CN116113641A - 新型内－β－N－乙酰氨基葡萄糖苷酶 - Google Patents

新型内－β－N－乙酰氨基葡萄糖苷酶 Download PDF

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CN116113641A
CN116113641A CN202180054259.2A CN202180054259A CN116113641A CN 116113641 A CN116113641 A CN 116113641A CN 202180054259 A CN202180054259 A CN 202180054259A CN 116113641 A CN116113641 A CN 116113641A
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西泽华子
小野泰典
岩本充广
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Daiichi Sankyo Co Ltd
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Abstract

本发明提供从属于海豚链球菌的菌株中克隆的内－β－N－乙酰氨基葡萄糖苷酶(Endo－Si)及其突变酶、编码该酶的基因、重组质粒、由该质粒转化而成的转化体及其用途、以及使用该酶的糖链重构抗体等的制造方法等。一种多肽，其具有：序列号2的第34～928位所记载的氨基酸序列；或在所述氨基酸序列中，存在包含对选自由第241位(D241)、第190位(T190)、第311位(Q311)以及第360位(E360)构成的组中的一个或两个以上氨基酸位点的突变的氨基酸序列，并且，所述多肽显示水解活性和/或糖链转移活性。

Description

新型内-β-N-乙酰氨基葡萄糖苷酶
技术领域
本发明涉及内－β－N－乙酰氨基葡萄糖苷酶(Endo－Si)、编码该酶的基因、重组质粒、由该质粒转化而成的转化体及其用途、以及使用了该酶的糖链重构抗体等的制造方法等。
背景技术
抗体是具有与位于重链分子中的Fc区的第297位的Asn侧链连接的N连接型糖链(N297连接糖链)的糖蛋白分子。抗体在基础研究、医疗领域中是重要的分子，特别是作为抗体医药的研究开发正在蓬勃开展，糖链的各种影响正在逐渐明确(非专利文献1)。目前，主要使用的医疗用抗体是IgG类的分子，这样的抗体通常使用以CHO细胞、NS0细胞为代表的培养动物细胞而产生，在这些动物细胞中产生的抗体的N297连接糖链是两天线(biantennary)复合型糖链，但会获取在核心岩藻糖、末端的唾液基、半乳糖基以及平分(bisecting)GlcNAc中不均匀的糖链(非专利文献2)。明确了抗体的N297连接糖链对包含抗体的ADCC活性(Antibody－Dependent Cell－Mediated Cytotoxicity：抗体依赖性细胞毒活性)、CDC活性(Complement－Dependent Cytotoxicity：补体依赖性细胞毒活性)的效应物(effector)活性造成大的影响(非专利文献3、非专利文献4)，指出有可能对抗体的血浆半衰期也造成影响(非专利文献5)。此外，明确了N297连接糖链的非还原末端经2，6－唾液酸基化的抗体是IVIG(intravenous immunoglobulin：静脉注射免疫球蛋白)中的主要药效成分(非专利文献6)。此外，认为在IgG、包含Fc片段的医疗用分子中，N297连接糖链的不均匀性对作为有效成分的性质、品质产生大的影响，无法否定不均匀的糖链修饰分子的微量混入会大幅改变最终产物的特性的可能性。
根据这样的现状，在医疗用的抗体、包含抗体Fc区的糖蛋白分子的制造中，正在开发使糖链均匀化的技术。作为使添加于糖蛋白的糖链均匀的方法，已知使用酶的糖链转移反应(非专利文献7～9)。这是由体外环境下的糖链的切断(水解反应)和其他糖链的缩合(糖链转移反应)构成的多阶段工艺。特别是在以N型糖链的转换为目的的情况下，使用被称为内－β－N－乙酰氨基葡萄糖苷酶(ENGase)的一组酶家族。作为该酶的特性，要求1)作为底物特异性具有对复合型糖链进行水解反应的能力；以及2)具有对特定的结构进行糖链转移反应的能力。在糖链转移反应中，已知使用单独的ENGase将还原末端经噁唑啉化的糖链转移至GlcNAc(N－乙酰葡糖胺)受体的方法(非专利文献7～8)和使用两种ENGase将糖链直接转移至GlcNAc受体的一锅法(非专利文献9、专利文献1)。ENGase从各种生物种中分离，根据作为底物的糖链的种类而区分使用野生型、其突变酶。
作为ENGase，已知Endo－A(源自原玻璃蝇节杆菌(Arthrobacter protophormiae)的酶)(非专利文献10)、Endo－D(源自肺炎链球菌(Streptococcus pneumoniae)的酶)(非专利文献11)、Endo－M(源自冻土毛霉(Mucor hiemalis)的酶)(非专利文献12)、Endo－H(非专利文献13)、Endo－F2(源自脑膜脓毒性黄杆菌(Flavobacterium meningosepticum)的酶)、Endo－F3(源自脑膜脓毒性黄杆菌的酶)(非专利文献14)、Endo－E(源自粪肠球菌(Enterococcus faecalis)的酶)(非专利文献15)、Endo－S(源自酿脓链球菌(Streptococcus pygenes)的酶)(非专利文献16)、Endo－Tsp1006(源自坦氏菌(Tannerella)属细菌)、Endo－Tsp1263(源自坦氏菌属细菌)、Endo－Bno1263(源自拟杆菌(Bacteroides)属细菌)、Endo－Tsp1457(源自坦氏菌属细菌)、Endo－Bac1008(源自鼠尾草菌(Muribaculum)属细菌)、Endo－Tsp1603(源自坦氏菌属细菌)、Endo－Tsp1263(源自坦氏菌属细菌)(专利文献2)、源自鞘鞍醇杆菌(Sphingobacterium)属细菌的内－β－N－乙酰氨基葡萄糖苷酶(ORF1152)、源自鞘鞍醇杆菌属细菌的内－β－N－乙酰氨基葡萄糖苷酶(ORF1188))、源自鞘鞍醇杆菌属细菌的内－β－N－乙酰氨基葡萄糖苷酶(ORF3046)、源自鞘鞍醇杆菌属细菌的内－β－N－乙酰氨基葡萄糖苷酶(ORF3750)、源自虫草菌(Cordyceps)属线形菌的内－β－N－乙酰氨基葡萄糖苷酶、源自白僵菌(Beauveria)属线形菌的内－β－N－乙酰氨基葡萄糖苷酶(非专利文献17)、Endo－CC1(源自灰盖鬼伞(Coprinopsiscinerea)的酶)、Endo－CC2(源自灰盖鬼伞的酶)(非专利文献18)、Endo－Om(源自Ogataeaminuta的酶)(非专利文献19)、Endo－CE(源自秀丽隐杆线虫(Caenorhabditis elegans)的酶)(非专利文献20)、Endo－BH(源自耐盐芽孢杆菌(Bacillus halodurans)C－125的酶)(非专利文献21)、EndoSd(源自停乳链球菌(Streptococcus dysgalactiae)的酶)、EndoS2d(源自停乳链球菌的酶)(非专利文献22)、EndoSe(源自马链球菌兽疫亚种(Streptococcusequi subsp.equi)的酶)(非专利文献23)、Endo－Rp(源自微小根毛霉(Rhizomucorpusillus)的酶)(专利文献3)、EndoS2或EndoS49(非专利文献24)等。
其中，作为以抗体的具有核心岩藻糖的复合型的N297连接糖链为底物、能确认兼具水解活性和糖链转移活性的酶，已知EndoS(非专利文献25)、EndoS2(非专利文献26)、Endo－F3(非专利文献27)。
已知在EndoS中，作为其突变酶的EndoS D233Q在一定程度上抑制水解活性。已知该突变酶在反应体系中大量存在将糖链的还原末端噁唑啉化的中间体的条件下，选择性地进行糖链转移反应(专利文献4、非专利文献8)。
此外，已知通过在EndoS D233Q中追加并导入进一步的突变，与EndoS D233Q酶相比较，糖链转移活性变高、或者水解活性变低(专利文献5)。
已知与野生型EndoS2酶相比，EndoS2突变酶(D184Q)显示出增加的糖链转移活性和减少的水解活性(专利文献6～8、非专利文献28)。
已知与野生型Endo－F3酶相比，Endo－F3突变酶(D165A或D165Q)对产物的水解活性减少，糖噁唑啉的糖链转移活性变高。此外，Endo－F3突变酶可以使用两天线和三天线糖链噁唑啉作为糖链转移反应的底物(非专利文献27)。
作为将糖链直接转移至GlcNAc受体的一锅法中使用的ENGase，已知EndoS/EndoS突变酶与Endo－M/Endo－M突变酶或Endo－CC/Endo－CC突变酶这两种酶的组合(专利文献1、非专利文献9)。
已知海豚链球菌(Streptococcus iniae)(非专利文献29)为鱼的病原菌，在日本，在比目鱼、真鲷的养殖中受害大(非专利文献30)。因此，作为比目鱼β溶血性链球菌症失活疫苗，市售有M-Vac Iniae(松研药品工业株式会社)。作为对人、农林水产业产生严重影响的病原菌的链球菌属的菌株通过基因组分析进行的分类非常盛行(非专利文献31)，对于S.iniae也已知ENGase序列的存在，但没有研究酶活性(非专利文献24)。
现有技术文献
专利文献
专利文献1：WO2018/003983
专利文献2：JP2020－022440
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专利文献5：WO2017/010559
专利文献6：WO2017/124084
专利文献7：WO2018/039373
专利文献8：JP2020－500549
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发明内容
发明所要解决的问题
本发明的目的在于提供新型内－β－N－乙酰氨基葡萄糖苷酶，其具有对糖蛋白的N297连接糖链的水解活性和/或糖链转移活性。
用于解决问题的方案
本发明人等为了解决上述问题而反复进行了深入研究，结果发现了如下事实，从而完成了本发明，即，从属于海豚链球菌的菌株中克隆的内－β－N－乙酰氨基葡萄糖苷酶(Endo－Si)具有对N297连接糖链的水解活性，并且通过在Endo－Si中引入突变，水解活性进一步得到抑制且具有一定程度以上的糖链转移活性。
本发明提供以下的发明。
[1]一种多肽，其具有：序列号2的第34～928位所记载的氨基酸序列；或在所述氨基酸序列中，存在包含对选自由第241位(D241)、第190位(T190)、第311位(Q311)以及第360位(E360)的氨基酸构成的组中的一个或两个以上氨基酸位点的突变的氨基酸序列，并且，所述多肽显示糖链水解活性和/或糖链转移活性。
[2]根据[1]的多肽，其特征在于，对于突变，在选自由D241、T190、Q311以及E360的氨基酸构成的组中的1～3个氨基酸位点具有突变。
[3]根据[1]或[2]的多肽，其中，具有选自由以下的(A)～(D)构成的组中的一个或两个以上的突变，(A)：在序列号2的氨基酸序列中，第241位的氨基酸(D241)突变为谷氨酰胺(D241Q)、第241位的氨基酸(D241)突变为蛋氨酸(D241M)、或第241位的氨基酸(D241)突变为丙氨酸(D241A)；(B)：在序列号2的氨基酸序列中，第190位的氨基酸(T190)突变为谷氨酰胺(T190Q)；(C)：在序列号2的氨基酸序列中，第311位的氨基酸(Q311)突变为亮氨酸(Q311L)；以及(D)：在序列号2的氨基酸序列中，第360位的氨基酸(E360)突变为谷氨酰胺(E360Q)、第360位的氨基酸(E360)突变为丙氨酸(E360A)、第360位的氨基酸(E360)突变为天冬酰胺(E360N)、或第360位的氨基酸(E360)突变为天冬氨酸(E360D)。
[4]根据[1]～[3]中任一项的多肽，其中，具有选自由以下的(A)～(D)构成的组中的一个或两个以上的突变，(A)：D241Q或D241M；(B)：T190Q；(C)：Q311L；以及(D)：E360Q。
[5]根据[1]～[4]中任一项的多肽，其中，包含以下的(A)～(C)所述的氨基酸序列，(A)：选自由序列号3、序列号4、序列号5、序列号6、序列号7、序列号8、序列号9、序列号10以及序列号11构成的组中的氨基酸序列；(B)：与(A)的各序列中的第241位、第190位、第311位、或第360位的氨基酸以外的氨基酸序列具有至少90％以上的同源性或同一性的氨基酸序列；或(C)：在(A)的序列中第241位、第190位、第311位、或第360位的氨基酸以外的氨基酸序列中缺失、取代和/或添加了一个或数个氨基酸的氨基酸序列。
[6]根据[1]～[5]中任一项的多肽，其中，所述多肽对N连接型糖链显示水解活性和/糖链转移活性。
[7]根据[6]的多肽，其中，N连接型糖链是糖蛋白中的N连接型糖链。
[8]根据[6]或[7]的多肽，其中，糖蛋白是抗体或包含抗体的Fc区的分子(含Fc区的分子)。
[9]根据[6]～[8]中任一项的多肽，其中，N连接型糖链是与抗体的第297位的Asn连接的N连接型糖链(N297连接糖链)。
[10]根据[9]的多肽，其中，N297连接糖链是非还原末端任选地被化学修饰的复合型糖链。
[11]根据[9]或[10]的多肽，其中，N297连接糖链是在核心GlcNAc任选地添加岩藻糖的N297连接糖链。
[12]一种多核苷酸，其编码[1]～[11]中任一项的多肽。
[13]一种表达载体，其包含[12]的多核苷酸。
[14]一种宿主细胞，其通过[13]的表达载体而被转化。
[15]一种[1]～[11]中任一项的多肽的制造方法，其特征在于，包括：培养[14]的宿主细胞的工序；以及从该工序中得到的培养物中采集目标多肽的工序。
[16]一种多肽，其通过[15]的制造方法而得到。
[17]一种抗体或含其Fc区的分子的制造方法，其特征在于，在[1]～[11]中任一项的多肽的存在下，使受体分子与包含还原末端经活化的GlcNAc的糖链供体分子进行反应，其中，所述受体分子是具有任选地添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。
[18]根据[17]的制造方法，其中，还原末端经活化的GlcNAc是经噁唑啉化的GlcNAc。
[19]根据[17]或[18]的制造方法，其中，糖链供体分子是非还原末端任选地被化学修饰的复合型糖链。
[20]根据[17]～[19]中任一项的制造方法，其中，糖链供体分子是非还原末端任选地被化学修饰的SG(10)－Ox、MSG1(9)－Ox、MSG2(9)－Ox或MSG1(9)－Ox与MSG2(9)－Ox的混合物。
[21]根据[17]～[20]中任一项的制造方法，其中，糖链供体分子是[N3－PEG(3)]2－SG(10)－Ox、[N3－PEG(3)]－MSG1(9)－Ox、[N3－PEG(3)]－MSG2(9)－Ox、或[N3－PEG(3)]－MSG1(9)－Ox与[N3－PEG(3)]－MSG2(9)－Ox的混合物。
[22]根据[21]的制造方法，其中，还包括：使叠氮基(N3－)与具有炔烃结构的分子进行反应的工序。
[23]根据[22]的制造方法，其中，具有炔烃结构的分子选自化学治疗剂、分子靶向药、免疫活化剂、毒素、抗菌剂、抗病毒剂、诊断用药剂、蛋白质、肽、氨基酸、核酸、抗原、脂质、脂质体、维生素以及激素中。
[24]根据[23]的制造方法，其中，化学治疗剂选自喜树碱、吡咯并苯并二氮杂卓、阿霉素、澳瑞他汀、紫杉烷或其衍生物中。
[25]根据[23]的制造方法，其中，免疫活化剂选自STING激动剂、TLR激动剂、A2AR拮抗剂、IDO抑制剂、CTLA－4、LAG－3以及PD－1途径的拮抗剂、检查点抑制剂、血管内皮生长因子(VEGF)受体抑制剂、平滑蛋白(smoothen)抑制剂、烷基化剂、代谢拮抗剂、类视黄醇、抗癌疫苗以及佐剂中。
[26]根据[23]～[25]中任一项的制造方法，其中，具有炔烃结构的分子选自由(A)～(E)构成的组中。
(A)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、(B)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－[4－({[(11’S，11’aS)－11’－羟基－7’－甲氧基－8’－(3－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}丙氧基)－5’－桥氧基－11’，11’a－二氢－1’H，3’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－羰基]氧基}甲基)苯基]－L－丙氨酰胺、(C)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、(D)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，10’，11’，11a’－四氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、以及(E)：(双(N，N－二乙基乙铵)N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－苯丙氨酰基－N－[(2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R)－15－氟－16－羟基－2，10－二桥氧基－2，10－二硫－14－(6，7，8，9－四氢－2H－2，3，5，6－四氮杂苯并[cd]薁－2－基)八氢－2H，10H，12H－5，8－桥亚甲基－2λ5，10λ5－呋喃并[3，2－l][1，3，6，9，11，2，10]五氧杂二膦环十四炔－7－基]－6－桥氧基－6，9－二氢－1H－嘌呤－1－基}乙氧基)甲基]甘氨酰胺。
[27]根据[17]～[26]中任一项的制造方法，其中，受体分子是具有由任选地添加岩藻糖的核心GlcNAc构成的N297连接糖链的抗体或含Fc区的分子。
