.制备氨基酸的方法，其包括如下步骤：
(a)向反应介质提供权利要求1-5任一项的乙内酰脲酶的乙内酰脲酶活性及氨甲酰水解酶活性和至少一种5-取代的乙内酰脲，及向所述反应介质提供乙内酰脲消旋酶活性；
(b)温育所述反应介质，以使得通过在(a)中提供的酶活性将5-取代的乙内酰脲转化为相应的氨基酸；及
(c)从所述反应介质中回收得自相应的5-取代的乙内酰脲的酶促转化的氨基酸，
其中所述5-取代的乙内酰脲选自D-5-吲哚基甲基乙内酰脲、L-5-吲哚基甲基乙内酰脲和D,L-5-吲哚基甲基乙内酰脲。
12.权利要求10或11的方法，其中要制备的氨基酸是D-色氨酸，及其中5-取代的乙内酰脲选自D-5-吲哚基甲基乙内酰脲、L-5-吲哚基甲基乙内酰脲或者D,L-5-吲哚基甲基乙内酰脲。
13.权利要求12的方法，其中所述反应介质是部分或全部由细胞培养基组成的介质，其中乙内酰脲酶活性由权利要求8或9的宿主细胞提供，以及其中所述宿主细胞在所述反应介质中培养。
14.权利要求10或11的方法，其中所述反应介质是部分或全部由细胞培养基组成的介质，所述氨甲酰水解酶活性由权利要求8或9的宿主细胞提供，以及其中所述宿主细胞在所述反应介质中培养。
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