[28]一种抗体或含Fc区的分子的制造方法，其特征在于，在[1]～[11]中任一项的多肽和酶A(也记载为Enzyme A)的存在下，使受体分子与包含还原末端未被活化的GlcNAc的糖链供体分子进行反应，其中，所述酶A是以还原末端未被活化的糖链供体分子的复合型糖链为底物但不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶，所述受体分子是具有任选地添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。
[29]根据[27]的制造方法，其特征在于，使[1]～[11]中任一项的多肽、酶A、受体分子以及糖链供体分子在同一反应液中进行反应。
[30]根据[28]或[29]的制造方法，其中，糖链供体分子是非还原末端任选地被化学修饰的复合型糖链。
[31]根据[28]～[30]中任一项的制造方法，其中，糖链供体分子是非还原末端任选地被化学修饰的SGP、(SG－)Asn、(MSG1－)Asn、(MSG2－)Asn、(MSG1－)Asn与(MSG2－)Asn的混合物。
[32]根据[28]～[31]中任一项的制造方法，其中，糖链供体分子是([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3、或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3与([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3的混合物。
[33]根据[32]的制造方法，其中，还包括：使叠氮基(N3－)与具有炔烃结构的分子进行反应的工序。
[34]根据[33]的制造方法，其中，具有炔烃结构的分子选自化学治疗剂、分子靶向药、免疫活化剂、毒素、抗菌剂、抗病毒剂、诊断用药剂、蛋白质、肽、氨基酸、核酸、抗原、脂质、脂质体、维生素以及激素中。
[35]根据[34]的制造方法，其中，化学治疗剂选自喜树碱、吡咯并苯并二氮杂卓、阿霉素、澳瑞他汀、紫杉烷或其衍生物中。
[36]根据[34]的制造方法，其中，免疫活化剂选自STING激动剂、TLR激动剂、A2AR拮抗剂、IDO抑制剂、CTLA－4、LAG－3以及PD－1途径的拮抗剂、检查点抑制剂、血管内皮生长因子(VEGF)受体抑制剂、平滑蛋白(smoothen)抑制剂、烷基化剂、代谢拮抗剂、类视黄醇、抗癌疫苗以及佐剂中。
[37]根据[34]～[36]中任一项的制造方法，其中，具有炔烃结构的分子选自由(A)～(E)构成的组中。
(A)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、(B)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－[4－({[(11’S，11’aS)－11’－羟基－7’－甲氧基－8’－(3－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}丙氧基)－5’－桥氧基－11’，11’a－二氢－1’H，3’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－羰基]氧基}甲基)苯基]－L－丙氨酰胺、(C)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、(D)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，10’，11’，11a’－四氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、以及(E)：(双(N，N－二乙基乙铵)N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－苯丙氨酰基－N－[(2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R)－15－氟－16－羟基－2，10－二桥氧基－2，10－二硫－14－(6，7，8，9－四氢－2H－2，3，5，6－四氮杂苯并[cd]薁－2－基)八氢－2H，10H，12H－5，8－桥亚甲基－2λ5，10λ5－呋喃并[3，2－l][1，3，6，9，11，2，10]五氧杂二膦环十四炔－7－基]－6－桥氧基－6，9－二氢－1H－嘌呤－1－基}乙氧基)甲基]甘氨酰胺。
[38]根据[28]～[37]中任一项的制造方法，其中，受体分子是具有由任选地添加岩藻糖的核心GlcNAc构成的N297连接糖链的抗体或或含Fc区的分子。
[39]根据[28]～[38]中任一项的制造方法，其中，酶A是具有从SGP向具有GlcNAc的受体的糖链转移活性的酶。
[40]根据[28]～[39]中任一项的制造方法，其中，酶A是Endo－M、Endo－Rp、Endo－Om、Endo－CC、或使它们的水解活性降低的突变酶。
[41]根据[40]的制造方法，其中，使水解活性降低的突变酶选自由Endo－RpN172Q、Endo－Rp N172H、Endo－Rp N172A、Endo－Rp N172C、Endo－Rp N172D、Endo－RpN172E、Endo－Rp N172G、Endo－Rp N172I、Endo－Rp N172L、Endo－Rp N172M、Endo－RpN172P、Endo－Rp N172S、Endo－Rp N172T、Endo－Rp N172V、Endo－Rp W278F/S216V、Endo－Rp W278F/N246D、Endo－Rp W278F/D276N、Endo－Rp W278F/A310D、Endo－RpW278F/N172D/F307Y、Endo－Rp W278F/N172D/F307H、Endo－Rp W278F/N172D/A310D、Endo－Rp W214F/F307Y/L306I、Endo－M N175Q、Endo－CC N180H以及Endo－Om N194Q构成的组中。
[42]一种抗体或含Fc区的分子，其通过[17]～[41]中任一项的制造方法而得到。
[43]一种仅具有任选地添加岩藻糖的核心GlcNAc的抗体或含Fc区的分子的制造方法，其特征在于，使具有序列号2的第34～928位所记载的氨基酸序列的多肽作用于抗体或含Fc区的分子。
[44]一种仅具有核心GlcNAc的抗体或或含Fc区的分子，其通过[43]的制造方法而得到。
本说明书包括作为本申请的优先权基础的日本专利申请号2020－147745号的公开内容。
发明效果
本发明的Endo－Si酶具有良好的水解活性，作用于糖蛋白的包含N297键的N连接型糖链，能高效地切断存在于糖链中的核心壳二糖结构的GlcNAc间的β1，4－糖苷键。游离的糖链可以用作用于糖蛋白的糖链结构分析的试样和糖链衍生物的原料。在将糖蛋白用于底物的情况下，糖链被水解的糖蛋白可以用作糖链重构的受体分子。
此外，与野生型Endo－Si相比，在Endo－Si中引入了突变的Endo－Si突变酶的水解活性降低，且具有经增强的糖链转移活性，因此能通过糖链重构高效地或高纯度地获取具有均匀的糖链的抗体或含糖链分子(包括含Fc区的分子)。因此，还能削减糖链重构中使用的糖链供体分子量，因此降低经糖链重构的抗体或含糖链分子的制造成本。
附图说明
图1表示[N3－PEG(3)]－MSG1(9)－Ox的结构式。
图2表示SGP的结构式。
图3是表示Endo－Si(〇)和Endo－S(X)的、对曲妥珠单抗(mAb1)的水解活性的经时变化的图表。X轴表示反应开始后的经过时间，Y轴表示糖链水解率。
图4是使用Endo－Si或Endo－Si突变酶的、对抗体的N297连接糖链的水解反应的示意图。
图5是将糖链噁唑啉体作为供体，使用Endo－Si或Endo－Si突变酶的糖链转移反应的示意图。
图6是表示序列号1的序列(Endo－Si碱基序列)的图。
图7是表示序列号2的序列(Endo－Si氨基酸序列)的图。
图8是表示序列号3的序列(Endo－Si氨基酸序列D241Q)的图。
图9是表示序列号4的序列(Endo－Si氨基酸序列D241Q/Q311L)的图。
图10是表示序列号5的序列(Endo－Si氨基酸序列D241Q/E360Q)的图。
图11是表示序列号6的序列(Endo－Si氨基酸序列D241M)的图。
图12是表示序列号7的序列(Endo－Si氨基酸序列D241M/Q311L)的图。
图13是表示序列号8的序列(Endo－Si氨基酸序列D241M/E360Q)的图。
图14是表示序列号9的序列(Endo－Si氨基酸序列T190Q/D241Q)的图。
图15是表示序列号10的序列(Endo－Si氨基酸序列T190Q)的图。
图16是表示序列号11的序列(Endo－Si氨基酸序列T190Q/D241M)的图。
图17是将SGP作为供体，使用Endo－Si突变酶和酶A的糖链转移反应的示意图。
图18是表示Endo－Si和EndoS的反应温度与糖链水解率的关系的图。
图19是表示Endo－Si和EndoS的反应pH与糖链水解率的关系的图。
图20是表示Endo－Si、EndoS、PNGaseF对各种抗体的水解活性的比较的图。
图21是将([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3作为供体，使用Endo－Si突变酶和酶A的糖链转移反应的示意图。
图22是将([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3作为供体，使用Endo－Si突变酶和酶A的糖链转移反应的示意图。
具体实施方式
以下，对本发明详细地进行说明。
在本说明书中，分子中所含的氨基酸的标记法按照本领域的惯例，在表示突变位点的情况下，由野生型的氨基酸(或核酸)的单字标记及其编号(例如，如果是第241位的Asp，则为“D241”)表示。需要说明的是，在本说明书中，对氨基酸位点的突变表示使氨基酸取代、缺失、插入或添加，优选表示取代氨基酸。此外，关于突变，由野生型的氨基酸(或核酸)的单字标记、其编号以及突变后的氨基酸(或核酸)的单字标记(例如，第241位的Asp被取代为Gln的突变为“D241Q”)表示。此外，具有突变的特定的突变酶由分子名和突变(例如，Endo－Si的第241位Asp被取代为Gln的突变酶为“Endo－Si D241Q”)表示，在具有多个突变的情况下，表示为将突变之间用“/”划分的形式(例如，在Endo－Si D241Q中，具有第241位的Gln被取代为Leu的追加突变的突变酶为“Endo－Si D241Q/Q311L”)。
在本发明中，“N297连接糖链”是指与IgG重链的第297位Asn的侧链连接的N连接型糖链。在将IgG片段化的情况下，即使是在包含该Asn的肽片段中，与对应的Asn连接的糖链，也包含在N297连接糖链中。通常，动物等产生的IgG中的N297连接糖链具有由下述式(I)或(II)的结构构成的基本结构，其非还原末端可以进一步被化学修饰，例如可以添加半乳糖(Gal)、唾液酸(Sia)。
Figure BDA0004105389400000161
细胞产生的IgG的N297连接糖链大多具有糖链结构的多样性，该糖链结构在该基本结构中包含在其还原末端的GlcNAc(核心GlcNAc)、非还原末端、支链糖等中进一步连接了糖链的结构。也可以修饰为在核心GlcNAc的6位具有岩藻糖(Fuc)进行了α1，6键合的核心岩藻糖的结构((Fucα1，6)GlcNAc)。在作为支链糖的Man中，也有形成在其5位进一步键合有包含GlcNAc的糖链的三天线型的糖链的情况。在非还原末端的GlcNAc中，也有进一步键合有包含半乳糖、唾液酸的糖链的情况。
在本发明中，唾液酸聚糖(Sialyl Glycan，以下，称为“SG”)具有由下述的结构式和序列式构成的基本结构。
作为SG的代表性物质，可以举例示出鸡蛋的蛋黄中所含的唾液酸糖肽(SialylGlycoPeptide：以下，称为“SGP”)中所含的糖链。
Figure BDA0004105389400000171
(式中，“－(N/Q)”表示与Asn或Gln的侧链形成N糖苷键。)
市售有仅由在SG的糖链部分中缺失了一个还原末端的GlcNAc的糖链(以下称为“SG(10)”)构成的二唾液酸八糖(东京化成(株)制)等。在本说明书中，将仅在SG(10)的β甘露糖(β－Man)的支链中的任一者缺失了非还原末端的唾液酸的糖链结构称为MSG(9)，将仅在支链的1－3糖链具有唾液酸的糖链记为MSG1(9)，将仅在支链的1－6糖链具有唾液酸的糖链记为MSG2(9)(专利文献1，WO2019/065964)。
在本发明中，“糖链供体分子”是指具有糖链的还原末端经活化的GlcNAc、优选具有经噁唑啉化的GlcNAc的含糖链分子，可以使用多种糖链结构的分子。活化是指提高了糖端基异构体位置的反应性的状态，包括噁唑啉化或卤化。作为糖链供体分子的例子，可列举出实施例6中使用的[N3－PEG(3)]－MSG1(9)－Ox(图1)、SG(9)－Ox(噁唑啉)、MSG1(9)－Ox、MSG2(9)－Ox或MSG1(9)－Ox与MSG2(9)－Ox的混合物。
作为糖链供体分子的另一方案，可列举出具有糖链的还原末端未被活化的GlcNAc的含糖链分子，优选SGP(图2)、(SG－)Asn、(MSG1－)Asn、(MSG2－)Asn或(MSG1－)Asn、(MSG2－)Asn的混合物。
在本发明中，只要没有特别记载，在氨基酸的侧链中与糖链连结的情况的部分结构用括号表示侧链部分，例如，如“(SG－)Asn”那样记载。
糖链供体分子可以被化学修饰，例如，包含非还原末端经化学修饰的SGP、(SG－)Asn、(MSG1－)Asn、(MSG2－)Asn、(MSG1－)Asn与(MSG2－)Asn的混合物、SG(10)－Ox、MSG1(9)－Ox、MSG2(9)－Ox或MSG1(9)－Ox与MSG2(9)－Ox的混合物等。优选为([N3－PEG(3)]2－SG(10))－Ox、[N3－PEG(3)]－MSG1(9)－Ox、[N3－PEG(3)]－MSG2(9)－Ox、或[N3－PEG(3)]－MSG1(9)－Ox与[N3－PEG(3)]－MSG2(9)－Ox的混合物、或者([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3、或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3与([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3的混合物(专利文献1，WO2019/065964)等。
在用于以药物研发为目的的糖链重构的情况下，优选采用具有在用于人时问题少的人型糖链、或人适合型糖链的糖链供体。这样的糖链是已知在人的体内不显示抗原性的糖链，在N连接型糖链中，已知高甘露糖型、杂合(hybrid)型、复合(complex)型等。这三个具有共同的基本结构。高甘露糖型是在从位于靠近还原末端的位置的甘露糖(β甘露糖)分支的两个支链(1－3链、1－6链)具有多个甘露糖连续而成的富含甘露糖的结构的糖链。杂合型是从位于靠近还原末端的位置的甘露糖(β甘露糖)分支的两个支链(1－3链、1－6链)中的一个具有GlcNAc的结构而成的糖链。复合型是在从位于靠近还原末端的位置的甘露糖(β甘露糖)分支的两个支链(1－3链、1－6链)具有GlcNAc的结构，并具有包含有无半乳糖、有无唾液基、以及它们的键异构性、位置异构性的多样结构的糖链。复合型糖链已知有两天线型、三天线型、四天线型。
以下，示出高甘露糖型、杂合型以及复合型的结构的例子。
Figure BDA0004105389400000191
人型N连接糖链的种类
在本发明中，“受体分子”是指包含在非还原末端具有GlcNAc的糖结构的分子，通过在Endo－Si或其突变酶存在下与糖链供体分子反应，糖链供体分子的噁唑啉环或活性中间体(非专利文献9)与该非还原末端的GlcNAc的4位反应，从而能形成壳二糖结构。
作为受体分子，典型的是具有仅由源自单抗的、可结合核心Fuc的核心GlcNAc构成的N297连接糖链的IgG或其Fc片段。根据成为其来源的抗体或其产生方法，核心GlcNAc可以与核心Fuc结合，也可以不结合。作为受体分子的来源，可以利用各种单抗或含糖链分子或含Fc区的分子(Fc、仅由从重链中缺失可变区的恒定区构成的CH与仅由轻链的恒定区构成的CL组合而成的CLCH等)，优选列举出(Fucα1，6)－GlcNAc－IgG(例如，图4的(Fucα1，6)GlcNAc－mAb1)、(Fucα1，6)－GlcNAc－Fc、(Fucα1，6)－GlcNAc－CLCH等(专利文献1)。
在本发明中，“Endo－Si”是源自海豚链球菌的内－β－N－乙酰氨基葡萄糖苷酶(ENGase：Endo－β－N－acetylglucosaminidase)的一种，序列号1表示碱基序列，序列号2表示氨基酸序列。其是由在序列号2的第34～928位(第1～33位的氨基酸表示信号序列。在信号序列的预测中，利用作为CBS提供的工具的SignalP－5.0)的氨基酸序列中，第241位的氨基酸为Asp的氨基酸序列构成的酶(EC 3.2.1.96、GH18)。Endo－Si特异性地识别N连接型糖链(例如N297连接糖链)，兼具水解活性和糖链转移活性。
Endo－Si的水解活性是特异性地水解具有上述基本结构的N连接型糖链的核心壳二糖中所含的β1，4糖苷键的活性(在本说明书中，只要没有特别提及，“水解活性”就是指该活性。将反应示意图示于图4。)。
Endo－Si的糖链转移活性是使上述糖链供体分子(具有还原末端经活化的GlcNAc、或还原末端未被活化的GlcNAc的含糖链分子)的还原末端与包含在N297仅具有核心GlcNAc(可以添加核心岩藻糖，也可以不添加核心岩藻糖)的Fc位点的受体分子进行糖苷键合的活性(以下，称为“糖链转移活性”。将反应示意图示于图5或图17。)。
Endo－Si对各种抗体的底物特异性如下。对IgG的四个亚类(subclass)全部显示糖链水解活性，但不显示对IgA和IgE的糖链水解活性。此外，对各种N连接型糖链的底物特异性如下。对高甘露糖型糖链和复合型两天线糖链均显示水解活性，但与高甘露糖型糖链相比，对复合型两天线糖链的特异性高，其中对G0糖链的水解活性最高。进而，对唾液酸糖链、岩藻糖基化糖链也显示水解活性，但不显示对复合型三天线糖链的水解活性。
需要说明的是，G0糖链是指非还原末端键合于两个支链(1－3链、1－6链)的GlcNAc，称为不具有半乳糖残基的两天线复合型糖链。
本发明的酶只要具有上述特性，就不限定于实施例中获取到的具体序列的酶，可以是从天然分离到的酶，也可以是基于本发明的酶的序列信息而人为制作或改造的酶。在从天然分离到的情况下，作为其分离源的生物种类没有特别限定，优选为细菌，更优选为链球菌(Streptococcus)属的细菌，进一步优选为属于海豚链球菌的细菌。
Endo－Si的活性结构域和碳水化合物结合组件(CBM：Carbohydrate－bindingmodule)根据与进行晶体结构分析的EndoS(B.Trastoy et al.，PNAS(2014)vol111，No.18，pp6714－6719)的序列比较，分别推测为序列号2的第106～447位和第762～897位的区域，认为这两个是对于水解活性和/或转移活性与抗体的相互作用而言重要的位点。因此，作为本发明的酶，可列举出：含有序列号2的第106～447位和/或第762～897位所记载的氨基酸序列、优选含有序列号2的第106～897位所记载的氨基酸序列、更优选含有序列号2的第106～928位所记载的氨基酸序列、进一步优选含有序列号2的第34～928位所记载的氨基酸序列并且显示水解活性和/或糖链转移活性的多肽。
＜本发明的突变酶＞
本发明提供一种Endo－Si的突变酶，其特征在于，具有：在序列号2的第34～928位所记载的氨基酸序列中，存在包含对选自由第241位(D241)、第190位(T190)、第311位(Q311)以及第360位(E360)的氨基酸构成的组中的一个或两个以上的氨基酸位点的突变的氨基酸序列，并且显示糖链水解活性和/或糖链转移活性，优选如实施例6所示，提供一种Endo－Si的突变酶，其特征在于，在序列号2的第34～928位所记载的氨基酸序列中，含有糖链转移活性所需的区，且作为对IgG的N297连接糖链的活性，与Endo－Si WT(以下，将在氨基酸序列中未引入突变的野生株称为“WT”)相比较，水解活性降低，糖链转移活性提高。
作为本发明的氨基酸的取代/突变，为显示上述特征的取代/突变，优选为选自序列号2的T190、D241、Q311、E360中的至少一个或多个氨基酸位点的取代/突变，更优选为D241Q、D241M、D241A、T190Q、Q311L、E360Q、E360A、E360N、或E360D，进一步优选为D241Q、D241M、T190Q、Q311L、E360Q，最优选为D241Q、D241Q/Q311L、D241Q/E360Q、D241M、D241M/Q311L、D241M/E360Q、T190Q/D241Q、T190Q、T190Q/D241M。需要说明的是，只要显示上述特性，则除了序列号2的T190、D241、Q311、E360中的取代/突变以外，还可以包含进一步的取代/突变。
内－β－N－乙酰氨基葡萄糖苷酶兼具水解活性和糖链转移活性(以下，将兼具两种活性称为具有“本酶活性”)。因此，保持强的水解活性的酶有时也将通过糖链转移活性而转移至受体分子(具有核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子)的核心GlcNAc的糖链作为底物进行水解，无法适当地获得所期望的糖链转移体。因此，在糖链重构抗体等或糖链修饰化合物的合成中，提高了糖链转移活性的突变酶是有用的。本发明的突变酶的特征在于，兼具比Endo－Si WT降低的水解活性和增强的糖链转移活性。
突变酶的糖链转移活性可以通过后述的实施例6、或实施例7的方法进行评价。
本发明的突变酶所具有的本酶活性中，糖链转移活性高于Endo－Si WT的糖链转移活性。即，在pH7～8(例如，pH7.5)、糖链供体(例如，包含还原末端经活化的GlcNac(经噁唑啉化的GlcNAc等)的糖链)存在受体分子的5～10当量(例如，8当量)的条件下的糖链转移率在从反应开始至1～24或48小时后为止的任一时间点以后，示出超过Endo－Si WT的糖链转移率。优选在反应开始后24小时或48小时之前糖链转移率超过50％，更优选在反应开始后24小时之前糖链转移率超过60％，进一步优选在反应开始后24小时之前糖链转移率超过80％，更进一步优选在反应开始后24小时之前糖链转移率超过95％。
作为本发明的突变酶所具有的糖链转移活性的另一方案，可列举出超过Endo－SiWT的糖链转移活性的方案。即，在pH7～8(例如，pH7.5)、包含还原末端未被活化的GlcNac的糖链供体存在受体分子的10～100当量(例如，50当量)的条件下的糖链转移率在从反应开始至1～48小时后为止的任一时间点以后，示出与Endo－Si WT的糖链转移率同等或其以上的糖链转移率。优选在反应开始后24小时或48小时之前糖链转移率超过50％，更优选在反应开始后48小时之前糖链转移率超过60％，进一步优选在反应开始后48小时之前糖链转移率超过80％，更进一步优选在反应开始后48小时之前糖链转移率超过90％。
本发明的突变酶只要在序列号2的第34～928位的氨基酸序列中具有对选自由D241、T190、Q311以及E360构成的组中的1个或2个以上(优选为1～3个，更优选为1个或2个)氨基酸位点的突变，并且保持对于Endo－Si的糖链转移活性而言重要的区，就无需是其全长序列。由EndoS的结构域分析可知，催化结构域(序列号2的第106～447位)和/或CBM(序列号2的第762～897位)是重要的，只要包含它们，就可以用作本发明的突变酶。
本发明的突变酶是包含上述“本发明的氨基酸的取代/突变”中记载的突变的多肽，具体而言，可列举出包含选自由序列号3(Endo－Si D241Q)、序列号4(Endo－Si D241Q/Q311L)、序列号5(Endo－Si D241Q/E360Q)、序列号6(Endo－Si D241M)、序列号7(Endo－SiD241M/Q311L)、序列号8(Endo－Si D241M/E360Q)、序列号9(Endo－Si T190Q/D241Q)、序列号10(Endo－Si T190Q)以及序列号11(Endo－Si T190Q/D241M)构成的组中的氨基酸序列的多肽。
在本发明的突变酶的氨基酸序列中，在不影响本酶活性的范围内，可以在必需突变(D241、T190、Q311或E360)以外的位点，取代、缺失、插入和/或添加1～数个氨基酸。作为这样的氨基酸突变的位点，只要不影响本酶活性，就可以选择所有位点，优选为第241位、第190位、第311位或第360位以外的氨基酸，更优选为催化结构域(序列号2的第106～447位)、以及CBM(序列号2的第762～897位)以外的位点，进一步优选为序列号2的第34～105位或第898～928位的区域中包含的位点。
在本发明中，数个为30个或20个以下，优选为10个以下，进一步优选为5个以下，最优选为4个、3个、2个或1个。
在本发明中，由突变引起的取代后的氨基酸只要最终得到的突变酶具有本酶活性就没有特别限定，可以采用天然存在的氨基酸、人工合成的氨基酸、它们的修饰氨基酸等各种氨基酸，优选为天然存在的氨基酸，更优选为天然存在的L－氨基酸，进一步优选为必需氨基酸。
可列举出：在本发明的突变酶的氨基酸序列中，在不影响本酶活性的范围内，与必需突变(D241、T190、Q311或E360)的氨基酸以外的氨基酸序列具有至少80％以上、优选85％以上、更优选90％以上、进一步优选95％、96％、97％、98％或99％以上的同源性或同一性的氨基酸序列。
两种氨基酸序列间的同一性或者同源性、两种氨基酸序列间的同一性或者同源性可以通过使用Blast算法版本2.2.2Blast algorithm version 2.2.2(Altschul，SF，etal.，Nucleic Acids Res.1997，25，3389－3402)的系统内定参数(default parameter)来决定。Blast算法例如也可以通过在网络上访问http：//blast.ncbi.nlm.nih.gov/来使用。
＜基因、宿主细胞、酶产生方法＞
本发明还提供编码上述Endo－Si(序列号1)或Endo－Si突变酶的重组基因、包含该重组基因的质粒、表达载体等基因构建体、由该基因构建体转化的宿主细胞、包括从该宿主细胞的培养物中回收本发明的Endo－Si或Endo－Si突变酶的工序的本发明的酶的制造方法等。这些重组基因、基因构建体、宿主细胞等可以基于本发明的突变酶的氨基酸序列，按照公知的基因工程方法来制作。将大肠杆菌用的Endo－Si的碱基序列示于序列号16。
通过引入编码本发明的酶的基因而转化的宿主细胞(可以适当选择动物细胞、植物细胞、大肠杆菌、酵母等通常用于蛋白质产生的细胞等)可以根据细胞的种类在适当的条件下培养，从该培养物中回收本发明的酶。酶的回收利用该酶的物性，适当组合通常的纯化方法来进行，但为了简便地回收，以预先使His标签、GST标签等标签肽以与酶连结的形式表达的方式设计基因构建体，由此可以进行利用该标签肽的亲和性的回收。标签肽可以在纯化后去除，但在不影响酶活性的情况下，也可以将连结有标签肽的状态下的酶用于糖链重构等反应。本发明的酶中包含这样的具有连结有标签肽的氨基酸序列的酶。
＜糖链重构＞
本发明提供使用了本发明的Endo－Si或Endo－Si突变酶的、糖蛋白的糖链的糖链重构方法、以及通过该糖链重构制造的具有由实质上均匀的结构构成的糖链的糖蛋白。此外，提供通过该糖链重构来制造具有由实质上均匀的结构构成的糖链的糖蛋白的方法。
本发明的一个实施方式提供使用了本发明的Endo－Si或Endo－Si突变酶的、抗体或含其Fc区的分子中的包含N297键的N连接型糖链的糖链重构方法、以及通过该糖链重构制造的具有由实质上均匀的结构构成的包含N297键的N连接型糖链的糖蛋白、优选抗体或含Fc区的分子。此外，提供通过该糖链重构来制造具有由实质上均匀的结构构成的包含N297键的N连接型糖链的糖蛋白、优选抗体或含Fc区的分子的方法。抗体优选为IgG。以下，对IgG进行说明。需要说明的是，在本发明中，糖蛋白是指存在于动植物的组织、真核微生物的细胞膜、细胞壁等中的、在蛋白质的氨基酸序列中结合有至少一个以上O连接型糖链或N连接型糖链的蛋白质，可以是源自天然的，也可以是合成的，例如是指单抗的IgG或IgG的Fc片段、仅由恒定区构成的CLCH(专利文献1、WO2018/003983)等含Fc区的分子等。
“糖链重构”是指以下的方法：首先，制作将特定的糖蛋白、例如单抗的IgG或IgG的Fc片段、仅由恒定区构成的CLCH等含Fc区的分子的N297连接糖链以保留核心GlcNAc(也可以添加核心岩藻糖)的方式切除而得到的受体分子，接着，对于该受体分子的核心GlcNAc，利用本发明的Endo－Si突变酶的糖链转移活性，使源自糖链供体的糖链转移，由此制造N297连接糖链为源自糖链供体的均匀的糖链结构的IgG或含其Fc区的分子。
糖链重构中使用的IgG或含Fc区的分子优选为源自由同一氨基酸序列构成的IgG重链、以具有N297连接糖链的形式产生的分子即可。其产生方法没有限定，可以利用由通常已知的单抗的生产方法产生的IgG、IgG的CLCH、或对其进行酶处理而得到的Fc片段等。此外，这样的IgG或Fc片段也可以采用通过不同的生产方法、不同批次得到的样品的混合物。
作为糖链重构中使用的受体分子的制备方法，可以通过利用保持了特异性地水解N297连接糖链的核心壳二糖结构中的GlcNAc间的1，4－糖苷键(GlcNAcβ1－4GlcNAc)的活性的ENGase对上述的IgG或含Fc区的分子进行处理来制备。在该情况下，作为ENGase，可以采用以Endo－Si WT、Endo－A、Endo－D、Endo－E、Endo－F3、Endo－H、EndoS、EndoS2为首的各种ENGase。
作为糖链重构中使用的糖链供体分子，可以采用各种糖链结构的物质，但在以将重构后的抗体作为抗体医药为目的的情况下，优选采用具有与人保有的糖链结构类似或相同的人型糖链或人适合型糖链结构的糖链供体。
作为这样的糖链供体分子的代表性分子，可列举出在上述的N连接型糖链的基本结构中去除核心GlcNAc、从还原末端起第二位的GlcNAc被活化的分子。例如，可列举出SG(10)－Ox、([N3－PEG(3)]2－SG(10))－Ox、[N3－PEG(3)]－MSG1(9)－Ox、[N3－PEG(3)]－MSG2(9)－Ox、或[N3－PEG(3)]－MSG1(9)－Ox与[N3－PEG(3)]－MSG2(9)－Ox的混合物(WO2019/065964)。
作为糖链供体分子的其他方案，可列举出从还原末端起第二位的GlcNAc未被活化的分子。例如，可列举出([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3、或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3与([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3的混合物(专利文献1，WO2019/065964)。
此时，通过使以糖链供体分子的复合型糖链为底物但不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶(酶A)与本发明的Endo－Si或Endo－Si突变酶、优选Endo－Si突变酶共存，使供体分子的糖链与切断了作为受体分子的糖链的IgG或含Fc区的分子的核心GlcNAc残基结合。在此使用的酶A具有从作为糖链供体的SGP向具有GlcNAc的受体的糖链转移活性的情况下，酶A在一锅法中示出高糖链转移效率。即，作为酶A，可以从以还原末端未被活化的糖链供体分子的复合型糖链为底物但不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶中，以向具有GlcNAc的受体的糖链转移活性为指标进行选择。作为不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶，可列举出Endo－M、Endo－Rp、Endo－Om、Endo－CC、或降低了它们的水解活性的突变酶。作为使水解活性降低的突变酶，可列举出公知的Endo－Rp N172Q、Endo－Rp N172H(专利文献3)、Endo－M N175Q(Umekawa M.et al.，J Biol Chem.2010，285，511－521)、Endo－CC N180H或Endo－OmN194Q(Chiba Y.Kagaku to Seibutsu 2015，53，236－244)等。优选可列举出Endo－RpN172Q、Endo－Rp N172H、Endo－Rp N172A、Endo－Rp N172C、Endo－Rp N172D、Endo－RpN172E、Endo－Rp N172G、Endo－Rp N172I、Endo－Rp N172L、Endo－Rp N172M、Endo－RpN172P、Endo－Rp N172S、Endo－Rp N172T、Endo－Rp N172V。此外，作为两个氨基酸被取代的突变酶，可列举出Endo－Rp W278F/S216V、Endo－Rp W278F/N246D、Endo－Rp W278F/D276N、Endo－Rp W278F/A310D。进而，作为三个氨基酸被取代的突变酶，可列举出Endo－RpW278F/N172D/F307Y、Endo－Rp W278F/N172D/F307H、Endo－Rp W278F/N172D/A310D、Endo－Rp W214F/F307Y/L306I。
将Endo－Rp的突变酶的氨基酸序列示于序列号17～43，将Endo－M的氨基酸序列示于序列号44，将Endo－Om的氨基酸序列示于序列号45，将Endo－CC的氨基酸序列示于序列号46。
作为本发明的Endo－Si突变酶与酶A的组合，例如可列举出表1和表2的组合。
[表1]
Figure BDA0004105389400000271
Figure BDA0004105389400000281
Figure BDA0004105389400000291
Figure BDA0004105389400000301
Figure BDA0004105389400000311
Figure BDA0004105389400000321
Figure BDA0004105389400000331
Figure BDA0004105389400000341
[表2]
Figure BDA0004105389400000351
从通过糖链重构来制造具有由实质上均匀的结构构成的糖链的糖蛋白的观点考虑，优选列举出表3的组合作为本发明的Endo－Si突变酶与酶A的组合。
[表3]
Figure BDA0004105389400000361
在本发明的显示糖链转移活性的多肽和酶A的存在下，使受体分子与包含还原末端未被活化的糖链的糖链供体分子进行反应即可(图17)，其中，所述酶A是以还原末端未被活化的糖链供体分子的复合型糖链为底物但不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶，所述受体分子是具有可以添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。
糖链重构中的用于制备受体分子的水解反应的反应条件可以根据其他酶中已知的条件适当选择，此外，也可以考虑酶活性、抗体的性质、纯化工序中的回收率、作业时间等来选择。反应在缓冲液中进行，可以从柠檬酸缓冲液(pH3.5～5.5)、乙酸缓冲液(pH4.5～6.0)、磷酸缓冲液(pH6.0～7.5)、MOPS－NaOH缓冲液(pH6.5～8.0)、三羟甲基氨基甲烷盐酸(Tris－HCl)缓冲液(pH7.0～9.0)等通常的酶反应中使用的缓冲液中适当选择。优选为磷酸缓冲液(pH6.0～7.5)或三羟甲基氨基甲烷盐酸缓冲液(pH7.0～9.0)。出于使酶稳定的目的，可以在反应液中加入不阻碍酶反应的添加剂，也可以不添加。
反应温度可以在4℃～50℃之间适当选择，优选为15℃～45℃，更优选为18℃～40℃，更优选为20℃～35℃。
此外，Endo－Si的水解反应的反应pH可以在pH5.8～9.5之间适当选择，优选为pH6.2～pH8.0，更优选为pH6.5～pH7.5。
反应时间可以在10分钟至96小时之间适当选择，优选为0.5小时～80小时，更优选为1小时～60小时，更优选为8小时～48小时，更优选为12～24小时，可以经时地采集少量反应液，一边确认水解的进展度一边判断反应的结束。通常，糖链水解反应的进展度可以通过十二烷基硫酸钠－聚丙烯酰胺凝胶电泳(SDS－PAGE)、全自动电泳系统、或液相色谱－质谱联用仪(LC－MS)等进行监测。在本专利中，将市售抗体或糖链重构抗体片段化为重链和轻链后，使用全自动电泳系统，通过仅在添加有N297连接糖链的重链侧的保持时间发生变化来确认。
在糖链重构中将还原末端经活化的GlcNAc用作糖链供体(经噁唑啉化的GlcNAc等)或将未被活化的GlcNAc用作糖链供体的糖链转移反应的反应条件可以根据其他酶中已知的条件适当选择(专利文献1，WO2019/065964等)。
反应在缓冲液中进行，但理想的是不促进还原末端经活化或未被活化的GlcNAc作为糖链供体的分解的缓冲液，可以从磷酸缓冲液(pH6.0～7.5)、MOPS－NaOH缓冲液(pH6.5～8.0)、三羟甲基氨基甲烷盐酸缓冲液(pH7.0～9.0)等中适当选择。优选为三羟甲基氨基甲烷盐酸缓冲液(pH7.0～9.0)。出于使酶稳定的目的，可以在反应液中加入不阻碍酶反应的添加剂，也可以不添加。
反应温度可以在4℃～50℃之间适当选择，反应温度为15℃～45℃，更优选为20℃～40℃，更优选为25℃～40℃。
此外，Endo－Si的水解反应的反应pH可以在pH5.8～9.5之间适当选择，优选为pH6.2～pH8.0，更优选为pH6.5～pH7.5。
反应时间可以在10分钟至96小时之间适当选择，优选为0.5小时～80小时，更优选为2小时～70小时，更优选为12小时～60小时，更优选为16～48小时，更优选为16～28小时，可以经时地采集少量反应液，一边确认糖链转移反应的进展度一边判断反应的结束。通常，糖链转移反应的进展度可以通过十二烷基硫酸钠－聚丙烯酰胺凝胶电泳(SDS－PAGE)、全自动电泳系统、或液相色谱－质谱联用仪(LC－MS)等进行监测。在本专利中，将市售抗体或糖链重构抗体片段化为重链和轻链后，使用全自动电泳系统，通过仅在添加有N297连接糖链的重链侧的保持时间发生变化来确认。
此外，也可以通过一锅法来进行糖链重构，所述一锅法使用保持了特异性地水解N297连接糖链的核心壳二糖结构中的GlcNAc间的1，4－糖苷键(GlcNAcβ1－4GlcNAc)的活性的ENGase和上述酶A这两者，将糖链供体分子的糖链直接转移至作为受体分子的具有可以添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。作为保持了特异性地水解N297连接糖链的核心壳二糖结构中的GlcNAc间的1，4－糖苷键(GlcNAcβ1－4GlcNAc)的活性的ENGase，优选上述Endo－Si的突变酶，作为酶A，优选上述降低了水解活性的Endo－Rp的突变酶。在一锅法中，作为糖链供体分子，例如包含非还原末端经化学修饰的SGP、(SG－)Asn、(MSG1－)Asn、(MSG2－)Asn、(MSG1－)Asn与(MSG2－)Asn的混合物；SG(10)－Ox、MSG1(9)－Ox、MSG2(9)－Ox或MSG1(9)－Ox与MSG2(9)－Ox的混合物等。优选可以使用([N3－PEG(3)]2－SG(10))－Ox、[N3－PEG(3)]－MSG1(9)－Ox、[N3－PEG(3)]－MSG2(9)－Ox、或[N3－PEG(3)]－MSG1(9)－Ox与[N3－PEG(3)]－MSG2(9)－Ox的混合物、或者([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3、或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3与([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3的混合物(专利文献1，WO2019/065964)等。
通过糖链重构方法制造的糖蛋白(抗体或含Fc区的分子)可以进一步进行化学或生化修饰。例如，叠氮基(N3－)通过与(杂)环炔基(例如，DBCO(Dibenzocyclooctyne：二苯并环辛炔)等)等炔烃结构进行反应而形成1，2，3－三唑环(SPAAC(strain－promotedalkyne azide cycloaddition：Agard NJ，et al.，J Am Chem Soc.2004，126，46，15046-15047))。因此，通过将使用上述具有叠氮基(N3－)的供体分子而得到的糖链重构抗体与具有(杂)环炔基且具有所期望的活性的分子(具有药学活性的化合物(例如，化学治疗剂、分子靶向药、免疫活化剂(例如，STING激动剂(WO2020/050406、WO2014/099824、WO2014/179335、WO2014/189805、WO2014/189806、WO2015/074145、WO2015/185565、WO2016/096714、WO2016/012305、WO2016/145102、WO2017/027646、WO2017/027645、WO2017/075477、WO2017/093933、WO2017/100305、WO2017/123669、WO2017/161349、WO2017/175147、WO2017/175156、WO2018/009466、WO2018/045204、WO2018/060323、WO2018/067423、WO2018/065360、WO2014/093936、WO2018/009648、WO2018/100558)、TLR激动剂、A2AR拮抗剂、IDO抑制剂、CTLA－4、LAG－3以及PD－1途径的拮抗剂、检查点抑制剂、血管内皮生长因子(VEGF)受体抑制剂、平滑蛋白(smoothen)抑制剂、烷基化剂、代谢拮抗剂、类视黄醇以及抗癌疫苗、佐剂、脂质、脂质体、毒素、抗菌剂、抗病毒剂、诊断用药剂、蛋白质、肽、氨基酸、核酸、抗原、维生素、激素等))进行反应，可以得到进一步修饰后的具有所期望的活性的抗体(例如，抗体－药物偶联物等)。作为化学治疗剂或毒素，可列举出喜树碱(例如，WO2014/057687)、吡咯并苯并二氮杂卓(例如，WO2013/173496、WO2014/130879、WO2017/004330、WO2017/004025、WO2017/020972、WO2016/036804、WO2015/095124、WO2015/052322、WO2015/052534、WO2016/011519、WO2015/052321、WO2015/031693、WO2011/130613、WO2019/065964)、阿霉素(Doxorubicin)、澳瑞他汀、紫杉烷或其衍生物。
作为上述具有叠氮基(N3－)的供体分子，例如可列举出WO2020/050406、WO2019/065964所记载的药物连接子。例如可列举出N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺；N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－[4－({[(11’S，11’aS)－11’－羟基－7’－甲氧基－8’－(3－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}丙氧基)－5’－桥氧基－11’，11’a－二氢－1’H，3’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－羰基]氧基}甲基)苯基]－L－丙氨酰胺；N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺；N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，10’，11’，11a’－四氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺；(双(N，N－二乙基乙铵)N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－苯丙氨酰基－N－[(2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R)－15－氟－16－羟基－2，10－二桥氧基－2，10－二硫－14－(6，7，8，9－四氢－2H－2，3，5，6－四氮杂苯并[cd]薁－2－基)八氢－2H，10H，12H－5，8－桥亚甲基－2λ5，10λ5－呋喃并[3，2－l][1，3，6，9，11，2，10]五氧杂二膦环十四炔－7－基]－6－桥氧基－6，9－二氢－1H－嘌呤－1－基}乙氧基)甲基]甘氨酰胺。
实施例
以下，使用实施例对本发明进行具体说明。实施例所示的是本发明的实施方式的一个例子，本发明并不限定于此。
本说明书中记载的蛋白质浓度使用超微量分光光度计NanoDrop1000(ThermoFisher Scientific制)或NanoDrop2000(Thermo Fisher Scientific制)进行定量。
实施例中的[N3－PEG(3)]－MSG1(9)－Ox表示图1的化合物。SGP表示图2的化合物。mAb1表示市售的曲妥珠单抗(从中外制药购入)。(Fucα1，6)GlcNAc－mAb1表示曲妥珠单抗的糖链水解体。mAb2表示通过WO2019065964的实施例136中记载的方法制作的抗体。将mAb2的轻链和重链的氨基酸序列示于序列号12和序列号13。(Fucα1，6)GlcNAc－mAb2表示通过WO2019/065964的实施例61－工序1中记载的方法制作的糖链水解体(Fucα1，6)GlcNAc－抗CLDN6抗体(H1L1)。
使用蛋白质的凝胶电泳法确认了糖链水解和糖链转移反应的进行状况(专利文献4、非专利文献8)。作为蛋白质的全自动电泳系统，装置使用LabChip GX II(PerkinElmer制)，试剂使用Protein Express LabChip和Protein Express Reagent Kit(PerkinElmer制)。
＜实施例1＞源自S.iniae的内－β－N－乙酰氨基葡萄糖苷酶的获取
通过以下的方法，从S.iniae SIO1002株中获取内－β－N－乙酰氨基葡萄糖苷酶的基因序列。
首先，从S.iniae SIO1002株福尔马林灭活菌体(日本株，从共立制药株式会社购入)获取基因组DNA。将0.1％(w/v)福尔马林溶液1mL进行离心(6000rpm，10min，4℃)，用灭菌水1mL清洗沉淀。再次用相同条件进行离心，将沉淀悬浮于InstaGene DNA纯化基质(Bio－Rad制)200μL中。将该悬浮液在56℃下热处理30分钟，之后在99℃下热处理8分钟，进行离心(12000rpm，10min，4℃)，将所得到的上清液用作DNA提取液。
以DNA提取液为模板，使用引物1(序列号14)和引物2(序列号15)以及PrimeSTARMax DNA Polymerase(Takara Bio制)扩增编码内－β－N－乙酰氨基葡萄糖苷酶的基因，分析序列。该基因包含终止密码子在内由2787碱基(序列号1)组成，并编码由928氨基酸残基(序列号2)组成的分子量104644的蛋白质，将该蛋白质命名为Endo－Si。
＜实施例2＞使用大肠杆菌的Endo－Si的表达
以实施例1中得到的内－β－N－乙酰氨基葡萄糖苷酶基因为基础，设计在C末端添加了6×His标签的、最适于大肠杆菌的异种表达用的核酸序列(序列号16)，由EurofinsGenomics公司制作了人工合成基因。将其克隆到pET24b(+)载体中，转化到E.coli BL21(DE3)中。将转化后的菌液接种于12mL锥形管中的2mL LB培养基(1％(w/v)胰胨(Tryptone)，0.5％(w/v)酵母提取物(Yeast extract)，0.5％(w/v)NaCl，50μg/mL卡那霉素)，在37℃下进行振荡培养一晩(600rpm，O/N)。将该前培养液1.2mL植菌于500mL带挡板的烧瓶中的100mL TB培养基(1.2％(w/v)胰胨，2.4％(w/v)酵母提取物，0.94％(w/v)K2HPO4，0.22％(w/v)KH2PO4，50μg/mL卡那霉素，0.01％(w/v)止泡剂(anti foam)204，2mM MgSO4)，在37℃下开始振荡培养(210rpm)。在37℃下培养1.5小时后，将培养箱(incubator)的温度降至16℃继续培养1小时。确认培养液的温度下降至16℃后，以最终浓度成为0.2mM的方式添加IPTG，继续培养24小时。培养结束后通过离心分离来进行集菌。
将所集菌的菌体悬浮于5mL的结合缓冲液(50mM HEPES(pH 8.0)，0.5M NaCl，20mM咪唑，5％(w/v)甘油)，将进行了超声波破碎和离心分离的上清液用Ni Sepharose 6FastFlow(GE Healthcare制)纯化。产量(A280、吸光系数换算)为8.43mg/100mL broth。
＜实施例3＞Endo－Si对抗体糖链的水解活性
对于实施例2中获取到的酶，通过以下的方法测定水解活性。此时，将EndoS用作比较对象。
将mAb1 60mg溶解于灭菌水5mL中，一边用Vivaspin 20(30000MWCO，PES，Sartorius制)浓缩一边取代为50mM三羟甲基氨基甲烷盐酸缓冲液(pH7.5)。
制备包含该mAb1 0.1mg和10ng的酶的反应液(总容量50μL)，并在37℃下培养(incubate)。对反应0.5小时、1小时以及2小时的反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。根据所得到的色谱图，未反应物和水解体被确认为分离的峰。根据未反应物与水解体的峰面积比，通过下述计算式来计算糖链水解率。
糖链水解率(％)＝〔[源自(Fucα1，6)GlcNAc－mAb1的H链的峰面积]/{[源自mAb1的H链峰面积]+[源自(Fucα1，6)GlcNAc－mAb1的H链的峰面积]}〕×100
将反应产率的经时变化示于图3。0.5小时后的糖链水解率为Endo－Si为57.4％、EndoS为41.8％，Endo－Si示出比EndoS强的水解活性。
＜实施例4＞Endo－Si的反应条件的研究
研究了Endo－Si的反应温度和pH。
(4－1)Endo－Si的反应温度的评价
Endo－Si和EndoS的各温度下的糖链水解率如下测定。制备包含0.1mg mAb1、以及10ng Endo－Si WT或EndoS WT的50mM三羟甲基氨基甲烷盐酸缓冲液(pH7.5)50μL，在27℃、29℃、31℃、35℃、37℃、40℃、43℃、46℃、48℃以及50℃各温度下培养。对反应0.5小时的反应液进行取样，通过上述方法计算出糖链水解率。
将结果示于图18。Endo－Si在水解反应中的最适温度(酶良好地发挥功能的温度)为25℃～45℃的范围，更优选为30℃～42℃的范围，特别是在37℃附近的35℃～39℃示出良好的水解性，此外，确认了各温度下的水解活性高于EndoS。
(4－2)Endo－Si的反应pH的评价
Endo－Si和EndoS的各pH下的糖链水解率如下测定。制备包含0.1mg mAb1、以及10ng Endo－Si WT或EndoS WT的50mM柠檬酸－磷酸钠缓冲液(pH5.0或5.5)、或磷酸钠缓冲液(pH6.0、6.5或7.0)、或三羟甲基氨基甲烷盐酸缓冲液(pH7.5、8.0、8.5或9.0)50μL，在37℃下培养。对反应0.5小时的反应液进行取样，通过上述方法计算出糖链水解率。
将结果示于图19。Endo－Si在水解反应中的最适pH(酶良好地发挥作用的pH)为pH6.3～9.0的范围，进一步优选为pH6.7～8.8的范围，特别是在pH7.5附近的pH7.2～8.0示出良好的水解性。确认了与EndoS相比，Endo－Si在pH6.7附近以上的pH条件下的活性更高。
＜实施例5＞Endo－Si的底物特异性
(5－1)各种抗体中的Endo－Si的底物特异性评价
Endo－Si、EndoS、PNGaseF对各种抗体的水解活性如下测定。制备包含10μg的各种底物和1μg Endo－Si WT的50mM三羟甲基氨基甲烷盐酸缓冲液(pH7.5)50μL，在37℃下培养。底物使用人IgG1－4、IgA以及IgE(全部为Sigma-Aldrich制)。作为对照，使用1μg EndoSWT和500U PNGase F PRIME(N－zyme scientifics制)。2小时后对反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。
将结果示于图20。在通过添加酶而使糖链水解的情况下，与不添加酶的情况相比较，蛋白质的谱带向低分子量侧位移。实验的结果是，Endo－Si WT对IgG的四个亚类全部显示活性。另一方面，对于IgA和IgE未显示水解活性。在对照中使用的EndoS WT中，也确认到显示同样的底物特异性。
(5－2)各种糖链中的Endo－Si的底物特异性评价
各种糖链中的Endo－Si的底物特异性如下测定。制备了包含5pmol的各种2－AB标记糖链(Agilent Technologies制)和20μg Endo－Si WT的50mM三羟甲基氨基甲烷盐酸缓冲液(pH7.5)10μL。在37℃下培养24小时，之后在95℃下处理5分钟，由此使反应停止。在以下条件下对反应液进行HPLC分析。
[HPLC分析条件]
HPLC装置：1200Infinity LC(Agilent Technologies制)
柱：ACQUITY UPLC Glycan Amide
Figure BDA0004105389400000441
1.7μm，2.1x 150mm(Waters制)
柱温：40℃
检测器：荧光检测器RF－20Axs(岛津制作所制)
移动相A：H2O+0.1％HCOOH
移动相B：乙腈+0.1％HCOOH
梯度(移动相B％)：90％(0分钟)、40％(25分钟)
流速：0.2mL/min
根据底物及其水解物即GlcNAc-2AB的峰面积比计算酶活性。将对G0糖链的活性设为100％的情况的、对各种糖链的相对活性示于表4。Endo-Si对高甘露糖型糖链和复合型两天线糖链均显示活性，但与高甘露糖型糖链相比，对复合型两天线糖链的特异性高，其中对G0糖链的活性最高。对唾液酸糖链、岩藻糖基化糖链也显示活性，另一方面，未显示对复合型三天线糖链的活性。
[表4]各种糖链中的Endo-Si的水解活性
Figure BDA0004105389400000451
将相对于GO糖链的活性设为100％来计算相对活性。
Figure BDA0004105389400000452
甘露糖，
Figure BDA0004105389400000453
GlcNAc，
Figure BDA0004105389400000454
半乳糖，
Figure BDA0004105389400000455
岩藻糖，
Figure BDA0004105389400000456
Neu5Ac.
＜实施例6＞Endo-Si的改造与转移活性的测定
(6-1)[N3-PEG(3)]-MSG1(9)-Ox的制备
在以后的实施例中用作糖链供体的[N3-PEG(3)]-MSG1(9)-Ox通过WO2019/065964的实施例56中记载的方法制造。
Figure BDA0004105389400000461
(6－2)Endo－Si的改造与糖链转移活性的确认
为了获得糖链转移活性高的Endo－Si突变酶，实施突变引入。基于EndoS的立体结构信息(PDB ID：4NUY)，设计表1所示的各种突变酶，测定其对抗体的糖链转移活性。
糖链转移活性的评价如下进行。制备包含(Fucα1，6)GlcNAc－mAb20.5mg、糖链噁唑啉体[N3－PEG(3)]－MSG1(9)－Ox 50.4μg(8eq.)、酶1.25μg的50mM三羟甲基氨基甲烷盐酸缓冲液(pH7.5)45μL，在28℃下培养。对反应1小时、2小时、4小时、6小时以及24小时的反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。根据所得到的色谱图，未反应物和糖链转移体mAb2－(MSG1－N3)2被确认为分离的峰。根据未反应物与糖链转移体的峰面积比，通过下述计算式来计算糖链转移率。
糖链转移率(％)＝〔[源自mAb2－(MSG1－N3)2的H链的峰面积]/{[源自(Fucα1，6)GlcNAc－mAb2的H链峰面积]+[源自mAb2－(MSG1－N3)2的H链的峰面积]}〕×100
同样地计算出各Endo－Si突变酶在各反应时间的糖链转移率(表5)。
[表5]将噁唑啉体用于糖链供体的Endo－Si WT和各突变酶的糖链转移率的经时变化
[表5]
Figure BDA0004105389400000471
除了Endo－Si WT以外，各Endo－Si突变酶均显示高的糖链转移活性。
＜实施例7＞将SGP用于糖链供体的糖链转移活性的测定
糖链转移活性的评价如下进行。制备包含(Fucα1，6)GlcNAc－mAb2 0.5mg、SGP0.485mg(50eq.)、Endo－M N175Q(东京化成工业制)2.5mU、以及各种Endo－Si突变酶10μg的50mM磷酸(钠)缓冲液(pH7.5)17μL，在23℃下培养。对反应2小时、4小时、6小时、24小时以及48小时的反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。根据所得到的色谱图，未反应物和糖链转移体mAb2－(SG)2被确认为分离的峰。根据未反应物与糖链转移体的峰面积比，通过下述计算式来计算糖链转移率。
糖链转移率(％)＝〔[源自mAb2－(SG)2的H链的峰面积]/{[源自(Fucα1，6)GlcNAc－mAb2的H链峰面积]+[源自mAb2－(SG)2的H链的峰面积]}]×100
同样地计算出各Endo－Si突变酶在各反应时间的糖链转移率(表6)。
[表6]将SGP用于糖链供体的Endo－Si WT和各突变酶的糖链转移率的经时变化
[表6]
Figure BDA0004105389400000481
除了Endo－Si WT以外，各Endo－Si突变酶均显示高的糖链转移活性。
＜实施例8＞基于与Endo－Rp的组合的一锅法的研究
将已知从作为糖链供体的SGP向GlcNAc衍生物的糖链转移的Endo－Rp用作酶A的代表例，研究了能与Endo－Si组合的酶A的性质。
利用了一锅法的对抗体的糖链转移活性的评价如下进行。制备包含(Fucα1，6)GlcNAc－mAb2 0.5mg、SGP 0.485mg(50eq.)、Endo－M N175Q(东京化成工业制)2.5mU或各种Endo－Rp突变酶8μg、以及Endo－Si D241Q 5μg的50mM磷酸(钠)缓冲液(pH 7.5)17μL，在28℃下培养。对反应2小时、4小时、6小时、24小时以及48小时的反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。根据所得到的色谱图，使用实施例5中记载的计算式来计算糖链转移率。
将结果示于表7。将SGP转移至GlcNAc衍生物的活性低的Endo－Rp突变酶，例如专利文献3中完全未观察到转移活性的N172F、N172K、N172L、N172R、N172W、N172Y突变酶在一锅法中也成为转移效率低的结果。
由上述可知，酶A自身的糖链转移活性与糖链原料活化能力相关，可认为以从SGP向GlcNAc衍生物的转移活性为指标，能确定具有糖链原料活化能力的适当的酶A。一般而言，可认为Endo－M、Endo－Om、Endo－CC具有与Endo－Rp同样的反应性，因此将Endo－Rp取代为Endo－M、Endo－Om、Endo－CC，能够确定对一锅法有效的酶A。进而，在与Endo－Si同样地对抗体具有糖链转移活性的Endo－S、Endo－S2的情况下，也能应用该方法。
如此，可认为上述确定法的应用范围并不限定于Endo－Si与Endo－Rp的组合，只要是具有与各个酶同样的性质的酶，就可以利用。
[表7]利用了一锅法的各种突变酶对抗体的糖链转移活性的经时变化
Figure BDA0004105389400000491
＜实施例9＞将经化学修饰的糖链衍生物用于供体的一锅法的研究
作为天然型糖链即SGP以外的糖链衍生物供体，准备非还原末端侧和还原末端氨基酸被叠氮化物修饰的衍生物，研究了一锅法中的糖链转移反应。
(9－1)([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3的制备
在以后的实施例中用作糖链供体的([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3通过WO2019065964的实施例154的工序3中记载的方法制造。
Figure BDA0004105389400000501
(9－2)([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3的制备
在以后的实施例中用作糖链供体的([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3通过WO2018003983的实施例1－12、工序1－12A中记载的方法制造。
Figure BDA0004105389400000502
(9－3)糖链转移活性评价
糖链转移活性的评价如下进行。制备(Fucα1，6)GlcNAc－mAb2 0.5mg、SGP0.485mg(50eq.)或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3 0.415mg(50eq.)或([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3 0.495mg(50eq.)、Endo－Rp N172H 8μg、以及Endo－SiD241M/Q311L 10μg/50mM磷酸(钠)缓冲液(pH7.5)17μL，在28℃下培养。对反应2小时、4小时、6小时、24小时以及48小时的反应液进行取样，用上述蛋白质的全自动电泳系统进行分析。作为转移反应物，在将SGP用于供体的情况生成实施例7中记载的mAb2－(SG)2，在将([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3用于供体的情况生成mAb2－(MSG1－N3)2(图21)，在将([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3用于供体的情况生成mAb2－[SG－(N3)2]2(图22、式中称为mAb2－(SG－N3)2)。根据所得到的色谱图，未反应物与各糖链转移体被确认为分离的峰。将SGP用于供体的情况的糖链转移率使用实施例5中记载的公式来计算。将([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3用于供体的情况的糖链转移率使用实施例4－2中记载的公式来计算。此外，将([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3用于供体的情况的糖链转移率使用以下的计算式来计算。
糖链转移率(％)＝〔[源自mAb2－(SG－N3)2的H链的峰面积]/{[源自(Fucα1，6)GlcNAc－mAb2的H链峰面积]+[源自mAb2－(SG－N3)2的H链的峰面积]}〕×100
将结果示于表8。不仅是天然型的糖链结构，在将经化学修饰的糖链用于供体的情况下，也确认到进行一锅转移反应。
[表8]利用了一锅法的各种糖链供体向抗体的糖链转移活性的经时变化
Figure BDA0004105389400000511
产业上的可利用性
能高效或高纯度地获取使用本发明的Endo－Si酶而得到的具有均匀的糖链的抗体或含糖链分子，能将该抗体等用作医药。
序列号1：Endo－Si碱基序列
序列号2：Endo－Si氨基酸序列
序列号3：Endo－Si氨基酸序列D241Q
序列号4：Endo－Si氨基酸序列D241Q/Q311L
序列号5：Endo－Si氨基酸序列D241Q/E360Q
序列号6：Endo－Si氨基酸序列D241M
序列号7：Endo－Si氨基酸序列D241M/Q311L
序列号8：Endo－Si氨基酸序列D241M/E360Q
序列号9：Endo－Si氨基酸序列T190Q/D241Q
序列号10：Endo－Si氨基酸序列T190Q
序列号11：Endo－Si氨基酸序列T190Q/D241M
序列号12：mAb2轻链氨基酸序列
序列号13：mAb2重链氨基酸序列
序列号14：引物1
序列号15：引物2
序列号16：Endo－Si大肠杆菌用序列
序列号17：Endo－Rp氨基酸序列N172Q
序列号18：Endo－Rp氨基酸序列N172H
序列号19：Endo－Rp氨基酸序列N172A
序列号20：Endo－Rp氨基酸序列N172C
序列号21：Endo－Rp氨基酸序列N172D
序列号22：Endo－Rp氨基酸序列N172E
序列号23：Endo－Rp氨基酸序列N172F
序列号24：Endo－Rp氨基酸序列N172G
序列号25：Endo－Rp氨基酸序列N172I
序列号26：Endo－Rp氨基酸序列N172K
序列号27：Endo－Rp氨基酸序列N172L
序列号28：Endo－Rp氨基酸序列N172M
序列号29：Endo－Rp氨基酸序列N172P
序列号30：Endo－Rp氨基酸序列N172R
序列号31：Endo－Rp氨基酸序列N172S
序列号32：Endo－Rp氨基酸序列N172T
序列号33：Endo－Rp氨基酸序列N172V
序列号34：Endo－Rp氨基酸序列N172W
序列号35：Endo－Rp氨基酸序列N172Y
序列号36：Endo－Rp氨基酸序列W278F/S216V
序列号37：Endo－Rp氨基酸序列W278F/N246D
序列号38：Endo－Rp氨基酸序列W278F/D276N
序列号39：Endo－Rp氨基酸序列W278F/A310D
序列号40：Endo－Rp氨基酸序列W278F/N172D/F307Y
序列号41：Endo－Rp氨基酸序列W278F/N172D/F307H
序列号42：Endo－Rp氨基酸序列W278F/N172D/A310D
序列号43：Endo－Rp氨基酸序列W278F/F307Y/L306I
序列号44：Endo－M氨基酸序列
序列号45：Endo－Om氨基酸序列
序列号46：Endo－CC氨基酸序列
本说明书中引用的全部刊物、专利以及专利申请通过原样地引用来引入至本说明书中。
序列表
<110> 第一三共株式会社
<120> 新型内－β－N－乙酰氨基葡萄糖苷酶
<130> PH-9000-PCT
<150> JP 2020-147745
<151> 2020-09-02
<160> 46
<170> PatentIn 3.5版
<210> 1
<211> 2787
<212> DNA
<213> 海豚链球菌
<400> 1
atgaacaaac gtttattggt taaacgcact ttcggttgtg tctgcgcagc agctatttta 60
ggtgttgccc ccctcagcca tccaacaatc gtcgaggcaa gagaagaatt gaagatgcca 120
aacggccttg aacaatcaat tgctgacgtc gaagctaaaa ttgatgcctt aacatatctt 180
tcaaagaata gtaaagatga atttaagcat tccatgtatg aaatcccgtc aaatcgtgaa 240
cacaaaccag tatcacccaa acaggccttg caaaacgcta aaaaagctga tgctcaagca 300
gaacgccttg ccaaaatgac cattcctaaa aaggaagaac taaaagcact cgaaggacca 360
ctttacggtg gctatttccg tacctggcaa gataaaactt ctgatcctac tgaaactaat 420
aaggtcaact cctttgggga gttgcctaaa gaagttgacc tagcatttgt ttttcacgac 480
tacactaaag actatagcct tttctgggag gaattagcga caaaacaagt tccgaaactc 540
aacaagcaag gaacacgtgt gattcgtacc attccatggc gtttcttaag tggtgctgac 600
catagtgata tctctgctga taaggagaaa ttccctaaca ctgaagctgg aaacaaagca 660
ctggctaaag ctatcgttga tgaatatgtt tacaaataca accttgatgg cttagacatc 720
gatatcgaac gtgatagtgt tcctaaagtt aatgacaaag aagatcccga agcactggct 780
cgcaccgttg aagtctttaa agaaatcggc aaattaattg gtgcaaatgg cgctgacaag 840
agccgcctct taatcatgga cacaacctat actgctgaag aaaacccatt gattaaagaa 900
acagcacaat acctaaactt actcttggtt caagtctatg gcttctctgg agaaaatgga 960
aattatttac atcacaagaa catattagac gaaacaagta gcatggaagg cagatggcaa 1020
ggctatagca aatacattcg tccagaacaa tacatggtcg gcttttcatt ctatgaagaa 1080
aaggatttca ataatcgttg gaaagatatt aatgaagaag atccttccga tccacatatt 1140
ggtgagaaaa tccaaggaac gcgtgctgaa cggtatgcta agtggcaacc taaaacaggt 1200
gggctaaaag gtggtctctt ctcttatgcc attgaccgcg atggggttgc acaaccaaaa 1260
caaaaaactg agcacccaga actagacaag atcgttaaat ctgaatacaa agtatctaaa 1320
gctttgaaaa aactcatgat gacagatgac caataccaac caattgatca atctgatttt 1380
cctgataagg cactccgtga aagcattatt aaacaggtcg ggacaagacg tggcgactta 1440
gaacgtttca agggtactct aagacttgac aatcctgaga ttaaagattt gacaggtctt 1500
aacaaactta aaagagtcgc taaactagaa ctcatcaacc ttccaaaaat cactaaaatt 1560
gataaggacg atctcccaca aaaccttaaa cctttaactg acaatcaaaa atctaacctc 1620
gaaataaaag gcacttacga tgattcaaaa ctatataagg atattccagc ctttgatttg 1680
gtcatttctg gtctaagtgg tcttgaatca ttggatattt caggccatca gcgtgataca 1740
cttagtggta ttgatgcttc cacacttcct tctttaaaag caatcaatat ttctgataat 1800
cactttgatt tggcacaagg aactgaaaat cgtcacattc ttgatactat cttagctaca 1860
cttgctaaaa atggtgcctc aacagctagc tttgataaac aaaaaccaaa aggcctatac 1920
cctgaaagct atagtactgc cccacttcac ctccaagttg gccaaggtaa aatcaatgtc 1980
attgatgacc ttatctttgg aactcgcacc aatcaaaata ccttaattaa tactgaaaat 2040
gactttgagg cctataaaga gcaaaccatt cagggtaaac cttttattgc ccctgattat 2100
ctctatgaca actttaaagt tagttataag gaatactctg cttcaatcgt tgactcaaca 2160
cttgctgaaa caactgataa aaccattgat accgctaaag ctgaaactta tcaggtcact 2220
gtctcaaaca aggatggaaa aactgttcac tcagttaaag ttatcgttgg tgacgaaaaa 2280
cccatgatgg ttaacttagc acaagatgca aaaatcattg gtactgacaa catgactcaa 2340
agtgcaaaag tttttgatgg gcaaaaagac caatttcttt taagttggaa taaagactcc 2400
tctgtcattt ttgaattaaa aacccctggt acagccaaac actggcgctt ctttgatgat 2460
ggcaagaatg actctgtaac cctatctgtc ttcaaagggg atgcatctaa ctttgaaact 2520
gaaaaagaca aagccgaaaa ttgggttgaa atcacaaaag acagccgcaa aaatgacgac 2580
aaagtattca gtagtccatt agaagttgac aatgccaaat atttgaaagt gacaataaaa 2640
aaagaagcta agtatatcta cttcaacgaa cttcaaatcc ttggttatcc aggtgtagtt 2700
gctaaaaaaa ctgctgatga cctcagacca acagaggcag acaagagcga tgacaaatcc 2760
gacaaaaatg acacagaagc caagtaa 2787
<210> 2
<211> 928
<212> PRT
<213> 海豚链球菌
<400> 2
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Asp Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 3
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 D241Q
<400> 3
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Gln Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 4
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 D241Q/Q311L
<400> 4
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Gln Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Leu Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 5
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si 氨基酸序列D241Q/E360Q
<400> 5
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Gln Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Gln Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 6
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 D241M
<400> 6
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Met Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 7
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si 氨基酸序列D241M/Q311L
<400> 7
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Met Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Leu Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 8
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 D241M/E360Q
<400> 8
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Thr Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Met Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Gln Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 9
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 T190Q/D241Q
<400> 9
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Gln Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Gln Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 10
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 T190Q
<400> 10
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Gln Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Asp Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 11
<211> 928
<212> PRT
<213> 人工
<220>
<223> Endo-Si氨基酸序列 T190Q/D241M
<400> 11
Met Asn Lys Arg Leu Leu Val Lys Arg Thr Phe Gly Cys Val Cys Ala
1 5 10 15
Ala Ala Ile Leu Gly Val Ala Pro Leu Ser His Pro Thr Ile Val Glu
20 25 30
Ala Arg Glu Glu Leu Lys Met Pro Asn Gly Leu Glu Gln Ser Ile Ala
35 40 45
Asp Val Glu Ala Lys Ile Asp Ala Leu Thr Tyr Leu Ser Lys Asn Ser
50 55 60
Lys Asp Glu Phe Lys His Ser Met Tyr Glu Ile Pro Ser Asn Arg Glu
65 70 75 80
His Lys Pro Val Ser Pro Lys Gln Ala Leu Gln Asn Ala Lys Lys Ala
85 90 95
Asp Ala Gln Ala Glu Arg Leu Ala Lys Met Thr Ile Pro Lys Lys Glu
100 105 110
Glu Leu Lys Ala Leu Glu Gly Pro Leu Tyr Gly Gly Tyr Phe Arg Thr
115 120 125
Trp Gln Asp Lys Thr Ser Asp Pro Thr Glu Thr Asn Lys Val Asn Ser
130 135 140
Phe Gly Glu Leu Pro Lys Glu Val Asp Leu Ala Phe Val Phe His Asp
145 150 155 160
Tyr Thr Lys Asp Tyr Ser Leu Phe Trp Glu Glu Leu Ala Thr Lys Gln
165 170 175
Val Pro Lys Leu Asn Lys Gln Gly Thr Arg Val Ile Arg Gln Ile Pro
180 185 190
Trp Arg Phe Leu Ser Gly Ala Asp His Ser Asp Ile Ser Ala Asp Lys
195 200 205
Glu Lys Phe Pro Asn Thr Glu Ala Gly Asn Lys Ala Leu Ala Lys Ala
210 215 220
Ile Val Asp Glu Tyr Val Tyr Lys Tyr Asn Leu Asp Gly Leu Asp Ile
225 230 235 240
Met Ile Glu Arg Asp Ser Val Pro Lys Val Asn Asp Lys Glu Asp Pro
245 250 255
Glu Ala Leu Ala Arg Thr Val Glu Val Phe Lys Glu Ile Gly Lys Leu
260 265 270
Ile Gly Ala Asn Gly Ala Asp Lys Ser Arg Leu Leu Ile Met Asp Thr
275 280 285
Thr Tyr Thr Ala Glu Glu Asn Pro Leu Ile Lys Glu Thr Ala Gln Tyr
290 295 300
Leu Asn Leu Leu Leu Val Gln Val Tyr Gly Phe Ser Gly Glu Asn Gly
305 310 315 320
Asn Tyr Leu His His Lys Asn Ile Leu Asp Glu Thr Ser Ser Met Glu
325 330 335
Gly Arg Trp Gln Gly Tyr Ser Lys Tyr Ile Arg Pro Glu Gln Tyr Met
340 345 350
Val Gly Phe Ser Phe Tyr Glu Glu Lys Asp Phe Asn Asn Arg Trp Lys
355 360 365
Asp Ile Asn Glu Glu Asp Pro Ser Asp Pro His Ile Gly Glu Lys Ile
370 375 380
Gln Gly Thr Arg Ala Glu Arg Tyr Ala Lys Trp Gln Pro Lys Thr Gly
385 390 395 400
Gly Leu Lys Gly Gly Leu Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
405 410 415
Ala Gln Pro Lys Gln Lys Thr Glu His Pro Glu Leu Asp Lys Ile Val
420 425 430
Lys Ser Glu Tyr Lys Val Ser Lys Ala Leu Lys Lys Leu Met Met Thr
435 440 445
Asp Asp Gln Tyr Gln Pro Ile Asp Gln Ser Asp Phe Pro Asp Lys Ala
450 455 460
Leu Arg Glu Ser Ile Ile Lys Gln Val Gly Thr Arg Arg Gly Asp Leu
465 470 475 480
Glu Arg Phe Lys Gly Thr Leu Arg Leu Asp Asn Pro Glu Ile Lys Asp
485 490 495
Leu Thr Gly Leu Asn Lys Leu Lys Arg Val Ala Lys Leu Glu Leu Ile
500 505 510
Asn Leu Pro Lys Ile Thr Lys Ile Asp Lys Asp Asp Leu Pro Gln Asn
515 520 525
Leu Lys Pro Leu Thr Asp Asn Gln Lys Ser Asn Leu Glu Ile Lys Gly
530 535 540
Thr Tyr Asp Asp Ser Lys Leu Tyr Lys Asp Ile Pro Ala Phe Asp Leu
545 550 555 560
Val Ile Ser Gly Leu Ser Gly Leu Glu Ser Leu Asp Ile Ser Gly His
565 570 575
Gln Arg Asp Thr Leu Ser Gly Ile Asp Ala Ser Thr Leu Pro Ser Leu
580 585 590
Lys Ala Ile Asn Ile Ser Asp Asn His Phe Asp Leu Ala Gln Gly Thr
595 600 605
Glu Asn Arg His Ile Leu Asp Thr Ile Leu Ala Thr Leu Ala Lys Asn
610 615 620
Gly Ala Ser Thr Ala Ser Phe Asp Lys Gln Lys Pro Lys Gly Leu Tyr
625 630 635 640
Pro Glu Ser Tyr Ser Thr Ala Pro Leu His Leu Gln Val Gly Gln Gly
645 650 655
Lys Ile Asn Val Ile Asp Asp Leu Ile Phe Gly Thr Arg Thr Asn Gln
660 665 670
Asn Thr Leu Ile Asn Thr Glu Asn Asp Phe Glu Ala Tyr Lys Glu Gln
675 680 685
Thr Ile Gln Gly Lys Pro Phe Ile Ala Pro Asp Tyr Leu Tyr Asp Asn
690 695 700
Phe Lys Val Ser Tyr Lys Glu Tyr Ser Ala Ser Ile Val Asp Ser Thr
705 710 715 720
Leu Ala Glu Thr Thr Asp Lys Thr Ile Asp Thr Ala Lys Ala Glu Thr
725 730 735
Tyr Gln Val Thr Val Ser Asn Lys Asp Gly Lys Thr Val His Ser Val
740 745 750
Lys Val Ile Val Gly Asp Glu Lys Pro Met Met Val Asn Leu Ala Gln
755 760 765
Asp Ala Lys Ile Ile Gly Thr Asp Asn Met Thr Gln Ser Ala Lys Val
770 775 780
Phe Asp Gly Gln Lys Asp Gln Phe Leu Leu Ser Trp Asn Lys Asp Ser
785 790 795 800
Ser Val Ile Phe Glu Leu Lys Thr Pro Gly Thr Ala Lys His Trp Arg
805 810 815
Phe Phe Asp Asp Gly Lys Asn Asp Ser Val Thr Leu Ser Val Phe Lys
820 825 830
Gly Asp Ala Ser Asn Phe Glu Thr Glu Lys Asp Lys Ala Glu Asn Trp
835 840 845
Val Glu Ile Thr Lys Asp Ser Arg Lys Asn Asp Asp Lys Val Phe Ser
850 855 860
Ser Pro Leu Glu Val Asp Asn Ala Lys Tyr Leu Lys Val Thr Ile Lys
865 870 875 880
Lys Glu Ala Lys Tyr Ile Tyr Phe Asn Glu Leu Gln Ile Leu Gly Tyr
885 890 895
Pro Gly Val Val Ala Lys Lys Thr Ala Asp Asp Leu Arg Pro Thr Glu
900 905 910
Ala Asp Lys Ser Asp Asp Lys Ser Asp Lys Asn Asp Thr Glu Ala Lys
915 920 925
<210> 12
<211> 214
<212> PRT
<213> 人工
<220>
<223> mAb2轻链氨基酸序列
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Phe Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Pro Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 13
<211> 452
<212> PRT
<213> 人工
<220>
<223> mAb2重链氨基酸序列
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Val Asn Pro Asn Ser Gly Asp Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Gly Gly Tyr Asp Val Gly Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 14
<211> 25
<212> DNA
<213> 人工
<220>
<223> 引物1
<400> 14
agcttcgatg caatttactg gtaag 25
<210> 15
<211> 26
<212> DNA
<213> 人工
<220>
<223> 引物2
<400> 15
ggactttagt ctggcaaaac atactc 26
<210> 16
<211> 2805
<212> DNA
<213> 人工
<220>
<223> EndoSi 大肠杆菌用序列
<400> 16
atgaacaaac gcctgctggt taaacgtacc tttggttgtg tttgtgcagc agcaattctg 60
ggtgttgcac cgctgagcca tccgaccatt gttgaagcac gtgaagaact gaaaatgccg 120
aatggtctgg aacagagcat tgcagatgtt gaagccaaaa ttgatgcact gacctatctg 180
agcaaaaaca gcaaagatga attcaaacac agcatgtatg aaattccgag caaccgtgaa 240
cataaaccgg ttagcccgaa acaggcactg cagaatgcaa aaaaagcaga tgcacaggca 300
gaacgtctgg caaaaatgac cattccgaaa aaagaggaac tgaaagcact ggaaggtccg 360
ctgtatggtg gttattttcg tacctggcag gataaaacca gcgatccgac cgaaaccaat 420
aaagttaata gctttggtga actgccgaaa gaagttgatc tggcctttgt gtttcacgat 480
tataccaaag attatagcct gttttgggaa gaactggcaa ccaaacaggt tccgaaactg 540
aataaacagg gcacccgtgt tattcgtacc attccgtggc gttttctgag cggtgcagat 600
catagcgata ttagcgcaga taaagaaaaa tttccgaata ccgaagccgg taataaagcc 660
ctggcaaaag caattgttga tgagtacgtg tacaaatata acctggatgg cctggatatt 720
gatattgaac gtgatagcgt tccgaaggtg aacgataaag aagatccgga agcactggca 780
cgtaccgttg aagtttttaa agaaattggc aaactgatcg gtgcaaacgg tgccgataaa 840
agccgtctgc tgattatgga taccacctat accgcagaag aaaacccgct gattaaagaa 900
accgcacagt atctgaatct gctgctggtt caggtttatg gttttagcgg tgaaaatggc 960
aactatctgc atcacaaaaa catcctggat gaaaccagca gtatggaagg tcgttggcag 1020
ggttatagca aatatatccg tccggaacag tatatggtgg gctttagctt ttatgaagag 1080
aaagatttta acaaccgctg gaaggatatc aatgaagagg atccgagcga tccgcatatt 1140
ggtgaaaaaa ttcagggtac acgtgcagaa cgttatgcaa aatggcagcc gaaaaccggt 1200
ggtctgaaag gtggtctgtt tagctatgca attgatcgtg atggtgttgc ccagccgaaa 1260
cagaaaaccg aacatccgga actggataag attgtgaaaa gcgaatacaa agttagcaaa 1320
gccctgaaaa aactgatgat gaccgatgat cagtatcagc cgattgatca gagcgatttt 1380
ccggataaag cactgcgtga aagcatcatt aaacaggttg gcacccgtcg tggtgatctg 1440
gaacgtttta aaggcaccct gcgtctggat aatccggaaa ttaaagatct gaccggtctg 1500
aacaaactga aacgtgttgc aaaactggaa ctgattaacc tgccgaaaat caccaaaatc 1560
gataaagatg acctgccgca gaatctgaaa ccgctgacag ataatcagaa aagcaacctg 1620
gaaatcaaag gcacctatga tgatagcaaa ctgtacaaag atattccggc atttgatctg 1680
gttattagtg gtctgtcagg tctggaaagt ctggatattt caggtcatca gcgtgatacc 1740
ctgagcggta ttgatgcaag caccctgccg agcctgaaag caattaacat tagcgataac 1800
catttcgatc tggcccaggg caccgaaaat cgtcatattc tggataccat tctggcaacc 1860
ctggccaaaa atggtgccag caccgcaagc tttgataaac agaaacctaa aggtctgtac 1920
ccggaaagct atagcaccgc accgctgcat ctgcaggttg gtcagggtaa aatcaatgtt 1980
attgacgatc tgatttttgg cacgcgcacc aatcagaata ccctgattaa taccgaaaac 2040
gatttcgagg cctataaaga acagacaatt cagggcaaac cgtttattgc accggattac 2100
ctgtatgaca atttcaaggt tagctataaa gagtacagcg ccagcattgt ggatagcacc 2160
ctggcagaaa ccaccgataa aacaattgat accgcaaaag ccgaaaccta tcaggttacc 2220
gtgagcaata aagatggtaa aaccgttcat agcgtgaaag tgattgtggg tgatgaaaaa 2280
ccaatgatgg ttaatctggc acaggatgca aaaatcattg gcaccgataa tatgacccag 2340
agcgcaaaag tttttgatgg tcagaaagat cagtttctgc tgagctggaa taaagatagc 2400
agcgtgattt ttgagctgaa aacccctggc accgcaaaac attggcgttt ttttgatgat 2460
ggcaaaaacg atagcgttac gctgagcgtg tttaaaggtg atgccagcaa ttttgaaacc 2520
gagaaagata aagcggaaaa ctgggtcgaa attacgaaag atagccgtaa aaacgacgac 2580
aaagttttta gcagccctct ggaagttgat aacgccaaat atctgaaagt gacgatcaag 2640
aaagaggcca agtatatcta tttcaacgaa ctgcagattc tgggttatcc gggtgttgtt 2700
gccaaaaaaa ccgcagacga tctgcgtccg actgaagcag ataaaagtga tgataaaagc 2760
gacaaaaatg ataccgaggc caaacaccac catcaccatc cactga 2805
<210> 17
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172Q
<400> 17
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Gln Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 18
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172H
<400> 18
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu His Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 19
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172A
<400> 19
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Ala Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 20
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172C
<400> 20
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Cys Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 21
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172D
<400> 21
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asp Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 22
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172E
<400> 22
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Glu Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 23
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172F
<400> 23
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Phe Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 24
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172G
<400> 24
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Gly Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 25
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172I
<400> 25
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Ile Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 26
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172K
<400> 26
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Lys Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 27
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172L
<400> 27
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Leu Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 28
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172M
<400> 28
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Met Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 29
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172P
<400> 29
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Pro Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 30
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172R
<400> 30
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Arg Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 31
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172S
<400> 31
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Ser Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 32
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 N172T
<400> 32
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Thr Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 33
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172V
<400> 33
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Val Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 34
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172W
<400> 34
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Trp Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 35
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列N172Y
<400> 35
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Tyr Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 36
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 W278F/S216V
<400> 36
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asn Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Val Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 37
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/N246D
<400> 37
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asn Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asp Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 38
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP氨基酸序列 W278F/D276N
<400> 38
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asn Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asn Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 39
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/A310D
<400> 39
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asn Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Asp Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 40
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/N172D/F307Y
<400> 40
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asp Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Tyr Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 41
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/N172D/F307H
<400> 41
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asp Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu His Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 42
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/N172D/A310D
<400> 42
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asp Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Leu Phe Gly Thr Asp Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 43
<211> 696
<212> PRT
<213> 人工
<220>
<223> Endo-RP 氨基酸序列W278F/F307Y/L306I
<400> 43
Met Pro Ser Leu Glu Leu Gln Gln Ala Ala Asp Thr Arg Leu Phe Glu
1 5 10 15
Ser Met Pro Leu Gln Thr Met Asn Glu Leu Gly Ser Trp Glu Pro Ser
20 25 30
Asn Ala Ser Arg Ala Asn Ile Ala Thr Ile Pro Leu His Gln Arg Ser
35 40 45
Asn Leu Asp Pro Ala Glu Pro Arg Leu Ile Val Thr His Asp Met Ala
50 55 60
Gly Gly Tyr Lys Glu Asp Ser Asn Ile Gln Gly Asn Thr Tyr Asp Thr
65 70 75 80
Ile Tyr Ser Cys Gln Tyr Trp Gln Tyr Val Asp Thr Phe Ile Tyr Phe
85 90 95
Ser His His Arg Val Thr Ile Pro Pro Val Asn Trp Ile Asn Ala Cys
100 105 110
His Arg Asn Gly Val Lys Thr Leu Gly Thr Phe Ile Val Glu Gly Ala
115 120 125
Ala Gly Met Phe Ala Leu Glu Arg Phe Val Tyr Gly Pro Glu Pro Gly
130 135 140
Gln Arg Asn Ser Trp Ser Pro Tyr Tyr Ala Asp Lys Leu Val Asp Ile
145 150 155 160
Ala Glu Phe Tyr Gly Phe Asp Gly Trp Leu Leu Asn Ile Glu Ser Asp
165 170 175
Phe Phe Pro Leu Tyr Arg Asn Pro Ser Leu Lys Ala Ile His Leu Ala
180 185 190
Lys Leu Leu Arg Tyr Leu Lys Asn Ala Met His Ala Arg Val Pro Gly
195 200 205
Ser Glu Ile Ile Trp Tyr Asp Ser Met Thr Thr Asn Gly Ser Val Gln
210 215 220
Trp Gln Asn Asn Ile Thr Pro Lys Asn Ser Ile Phe Phe Glu Ala Ala
225 230 235 240
Asp Gly Ile Phe Leu Asn Tyr Trp Trp Asn Ala Thr Val Pro Pro Leu
245 250 255
Ala Leu Gln Val Ala His Arg Leu Gly Arg Gln Gly Ser Asp Val Tyr
260 265 270
Phe Gly Thr Asp Val Trp Gly Arg Gly Thr Phe Gly Gly Gly Gly Phe
275 280 285
Asp Ser Tyr Leu Ala Val Gly Thr Ala Arg Ala Phe Lys Thr Ser Ser
290 295 300
Ala Ile Tyr Gly Thr Ala Trp Ile Tyr Glu His Phe Gly Lys Lys Asp
305 310 315 320
Phe Glu Leu Met Asp Arg Leu Leu Trp Leu Gly Gly Asp Gln Ser Glu
325 330 335
Tyr Pro Ala Gln Glu Gly Glu Gln Asn Arg Thr Val Lys Val Thr Ser
340 345 350
His Leu Gly Arg His Pro Gly Ile Ala Asp Val Ser Pro Val Arg Ser
355 360 365
Ala Pro Gly Lys Thr Trp Phe Ala Thr Trp Phe Asp Arg Gly Tyr Gly
370 375 380
Thr Gly Phe Tyr Tyr Gln Gly Lys Lys Leu Leu Ser Gln Pro Trp Ser
385 390 395 400
His Leu Ser His Gln Ser Ile Pro Pro Asn Leu Ile Ala Arg Leu Gln
405 410 415
Arg Glu Glu Asn His Gly Leu Ser Tyr Phe Leu Ala Asp Asp Asp Ala
420 425 430
Tyr Ile Gly Gly Thr Ser Leu Leu Ile Ala Ala Glu Ile Thr Gln Glu
435 440 445
Arg Gln Leu Pro Leu Tyr Gln Leu Glu Tyr Asp Val Thr Glu Gly Cys
450 455 460
Glu Val Gln Phe Ile Tyr Lys Ser Pro Glu Pro Asp Met Gln Gly Lys
465 470 475 480
Ile Asp Ile Tyr Leu Asn Leu Gln Val Thr Asp Ile Leu Pro Asp Glu
485 490 495
Leu Ala Phe Tyr Trp Gln Asp Val Thr Asp Ala Ser Ser Gln Ala Asp
500 505 510
Ala Thr Thr Ala Met Arg Leu Tyr Leu Asn Glu Asn Thr Val Ile Tyr
515 520 525
Leu Lys Pro Ser Arg Lys Gln Glu Leu Ala Glu Gly Trp Leu Leu Cys
530 535 540
Ser Val Arg Val Pro Pro Thr Tyr Pro Leu Gly Ile Ala Thr Ile Lys
545 550 555 560
Glu Leu Gly Ile His Val Asp Gly Lys Glu Thr Val Leu Phe Arg Leu
565 570 575
Gly Leu Leu Thr Ile Ile Pro Leu Gly Asp Ala Pro Ser Ala Leu Ser
580 585 590
Arg Ile Thr Gln Val Gln Leu Gln Arg Asp Glu Asp Ile His Ser Lys
595 600 605
Cys Pro Ser Ser Ser Cys Glu Leu Trp Ala Thr Leu Ser Trp Met Met
610 615 620
Glu His Asn Ser Lys Glu Asp Trp Asp Gln Val Asp His Tyr Met Ile
625 630 635 640
Phe Phe Lys Asn Val Asp Ser Lys Ala Glu Pro Ile Phe Leu Gly Thr
645 650 655
Ser Phe Ser Thr Glu Tyr Arg Ile Ser Gly Leu Glu Ile Lys Lys His
660 665 670
Gly Asn Ser Ile Glu Ile Trp Ala Val Asn Arg Leu Gly Thr Val Ile
675 680 685
Ala Arg Gln Asp Ile Asp Ile Gln
690 695
<210> 44
<211> 744
<212> PRT
<213> 人工
<220>
<223> Endo-M氨基酸序列
<400> 44
Met Pro Ser Leu Gln Leu Gln Pro Asp Asp Lys Leu Ala Pro Val Ser
1 5 10 15
Phe Ala Leu Lys Ser Met Asn Glu Leu Arg Asp Trp Thr Pro Asp Glu
20 25 30
Lys Ile Lys Phe Asn Val Ser Ser Val Ala Leu Gln Pro Arg Val Lys
35 40 45
Asn Ala Leu Lys Pro Gln Leu Leu Leu Thr His Asp Met Ala Gly Gly
50 55 60
Tyr Lys Glu Asp Lys Asn Ile Gln Gly Asn Asn Tyr Lys Asp Ile Tyr
65 70 75 80
Asn Ile Gln Tyr Trp His Leu Ala Asp Thr Phe Val Tyr Phe Ser His
85 90 95
Glu Arg Val Ser Ile Pro Pro Val Asn Trp Thr Asn Ala Cys His Arg
100 105 110
Asn Gly Val Lys Cys Leu Gly Thr Phe Leu Val Glu Gly Asn Asn Gln
115 120 125
Met His Glu Met Glu Ala Leu Leu His Gly Pro Pro Leu Leu Asn Asn
130 135 140
Thr Asp Asp Pro Met Arg Leu Trp Ser Pro Tyr Tyr Ala Asp Gln Leu
145 150 155 160
Val Ala Ile Ala Lys His Tyr Gly Phe Asp Gly Trp Leu Phe Asn Ile
165 170 175
Glu Cys Glu Phe Phe Pro Phe Pro Thr Asn Pro Lys Phe Lys Ala Glu
180 185 190
Glu Leu Ala Lys Phe Leu His Tyr Phe Lys Glu Lys Leu His Asn Glu
195 200 205
Ile Pro Gly Ser Gln Leu Ile Trp Tyr Asp Ser Met Thr Asn Glu Gly
210 215 220
Glu Ile His Trp Gln Asn Gln Leu Thr Trp Lys Asn Glu Leu Phe Phe
225 230 235 240
Lys Asn Thr Asp Gly Ile Phe Leu Asn Tyr Trp Trp Lys Lys Glu Tyr
245 250 255
Pro Glu Met Ala Arg Arg Val Ala Glu Gly Ile Gly Arg Ser Gly Leu
260 265 270
Glu Val Tyr Phe Gly Thr Asp Val Trp Gly Arg His Thr Tyr Gly Gly
275 280 285
Gly Gly Phe Lys Ser Tyr Lys Gly Val Lys Thr Ala Tyr Ser Ala Met
290 295 300
Thr Ser Ser Ala Leu Phe Gly Met Ala Trp Thr Tyr Glu His Phe Glu
305 310 315 320
Lys Ser Glu Phe Glu Lys Met Asp Arg Leu Phe Trp Cys Gly Gly Lys
325 330 335
Tyr Ser Asp Tyr Pro Pro Pro Pro Pro Lys Asn Pro Asp Asp Glu Lys
340 345 350
Glu Val Glu Ser Asp Asp Ser Glu Asp Glu Leu Met Tyr Gly His Lys
355 360 365
Lys Gly Ile Ala Asp Thr Val Glu Ser Ile Pro Val Pro Gly Thr Asp
370 375 380
Trp Phe Val Thr Asn Phe Asp Arg Gly Phe Gly Asn Arg Phe Tyr Tyr
385 390 395 400
Arg Gly Lys Arg Leu Leu Ser Gln Pro Trp Ser His Leu Ser His Gln
405 410 415
Ala Ile Leu Pro Asn Lys Ser Tyr Arg Asn Pro Glu Ile Tyr Pro Thr
420 425 430
Asp Gln Asn Ile Lys Ile Thr Ser Ser Leu Asp Cys Asp His Gly Ala
435 440 445
Phe Leu Gly Gly Thr Ser Leu Ile Ile Lys Gly Gln Arg Phe Asn His
450 455 460
Arg Glu Ser His Asp Val Glu Thr Glu Ile Ser Ile Pro Leu Tyr Lys
465 470 475 480
Leu Ser Leu Asp Ala Ser Lys Gly Cys Ser Leu Arg Tyr Ile Tyr Arg
485 490 495
Thr Leu Leu Met Lys Asp Val Lys Leu Thr Val Ala Cys His Phe Ser
500 505 510
Leu Lys Thr Asn Asp Ser Val Asn Phe Phe Lys Val Trp Gln Pro Asp
515 520 525
Glu Asn Phe Ser Phe Glu Tyr Asp Asp Gly Met Arg Ala Thr Val Thr
530 535 540
Thr Glu Asn Ser Thr Glu Ser Arg Cys Phe Leu Leu Arg Thr Thr Glu
545 550 555 560
Glu Asp Thr Gly Glu Asn Asp Trp Ile Thr Lys Thr Ile Asn Val Pro
565 570 575
Ala Val Pro Glu Gly Ser Gln Leu Tyr Ile Thr Arg Leu Glu Val Ser
580 585 590
Val Val Leu Asp Thr Ala Gly Leu Val Gly Leu Val Asn Gln Val Ile
595 600 605
Ala Cys Leu Gly Tyr Ile Ser Ile Ile Pro Thr Ile Asn Ser Gly Ile
610 615 620
Lys Thr Asp Ser Ser Arg Ile Ile Gln Asp Leu Phe Trp Lys Asp Gln
625 630 635 640
Lys Tyr Thr Lys Ile Gly Lys Glu Ser Leu Asp Asp Ile Ala Gln Glu
645 650 655
Glu Val His Arg Tyr Tyr Gly Thr Leu Asn Trp Glu Asn Thr Ala Asn
660 665 670
Val Val Asn Ala Trp Glu Glu Ile Asp Tyr Tyr Asn Val Phe Tyr Lys
675 680 685
Glu Ser Asp Asp Ser Ala Thr Arg Ile Phe Leu Gly Thr Ala Phe Cys
690 695 700
Asn Gln Phe Arg Val Ser Gly Leu Asp Ile Ile Leu Ser Lys Leu Pro
705 710 715 720
Lys Ile Val Ile Glu Ala Val Asn Lys Glu Gly Tyr Ile Ser Ser Ser
725 730 735
Gly Ser Ile Asp Leu Ser Leu Asn
740
<210> 45
<211> 772
<212> PRT
<213> 人工
<220>
<223> Endo-Om氨基酸序列
<400> 45
Met Ala Gln Ser Gln Leu Leu Gly Gly Ala Val Arg Pro Val Phe Phe
1 5 10 15
Asp Lys Leu Glu Glu Leu Arg Arg Trp His Thr Gln Ser Ala Asn Leu
20 25 30
Ser Arg Glu Ser Glu Leu Asp Ser Leu Asn Val Ala Thr Glu Pro Phe
35 40 45
Ser Ser Tyr Glu Arg Ala Gln Thr Gly Ser Gly Ser Arg Ser Ser Glu
50 55 60
Pro Val Pro Gly Asp Lys Glu Asp Pro Pro Ile Lys Leu Met Val Cys
65 70 75 80
His Asp Phe Lys Gly Gly Tyr Gln Asp Tyr Glu Asp Ala Gln Pro Leu
85 90 95
Gly Tyr Phe Pro His Pro Thr Gly Ser Arg Tyr Phe Leu Gln Tyr Pro
100 105 110
Gln Leu Ile Asp Gln Phe Val Tyr Phe Ser His His Arg Val Thr Val
115 120 125
Pro Pro Val Asn Trp Ile Asn Phe Cys His Arg Asn Gly Ile Lys Cys
130 135 140
Phe Gly Thr Val Ile Phe Glu Gly Asn Ala Ser Lys Asp Phe Glu Glu
145 150 155 160
Leu Asp Arg Leu Val Ser Arg Asp Glu Lys Gly Asp Phe Val Phe Val
165 170 175
Asp Ala Leu Ile Lys Leu Ala Ala His Tyr Gly Phe Asp Gly Tyr Leu
180 185 190
Leu Asn Ile Glu Thr Thr Phe Ser Asn Thr Lys Ile Ala Ala Asp Leu
195 200 205
Glu Pro Phe Ala Glu Gln Leu Lys Ser Gly Leu His Cys Leu Asp Ser
210 215 220
Lys Asn Glu Leu Ile Trp Tyr Asp Ser Tyr Val Phe Pro Ala Asn Lys
225 230 235 240
Val Ser Tyr Thr Asn Gly Val Thr Glu Ser Asn Tyr Asn Phe Phe Ser
245 250 255
Leu Ser Asp Ala Phe Phe Ser Asn Tyr Trp Trp Asn Ile Lys Asn Leu
260 265 270
Gln Glu Asn Ile Lys Asn Val Gly Val Leu Gly Val Gln Lys Lys Ile
275 280 285
Tyr Val Gly Tyr Asp Val Trp Gly Arg Gly Thr Leu Val Gly Lys Gly
290 295 300
Gly Phe Asp Ser Ser Leu Ala Cys Lys Met Ile Ala Lys Phe Lys Ser
305 310 315 320
Asn Val Ala Leu Phe Ala Pro Ala Trp Thr Tyr Glu Ser Leu Gly Pro
325 330 335
Lys Asp Phe Asn Gln Asn Asp Ala Arg Phe Trp Ile Gly Leu Phe Glu
340 345 350
Asn Glu Ser Ser Ile Ser Ser Thr Val Pro Pro His Ser Ser Ala Val
355 360 365
Tyr Lys Ile Asn Glu Ser Ser Phe Ile Phe Tyr Thr Asn Phe Ser Ser
370 375 380
Gly Glu Gly Asn Arg Phe Phe Ser Lys Gly Ser Glu Val Tyr Arg Lys
385 390 395 400
Asn Trp Val Asn Gly Ser Leu Gln Phe Asp Leu Pro Ile Asp Leu His
405 410 415
Arg Lys Asp Lys Asn Gly Leu Gln Trp Ala Leu Asp Lys Ser Asp Ala
420 425 430
Phe His Gly Gly Ala Cys Leu Glu Ile Lys Tyr Ser Glu Ile Lys Asp
435 440 445
Glu Asn Gly Tyr Gln Ile Phe Asn Asn Gln Met Val Ser Asp Phe Thr
450 455 460
Leu Phe Asn Phe Thr Lys Glu Cys His Phe Pro Thr Val Asn Val Lys
465 470 475 480
Val Thr Tyr Lys Leu Asn His Lys Thr Lys Ser Thr Phe Lys Ile Lys
485 490 495
Ile Lys Tyr Ile Ile Glu Arg Arg Phe Arg Ser Val Gln Thr Val Arg
500 505 510
Thr Gly Tyr Leu Thr Ile Pro Leu Leu Ser Thr Ser Gly Lys Trp Phe
515 520 525
Thr Val Glu Glu Ser Phe Gln Ile Asn Leu Gln Thr Ser His Glu Tyr
530 535 540
Ile Val Leu Glu Ser Ala His Val Thr Tyr Asp Glu Asp Arg Ser Ala
545 550 555 560
Asp Ser Phe Phe Arg Ser Tyr Ile Val Glu Asp Ser Ala Ile Thr Ser
565 570 575
Val Ile Asp Asn Glu Glu Tyr Glu Lys Leu Ile Asn Ser Glu Ile Tyr
580 585 590
Asn Asp Asp Glu Asp Glu Asp Trp Ile Leu Val Pro Ser Asp Val Ser
595 600 605
Ile Ser Ser Ser Glu Ser Gln Ser Asn Asp Ser Lys Thr Gln Tyr Leu
610 615 620
Gly Arg Lys Leu Phe Gly Asn Lys Ser Thr Pro Lys Thr Arg Thr Leu
625 630 635 640
Glu Gly Thr Ala Pro Leu Leu Arg Ile Gly Glu Phe Ala Ile Ile Ser
645 650 655
Ala Asn Asn Tyr Pro Ser Ser Asn Phe Leu Ala Val Thr Ser Val Lys
660 665 670
Ser Ile Glu Ser Ser Arg Leu Glu Gly Asp Ser Leu Val Leu Leu Asn
675 680 685
Trp Gln Val Gly Glu Gly His Gln Lys Gly Val Cys Tyr Tyr Ile Ile
690 695 700
Tyr Val Asn Gly Ala Val Val Gly Leu Ser Val Ala Pro Lys Phe Ile
705 710 715 720
Tyr Gln Asp Thr Glu Leu Ala Ser Glu Asn Ser Ala Ser Ala Arg Ser
725 730 735
Asn Tyr Lys Lys Ser Gly Leu Gly Ser Ser Ser Asp Arg Lys Ser Lys
740 745 750
Val Arg Val Asp Ser Val Asp Lys Leu Gly Asn Val Phe Thr Gly Ser
755 760 765
Glu Val Trp Val
770
<210> 46
<211> 787
<212> PRT
<213> 人工
<220>
<223> Endo-CC氨基酸序列
<400> 46
Met Pro Ile Ala Gly Lys Lys Phe His Pro Arg Ala Leu Pro Glu Phe
1 5 10 15
Trp Arg Thr Phe Arg Glu Met Asp Glu Trp Arg Ala Thr Gln Thr Gly
20 25 30
Pro Gln Ala Arg Pro Ala Glu Gly Ile Leu Lys Tyr Val Pro Arg Lys
35 40 45
Ile Arg Pro Ala Asp Ile Ala Gly Lys Gly Arg Leu Leu Val Ser His
50 55 60
Asp Tyr Lys Gly Gly Tyr Val Glu Asp Pro Phe Ser Lys Ser Tyr Ser
65 70 75 80
Phe Asn Trp Trp Phe Ser Thr Asp Ser Phe Asn Tyr Phe Ala His His
85 90 95
Arg Ile Thr Ile Pro Pro Pro Glu Trp Ile Asn Ala Ala His Arg Gln
100 105 110
Gly Val Pro Ile Leu Gly Thr Ile Ile Phe Glu Gly Gly Ser Asp Glu
115 120 125
Asp Ile Leu Arg Met Val Ile Gly Lys Thr Pro Gly Ser Thr Ser Asn
130 135 140
Phe His Ala Glu Arg Asn Ala Glu Tyr Thr Val Pro Val Ser Ser Tyr
145 150 155 160
Tyr Ala Glu Leu Phe Ala Asp Leu Ala Val Glu Arg Gly Phe Asp Gly
165 170 175
Trp Leu Leu Asn Val Glu Ile Gly Leu Gln Gly Gly Ser Glu Gln Ala
180 185 190
Arg Gly Leu Ala Ala Trp Val Ala Leu Leu Gln Gln Glu Val Leu Lys
195 200 205
Lys Val Gly Pro His Gly Leu Val Ile Trp Tyr Asp Ser Val Thr Val
210 215 220
Arg Gly Asp Leu Trp Trp Gln Asp Arg Leu Asn Ala Phe Asn Leu Pro
225 230 235 240
Phe Phe Leu Asn Ser Ser Gly Ile Phe Thr Asn Tyr Trp Trp Tyr Asn
245 250 255
Asp Ala Pro Gln Lys Gln Ile Asp Phe Leu Ser Arg Val Asp Pro Asn
260 265 270
Leu Thr Gly Gln Thr Ala Glu Pro His Gln Tyr Asn Leu Gln Lys Thr
275 280 285
Ile Gln Asp Ile Tyr Ile Gly Val Asp Val Trp Gly Arg Gly Ser His
290 295 300
Gly Gly Gly Gly Phe Gly Ala Tyr Lys Ala Ile Glu His Ala Asp Pro
305 310 315 320
Lys Gly Leu Gly Phe Ser Val Ala Leu Phe Ala Gln Gly Trp Thr Trp
325 330 335
Glu Thr Glu Glu Glu Lys Pro Gly Trp Asn Trp Ala Gln Phe Trp Asp
340 345 350
Tyr Asp Ser Lys Leu Trp Val Gly Pro Pro Gly Val Val Glu Ala Pro
355 360 365
Asp His Thr Val Lys Pro Gly Glu Tyr Pro Cys Val His Gly Pro Phe
370 375 380
Gln Pro Ile Ser Ser Phe Phe Leu Thr Tyr Pro Pro Pro Asp Pro Leu
385 390 395 400
Asp Leu Pro Phe Tyr Thr Asn Phe Cys Pro Gly Ile Gly Asp Ala Trp
405 410 415
Phe Val Glu Gly Lys Glu Val Phe Arg Ser Glu Thr Gly Trp Thr Asp
420 425 430
Met Asp Lys Gln Thr Thr Val Gly Asp Leu Val Trp Pro Arg Pro Lys
435 440 445
Ile Tyr Asp Leu Pro Ser Gln Asn Ala Ser Gln Ala Thr Leu Asn Ala
450 455 460
Ala Phe Asn Phe Asn Asp Ala Trp Asn Gly Gly Asn Ser Leu Gln Ile
465 470 475 480
Asn Leu Thr Val Pro Gly Gly Ala Thr Thr Tyr Gly Ala Tyr Trp Val
485 490 495
Pro Ile Gln Thr Phe Thr Phe Ser Ser Arg Arg Gln Tyr Glu Ala Ser
500 505 510
Ile Val Tyr Lys Pro Gly Leu Ser Gly Lys Thr Arg Phe Asp Ala Lys
515 520 525
Tyr Glu Val Gly Ile Arg Thr Ile Thr Gly Glu Asp Gln Gly Lys Ile
530 535 540
Ile Ser Asn Thr Thr Thr Glu Val Gly Asn Gly Trp Arg Lys Val His
545 550 555 560
Ile Leu Phe Glu Ile Glu Thr Pro Val Glu Gly Gly Ser Ile Ile Val
565 570 575
Pro Ser Ser Ile Gly Leu Val Ile Ala Val Ser Asn Val Ser Thr Thr
580 585 590
Glu Gln Phe Glu Phe Pro Phe Leu Val Gly Gln Ile Thr Ile His Pro
595 600 605
His Leu Pro Asp Arg Tyr Lys Glu Phe Lys Pro Ala Leu Leu Trp Leu
610 615 620
Leu Phe Thr Pro Ser Ala Gly Thr Asn Ser Leu Asp Gly Thr Leu Thr
625 630 635 640
Trp Asp Val Val Ala Ala Ile Glu Arg Pro Pro Pro Val Glu Ile Asn
645 650 655
Asn Pro Asp Asp Ala Gln Ile Pro Trp Asn Leu Gln Pro Thr Lys Gln
660 665 670
Glu Trp Phe Pro Asp Phe Leu Tyr Phe Asn Val Tyr Val Leu Glu Leu
675 680 685
Leu Asp Gly Gly Gly Gln Gly Pro Pro Gln Trp Ile Gly Thr Thr Gly
690 695 700
Tyr Asp Gly Glu Lys Lys Arg Phe Phe Ile Tyr Asp Glu Ser Leu Pro
705 710 715 720
Pro Thr Ser Gly Leu Arg Arg Phe Thr Phe Gln Ile Glu Gly Val Leu
725 730 735
Glu Thr Gly Glu Ser Thr His Trp Tyr Asp Ala Pro Ala Ala Pro Ser
740 745 750
Ala Thr Ala Gly Gly Glu Gln Lys Arg Thr Arg Arg Thr Ser Leu Lys
755 760 765
Ser Val Leu Ser Pro Leu Arg Arg Lys Lys Ser Lys Gly Asp Ile Ser
770 775 780
Val Ala Lys
785

Claims (44)

1.一种多肽，其具有：
序列号2的第34～928位所记载的氨基酸序列；或
在所述氨基酸序列中，存在包含对选自由第241位即D241、第190位即T190、第311位即Q311以及第360位即E360的氨基酸构成的组中的一个或两个以上氨基酸位点的突变的氨基酸序列，并且，
所述多肽显示糖链水解活性和/或糖链转移活性。
2.根据权利要求1所述的多肽，其特征在于，
对于突变，在选自由D241、T190、Q311以及E360的氨基酸构成的组中的1～3个氨基酸位点具有突变。
3.根据权利要求1或2所述的多肽，其中，
具有选自由以下的(A)～(D)构成的组中的一个或两个以上的突变，
(A)：在序列号2的氨基酸序列中，第241位的氨基酸D241突变为谷氨酰胺D241Q、第241位的氨基酸D241突变为蛋氨酸D241M或第241位的氨基酸D241突变为丙氨酸D241A；
(B)：在序列号2的氨基酸序列中，第190位的氨基酸T190突变为谷氨酰胺T190Q；
(C)：在序列号2的氨基酸序列中，第311位的氨基酸Q311突变为亮氨酸Q311L；以及
(D)：在序列号2的氨基酸序列中，第360位的氨基酸E360突变为谷氨酰胺E360Q、第360位的氨基酸E360突变为丙氨酸E360A、第360位的氨基酸E360突变为天冬酰胺E360N或第360位的氨基酸E360突变为天冬氨酸E360D。
4.根据权利要求1～3中任一项所述的多肽，其中，
具有选自由以下的(A)～(D)构成的组中的一个或两个以上的突变，
(A)：D241Q或D241M；
(B)：T190Q；
(C)：Q311L；以及
(D)：E360Q。
5.根据权利要求1～4中任一项所述的多肽，其中，
包含以下的(A)～(C)中任一项所述的氨基酸序列，
(A)：选自由序列号3、序列号4、序列号5、序列号6、序列号7、序列号8、序列号9、序列号10以及序列号11构成的组中的氨基酸序列；
(B)：与(A)的各序列中的第241位、第190位、第311位、或第360位的氨基酸以外的氨基酸序列具有至少90％以上的同源性或同一性的氨基酸序列；或
(C)：在(A)的序列中第241位、第190位、第311位、或第360位的氨基酸以外的氨基酸序列中缺失、取代和/或添加了一个或数个氨基酸的氨基酸序列。
6.根据权利要求1～5中任一项所述的多肽，其中，
所述多肽对N连接型糖链显示水解活性和/或糖链转移活性。
7.根据权利要求6所述的多肽，其中，
N连接型糖链是糖蛋白中的N连接型糖链。
8.根据权利要求6或7所述的多肽，其中，
糖蛋白是抗体或包含抗体的Fc区的分子即含Fc区的分子。
9.根据权利要求6～9中任一项所述的多肽，其中，
N连接型糖链是与抗体的第297位的Asn连接的N连接型糖链即N297连接糖链。
10.根据权利要求9所述的多肽，其中，
N297连接糖链是非还原末端任选地被化学修饰的复合型糖链。
11.根据权利要求9或10所述的多肽，其中，
N297连接糖链是在核心GlcNAc任选地添加岩藻糖的N297连接糖链。
12.一种多核苷酸，其编码如权利要求1～11中任一项所述的多肽。
13.一种表达载体，其包含如权利要求12所述的多核苷酸。
14.一种宿主细胞，其通过如权利要求13所述的表达载体而被转化。
15.一种如权利要求1～11中任一项所述的多肽的制造方法，其特征在于，包括：
培养如权利要求14所述的宿主细胞的工序；以及
从该工序中得到的培养物中采集目标多肽的工序。
16.一种多肽，其通过如权利要求15所述的制造方法而得到。
17.一种抗体或含其Fc区的分子的制造方法，其特征在于，
在如权利要求1～11中任一项所述的多肽的存在下，使受体分子与包含还原末端经活化的GlcNAc的糖链供体分子进行反应，
其中，所述受体分子是具有任选地添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。
18.根据权利要求17所述的制造方法，其中，
还原末端经活化的GlcNAc是经噁唑啉化的GlcNAc。
19.根据权利要求17或18所述的制造方法，其中，
糖链供体分子是非还原末端任选地被化学修饰的复合型糖链。
20.根据权利要求17～19中任一项所述的制造方法，其中，
糖链供体分子是非还原末端任选地被化学修饰的SG(10)－Ox、MSG1(9)－Ox、MSG2(9)－Ox或MSG1(9)－Ox与MSG2(9)－Ox的混合物。
21.根据权利要求17～20中任一项所述的制造方法，其中，
糖链供体分子是[N3－PEG(3)]2－SG(10)－Ox、[N3－PEG(3)]－MSG1(9)－Ox、[N3－PEG(3)]－MSG2(9)－Ox、或[N3－PEG(3)]－MSG1(9)－Ox与[N3－PEG(3)]－MSG2(9)－Ox的混合物。
22.根据权利要求21所述的制造方法，其中，还包括：
使叠氮基N3－与具有炔烃结构的分子进行反应的工序。
23.根据权利要求22所述的制造方法，其中，
具有炔烃结构的分子选自化学治疗剂、分子靶向药、免疫活化剂、毒素、抗菌剂、抗病毒剂、诊断用药剂、蛋白质、肽、氨基酸、核酸分子、核酸、抗原、脂质、脂质体、维生素以及激素中。
24.根据权利要求23所述的制造方法，其中，
化学治疗剂选自喜树碱、吡咯并苯并二氮杂卓、阿霉素、澳瑞他汀、紫杉烷或其衍生物中。
25.根据权利要求23所述的制造方法，其中，
免疫活化剂选自STING激动剂、TLR激动剂、A2AR拮抗剂、IDO抑制剂、CTLA－4、LAG－3以及PD－1途径的拮抗剂、检查点抑制剂、血管内皮生长因子VEGF受体抑制剂、平滑蛋白抑制剂、烷基化剂、代谢拮抗剂、类视黄醇、抗癌疫苗以及佐剂中。
26.根据权利要求23～25中任一项所述的制造方法，其中，
具有炔烃结构的分子选自由(A)～(E)构成的组中，
(A)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、
(B)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－[4－({[(11’S，11’aS)－11’－羟基－7’－甲氧基－8’－(3－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}丙氧基)－5’－桥氧基－11’，11’a－二氢－1’H，3’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－羰基]氧基}甲基)苯基]－L－丙氨酰胺、
(C)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、
(D)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，10’，11’，11a’－四氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、以及
(E)：(双(N，N－二乙基乙铵)N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－苯丙氨酰基－N－[(2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R)－15－氟－16－羟基－2，10－二桥氧基－2，10－二硫－14－(6，7，8，9－四氢－2H－2，3，5，6－四氮杂苯并[cd]薁－2－基)八氢－2H，10H，12H－5，8－桥亚甲基－2λ5，10λ5－呋喃并[3，2－l][1，3，6，9，11，2，10]五氧杂二膦环十四炔－7－基]－6－桥氧基－6，9－二氢－1H－嘌呤－1－基}乙氧基)甲基]甘氨酰胺。
27.根据权利要求17～26中任一项所述的制造方法，其中，
受体分子是具有由任选地添加岩藻糖的核心GlcNAc构成的N297连接糖链的抗体或含Fc区的分子。
28.一种抗体或含Fc区的分子的制造方法，其特征在于，
在如权利要求1～11中任一项所述的多肽和酶A的存在下，使受体分子与包含还原末端未被活化的GlcNAc的糖链供体分子进行反应，
其中，所述酶A是以还原末端未被活化的糖链供体分子的复合型糖链为底物但不以N297连接糖链为底物的内－β－N－乙酰氨基葡萄糖苷酶，
所述受体分子是具有任选地添加岩藻糖的核心GlcNAc作为N297连接糖链的抗体或含其Fc区的分子。
29.根据权利要求28所述的制造方法，其特征在于，
使如权利要求1～11中任一项所述的多肽、酶A、受体分子以及糖链供体分子在同一反应液中进行反应。
30.根据权利要求28或29所述的制造方法，其中，
糖链供体分子是非还原末端任选地被化学修饰的复合型糖链。
31.根据权利要求28～30中任一项所述的制造方法，其中，
糖链供体分子是非还原末端任选地被化学修饰的SGP、(SG－)Asn、(MSG1－)Asn、(MSG2－)Asn、(MSG1－)Asn与(MSG2－)Asn的混合物。
32.根据权利要求28～31中任一项所述的制造方法，其中，
糖链供体分子是([N3－PEG(3)]2－SG－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3、([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3、或([N3－PEG(3)]－MSG1－)Asn－PEG(3)－N3与([N3－PEG(3)]－MSG2－)Asn－PEG(3)－N3的混合物。
33.根据权利要求32所述的制造方法，其中，还包括：
使叠氮基N3－与具有炔烃结构的分子进行反应的工序。
34.根据权利要求33所述的制造方法，其中，
具有炔烃结构的分子选自化学治疗剂、分子靶向药、免疫活化剂、毒素、抗菌剂、抗病毒剂、诊断用药剂、蛋白质、肽、氨基酸、核酸、抗原、脂质、脂质体、维生素以及激素中。
35.根据权利要求34所述的制造方法，其中，
化学治疗剂选自喜树碱、吡咯并苯并二氮杂卓、阿霉素、澳瑞他汀、紫杉烷或其衍生物中。
36.根据权利要求35所述的制造方法，其中，
免疫活化剂选自STING激动剂、TLR激动剂、A2AR拮抗剂、IDO抑制剂、CTLA－4、LAG－3以及PD－1途径的拮抗剂、检查点抑制剂、血管内皮生长因子VEGF受体抑制剂、平滑蛋白抑制剂、烷基化剂、代谢拮抗剂、类视黄醇、抗癌疫苗以及佐剂中。
37.根据权利要求34～36中任一项所述的制造方法，其中，
具有炔烃结构的分子选自由(A)～(E)构成的组中，
(A)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、
(B)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－[4－({[(11’S，11’aS)－11’－羟基－7’－甲氧基－8’－(3－{[(11aS)－7－甲氧基－2－(4－甲氧基苯基)－5－桥氧基－5，10，11，11a－四氢－1H－吡咯并[2，1－c][1，4]苯并二氮杂卓－8－基]氧基}丙氧基)－5’－桥氧基－11’，11’a－二氢－1’H，3’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－羰基]氧基}甲基)苯基]－L－丙氨酰胺、
(C)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、
(D)：N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－缬氨酰基－N－{4－[({[(11’S，11a’S)－11’－羟基－7’－甲氧基－8’－[(5－{[(11a’S)－7’－甲氧基－5’－桥氧基－5’，10’，11’，11a’－四氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－8’－基]氧基}戊基)氧基]－5’－桥氧基－11’，11a’－二氢－1’H－螺[环丙烷－1，2’－吡咯并[2，1－c][1，4]苯并二氮杂卓]－10’(5’H)－基]羰基}氧基)甲基]苯基}－L－丙氨酰胺、以及
(E)：(双(N，N－二乙基乙铵)N－[4－(11，12－二脱氢二苯并[b，f]氮杂环辛－5(6H)－基)－4－桥氧基丁酰基]甘氨酰基甘氨酰基－L－苯丙氨酰基－N－[(2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R)－15－氟－16－羟基－2，10－二桥氧基－2，10－二硫－14－(6，7，8，9－四氢－2H－2，3，5，6－四氮杂苯并[cd]薁－2－基)八氢－2H，10H，12H－5，8－桥亚甲基－2λ5，10λ5－呋喃并[3，2－l][1，3，6，9，11，2，10]五氧杂二膦环十四炔－7－基]－6－桥氧基－6，9－二氢－1H－嘌呤－1－基}乙氧基)甲基]甘氨酰胺。
38.根据权利要求28～37中任一项所述的制造方法，其中，
受体分子是具有由任选地添加岩藻糖的核心GlcNAc构成的N297连接糖链的抗体或或含Fc区的分子。
39.根据权利要求28～38中任一项所述的制造方法，其中，
酶A是具有从SGP向具有GlcNAc的受体的糖链转移活性的酶。
40.根据权利要求28～39中任一项所述的制造方法，其中，
酶A是Endo－M、Endo－Rp、Endo－Om、Endo－CC、或使它们的水解活性降低的突变酶。
41.根据权利要求40所述的制造方法，其中，
使水解活性降低的突变酶选自由Endo－Rp N172Q、Endo－Rp N172H、Endo－Rp N172A、Endo－Rp N172C、Endo－Rp N172D、Endo－Rp N172E、Endo－Rp N172G、Endo－Rp N172I、Endo－Rp N172L、Endo－Rp N172M、Endo－Rp N172P、Endo－Rp N172S、Endo－Rp N172T、Endo－Rp N172V、Endo－Rp W278F/S216V、Endo－Rp W278F/N246D、Endo－Rp W278F/D276N、Endo－Rp W278F/A310D、Endo－Rp W278F/N172D/F307Y、Endo－Rp W278F/N172D/F307H、Endo－Rp W278F/N172D/A310D、Endo－Rp W214F/F307Y/L306I、Endo－M N175Q、Endo－CC N180H以及Endo－Om N194Q构成的组中。
42.一种抗体或含Fc区的分子，其通过如权利要求17～41中任一项所述的制造方法而得到。
43.一种仅具有任选地添加岩藻糖的核心GlcNAc的抗体或含Fc区的分子的制造方法，其特征在于，
使具有序列号2的第34～928位所记载的氨基酸序列的多肽作用于抗体或含Fc区的分子。
44.一种仅具有核心GlcNAc的抗体或含Fc区的分子，其通过如权利要求43所述的制造方法而得到。
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